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2.  Site-Specific Conjugation of RAFT Polymers to Proteins via Expressed Protein Ligation 
Site-specific protein conjugates with RAFT polymers were synthesized using expressed protein ligation. Stable micelles were formed from both linear block copolymer and Y-shaped conjugates.
doi:10.1039/c3cc38976f
PMCID: PMC4059823  PMID: 23423478
3.  Solid-State Nanostructured Materials from Self-Assembly of a Globular Protein-Polymer Diblock Copolymer 
ACS nano  2011;5(7):5697-5707.
Self-assembly of three-dimensional solid-state nanostructures containing approximately 33% by weight globular protein is demonstrated using a globular protein-polymer diblock copolymer, providing a route to direct nanopatterning of proteins for use in bioelectronic and biocatalytic materials. A mutant red fluorescent protein, mCherryS131C, was prepared by incorporation of a unique cysteine residue and site-specifically conjugated to end-functionalized poly(N-isopropylacrylamide) through thiol-maleimide coupling to form a well-defined model protein-polymer block copolymer. The block copolymer was self-assembled into bulk nanostructures by solvent evaporation from concentrated solutions. Small-angle X-ray scattering and transmission electron microscopy illustrated the formation of highly disordered lamellae or hexagonally perforated lamellae depending upon the selectivity of the solvent during evaporation. Solvent annealing of bulk samples resulted in a transition towards lamellar nanostructures with mCherry packed in a bilayer configuration and a large improvement in long range ordering. Wide-angle X-ray scattering indicated that mCherry did not crystallize within the block copolymer nanodomains and that the β-sheet spacing was not affected by self-assembly. Circular dichroism showed no change in protein secondary structure after self-assembly, while UV-vis spectroscopy indicated approximately 35% of the chromophore remained optically active.
doi:10.1021/nn2013673
PMCID: PMC4059825  PMID: 21696135
self-assembly; block copolymer; mCherry; PNIPAM; bioconjugation
4.  Kinetically Controlled Nanostructure Formation in Self-Assembled Globular Protein-Polymer Diblock Copolymers 
Biomacromolecules  2012;13(9):2781-2792.
Aqueous processing of globular protein-polymer diblock copolymers into solid-state materials and subsequent solvent annealing enables kinetic and thermodynamic control of nanostructure formation to produce block copolymer morphologies that maintain a high degree of protein fold and function. Using model diblock copolymers composed of mCherry-b-poly(N-isopropylacrylamide), orthogonal control over solubility of the protein block through changes in pH and the polymer block through changes in temperature is demonstrated during casting and solvent annealing. Hexagonal cylinders, perforated lamellae, lamellae, or hexagonal and disordered micellar phases are observed depending upon the coil fraction of the block copolymer and the kinetic pathway used for self-assembly. Good solvents for the polymer block produce ordered structures reminiscent of coil-coil diblock copolymers, while an unfavorable solvent results in kinetically trapped micellar structures. Decreasing solvent quality for the protein improves long-range ordering, suggesting that the strength of protein interactions influences nanostructure formation. Subsequent solvent annealing results in evolution of the nanostructures, with the best ordering and the highest protein function observed when annealing in a good solvent for both blocks. While protein secondary structure was found to be almost entirely preserved for all processing pathways, UV-vis spectroscopy of solid-state films indicates that using a good solvent for the protein block enables up to 70% of the protein to be retained in its functional form.
doi:10.1021/bm300763x
PMCID: PMC4059826  PMID: 22924842
block copolymer; self-assembly; protein-polymer conjugate; mCherry; PNIPAM
5.  Nanopatterned Protein Films Directed by Ionic Complexation with Water-Soluble Diblock Copolymers 
Macromolecules  2012;45(11):4572-4580.
