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1.  DNA Hairpin Stabilization on a Hydrophobic Surface 
DNA hybridization in the vicinity of surfaces is a fundamental process for self-assembled nanoarrays, nanocrystal superlattices, and biosensors. It is widely recognized that solid surfaces alter molecular forces governing hybridization relative to bulk solution, and these effects can either favor or disfavor the hybridized state depending on the specific sequence and surface. Results presented here provide new insights into the dynamics of DNA hairpin-coil conformational transitions in the vicinity of hydrophilic oligo(ethylene glycol) (OEG) and hydrophobic trimethylsilane (TMS) surfaces. Single-molecule methods are used to observe the forward and reverse hybridization hairpin-coil transition of adsorbed species while simultaneously measuring molecular surface diffusion in order to gain insight into surface interactions with individual DNA bases. At least 35,000 individual molecular trajectories are observed on each type of surface. We find that unfolding slows and the folding rate increases on TMS relative to OEG despite stronger attractions between TMS and unpaired nucleobases. These rate differences lead to nearly complete hairpin formation on hydrophobic TMS and significant unfolding on hydrophilic OEG, resulting in the surprising conclusion that hydrophobic surface coatings are preferable for nanotechnology applications that rely on DNA hybridization near surfaces.
doi:10.1002/smll.201202335
PMCID: PMC3741999  PMID: 23184340
conformational change; molecular diffusion; poly(ethylene glycol); self-assembly; single-molecule tracking
2.  Effects of Molecular Size and Surface Hydrophobicity on Oligonucleotide Interfacial Dynamics 
Biomacromolecules  2012;13(12):4002-4011.
Single-molecule total internal reflection fluorescence microscopy was used to observe the dynamic behavior of (poly)-cytosine ssDNA (1–50 nucleotides long) at the interface between aqueous solution and hydrophilic (oligoethylene oxide-modified fused silica, OEG) and hydrophobic (octadecyltriethoxysilane-modified fused silica, OTES) solid surfaces. High throughput molecular tracking was used to determine >75,000 molecular trajectories for each molecular length, which were then used to calculate surface residence time and squared displacement (i.e. “step-size”) distributions. On hydrophilic OEG surfaces, the surface residence time increased systematically with ssDNA chain length, as expected due to increasing molecule-surface interactions. Interestingly, the residence time decreased with increasing ssDNA length on the hydrophobic OTES surface, particularly for longer chains. Similarly, the interfacial mobility of polynucleotides slowed with increasing chain length on OEG, but became faster on OTES. On OTES surfaces, the rates associated with desorption and surface diffusion exhibited the distinctive anomalous temperature dependence that is characteristic of hydrophobic interactions for short chain species but not for longer chains. These combined observations suggest that long oligonucleotides adopt conformations minimizing hydrophobic interactions, e.g. by internal sequestration of hydrophobic nucleobases.
doi:10.1021/bm301289n
PMCID: PMC3518566  PMID: 23127250
3.  Identifying Mechanisms of Interfacial Dynamics Using Single-Molecule Tracking 
The “soft” (i.e. non-covalent) interactions between molecules and surfaces are complex and highly-varied (e.g. hydrophobic, hydrogen bonding, ionic) often leading to heterogeneous interfacial behavior. Heterogeneity can arise either from spatial variation of the surface/interface itself or from molecular configurations (i.e. conformation, orientation, aggregation state, etc.). By observing adsorption, diffusion, and desorption of individual fluorescent molecules, single-molecule tracking can characterize these types of heterogeneous interfacial behavior in ways that are inaccessible to traditional ensemble-averaged methods. Moreover, the fluorescence intensity or emission wavelength (in resonance energy transfer experiments) can be used to simultaneously track molecular configuration and directly relate this to the resulting interfacial mobility or affinity. In this feature article, we review recent advances involving the use of single-molecule tracking to characterize heterogeneous molecule-surface interactions including: multiple modes of diffusion and desorption associated with both internal and external molecular configuration, Arrhenius activated interfacial transport, spatially dependent interactions, and many more.
doi:10.1021/la3017134
PMCID: PMC3429661  PMID: 22716995
4.  Distinguishing Positional Uncertainty from True Mobility in Single-Molecule Trajectories That Exhibit Multiple Diffusive Modes 
Although imperfect spatial localization in single-molecule object tracking experiments has long been recognized to induce apparent motion in an immobile population of molecules, this effect is often ignored or incorrectly analyzed for mobile molecules. In particular, apparent motion due to positional uncertainty is often incorrectly assigned as a distinct diffusive mode. Here we show that, due to both static and dynamic contributions, positional uncertainty does not introduce a new apparent diffusive mode into trajectories, but instead causes a systematic shift of each measured diffusion coefficient. This shift is relatively simple: a factor of σ2/Δt is added to each diffusion coefficient, where σ is the positional uncertainty length scale and Δt is the time interval between observations. Therefore, by calculating the apparent diffusion coefficients as a function of Δt, it is straightforward to separate the true diffusion coefficients from the effective positional uncertainty. As a concrete demonstration, we apply this approach to the diffusion of the protein fibrinogen adsorbed to a hydrophobic surface, a system that exhibits three distinct modes of diffusion.
