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1.  Joint Associations of Diet, Lifestyle, and Genes with Age-Related Macular Degeneration 
Ophthalmology  2015;122(11):2286-2294.
Purpose
Healthy diets and lifestyles are thought to protect against age-related macular degeneration (AMD), but whether the benefits vary across high risk AMD genotypes is unknown. The objective is to investigate the joint effects of healthy diet and lifestyle with genetic risk on the odds for AMD.
Design
Healthy lifestyles scores and their interactions with AMD risk genotypes were studied in relation to the prevalence of AMD, assessed six years later.
Participants
Women 50–79 years of age in the Carotenoids in Age-Related Eye Disease Study (CAREDS) with exposure and AMD data available (N=1,663).
Methods
Healthy lifestyle scores (0–6 points) were assigned based on Healthy Eating Index scores, physical activity (MetHrs/week), and pack years of smoking assessed between 1994–1998. Genetic risk was based on Y402H in complement factor H (CFH) and A69S in age-related maculopathy susceptibility locus 2 (ARMS2). Interactions between healthy lifestyle score and genotype in relation to the odds of AMD were assessed.
Main Outcome
Stereoscopic fundus photographs were taken and graded for AMD six years after exposure assessment (2001–2004). A total of 308 women had early AMD and 29 had late AMD).
Results
The odds of AMD were 3.3 times greater in women with both low healthy lifestyle score (0–2) and high risk CFH genotype (CC), relative to those who had low genetic risk (TT) and healthy lifestyle scores of 4–6 (95% CI:1.8–6.1). There were no significant additive (SI=1.08, 95% CI: 0.70–1.67) or multiplicative (Pinteraction=0.94) interactions in the full sample. Limiting the sample to those with stable diets prior to AMD assessment (n=728) strengthened the joint effects (OR=4.6, 95% CI: 1.85–11.6) and suggested high risk genotype and low lifestyle score combined had a stronger association than expected by simply adding the two effects (SI=1.34, 95% CI: 1.05–1.70). Adjusting for dietary lutein and zeaxanthin attenuated, and therefore partially explained the joint association. There was no significant evidence of additive or multiplicative interactions for ARMS2 and lifestyle score.
Conclusions
These results, in a sample in which the majority of AMD cases were early, suggest the effects of high risk Y402H genotype and poor diets and lifestyles combine in at least an additive manner to influence odds for AMD, but may combine to be more than the sum of their individual effects.
doi:10.1016/j.ophtha.2015.07.029
PMCID: PMC4714866  PMID: 26354764
2.  Membrane Protein Structure, Function and Dynamics: A Perspective from Experiments and Theory 
The Journal of membrane biology  2015;248(4):611-640.
Membrane proteins mediate processes that are fundamental for the flourishing of biological cells. Membrane-embedded transporters move ions and larger solutes across membranes, receptors mediate communication between the cell and its environment and membrane-embedded enzymes catalyze chemical reactions. Understanding these mechanisms of action requires knowledge of how the proteins couple to their fluid, hydrated lipid membrane environment. We present here current studies in computational and experimental membrane protein biophysics, and show how they address outstanding challenges in understanding the complex environmental effects on the structure, function and dynamics of membrane proteins.
doi:10.1007/s00232-015-9802-0
PMCID: PMC4515176  PMID: 26063070
membrane proteins; lipids; protein structure; protein function; protein dynamics; membrane-mediated interactions
3.  Functional studies and modeling of pore-lining residue mutants of the influenza A virus M2 ion channel† 
Biochemistry  2010;49(4):696-708.
The A/M2 protein of influenza A virus forms a tetrameric proton selective pH-gated ion channel. The H37xxxW41 motif located in the channel pore is responsible for its gating and proton selectivity. Channel activation most likely involves protonation of the H37 residues, while the conductive state of the channel is characterized by two or three charged His residues in a tetrad. A/M2 channel activity is inhibited by the anti-viral drug amantadine. Although a large number of functional amantadine-resistant mutants of A/M2 have been observed in vitro, only a few are observed in highly transmissible viruses in the presence or absence of amantadine. We therefore examined 49 point mutants of the pore-lining residues, representing both natural and non-natural variants. Their ion selectivity, amantadine sensitivity, specific activity, and pH dependent conductance were measured in Xenopus oocytes. These measurements showed how variations in the sequence lead to variations in the proton conduction. The results are consistent with a multi-step mechanism that allows the protein to fine-tune its pH-rate profile over a wide range of proton concentrations, hypothesized to arise from different protonation states of the H37 tetrad. Mutations that give native-like conductance at low pH as well as minimal leakage current at pH 7.0 were surprisingly rare. Moreover, the results are consistent with a location of the amantadine-binding site inside the channel pore. These findings have helped to define the set of functionally fit mutants that should be targeted when considering the design of novel drugs that inhibit amantadine-resistant strains of influenza A virus.