The use of ionic interactions to direct both protein templating and block copolymer self-assembly into nanopatterned films with only aqueous processing conditions is demonstrated using block copolymers containing both thermally responsive and pH responsive blocks. Controlled reversible addition-fragmentation chain-transfer (RAFT) polymerization is employed to synthesize poly(N-isopropylacrylamide-b-2-(dimethylamino)ethyl acrylate) (PNIPAM-b-PDMAEA) diblock copolymers. The pH-dependent ionic complexation between the fluorescent protein, mCherry, and the ionic PDMAEA block is established using dynamic light scattering (DLS) and UV-Vis spectroscopy. DLS shows that the size of the resulting coacervate micelles depends strongly on pH, while UV-Vis spectroscopy shows a correlation between the protein’s absorption maximum and the ionic microenvironment. Zeta potential measurements clearly indicate the ionic nature of the complex-forming interactions. Spin casting was used to prepare nanostructured films from the protein-block copolymer coacervates. After film formation, the lower critical solution temperature (LCST) of the PNIPAM blocks allows the nanomaterial to be effectively immobilized in aqueous environments at physiological temperatures, enabling potential use as a controlled protein release material or polymer matrix for protein immobilization. At pH 9.2 and 7.8, the release rates are at least 10 times faster than that at pH 6.4 due to weaker interaction between protein and PNIPAM-b-PDMAEA (PND) diblock copolymer. Due to the ionic environment in which protein is confined, the majority of the protein (80%) remains active, independent of pH, even after having been dehydrated in vacuum and confined in the films.
doi:10.1021/ma2024914
PMCID: PMC4043372  PMID: 24904186
Stimuli-sensitive polymer; Block copolymer; Self-assembly; Protein delivery
6.  Effect of Bridging Anions on the Structure and Stability of Phenoxide Bridged Zinc Dipicolylamine Coordination Complexes 
Supramolecular chemistry  2013;25(6):315-322.
A series of four related phenol derivatives, with 2,2'-dipicolylamine substituents at the ortho positions, were prepared and their Zn2+ coordination complexes studied by spectroscopic methods. X-ray crystal diffraction analysis of a dinuclear zinc complex with two bridging acetate anions showed a ternary structure with highly charged interior and lipophilic exterior, which helps explain why this class of water-soluble complexes can effectively diffuse through cell membranes. The stability of the dinuclear zinc complexes in aqueous solution was found to be strongly anion dependent; that is, bridging oxyanions, such as acetate and pyrophosphate, lock the two Zn2+ cations to the surrounding ligand and greatly enhance ligand/zinc affinity. Overall, the results provide new insight into the structural and mechanistic factors that control the recognition and chemosensing performance of phenoxide bridged dipicolylamine molecular probes.
doi:10.1080/10610278.2013.776170
PMCID: PMC3728083  PMID: 23914128
Anion recognition; zinc coordination; nuclear magnetic resonance; X-ray crystallography; titration; spectroscopic analysis
7.  Water-Soluble, Deep-Red Fluorescent Squaraine Rotaxanes† 
Organic & biomolecular chemistry  2011;10(30):5769-5773.
Eight fluorescent squaraine rotaxanes with deep-red absorption/emission wavelengths were prepared and assessed for chemical stability and suitability as water-soluble, fluorescent tracers. The most stable squaraine rotaxanes have four large stopper groups attached to the ends of the encapsulated squaraine, and two members of this structural class have promise as highly fluorescent tracers with rapid renal clearance and very low tissue uptake in living mice.
doi:10.1039/c2ob06783h
PMCID: PMC4015105  PMID: 22159917
8.  TLR-Independent and P2X7-Dependent Signaling Mediate Alu RNA-Induced NLRP3 Inflammasome Activation in Geographic Atrophy 
Purpose.