doi:10.1017/S1431927612000438
PMCID: PMC3410552  PMID: 22849801
diffusion; total internal reflection fluorescence microscopy (TIRFM); fibrinogen; static error; dynamic error; single molecule
5.  Microcystic macular oedema in multiple sclerosis is associated with disease severity 
Brain  2012;135(6):1786-1793.
Macular oedema typically results from blood–retinal barrier disruption. It has recently been reported that patients with multiple sclerosis treated with FTY-720 (fingolimod) may exhibit macular oedema. Multiple sclerosis is not otherwise thought to be associated with macular oedema except in the context of comorbid clinical uveitis. Despite a lack of myelin, the retina is a site of inflammation and microglial activation in multiple sclerosis and demonstrates significant neuronal and axonal loss. We unexpectedly observed microcystic macular oedema using spectral domain optical coherence tomography in patients with multiple sclerosis who did not have another reason for macular oedema. We therefore evaluated spectral domain optical coherence tomography images in consecutive patients with multiple sclerosis for microcystic macular oedema and examined correlations between macular oedema and visual and ambulatory disability in a cross-sectional analysis. Participants were excluded if there was a comorbidity that could account for the presence of macular oedema, such as uveitis, diabetes or other retinal disease. A microcystic pattern of macular oedema was observed on optical coherence tomography in 15 of 318 (4.7%) patients with multiple sclerosis. No macular oedema was identified in 52 healthy controls assessed over the same period. The microcystic oedema predominantly involved the inner nuclear layer of the retina and tended to occur in small, discrete patches. Patients with multiple sclerosis with microcystic macular oedema had significantly worse disability [median Expanded Disability Score Scale 4 (interquartile range 3–6)] than patients without macular oedema [median Expanded Disability Score Scale 2 (interquartile range 1.5–3.5)], P = 0.0002. Patients with multiple sclerosis with microcystic macular oedema also had higher Multiple Sclerosis Severity Scores, a measure of disease progression, than those without oedema [median of 6.47 (interquartile range 4.96–7.98) versus 3.65 (interquartile range 1.92–5.87), P = 0.0009]. Microcystic macular oedema occurred more commonly in eyes with prior optic neuritis than eyes without prior optic neuritis (50 versus 27%) and was associated with lower visual acuity (median logMAR acuity of 0.17 versus −0.1) and a thinner retinal nerve fibre layer. The presence of microcystic macular oedema in multiple sclerosis suggests that there may be breakdown of the blood–retinal barrier and tight junction integrity in a part of the nervous system that lacks myelin. Microcystic macular oedema may also contribute to visual dysfunction beyond that explained by nerve fibre layer loss. Microcystic changes need to be assessed, and potentially adjusted for, in clinical trials that evaluate macular volume as a marker of retinal ganglion cell survival. These findings also have implications for clinical monitoring in patients with multiple sclerosis on sphingosine 1-phosphate receptor modulating agents.
doi:10.1093/brain/aws098
PMCID: PMC3359753  PMID: 22539259
multiple sclerosis; optical coherence tomography; retina; macular oedema
6.  Discounting of delayed rewards and executive dysfunction in individuals infected with hepatitis C 
Objective
Determine whether adults with hepatitis C, regardless of substance use disorder, are more likely to discount delayed rewards than adults without hepatitis C, and explore the relationship between delay discounting and neuropsychological functioning.
Methods
Procedures included clinical interviews, neuropsychological testing, and a delay discounting task.
Results
Regardless of substance abuse history, adults with hepatitis C were significantly more likely to choose smaller immediate rewards over larger delayed rewards. Delay discounting correlated with performance on executive functioning tasks.
Conclusions
Increased discounting is associated with broad executive dysfunction, suggesting that HCV associated executive dysfunction may lead to altered decision making style.
doi:10.1080/13803395.2010.499355
PMCID: PMC3615977  PMID: 20694872
hepatitis C; neuropsychology; impulsive behavior; substance-related disorders; delay discounting
7.  Biomineralization and Size Control of Stable Calcium Phosphate Core Protein Shell Nanoparticles: Potential for Vaccine Applications 
Bioconjugate Chemistry  2012;23(3):610-617.