doi:10.1021/bi901799k
PMCID: PMC4924815  PMID: 20028125
Influenza A virus; M2 channel; proton channel; channel gating; amantadine; MD simulations
4.  A large genome-wide association study of age-related macular degeneration highlights contributions of rare and common variants 
Fritsche, Lars G. | Igl, Wilmar | Cooke Bailey, Jessica N. | Grassmann, Felix | Sengupta, Sebanti | Bragg-Gresham, Jennifer L. | Burdon, Kathryn P. | Hebbring, Scott J. | Wen, Cindy | Gorski, Mathias | Kim, Ivana K. | Cho, David | Zack, Donald | Souied, Eric | Scholl, Hendrik P. N. | Bala, Elisa | Lee, Kristine E. | Hunter, David J. | Sardell, Rebecca J. | Mitchell, Paul | Merriam, Joanna E. | Cipriani, Valentina | Hoffman, Joshua D. | Schick, Tina | Lechanteur, Yara T. E. | Guymer, Robyn H. | Johnson, Matthew P. | Jiang, Yingda | Stanton, Chloe M. | Buitendijk, Gabriëlle H. S. | Zhan, Xiaowei | Kwong, Alan M. | Boleda, Alexis | Brooks, Matthew | Gieser, Linn | Ratnapriya, Rinki | Branham, Kari E. | Foerster, Johanna R. | Heckenlively, John R. | Othman, Mohammad I. | Vote, Brendan J. | Liang, Helena Hai | Souzeau, Emmanuelle | McAllister, Ian L. | Isaacs, Timothy | Hall, Janette | Lake, Stewart | Mackey, David A. | Constable, Ian J. | Craig, Jamie E. | Kitchner, Terrie E. | Yang, Zhenglin | Su, Zhiguang | Luo, Hongrong | Chen, Daniel | Ouyang, Hong | Flagg, Ken | Lin, Danni | Mao, Guanping | Ferreyra, Henry | Stark, Klaus | von Strachwitz, Claudia N. | Wolf, Armin | Brandl, Caroline | Rudolph, Guenther | Olden, Matthias | Morrison, Margaux A. | Morgan, Denise J. | Schu, Matthew | Ahn, Jeeyun | Silvestri, Giuliana | Tsironi, Evangelia E. | Park, Kyu Hyung | Farrer, Lindsay A. | Orlin, Anton | Brucker, Alexander | Li, Mingyao | Curcio, Christine | Mohand-Saïd, Saddek | Sahel, José-Alain | Audo, Isabelle | Benchaboune, Mustapha | Cree, Angela J. | Rennie, Christina A. | Goverdhan, Srinivas V. | Grunin, Michelle | Hagbi-Levi, Shira | Campochiaro, Peter | Katsanis, Nicholas | Holz, Frank G. | Blond, Frédéric | Blanché, Hélène | Deleuze, Jean-François | Igo, Robert P. | Truitt, Barbara | Peachey, Neal S. | Meuer, Stacy M. | Myers, Chelsea E. | Moore, Emily L. | Klein, Ronald | Hauser, Michael A. | Postel, Eric A. | Courtenay, Monique D. | Schwartz, Stephen G. | Kovach, Jaclyn L. | Scott, William K. | Liew, Gerald | Tƒan, Ava G. | Gopinath, Bamini | Merriam, John C. | Smith, R. Theodore | Khan, Jane C. | Shahid, Humma | Moore, Anthony T. | McGrath, J. Allie | Laux, Reneé | Brantley, Milam A. | Agarwal, Anita | Ersoy, Lebriz | Caramoy, Albert | Langmann, Thomas | Saksens, Nicole T. M. | de Jong, Eiko K. | Hoyng, Carel B. | Cain, Melinda S. | Richardson, Andrea J. | Martin, Tammy M. | Blangero, John | Weeks, Daniel E. | Dhillon, Bal | van Duijn, Cornelia M. | Doheny, Kimberly F. | Romm, Jane | Klaver, Caroline C. W. | Hayward, Caroline | Gorin, Michael B. | Klein, Michael L. | Baird, Paul N. | den Hollander, Anneke I. | Fauser, Sascha | Yates, John R. W. | Allikmets, Rando | Wang, Jie Jin | Schaumberg, Debra A. | Klein, Barbara E. K. | Hagstrom, Stephanie A. | Chowers, Itay | Lotery, Andrew J. | Léveillard, Thierry | Zhang, Kang | Brilliant, Murray H. | Hewitt, Alex W. | Swaroop, Anand | Chew, Emily Y. | Pericak-Vance, Margaret A. | DeAngelis, Margaret | Stambolian, Dwight | Haines, Jonathan L. | Iyengar, Sudha K. | Weber, Bernhard H. F. | Abecasis, Gonçalo R. | Heid, Iris M.
Nature genetics  2015;48(2):134-143.