Accumulation of Alu RNA transcripts due to DICER1 deficiency in the retinal pigmented epithelium (RPE) promotes geographic atrophy. Recently we showed that Alu RNA activated the NLRP3 inflammasome, leading to RPE cell death via interleukin-18 (IL-18)-mediated MyD88 signaling. However, the molecular basis for NLRP3 inflammasome activation by Alu RNA is not well understood. We sought to decipher the key signaling events triggered by Alu RNA that lead to priming and activation of the NLRP3 inflammasome and, ultimately, to RPE degeneration by investigating the roles of the purinoreceptor P2X7, the transcription factor NF-κB, and the Toll-like receptors (TLRs) in these processes.
Methods.
Human and mouse RPE cells were transfected with a plasmid encoding an Alu element (pAlu) or an in vitro-transcribed Alu RNA. Inflammasome priming was assessed by measuring NLRP3 and IL18 mRNA levels by real-time quantitative PCR. Using immunoblotting, we assessed NF-κB activation by monitoring phosphorylation of its p65 subunit, and inflammasome activation by monitoring caspase-1 cleavage into its active form. RPE degeneration was induced in mice by subretinal transfection of pAlu or Alu RNA. The NF-κB inhibitor BAY 11-7082, the P2X7 receptor antagonist A-740003, and the NLRP3 inflammasome inhibitor glyburide were delivered by intravitreous injections. We studied wild-type (WT) C57Bl/6J, P2rx7−/−, Nfkb1−/−, and Tlr23479−/− mice. RPE degeneration was assessed by fundus photography and zonula occludens-1 (ZO-1) staining of mouse RPE.
Results.
Alu RNA-induced NF-κB activation, independent of TLR-1, -2, -3, -4, -6, -7, and -9 signaling, was required for priming the NLRP3 inflammasome. Nfkb1−/− and P2rx7−/− mice and WT mice treated with the pharmacological inhibitors of NF-κB, P2X7, or NLRP3, were protected against Alu RNA-induced RPE degeneration.
Conclusions.
NF-κB and P2X7 are critical signaling intermediates in Alu RNA-induced inflammasome priming and RPE degeneration. These molecules are novel targets for rational drug development for geographic atrophy.
Our studies provide comprehensive analysis of critical signaling pathways regulating Alu RNA-induced inflammasome activation and RPE degeneration. Our data demonstrate that NF-κB and P2X7 are critical signaling intermediates in Alu RNA-induced inflammasome priming and RPE degeneration.
doi:10.1167/iovs.13-12500
PMCID: PMC3825570  PMID: 24114535
AMD; inflammasome; NLRP3
9.  In vivo imaging using polymeric nanoparticles stained with near-infrared chemiluminescent and fluorescent squaraine catenane endoperoxide† 
Polystyrene nanoparticles stained with squaraine catenane endoperoxide exhibit remarkably high chemiluminescence and enable optical imaging of biodistribution in living mice. Whole-body chemiluminescence imaging was much more effective than fluorescence at identifying lung accumulation of the nanoparticles.
doi:10.1039/c3cc40630j
PMCID: PMC3633569  PMID: 23467338
10.  SUPPORTING CHILDREN IN U.S. LEGAL PROCEEDINGS 
We conducted a national survey of 786 victim/witness assistants (VWAs) to provide descriptive and attitudinal information about support person use in U.S. legal proceedings involving children. VWAs (N = 414) from 46 states returned completed surveys (response rate = 53%). Prosecutor-based VWAs or parents/guardians most frequently served as support persons. One support person was almost always or often used with child victims and/or witnesses of all ages. Support persons were extremely common in cases involving child sexual abuse, physical abuse, neglect, and adult domestic violence. Overall, support persons provided more informational than emotional support. The most common informational support was to provide referrals to community resources, conduct courtroom visit/orientation, and disseminate relevant procedural information. The most common emotional support was to accompany the child to trial. Support persons rarely or never questioned children directly during investigative interviews or in court. Respondents believed support persons decrease children’s stress and increase accuracy and credibility; however, this effect varied as a function of who provided support, child age, case type, and type of emotional or informational support. Respondents believed that support person presence at trial probably does not prejudice jurors against defendants. These survey data provide a benchmark for legal professionals and a foundation for future social scientific research examining the effects of support person use on children.