Calcium phosphate (CaP) polymorphs are nontoxic, biocompatible and hold promise in applications ranging from hard tissue regeneration to drug delivery and vaccine design. Yet, simple and robust routes for the synthesis of protein-coated CaP nanoparticles in the sub-100 nm size range remain elusive. Here, we used cell surface display to identify disulfide-constrained CaP binding peptides that, when inserted within the active site loop of E. coli Thioredoxin 1 (TrxA), readily and reproducibly drive the production of nanoparticles that are 50–70 nm in hydrodynamic diameter and consist of an approximately 25 nm amorphous calcium phosphate (ACP) core stabilized by the protein shell. Like bone and enamel proteins implicated in biological apatite formation, peptides supporting nanoparticle production were acidic. They also required presentation in a loop for high affinity ACP binding since elimination of the disulfide bridge caused a nearly 3-fold increase in hydrodynamic diameters. When compared to a commercial aluminum phosphate adjuvant, the small core-shell assemblies led to a 3-fold increase in mice anti-TrxA titers three weeks post-injection, suggesting that they might be useful vehicles for adjuvanted antigen delivery to dendritic cells.
doi:10.1021/bc200654v
PMCID: PMC3310268  PMID: 22263898
8.  Spontaneous epiretinal membrane separation 
Oman Journal of Ophthalmology  2013;6(1):56-57.
doi:10.4103/0974-620X.111921
PMCID: PMC3678201  PMID: 23772129
9.  Connecting Rare DNA Conformations and Surface Dynamics using Single-Molecule Resonance Energy Transfer 
ACS nano  2011;5(12):9861-9869.
A mechanistic understanding of single-stranded DNA (ssDNA) behavior in the near-surface environment is critical to advancing DNA-directed self-assembled nanomaterials. A new approach is described that uses total internal reflection fluorescence microscopy to measure resonance energy transfer at the single-molecule level, providing a mechanistic understanding of the connection between molecular conformation and interfacial dynamics near amine-modified surfaces. Large numbers (>105) of ssDNA trajectories were observed, permitting dynamic correlation of molecular conformation with desorption and surface mobility. On the basis of dynamic behavior, molecules could be designated as members of the more common coiled population or a rare, weakly bound conformation. Molecules in the coiled state generally exhibited slow diffusion and conformational fluctuations that decreased with increasing average end-to-end distance. Lattice simulations of adsorbed self-avoiding polymers successfully predicted these trends. In contrast, the weakly bound conformation, observed in about 5% of molecules, had a large end-to-end distance but demonstrated conformational fluctuations that were much higher than predicted by simulations for adsorbed flexible chains. This conformation correlated positively with desorption events and led to fast diffusion, indicating weak surface associations. Understanding the role of the weakly bound conformation in DNA hybridization, and how solution conditions and surface properties may favor it, could lead to improved self-assembled nanomaterials.
doi:10.1021/nn2035389
PMCID: PMC3246573  PMID: 21942411
Single-molecule; resonance energy transfer; DNA; diffusion; adsorption; desorption; conformation
10.  Using Bacteria to Determine Protein Kinase Specificity and Predict Target Substrates 
PLoS ONE  2012;7(12):e52747.
The identification of protein kinase targets remains a significant bottleneck for our understanding of signal transduction in normal and diseased cellular states. Kinases recognize their substrates in part through sequence motifs on substrate proteins, which, to date, have most effectively been elucidated using combinatorial peptide library approaches. Here, we present and demonstrate the ProPeL method for easy and accurate discovery of kinase specificity motifs through the use of native bacterial proteomes that serve as in vivo libraries for thousands of simultaneous phosphorylation reactions. Using recombinant kinases expressed in E. coli followed by mass spectrometry, the approach accurately recapitulated the well-established motif preferences of human basophilic (Protein Kinase A) and acidophilic (Casein Kinase II) kinases. These motifs, derived for PKA and CK II using only bacterial sequence data, were then further validated by utilizing them in conjunction with the scan-x software program to computationally predict known human phosphorylation sites with high confidence.
doi:10.1371/journal.pone.0052747
PMCID: PMC3530509  PMID: 23300758
11.  GLOBAL AND LOCAL MORPHOMETRIC DIFFERENCES IN RECENTLY ABSTINENT METHAMPHETAMINE-DEPENDENT INDIVIDUALS 
NeuroImage  2010;50(4):1392-1401.