Advanced age-related macular degeneration (AMD) is the leading cause of blindness in the elderly with limited therapeutic options. Here, we report on a study of >12 million variants including 163,714 directly genotyped, most rare, protein-altering variant. Analyzing 16,144 patients and 17,832 controls, we identify 52 independently associated common and rare variants (P < 5×10–8) distributed across 34 loci. While wet and dry AMD subtypes exhibit predominantly shared genetics, we identify the first signal specific to wet AMD, near MMP9 (difference-P = 4.1×10–10). Very rare coding variants (frequency < 0.1%) in CFH, CFI, and TIMP3 suggest causal roles for these genes, as does a splice variant in SLC16A8. Our results support the hypothesis that rare coding variants can pinpoint causal genes within known genetic loci and illustrate that applying the approach systematically to detect new loci requires extremely large sample sizes.
doi:10.1038/ng.3448
PMCID: PMC4745342  PMID: 26691988
5.  New Glycosides and Trypanocidal Metabolites from Vangueria edulis 
Natural product communications  2015;10(11):1897-1900.
A new iridoid glucoside, 10-methoxy apodanthoside (1), and a new monoterpene glycoside, (3S,6S)-cis linalool-3,7-oxide O-β-D-glucopyranosyl-(1″→5′)-β-D-xylofuranoside (2), were isolated from V. edulis (Rubiaceae), along with eighteen known compounds (3–20), including monoterpenes, iridoid glycosides, and a lignin, which were encountered for the first time in the genus Vangueria,. The structural elucidation of the isolates was based on the analysis of spectroscopic (1D and 2D NMR) and HR-ESI-MS data. Detailed stereochemical studies of 1 and related iridoid glucosides (compounds 3, 4 and 8) were made by matching the calculated ECD peaks with the experimental ones. All isolates were tested for their antiprotozoal, antifungal, and antiplasmodial activities. Compounds 9, 15 and 16 showed good trypanocidal activities against Trypanosoma brucei brucei with IC50 values of 8.18, 9.02 and 7.80 μg/mL, respectively and IC90 values of >10, >10 and 9.76 μg/mL, respectively. Compound 16 showed a moderate activity against Candida glabrata with an IC50 value of 8.66 μg/mL. Compound 20 showed a weak antiplasmodial activity against chloroquine-sensitive (D6) and resistant (W2) Plasmodium falciparum with IC50 values of 3.29 (SI, >1.4) and 4.53 (SI, >1) μg/mL, respectively.
PMCID: PMC4887451  PMID: 26749819
Vangueria edulis; Rubiaceae; Iridoid glucosides; Monoterpene glycosides; ECD spectra; Trypanocidal
6.  Combined Computational and Experimental Analysis of a Complex of Ribonuclease III and the Regulatory Macrodomain Protein, YmdB 
Proteins  2015;83(3):459-472.
Ribonuclease III is a conserved bacterial endonuclease that cleaves double-stranded(ds) structures in diverse coding and noncoding RNAs. RNase III is subject to multiple levels of control that in turn confer global post-transcriptional regulation. The Escherichia coli macrodomain protein YmdB directly interacts with RNase III, and an increase in YmdB amount in vivo correlates with a reduction in RNase III activity. Here, a computational-based structural analysis was performed to identify atomic-level features of the YmdB-RNase III interaction. The docking of monomeric E. coli YmdB with a homology model of the E. coli RNase III homodimer yields a complex that exhibits an interaction of the conserved YmdB residue R40 with specific RNase III residues at the subunit interface. Surface Plasmon Resonance (SPR) analysis provided a KD of 61 nM for the complex, corresponding to a binding free energy (ΔG) of −9.9 kcal/mol. YmdB R40 and RNase III D128 were identified by in silico alanine mutagenesis as thermodynamically important interacting partners. Consistent with the prediction, the YmdB R40A mutation causes a 16-fold increase in KD (ΔΔG = +1.8 kcal/mol), as measured by SPR, and the D128A mutation in both RNase III subunits (D128A/D128'A) causes an 83-fold increase in KD (ΔΔG = +2.7 kcal/mol). The greater effect of the D128A/D128'A mutation may reflect an altered RNase III secondary structure, as revealed by CD spectroscopy, which also may explain the significant reduction in catalytic activity in vitro. The features of the modeled complex relevant to potential RNase III regulatory mechanisms are discussed.
doi:10.1002/prot.24751
PMCID: PMC4329070  PMID: 25546632
RNase III; Macrodomain; RNA processing; ADP-ribose; double-stranded RNA; post-transcriptional regulation
8.  Comparative sequence analysis suggests a conserved gating mechanism for TRP channels 
Comparative sequence analysis indicates that a common mechanism underlying polymodal gating of TRP channels involves the relaxation of a structural distortion of pore-lining helices.