doi:10.1037/a0027879
PMCID: PMC3985335  PMID: 24741286
support persons; victim/witness assistants; alternative testimony; child witness
12.  Factors affecting stress experienced by surrogate decision-makers for critically ill patients: implications for nursing practice 
Objectives
This study explores surrogate decision-makers’ (SDMs) challenges making decisions related to the care of patients in critical care, to 1) characterize the SDM stress 2) identify personal, social, care-related factors influencing stress and 3) consider implications of findings to improving critical care practice.
Methodology
Semi-structured interviews were conducted with SDMs of critically ill patients receiving care in two tertiary care institutions. Transcripts were analyzed using a grounded theory approach. Domains explored were: stress characteristics, stress mitigators, coping strategies, social networks, SDM decision-making role, decision-making concordance, knowledge of patient's preferences, experience with provider team, SDM-provider communication, patient outcome certainty.
Main Outcomes
We interviewed 34 SDMs. Most were female and described long-term relationships with patients. SDMs described the strain of uncertain outcomes and decision-making without clear, consistent information from providers. Decision-making anxiety was buffered by SDMs’ active engagement of social networks, faith and access to clear communication from providers.
Conclusion
Stress is a very real factor influencing SDMs confidence and comfort making decisions. These findings suggest that stress can be minimized by improving communication between SDMs and medical providers. Nurses central role in ICU make them uniquely poised to spearhead interventions to improve provider-SDM communication and reduce SDM decision-making anxiety.
doi:10.1016/j.iccn.2013.08.008
PMCID: PMC3946606  PMID: 24211047
Surrogate Decision-maker; Critical Illness; Provider-SDM Communication; Qualitative Data Collection; Social Science Research
13.  4D flow imaging with MRI 
Magnetic resonance imaging (MRI) has become an important tool for the clinical evaluation of patients with cardiovascular disease. Since its introduction in the late 1980s, 2-dimensional phase contrast MRI (2D PC-MRI) has become a routine part of standard-of-care cardiac MRI for the assessment of regional blood flow in the heart and great vessels. More recently, time-resolved PC-MRI with velocity encoding along all three flow directions and three-dimensional (3D) anatomic coverage (also termed ‘4D flow MRI’) has been developed and applied for the evaluation of cardiovascular hemodynamics in multiple regions of the human body. 4D flow MRI allows for the comprehensive evaluation of complex blood flow patterns by 3D blood flow visualization and flexible retrospective quantification of flow parameters. Recent technical developments, including the utilization of advanced parallel imaging techniques such as k-t GRAPPA, have resulted in reasonable overall scan times, e.g., 8-12 minutes for 4D flow MRI of the aorta and 10-20 minutes for whole heart coverage. As a result, the application of 4D flow MRI in a clinical setting has become more feasible, as documented by an increased number of recent reports on the utility of the technique for the assessment of cardiac and vascular hemodynamics in patient studies. A number of studies have demonstrated the potential of 4D flow MRI to provide an improved assessment of hemodynamics which might aid in the diagnosis and therapeutic management of cardiovascular diseases. The purpose of this review is to describe the methods used for 4D flow MRI acquisition, post-processing and data analysis. In addition, the article provides an overview of the clinical applications of 4D flow MRI and includes a review of applications in the heart, thoracic aorta and hepatic system.
doi:10.3978/j.issn.2223-3652.2014.01.02
PMCID: PMC3996243  PMID: 24834414
4D flow magnetic resonance imaging (4D flow MRI); phase contrast magnetic resonance imaging (PC-MRI); blood flow; hemodynamics; PC-VIPR; visualization; quantification; carotid bifurcation; aorta; heart; pulmonary arteries; renal arteries; liver hemodynamics; splanchnic vessel system; peripheral arteries
14.  Catalase (KatA) Plays a Role in Protection against Anaerobic Nitric Oxide in Pseudomonas aeruginosa 
PLoS ONE  2014;9(3):e91813.