Methamphetamine (MA) is associated with behavioral and cognitive deficits that may be related to macrostructural abnormalities. Quantitative anatomical comparisons between controls and methamphetamine-dependent individuals have produced conflicting results. We examined local and global differences in brain structure in 61 abstinent methamphetamine-dependent individuals and 44 controls with voxel-based morphometry and tissue segmentation. We related regional differences in gray matter density and whole brain segmentation volumes to performance on a behavioral measure of impulsivity and group membership using multiple linear regression. Within the MA group, we related cortical and subcortical gray matter density to MA use history, length of abstinence and age of first use. Controls had greater density relative to MA in bilateral insula and left middle frontal gyrus. Impulsivity was higher in the MA group and, within all subjects, impulsivity was positively correlated with gray matter density in posterior cingulate cortex and ventral striatum and negatively correlated in left superior frontal gyrus. Length of abstinence from MA was associated with greater amygdalar density. Earlier age of first use of MA (in subjects who initiated use before age 21) was associated with smaller intracranial volume. The findings are consistent with multiple possible mechanisms including neuroadaptations due to addictive behavior, neuroinflammation as well as dopaminergic and serotonergic neurotoxicity.
doi:10.1016/j.neuroimage.2010.01.056
PMCID: PMC3478236  PMID: 20096794
12.  Hippocampal M1 receptor function associated with spatial learning and memory in aged female rhesus macaques 
Age  2010;33(3):309-320.
Of the acetylcholine muscarinic receptors, the type 1 (M1) and type 2 (M2) receptors are expressed at the highest levels in the prefrontal cortex (PFC) and hippocampus, brain regions important for cognition. As equivocal findings of age-related changes of M1 and M2 in the nonhuman primate brain have been reported, we first assessed age-related changes in M1 and M2 in the PFC and hippocampus using saturation binding assays. Maximum M1 receptor binding, but not affinity of M1 receptor binding, decreased with age. In contrast, the affinity of M2 receptor binding, but not maximum M2 receptor binding, increased with age. To determine if in the elderly cognitive performance is associated with M1 or M2 function, we assessed muscarinic function in elderly female rhesus macaques in vivo using a scopolamine challenge pharmacological magnetic resonance imaging and in vitro using saturation binding assays. Based on their performance in a spatial maze, the animals were classified as good spatial performers (GSP) or poor spatial performers (PSP). In the hippocampus, but not PFC, the GSP group showed a greater change in T2*-weighted signal intensity after scopolamine challenge than the PSP group. The maximum M1 receptor binding and receptor binding affinity was greater in the GSP than the PSP group, but no group difference was found in M2 receptor binding. Parameters of circadian activity positively correlated with the difference in T2*-weighted signal intensity before and after the challenge, the maximum M1 receptor binding, and the M1 receptor binding affinity. Thus, while in rhesus macaques, there are age-related decreases in M1 and M2 receptor binding, in aged females, hippocampal M1, but not M2, receptor function is associated with spatial learning and memory and circadian activity.
doi:10.1007/s11357-010-9184-2
PMCID: PMC3168603  PMID: 20890730
M1 receptor; Scopolamine phMRI; Spatial maze
13.  Human Resistin: Found in Translation From Mouse to Man 
The discovery of resistin ten years ago as a fat cell-secreted factor that modulates insulin resistance suggested a link to the obesity-associated epidemics of diabetes and cardiovascular disease that are major human health concerns. While adipocyte-derived resistin is indisputably linked to insulin resistance in rodent models, the relevance of human resistin is complicated because human resistin is secreted by macrophages rather than adipocytes, and because of the descriptive nature of human epidemiology. Here we review the recent and growing evidence that human resistin is an inflammatory biomarker and potential mediator of diabetes and cardiovascular disease.
doi:10.1016/j.tem.2011.03.005
PMCID: PMC3130099  PMID: 21497511
14.  Noninvasive Imaging of the Foveal Avascular Zone with High-Speed, Phase-Variance Optical Coherence Tomography 
This study demonstrates two- and three-dimensional capillary networks of the human parafoveal region without use of any contrast agents or adaptive optics. Phase-variance optical coherence tomography provides precise measurement of the foveal avascular zone area from one single volumetric scan within 5-second acquisition time.
Purpose.
To demonstrate the application of phase-variance optical coherence tomography (pvOCT) for contrast agent–free in vivo imaging of volumetric retinal microcirculation in the human foveal region and for extraction of foveal avascular zone dimensions.
Methods.
A custom-built, high-speed Fourier-domain OCT retinal imaging system was used to image retinas of two healthy subjects and eight diabetic patients. Through the acquisition of multiple B-scans for each scan location, phase differences between consecutive scans were extracted and used for phase-variance contrast, identifying motion signals from within blood vessels and capillaries. The en face projection view of the inner retinal layers segmented out from volumetric pvOCT data sets allowed visualization of a perfusion network with the foveal avascular zone (FAZ). In addition, the authors presented 2D retinal perfusion maps with pseudo color-coded depth positions of capillaries.