The transient receptor potential (TRP) channel superfamily plays a central role in transducing diverse sensory stimuli in eukaryotes. Although dissimilar in sequence and domain organization, all known TRP channels act as polymodal cellular sensors and form tetrameric assemblies similar to those of their distant relatives, the voltage-gated potassium (Kv) channels. Here, we investigated the related questions of whether the allosteric mechanism underlying polymodal gating is common to all TRP channels, and how this mechanism differs from that underpinning Kv channel voltage sensitivity. To provide insight into these questions, we performed comparative sequence analysis on large, comprehensive ensembles of TRP and Kv channel sequences, contextualizing the patterns of conservation and correlation observed in the TRP channel sequences in light of the well-studied Kv channels. We report sequence features that are specific to TRP channels and, based on insight from recent TRPV1 structures, we suggest a model of TRP channel gating that differs substantially from the one mediating voltage sensitivity in Kv channels. The common mechanism underlying polymodal gating involves the displacement of a defect in the H-bond network of S6 that changes the orientation of the pore-lining residues at the hydrophobic gate.
doi:10.1085/jgp.201411329
PMCID: PMC4485022  PMID: 26078053
9.  Flipping in the Pore: Discovery of Dual Inhibitors That Bind in Different Orientations to the Wild-Type versus the Amantadine-Resistant S31N Mutant of the Influenza A Virus M2 Proton Channel 
Journal of the American Chemical Society  2014;136(52):17987-17995.
Influenza virus infections lead to numerous deaths and millions of hospitalizations each year. One challenge facing anti-influenza drug development is the heterogeneity of the circulating influenza viruses, which comprise several strains with variable susceptibility to antiviral drugs. For example, the wild-type (WT) influenza A viruses, such as the seasonal H1N1, tend to be sensitive to antiviral drugs, amantadine and rimantadine, while the S31N mutant viruses, such as the pandemic 2009 H1N1 (H1N1pdm09) and seasonal H3N2, are resistant to this class of drugs. Thus, drugs targeting both WT and the S31N mutant are highly desired. We report our design of a novel class of dual inhibitors along with their ion channel blockage and antiviral activities. The potency of the most active compound 11 in inhibiting WT and the S31N mutant influenza viruses is comparable with that of amantadine in inhibiting WT influenza virus. Solution NMR studies and molecular dynamics (MD) simulations of drug-M2 interactions supported our design hypothesis: namely, the dual inhibitor binds in the WT M2 channel with an aromatic group facing down toward the C-terminus, while the same drug binds in the S31N M2 channel with its aromatic group facing up toward the N-terminus. The flip-flop mode of drug binding correlates with the structure–activity relationship (SAR) and has paved the way for the next round of rational design of broad-spectrum antiviral drugs.
doi:10.1021/ja508461m
PMCID: PMC4286326  PMID: 25470189
10.  Rare and common variants in extracellular matrix gene Fibrillin 2 (FBN2) are associated with macular degeneration 
Human Molecular Genetics  2014;23(21):5827-5837.
Neurodegenerative diseases affecting the macula constitute a major cause of incurable vision loss and exhibit considerable clinical and genetic heterogeneity, from early-onset monogenic disease to multifactorial late-onset age-related macular degeneration (AMD). As part of our continued efforts to define genetic causes of macular degeneration, we performed whole exome sequencing in four individuals of a two-generation family with autosomal dominant maculopathy and identified a rare variant p.Glu1144Lys in Fibrillin 2 (FBN2), a glycoprotein of the elastin-rich extracellular matrix (ECM). Sanger sequencing validated the segregation of this variant in the complete pedigree, including two additional affected and one unaffected individual. Sequencing of 192 maculopathy patients revealed additional rare variants, predicted to disrupt FBN2 function. We then undertook additional studies to explore the relationship of FBN2 to macular disease. We show that FBN2 localizes to Bruch′s membrane and its expression appears to be reduced in aging and AMD eyes, prompting us to examine its relationship with AMD. We detect suggestive association of a common FBN2 non-synonymous variant, rs154001 (p.Val965Ile) with AMD in 10 337 cases and 11 174 controls (OR = 1.10; P-value = 3.79 × 10−5). Thus, it appears that rare and common variants in a single gene—FBN2—can contribute to Mendelian and complex forms of macular degeneration. Our studies provide genetic evidence for a key role of elastin microfibers and Bruch′s membrane in maintaining blood–retina homeostasis and establish the importance of studying orphan diseases for understanding more common clinical phenotypes.
doi:10.1093/hmg/ddu276
PMCID: PMC4189898  PMID: 24899048
11.  No Clinically Significant Association Between CFH and ARMS2 Genotypes and Response to Nutritional Supplements 
Ophthalmology  2014;121(11):2173-2180.
Objective
To determine whether genotypes at two major loci associated with late age-related macular degeneration (AMD), complement factor H (CFH) and Age-Related Maculopathy Susceptibility 2 (ARMS2), influenced the relative benefits of Age-Related Eye Disease Study (AREDS) supplements.
Design
Unplanned retrospective evaluation of a prospective, randomized, placebo-controlled clinical trial of vitamins and minerals for the treatment of AMD.