Pseudomonas aeruginosa (PA) is a common bacterial pathogen, responsible for a high incidence of nosocomial and respiratory infections. KatA is the major catalase of PA that detoxifies hydrogen peroxide (H2O2), a reactive oxygen intermediate generated during aerobic respiration. Paradoxically, PA displays elevated KatA activity under anaerobic growth conditions where the substrate of KatA, H2O2, is not produced. The aim of the present study is to elucidate the mechanism underlying this phenomenon and define the role of KatA in PA during anaerobiosis using genetic, biochemical and biophysical approaches. We demonstrated that anaerobic wild-type PAO1 cells yielded higher levels of katA transcription and expression than aerobic cells, whereas a nitrite reductase mutant ΔnirS produced ∼50% the KatA activity of PAO1, suggesting that a basal NO level was required for the increased KatA activity. We also found that transcription of the katA gene was controlled, in part, by the master anaerobic regulator, ANR. A ΔkatA mutant and a mucoid mucA22 ΔkatA bacteria demonstrated increased sensitivity to acidified nitrite (an NO generator) in anaerobic planktonic and biofilm cultures. EPR spectra of anaerobic bacteria showed that levels of dinitrosyl iron complexes (DNIC), indicators of NO stress, were increased significantly in the ΔkatA mutant, and dramatically in a ΔnorCB mutant compared to basal levels of DNIC in PAO1 and ΔnirS mutant. Expression of KatA dramatically reduced the DNIC levels in ΔnorCB mutant. We further revealed direct NO-KatA interactions in vitro using EPR, optical spectroscopy and X-ray crystallography. KatA has a 5-coordinate high spin ferric heme that binds NO without prior reduction of the heme iron (Kd ∼6 μM). Collectively, we conclude that KatA is expressed to protect PA against NO generated during anaerobic respiration. We proposed that such protective effects of KatA may involve buffering of free NO when potentially toxic concentrations of NO are approached.
doi:10.1371/journal.pone.0091813
PMCID: PMC3963858  PMID: 24663218
15.  Fluorine NMR Reporter for Phosphate Anions 
A fluorine-labelled zinc(II)-dipicolylamine coordination complex reports the presence of phosphate anions in aqueous solution, especially pyrophosphate and ADP, and is used to monitor the enzymatic hydrolysis of ATP.
doi:10.1039/c3cc42169d
PMCID: PMC3957275  PMID: 23619399
16.  Immunology of age-related macular degeneration 
Nature reviews. Immunology  2013;13(6):438-451.
Age-related macular degeneration (AMD) is a leading cause of blindness in aged individuals. Recent advances have highlighted the essential role of immune processes in the development, progression and treatment of AMD. In this Review we discuss recent discoveries related to the immunological aspects of AMD pathogenesis. We outline the diverse immune cell types, inflammatory activators and pathways that are involved. Finally, we discuss the future of inflammation-directed therapeutics to treat AMD in the growing aged population.
doi:10.1038/nri3459
PMCID: PMC3941009  PMID: 23702979
17.  Subtle signs, subtle designs: future change and BJR 
The British Journal of Radiology  2013;86(1023):20130078.
doi:10.1259/bjr.20130078
PMCID: PMC3608054  PMID: 23392198
18.  Convenient Synthesis of Multivalent Zinc(II)-Dipicolylamine Complexes for Molecular Recognition 
Tetrahedron letters  2013;54(8):861-864.