Results.
Retinal vascular imaging with pvOCT provides accurate measurements of the FAZ area and its morphology and a volumetric perfusion map of microcapillaries. In this study using two images from each fundus fluorescein angiography (FA) and pvOCT, the measured average areas of the FAZ from two healthy subjects were below 0.22 mm2, and each of eight diabetic patients had an enlarged FAZ area, larger than 0.22 mm2. Moreover, the FAZ areas demonstrated a significant correlation (r = 0.91) between measurements from FA and pvOCT.
Conclusions.
The high-speed pvOCT allows contrast agent–free visualization of capillary networks in the human foveal region that is analogous to fundus FA imaging. This could allow for noninvasive diagnosis and progression monitoring of diabetic retinopathy in clinical settings.
doi:10.1167/iovs.11-8249
PMCID: PMC3292386  PMID: 22125275
15.  Single-molecule resolution of interfacial fibrinogen behavior: Effects of oligomer populations and surface chemistry 
Using single-molecule total internal reflection fluorescence microscopy, the dynamic behavior of fibrinogen was observed at the interface between aqueous solution and various solid surfaces. Multiple populations of objects were observed, as characterized by surface residence times, interfacial diffusion, and fluorescence intensity. On all surfaces, populations exhibited direct links between surface residence time, rate of diffusion and fluorescence intensity. In particular longer-lived populations diffused more slowly and exhibited greater fluorescence intensity, leading to the conclusion that the objects represented fibrinogen monomers and discrete oligomer populations (dimers, trimers, etc.), and that these oligomer populations play an important role in the protein-surface interaction because of their long surface residence times. Two or three diffusive modes were observed for most populations, indicating that protein aggregates have multiple mechanisms for interaction with solid substrates. In addition, the fastest diffusive mode is believed to represent a hopping mode that often precedes desorption events. Surprisingly, a monolayer of PEG(5000) increased surface residence time and slowed diffusion of fibrinogen relative to bare fused silica or hydrophobically-modified fused silica, suggesting that the mechanism of PEG resistance to protein adhesion is more sophisticated than the simple repulsion of individual proteins.
doi:10.1021/ja110663u
PMCID: PMC3077424  PMID: 21391676
16.  Evaluation of deficiencies in current discharge summaries for dialysis patients in Canada 
Background
Deficits in the transfer of information between inpatient and outpatient physicians are common and pose a patient safety risk. This is particularly the case for vulnerable populations such as patients with end-stage renal disease requiring dialysis. These patients have unique and complex health care needs that may not be effectively communicated on standard discharge summaries, which may result in potential medical errors and adverse events.
Objective
To evaluate Canadian dialysis center directors’ perceptions of deficiencies in the content and quality of hospital discharge summaries for dialysis patients.
Methods
A web-based, cross-sectional survey of Canadian dialysis center directors was performed between September and November 2010. The survey consisted of three parts. The first part was designed to assess dialysis center directors’ attitudes on the quality of discharge summaries they receive. The second part was designed to elicit respondents’ preferences for discharge summary content, and the third part consisted of questions regarding demographic and practice information.
Results
Of 79 dialysis center directors, 21 (27%) completed the survey. Sixty-two percent felt that current discharge summaries inadequately communicate dialysis-specific information. Receipt of antibiotics for line sepsis or peritonitis, modifications to vascular access, and changes in target weight/dialysis prescription were rated as essential dialysis-specific information to include in discharge summaries by respondents.
Conclusion
Over three quarters of dialysis center directors find the current practice of transferring discharge information for hospitalized dialysis patients grossly inadequate. The inclusion of dialysis-specific information may improve the quality of discharge summaries for dialysis patients.
doi:10.2147/JMDH.S27572
PMCID: PMC3333802  PMID: 22536078
information transfer; dialysis patients; discharge information
18.  Virus-host swinging party in the oceans 
Mobile Genetic Elements  2012;2(2):88-95.