Subjects
AREDS participants (mean age of 69 years) who were at risk for developing late AMD and who were randomized to the 4 arms of the AREDS supplements.
Methods
Analyses were performed using the Cox proportional hazards model to predict progression to late AMD (neovascular or central geographic atrophy). Statistical models, adjusted for age, gender, smoking status and baseline AMD severity, were used to examine the influence of genotypes on the response to therapy with 4 randomly assigned arms of AREDS supplement components: placebo, antioxidants (vitamins C, E, beta-carotene), zinc with copper, or the combination.
Main Outcome Measures
The influence of the genotype on the relative treatment response to the randomized components of the AREDS supplement, measured as progression to late AMD.
Results
Of the 1237 genotyped AREDS participants of Caucasian ethnicity, 385 (31.1%) developed late AMD during the mean follow-up period of 6.6 years. As previously demonstrated, both CFH genotype (p=0.005), ARMS2 (<0.0001) and supplement were each individually associated with progression to late AMD. An interaction analysis found no evidence that the relative benefits of AREDS supplementation varied by genotype. Analysis of (1) CFH rs1061170 and rs1410996 combined with ARMS2 rs10490924, with the 4 randomly assigned arms of AREDS supplement and (2) analysis of the combination of CFH rs412852 and rs3766405 with ARMS2 c.372_815del443ins54 with the AREDS components resulted in no interaction (p=0.06 and p=0.45, respectively, before multiplicity adjustment).
Conclusions
AREDS supplements reduced the rate of AMD progression across all genotype groups. Furthermore, the genotypes at the CFH and ARMS2 loci did not statistically significantly alter the benefits of AREDS supplements. Genetic testing remains a valuable research tool, but analysis of AREDS study data suggests it provides no benefits in managing nutritional supplementation for patients at risk of late AMD.
doi:10.1016/j.ophtha.2014.05.008
PMCID: PMC4253656  PMID: 24974817
12.  Proton Release from the Histidine-Tetrad in the M2 Channel of the Influenza A Virus 
The Journal of Physical Chemistry. B  2014;118(44):12644-12651.
The activity of the M2 proton channel of the influenza A virus is controlled by pH. The tautomeric state and conformation of His37, a key residue in the M2 transmembrane four-helix bundle, controls the gating of the channel. Previously, we compared the energetics and dynamics of two alternative conformations of the doubly protonated state at neutral pH, namely, a 4-fold symmetric “histidine-box” and a 2-fold symmetric “dimer-of-dimers”, and proposed a multiconfiguration model for this charge state. Here, we elaborate this model by further studying configurations of the His37 tetrad in the triply protonated state and its subsequent deprotonation via quantum mechanics/molecular mechanics (QM/MM) molecular dynamics (MD) simulations, starting with the aforementioned configurations, to gain information about proton release in a viral membrane environment. Interestingly, the two configurations converge under acidic pH conditions. Protons can be transferred from one charged His37 to a neighboring water cluster at the C-terminal side of the channel when the Trp41 gate is open transiently. With limited backbone expansion, the free energy barrier for proton release to the viral interior at low pH is ∼6.5 kcal/mol in both models, which is much lower than at either neutral pH or for an isolated His37 cluster without a membrane environment. Our calculations also suggest that the M2 protein would seem to exclude the entrance of anions into the central channel through a special mechanism, due to the latter’s potential inhibitory effect on proton conduction.
doi:10.1021/jp5102225
PMCID: PMC4226308  PMID: 25317959
13.  Canine CNGA3 Gene Mutations Provide Novel Insights into Human Achromatopsia-Associated Channelopathies and Treatment 
PLoS ONE  2015;10(9):e0138943.
Cyclic nucleotide-gated (CNG) ion channels are key mediators underlying signal transduction in retinal and olfactory receptors. Genetic defects in CNGA3 and CNGB3, encoding two structurally related subunits of cone CNG channels, lead to achromatopsia (ACHM). ACHM is a congenital, autosomal recessive retinal disorder that manifests by cone photoreceptor dysfunction, severely reduced visual acuity, impaired or complete color blindness and photophobia. Here, we report the first canine models for CNGA3-associated channelopathy caused by R424W or V644del mutations in the canine CNGA3 ortholog that accurately mimic the clinical and molecular features of human CNGA3-associated ACHM. These two spontaneous mutations exposed CNGA3 residues essential for the preservation of channel function and biogenesis. The CNGA3-R424W results in complete loss of cone function in vivo and channel activity confirmed by in vitro electrophysiology. Structural modeling and molecular dynamics (MD) simulations revealed R424-E306 salt bridge formation and its disruption with the R424W mutant. Reversal of charges in a CNGA3-R424E-E306R double mutant channel rescued cGMP-activated currents uncovering new insights into channel gating. The CNGA3-V644del affects the C-terminal leucine zipper (CLZ) domain destabilizing intersubunit interactions of the coiled-coil complex in the MD simulations; the in vitro experiments showed incompetent trimeric CNGA3 subunit assembly consistent with abnormal biogenesis of in vivo channels. These newly characterized large animal models not only provide a valuable system for studying cone-specific CNG channel function in health and disease, but also represent prime candidates for proof-of-concept studies of CNGA3 gene replacement therapy for ACHM patients.
doi:10.1371/journal.pone.0138943
PMCID: PMC4583268  PMID: 26407004
14.  Gating pore currents are defects in common with two Nav1.5 mutations in patients with mixed arrhythmias and dilated cardiomyopathy 
The Journal of General Physiology  2015;145(2):93-106.