A pair of novel dipicolylamine ligands bearing isothiocyanate groups were used as conjugation reagents to prepare multivalent molecules with anionic recognition capability. The isothiocyanates were reacted with two classes of dendritic scaffolds bearing primary amines, squaraine rotaxanes and PAMAM dendrimers, and the products were converted into water soluble zinc(II) coordination complexes. The multivalent squaraine rotaxanes exhibit high fluorescence quantum yields in water and are very well suited for biological imaging applications.
doi:10.1016/j.tetlet.2012.11.103
PMCID: PMC3580864  PMID: 23459472
zinc dipicolylamine; multivalent; squaraine rotaxane; fluorescence; dendrimer
19.  The Francisella tularensis migR, trmE, and cphA Genes Contribute to F. tularensis Pathogenicity Island Gene Regulation and Intracellular Growth by Modulation of the Stress Alarmone ppGpp 
Infection and Immunity  2013;81(8):2800-2811.
The Francisella tularensis pathogenicity island (FPI) encodes many proteins that are required for virulence. Expression of these genes depends upon the FevR (PigR) regulator and its interactions with the MglA/SspA and RNA polymerase transcriptional complex. Experiments to identify how transcription of the FPI genes is activated have led to identification of mutations within the migR, trmE, and cphA genes that decrease FPI expression. Recent data demonstrated that the small alarmone ppGpp, produced by RelA and SpoT, is important for stabilizing MglA/SspA and FevR (PigR) interactions in Francisella. Production of ppGpp is commonly known to be activated by cellular and nutritional stress in bacteria, which indicates that cellular and nutritional stresses act as important signals for FPI activation. In this work, we demonstrate that mutations in migR, trmE, or cphA significantly reduce ppGpp accumulation. The reduction in ppGpp levels was similar for each of the mutants and correlated with a corresponding reduction in iglA reporter expression. In addition, we observed that there were differences in the ability of each of these mutants to replicate within various mammalian cells, indicating that the migR, trmE, and cphA genes are likely parts of different cellular stress response pathways in Francisella. These results also indicate that different nutritional and cellular stresses exist in different mammalian cells. This work provides new information to help understand how Francisella regulates its virulence genes in response to host cell environments, and it contributes to our growing knowledge of this highly successful bacterial pathogen.
doi:10.1128/IAI.00073-13
PMCID: PMC3719569  PMID: 23716606
20.  Assessing the impact of race, social factors and air pollution on birth outcomes: a population-based study 
Environmental Health  2014;13:4.
Background
Both air pollution exposure and socioeconomic status (SES) are important indicators of children’s health. Using highly resolved modeled predictive surfaces, we examine the joint effects of air pollution exposure and measures of SES in a population level analysis of pregnancy outcomes in North Carolina (NC).
Methods
Daily measurements of particulate matter <2.5 μm in aerodynamic diameter (PM2.5) and ozone (O3) were calculated through a spatial hierarchical Bayesian model which produces census-tract level point predictions. Using multilevel models and NC birth data from 2002–2006, we examine the association between pregnancy averaged PM2.5 and O3, individual and area-based SES indicators, and birth outcomes.
Results
Maternal race and education, and neighborhood household income were associated with adverse birth outcomes. Predicted concentrations of PM2.5 and O3 were also associated with an additional effect on reductions in birth weight and increased risks of being born low birth weight and small for gestational age.
Conclusions
This paper builds on and complements previous work on the relationship between pregnancy outcomes and air pollution exposure by using 1) highly resolved air pollution exposure data; 2) a five-year population level sample of pregnancies; and 3) including personal and areal level measures of social determinants of pregnancy outcomes. Results show a stable and negative association between air pollution exposure and adverse birth outcomes. Additionally, the more socially disadvantaged populations are at a greater risk; controlling for both SES and environmental stressors provides a better understanding of the contributing factors to poor children’s health outcomes.
doi:10.1186/1476-069X-13-4
PMCID: PMC3922656  PMID: 24476365
Air pollution; Exposure predictions; Pregnancy outcomes; Socioeconomic status

Results 1-25 (311)