Bacteria and their viruses (phages) are antagonists, yet have coexisted in nature for billions of years. Models proposed to explain the paradox of antagonistic coexistence generally reach two types of solutions: Arms race-like dynamics that lead to hosts and viruses with increasing resistance and infection ranges; and population fluctuations between diverse host and viral types due to a metabolic cost of resistance. Recently, we found that populations of the marine cyanobacterium, Prochlorococcus, consist of cells with extreme hypervariability in gene sequence and gene content in a viral susceptibility region of the genome. Furthermore, we found a novel cost of resistance where resistance to one set of viruses is accompanied by changes in infection dynamics by other viruses. In this combined mini-review and commentary paper we discuss these findings in the context of existing ecological, evolutionary and genetic models of host-virus coexistence. We suggest that this coexistence is governed mainly by fluctuations between microbial subpopulations with differing viral susceptibility regions and that these fluctuations are driven by both metabolic and enhanced infection costs of resistance. Furthermore, we suggest that enhanced infection leads to passive host-switching by viruses, preventing the development of hosts with universal resistance. These findings highlight the vital importance of community complexity for host-virus coexistence.
doi:10.4161/mge.20031
PMCID: PMC3429526  PMID: 22934242
antagonistic coexistence; arms race; fluctuating selection; kill the winner; matching-alleles; gene-for-gene; cost of resistance; virus; cyanobacteria; Prochlorococcus
19.  Beyond Natural Numbers: Negative Number Representation in Parietal Cortex 
Unlike natural numbers, negative numbers do not have natural physical referents. How does the brain represent such abstract mathematical concepts? Two competing hypotheses regarding representational systems for negative numbers are a rule-based model, in which symbolic rules are applied to negative numbers to translate them into positive numbers when assessing magnitudes, and an expanded magnitude model, in which negative numbers have a distinct magnitude representation. Using an event-related functional magnetic resonance imaging design, we examined brain responses in 22 adults while they performed magnitude comparisons of negative and positive numbers that were quantitatively near (difference <4) or far apart (difference >6). Reaction times (RTs) for negative numbers were slower than positive numbers, and both showed a distance effect whereby near pairs took longer to compare. A network of parietal, frontal, and occipital regions were differentially engaged by negative numbers. Specifically, compared to positive numbers, negative number processing resulted in greater activation bilaterally in intraparietal sulcus (IPS), middle frontal gyrus, and inferior lateral occipital cortex. Representational similarity analysis revealed that neural responses in the IPS were more differentiated among positive numbers than among negative numbers, and greater differentiation among negative numbers was associated with faster RTs. Our findings indicate that despite negative numbers engaging the IPS more strongly, the underlying neural representation are less distinct than that of positive numbers. We discuss our findings in the context of the two theoretical models of negative number processing and demonstrate how multivariate approaches can provide novel insights into abstract number representation.
doi:10.3389/fnhum.2012.00007
PMCID: PMC3277269  PMID: 22363276
number cognition; symbolic number comparisons; distance effect; integers; negative numbers, intraparietal sulcus; prefrontal cortex; representational similarity
20.  Transphosphorylation of E. coli Proteins during Production of Recombinant Protein Kinases Provides a Robust System to Characterize Kinase Specificity 
Protein kinase specificity is of fundamental importance to pathway regulation and signal transduction. Here, we report a convenient system to monitor the activity and specificity of recombinant protein kinases expressed in E. coli. We apply this to the study of the cytoplasmic domain of the plant receptor kinase BRASSINOSTEROID-INSENSITIVE 1 (BRI1), which functions in brassinosteroid (BR) signaling. Recombinant BRI1 is catalytically active and both autophosphorylates and transphosphorylates E. coli proteins in situ. Using enrichment approaches followed by LC-MS/MS, phosphosites were identified allowing motifs associated with auto- and transphosphorylation to be characterized. Four lines of evidence suggest that transphosphorylation of E. coli proteins by BRI1 is specific and therefore provides meaningful results: (1) phosphorylation is not correlated with bacterial protein abundance; (2) phosphosite stoichiometry, estimated by spectral counting, is also not related to protein abundance; (3) a transphosphorylation motif emerged with strong preference for basic residues both N- and C-terminal to the phosphosites; and (4) other protein kinases (BAK1, PEPR1, FLS2, and CDPKβ) phosphorylated a distinct set of E. coli proteins and phosphosites. The E. coli transphosphorylation assay can be applied broadly to protein kinases and provides a convenient and powerful system to elucidate kinase specificity.
doi:10.3389/fpls.2012.00262
PMCID: PMC3510383  PMID: 23226150
BRI1; BAK1; PEPR1; FLS2; CDPK; phosphorylation motif
21.  International practice patterns and factors associated with non-conventional hemodialysis utilization 
BMC Nephrology  2011;12:66.
Background
The purpose of our study was to determine characteristics that influence the utilization of non-conventional hemodialysis (NCHD) therapies and its subtypes (nocturnal (NHD), short daily (SDHD), long conventional (LCHD) and conventional hemodialysis (CHD) as well as provider attitudes regarding the evidence for NCHD use.