Nav1.5 channels bearing voltage-sensor domain mutations associated with atypical cardiac arrhythmias and dilated cardiomyopathy generate gating pore currents.
The gating pore current, also called omega current, consists of a cation leak through the typically nonconductive voltage-sensor domain (VSD) of voltage-gated ion channels. Although the study of gating pore currents has refined our knowledge of the structure and the function of voltage-gated ion channels, their implication in cardiac disorders has not been established. Two Nav1.5 mutations (R222Q and R225W) located in the VSD are associated with atypical clinical phenotypes involving complex arrhythmias and dilated cardiomyopathy. Using the patch-clamp technique, in silico mutagenesis, and molecular dynamic simulations, we tested the hypothesis that these two mutations may generate gating pore currents, potentially accounting for their clinical phenotypes. Our findings suggest that the gating pore current generated by the R222Q and R225W mutations could constitute the underlying pathological mechanism that links Nav1.5 VSD mutations with human cardiac arrhythmias and dilatation of cardiac chambers.
doi:10.1085/jgp.201411304
PMCID: PMC4306709  PMID: 25624448
15.  Characterization of the honeybee AmNaV1 channel and tools to assess the toxicity of insecticides 
Scientific Reports  2015;5:12475.
Pollination is important for both agriculture and biodiversity. For a significant number of plants, this process is highly, and sometimes exclusively, dependent on the pollination activity of honeybees. The large numbers of honeybee colony losses reported in recent years have been attributed to colony collapse disorder. Various hypotheses, including pesticide overuse, have been suggested to explain the disorder. Using the Xenopus oocytes expression system and two microelectrode voltage-clamp, we report the functional expression and the molecular, biophysical, and pharmacological characterization of the western honeybee’s sodium channel (Apis Mellifera NaV1). The NaV1 channel is the primary target for pyrethroid insecticides in insect pests. We further report that the honeybee’s channel is also sensitive to permethrin and fenvalerate, respectively type I and type II pyrethroid insecticides. Molecular docking of these insecticides revealed a binding site that is similar to sites previously identified in other insects. We describe in vitro and in silico tools that can be used to test chemical compounds. Our findings could be used to assess the risks that current and next generation pesticides pose to honeybee populations.
doi:10.1038/srep12475
PMCID: PMC4894402  PMID: 26202396
16.  Quantitative optical coherence tomography angiography of choroidal neovascularization in age-related macular degeneration 
Ophthalmology  2014;121(7):1435-1444.
Purpose
To detect and quantify choroidal neovascularization (CNV) in age-related macular degeneration (AMD) patients using optical coherence tomography (OCT) angiography.
Design
Observational, cross-sectional study.
Participants
Five normal subjects and five neovascular AMD patients were included.
Methods
Five eyes with neovascular AMD and five normal age-matched controls were scanned by a high-speed (100,000 A-scans/sec) 1050 nm wavelength swept-source OCT. The macular angiography scan covered a 3×3 mm area and comprised 200×200×8 A-scans acquired in 3.5 sec. Flow was detected using the split-spectrum amplitude-decorrelation angiography (SSADA) algorithm. Motion artifacts were removed by three dimensional (3D) orthogonal registration and merging of 4 scans. The 3D angiography was segmented into 3 layers: inner retina (to show retinal vasculature), outer retina (to identify CNV), and choroid. En face maximum projection was used to obtain 2D angiograms from the 3 layers. CNV area and flow index were computed from the en face OCT angiogram of the outer retinal layer. Flow (decorrelation) and structural data were combined in composite color angiograms for both en face and cross-sectional views.
Main Outcome Measurements
CNV angiogram, CNV area, and CNV flow index.
Results
En face OCT angiograms of CNVs showed sizes and locations that were confirmed by fluorescein angiography. OCT angiography provided more distinct vascular network patterns that were less obscured by subretinal hemorrhage. The en face angiograms also showed areas of reduced choroidal flow adjacent to the CNV in all cases and significantly reduced retinal flow in one case. Cross-sectional angiograms were used to visualize CNV location relative to the retinal pigment epithelium and Bruch’s layer and classify type I and type II CNV. A feeder vessel could be identified in one case. Higher flow indexes were associated with larger CNV and type II CNV.