Methods
An international cohort of subscribers of a nephrology education website http://www.nephrologynow.com was invited to participate in an online survey. Non-conventional hemodialysis was defined as any forms of hemodialysis delivered > 3 treatments per week and/or > 4 hours per session. NHD and SDHD included both home and in-centre. Respondents were categorized as CHD if their centre only offered conventional thrice weekly hemodialysis. Variables associated with NCHD and its subtypes were determined using multivariate logistic regression analysis. The survey assessed multiple domains regarding NCHD including reasons for initiating and discontinuing, for not offering and attitudes regarding evidence.
Results
544 surveys were completed leading to a 15.6% response rate. The final cohort was limited to 311 physicians. Dialysis modalities utilized among the respondents were as follows: NCHD194 (62.4%), NHD 83 (26.7%), SDHD 107 (34.4%), LCHD 81 (26%) and CHD 117 (37.6%). The geographic regions of participants were as follows: 11.9% Canada, 26.7% USA, 21.5% Europe, 6.1% Australia/New Zealand, 10% Africa/Middle East, 10.9% Asia and 12.9% South America. Variables associated with NCHD utilization included NCHD training (OR 2.47 CI 1.25-4.16), government physician reimbursement (OR 2.66, CI 1.11-6.40), practicing at an academic centre (OR 2.28 CI 1.25-4.16), higher national health care expenditure and number of ESRD patients per centre. Hemodialysis providers with patients on NCHD were significantly more likely to agree with the statements that NCHD improves quality of life, improves nutritional status, reduces EPO requirements and is cost effective. The most common reasons to initiate NCHD were driven by patient preference and the desire to improve volume control and global health outcomes.
Conclusion
Physician attitudes toward the evidence for NCHD differ significantly between NCHD providers and conventional HD providers. Interventions and health policy targeting these areas along with increased physician education and training in NCHD modalities may be effective in increasing its utilization.
doi:10.1186/1471-2369-12-66
PMCID: PMC3241205  PMID: 22142369
22.  Delay discounting behavior and white matter microstructure abnormalities in youth with a family history of alcoholism 
Background
Youth with family history of alcohol abuse have a greater risk of developing an alcohol use disorder (AUD). Brain and behavior differences may underlie this increased vulnerability. The current study examined delay discounting behavior and white matter microstructure in youth at high-risk for alcohol abuse, as determined by a family history of alcoholism (FH+), and youth without such family history (FH−).
Methods
Thirty-three healthy youth (FH+ = 15, FH− = 18), ages 11 to 15 years, completed a delay discounting task and underwent diffusion tensor imaging (DTI). Tract Based Spatial Statistics (Smith et al., 2006), as well as follow-up region-of-interest analyses, were performed in order to compare fractional anisotropy (FA) between FH+ and FH− youth.
Results
FH+ youth showed a trend toward increased discounting behavior and had significantly slower reaction times on the delay discounting paradigm compared to FH− youth. Group differences in FA were seen in several white matter tracts. Furthermore, lower FA in the left inferior longitudinal fasciculus and the right optic radiation statistically mediated the relationship between FH status and slower reaction times on the delay discounting task.
Conclusion
Youth with a family history of substance abuse have disrupted white matter microstructure, which likely contributes to less efficient cortical processing, and may act as an intrinsic risk-factor contributing to an increased susceptibility of developing AUD. In addition, FHP youth showed a trend toward greater impulsive decision making, possibly representing an inherent personal characteristic that may facilitate substance use onset and abuse in high-risk youth.
doi:10.1111/j.1530-0277.2010.01244.x
PMCID: PMC3206791  PMID: 20586754
adolescence; delay discounting; diffusion tensor imaging; alcohol abuse; family history
23.  In vivo volumetric imaging of human retinal circulation with phase-variance optical coherence tomography 
Biomedical Optics Express  2011;2(6):1504-1513.
We present in vivo volumetric images of human retinal micro-circulation using Fourier-domain optical coherence tomography (Fd-OCT) with the phase-variance based motion contrast method. Currently fundus fluorescein angiography (FA) is the standard technique in clinical settings for visualizing blood circulation of the retina. High contrast imaging of retinal vasculature is achieved by injection of a fluorescein dye into the systemic circulation. We previously reported phase-variance optical coherence tomography (pvOCT) as an alternative and non-invasive technique to image human retinal capillaries. In contrast to FA, pvOCT allows not only noninvasive visualization of a two-dimensional retinal perfusion map but also volumetric morphology of retinal microvasculature with high sensitivity. In this paper we report high-speed acquisition at 125 kHz A-scans with pvOCT to reduce motion artifacts and increase the scanning area when compared with previous reports. Two scanning schemes with different sampling densities and scanning areas are evaluated to find optimal parameters for high acquisition speed in vivo imaging. In order to evaluate this technique, we compare pvOCT capillary imaging at 3x3 mm2 and 1.5x1.5 mm2 with fundus FA for a normal human subject. Additionally, a volumetric view of retinal capillaries and a stitched image acquired with ten 3x3 mm2 pvOCT sub-volumes are presented. Visualization of retinal vasculature with pvOCT has potential for diagnosis of retinal vascular diseases.