Conclusions
OCT angiography provides depth-resolved information and detailed images of CNV in neovascular AMD. Quantitative information regarding CNV flow and area can be obtained. Further studies are needed to assess the role of quantitative OCT angiography in the evaluation and treatment of neovascular AMD.
doi:10.1016/j.ophtha.2014.01.034
PMCID: PMC4082740  PMID: 24679442
17.  Clinical and Genetic Factors Associated with Progression of Geographic Atrophy Lesions in Age-Related Macular Degeneration 
PLoS ONE  2015;10(5):e0126636.
Worldwide, age-related macular degeneration (AMD) is a serious threat to vision loss in individuals over 50 years of age with a pooled prevalence of approximately 9%. For 2020, the number of people afflicted with this condition is estimated to reach 200 million. While AMD lesions presenting as geographic atrophy (GA) show high inter-individual variability, only little is known about prognostic factors. Here, we aimed to elucidate the contribution of clinical, demographic and genetic factors on GA progression. Analyzing the currently largest dataset on GA lesion growth (N = 388), our findings suggest a significant and independent contribution of three factors on GA lesion growth including at least two genetic factors (ARMS2_rs10490924 [P < 0.00088] and C3_rs2230199 [P < 0.00015]) as well as one clinical component (presence of GA in the fellow eye [P < 0.00023]). These correlations jointly explain up to 7.2% of the observed inter-individual variance in GA lesion progression and should be considered in strategy planning of interventional clinical trials aimed at evaluating novel treatment options in advanced GA due to AMD.
doi:10.1371/journal.pone.0126636
PMCID: PMC4427438  PMID: 25962167
18.  Proton affinity of the histidine-tryptophan cluster motif from the influenza A virus from ab initio molecular dynamics 
Chemical physics  2013;422:156-164.
Ab initio molecular dynamics calculations have been used to compare and contrast the deprotonation reaction of a histidine residue in aqueous solution with the situation arising in a histidine-tryptophan cluster. The latter is used as a model of the proton storage unit present in the pore of the M2 proton conducting ion channel. We compute potentials of mean force for the dissociation of a proton from the Nδ and Nε positions of the imidazole group to estimate the pKa’s. Anticipating our results, we will see that the estimated pKa for the first protonation event of the M2 channel is in good agreement with experimental estimates. Surprisingly, despite the fact that the histidine is partially desolvated in the M2 channel, the affinity for protons is similar to that of a histidine in aqueous solution. Importantly, the electrostatic environment provided by the indoles is responsible for the stabilization of the charged imidazolium.
doi:10.1016/j.chemphys.2013.03.006
PMCID: PMC4407280  PMID: 25914436
19.  Hydrogen-Bonded Water Molecules in the M2 Channel of the Influenza A Virus Guide the Binding Preferences of Ammonium-Based Inhibitors 
The journal of physical chemistry. B  2014;119(3):1173-1183.
The tetrameric M2 proton channel of influenza A virus is an integral membrane protein responsible for the acidification of the viral interior. Drugs such as amantadine target the transmembrane region of wild type M2 by acting as pore blockers. However, a number of mutations affecting this domain confer drug resistance, prompting the need for alternative inhibitors. The availability of high-resolution structures of drug-bound M2, paired with computational investigations, revealed that inhibitors can bind at different sites, and provided useful insights in understanding the principles governing proton conduction. Here, we investigated by computation the energetic and geometric factors determining the relative stability of pore blockers at individual sites of different M2 strains. We found that local free energy minima along the translocation pathway of positively charged chemical species correspond to experimentally determined binding sites of inhibitors. Then, by examining the structure of water clusters hydrating each site, as well as of those displaced by binding of hydrophobic scaffolds, we predicted the binding preferences of M2 ligands. This information can be used to guide the identification of novel classes of inhibitors.
doi:10.1021/jp506807y
PMCID: PMC4386636  PMID: 25353315
20.  Combined computational and experimental analysis of a complex of ribonuclease III and the regulatory macrodomain protein, YmdB 
Proteins  2015;83(3):459-472.
Ribonuclease III is a conserved bacterial endonuclease that cleaves double-stranded(ds) structures in diverse coding and noncoding RNAs. RNase III is subject to multiple levels of control that in turn confer global post-transcriptional regulation. The Escherichia coli macrodomain protein YmdB directly interacts with RNase III, and an increase in YmdB amount in vivo correlates with a reduction in RNase III activity. Here, a computational-based structural analysis was performed to identify atomic-level features of the YmdB-RNase III interaction. The docking of monomeric E. coli YmdB with a homology model of the E. coli RNase III homodimer yields a complex that exhibits an interaction of the conserved YmdB residue R40 with specific RNase III residues at the subunit interface. Surface Plasmon Resonance (SPR) analysis provided a KD of 61 nM for the complex, corresponding to a binding free energy (ΔG) of −9.9 kcal/mol. YmdB R40 and RNase III D128 were identified by in silico alanine mutagenesis as thermodynamically important interacting partners. Consistent with the prediction, the YmdB R40A mutation causes a 16-fold increase in KD (ΔΔG = +1.8 kcal/mol), as measured by SPR, and the D128A mutation in both RNase III subunits (D128A/D128′A) causes an 83-fold increase in KD (ΔΔG = +2.7 kcal/mol). The greater effect of the D128A/D128′A mutation may reflect an altered RNase III secondary structure, as revealed by CD spectroscopy, which also may explain the significant reduction in catalytic activity in vitro. The features of the modeled complex relevant to potential RNase III regulatory mechanisms are discussed. Proteins 2015; 83:459–472. © 2014 The Authors. Proteins: Structure, Function, and Bioinformatics Published by Wiley Periodicals, Inc.