doi:10.1364/BOE.2.001504
PMCID: PMC3114219  PMID: 21698014
(110.4500) Optical coherence tomography; (170.0110) Imaging systems; (170.4470) Ophthalmology; (120.3890) Medical optics instrumentation
24.  An In Vitro Intact Globe Expansion Method for Evaluation of Cross-linking Treatments 
We have developed an intact globe expansion method (GEM) that stresses the tissue by using an in vivo loading geometry and is capable of quantifying the effects of cross-linking treatments on both the cornea and sclera. We introduce rabbit kit eyes as a model of diseased tissue that can be subsequently strengthened to the level of normal tissue, as demonstrated with riboflavin/UVA and glyceraldehyde treatments.
Purpose.
To measure the tissue mechanical response to elevated intraocular pressure (IOP) using intact globe expansion of rabbit eyes. This method examined rabbit kit (2–3 weeks old) eyes as a model for weakened tissue and evaluated riboflavin/UVA and glyceraldehyde cross-linking treatments.
Methods.
The ocular shape of enucleated eyes was photographed during a 24-hour period while a controlled IOP was imposed (either low IOP = 22 mm Hg or high IOP = 85 mm Hg). Untreated controls consisted of kit eyes tested at both low- and high IOP and adult eyes tested at high IOP. Treated kit eyes (dextran controls, riboflavin/UVA treatment of the cornea, and glyceraldehyde treatment of the entire globe) were tested at high IOP.
Results.
Low IOP elicited negligible creep of the sclera and very gradual creep of the cornea. In contrast, high IOP induced up to an 8% strain in the sclera and a 15% strain in the cornea of rabbit kit eyes. The expansion of adult eyes was less than one third that of kit eyes at the same, high IOP. Riboflavin/UVA treatment of corneas reduced expansion compared with that in both dextran-treated and untreated control corneas. Glyceraldehyde treatment prevented expansion of the cornea and sclera.
Conclusions.
The intact globe expansion method (GEM) imposes a loading geometry comparable to in vivo conditions and can quantify changes in mechanical stability as a function of testing conditions (e.g., IOP, tissue maturation, and therapeutic cross-linking) with small sample sizes and small variability. Rabbit kit eyes provide a model of weak tissue suitable for screening treatments that strengthen the cornea and sclera.
doi:10.1167/iovs.09-4001
PMCID: PMC2891471  PMID: 20071684
25.  IS DEPRESSION AFTER AN ACUTE CORONARY SYNDROME SIMPLY A MARKER OF KNOWN PROGNOSTIC FACTORS FOR MORTALITY? 
Psychosomatic medicine  2009;71(7):697-703.
Objective
Controversy remains over whether the association between depression and mortality in patients with acute coronary syndromes (ACS) is confounded by incomplete adjustment for measures of known prognostic markers. We assessed whether depression was associated with the most comprehensive empirically derived index of clinical mortality predictors: the Global Registry of Acute Coronary Events (GRACE) risk score for predicting 6-month mortality after discharge for ACS. We also assessed whether depression remained an independent predictor of all-cause mortality after adjustment for the GRACE score and left ventricular dysfunction.
Methods
We prospectively surveyed 457 ACS patients (aged 25–92 years; 41% women, 13% black, and 11% Hispanic), hospitalized between May 2003 and June 2005. Depressive symptoms were assessed with the Beck Depression Inventory (BDI) and diagnosis of major depressive disorder (MDD) by a structured psychiatric interview, within one week of hospitalization.
Results
Despite differences in individual components of the GRACE score between depressed and non-depressed participants, neither depression measure was associated with overall GRACE score. For participants with MDD, the mean (SD) GRACE score was 84 (33), compared to 92 (31) for those without MDD (p=0.09). Using Cox proportional hazardsregression analysis, MDD and depressive symptom severity each predicted mortality after controlling for GRACE score and left ventricular dysfunction (adjusted hazard ratio for MDD, 2.51; 95% CI 1.45–4.37).
Conclusion
Depression is not simply a marker of clinical indicators that predict all-cause mortality after ACS. This strengthens the assertion that there is something unique in the association between depression and post-ACS prognosis, independent of known prognostic markers.
doi:10.1097/PSY.0b013e3181ad2abd
PMCID: PMC2908084  PMID: 19592517
depression; myocardial infarction; unstable angina; prognosis

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