doi:10.1002/prot.24751
PMCID: PMC4329070  PMID: 25546632
RNase III; macrodomain; RNA processing; ADP-ribose; double-stranded RNA; post-transcriptional regulation
21.  Flipping in the Pore: Discovery of Dual Inhibitors That Bind in Different Orientations to the Wild-Type versus the Amantadine-Resistant S31N Mutant of the Influenza A Virus M2 Proton Channel 
Journal of the American Chemical Society  2014;136(52):17987-17995.
Influenza virus infections lead to numerous deaths and millions of hospitalizations each year. One challenge facing anti-influenza drug development is the heterogeneity of the circulating influenza viruses, which comprise several strains with variable susceptibility to antiviral drugs. For example, the wild-type (WT) influenza A viruses, such as the seasonal H1N1, tend to be sensitive to antiviral drugs, amantadine and rimantadine, while the S31N mutant viruses, such as the pandemic 2009 H1N1 (H1N1pdm09) and seasonal H3N2, are resistant to this class of drugs. Thus, drugs targeting both WT and the S31N mutant are highly desired. We report our design of a novel class of dual inhibitors along with their ion channel blockage and antiviral activities. The potency of the most active compound 11 in inhibiting WT and the S31N mutant influenza viruses is comparable with that of amantadine in inhibiting WT influenza virus. Solution NMR studies and molecular dynamics (MD) simulations of drug-M2 interactions supported our design hypothesis: namely, the dual inhibitor binds in the WT M2 channel with an aromatic group facing down toward the C-terminus, while the same drug binds in the S31N M2 channel with its aromatic group facing up toward the N-terminus. The flip-flop mode of drug binding correlates with the structure–activity relationship (SAR) and has paved the way for the next round of rational design of broad-spectrum antiviral drugs.
doi:10.1021/ja508461m
PMCID: PMC4286326  PMID: 25470189
22.  Risk of Geographic Atrophy in the Comparison of Age-related Macular Degeneration Treatments Trials 
Ophthalmology  2013;121(1):150-161.
Purpose
To describe risk factors for geographic atrophy (GA) in the Comparison of Age-related Macular Degeneration Treatments Trials (CATT).
Design
Cohort within a randomized clinical trial.
Participants
We analyzed 1024 CATT patients with no GA visible on color fundus photographs (CFPs) and/or fluorescein angiograms (FAs) at enrollment.
Methods
Eyes were assigned to ranibizumab (0.5 mg) or bevacizumab (1.25 mg) treatment and to a 2-year monthly or pro re nata (PRN) injection regimen, or monthly injections for 1 year and PRN for 1 year. Demographic, genetic, and baseline ocular characteristics and lesion features of CFP/FA and optical coherence tomography (OCT) were evaluated as risk factors for GA through 2 years of follow-up. Time-dependent Cox proportional hazard models were used to estimate adjusted hazard ratios (aHRs).
Main Outcome Measures
Development of GA.
Results
By 2 years, GA developed in 187 of 1024 patients (18.3%). Baseline risk factors for GA development included baseline visual acuity (VA) ≤20/200 (aHR, 2.65; 95% confidence interval [CI], 1.43–4.93), retinal angiomatous proliferation (RAP; aHR, 1.69; 95% CI, 1.16–2.47), GA in the fellow eye (aHR, 2.07; 95% CI, 1.40–3.08), and intraretinal fluid at the foveal center (aHR, 2.10; 95% CI, 1.34–3.31). Baseline factors associated with lower risk for GA development included blocked fluorescence (aHR, 0.49; 95% CI, 0.29–0.82), OCT measurements of subretinal fluid thickness of >25 μ (aHR, 0.52; 95% CI, 0.35–0.78), subretinal tissue complex thickness of >275 compared with ≤75 μ (aHR, 0.31; 95% CI, 0.19–0.50), and vitreomacular attachment (aHR, 0.55; 95% CI, 0.31–0.97). Ranibizumab compared with bevacizumab had a higher risk (aHR, 1.43; 95% CI, 1.06–1.93), and monthly dosing had a higher risk (aHR, 1.59; 95% CI, 1.17–2.16) than PRN dosing. There were no strong associations between development of GA and the presence of risk alleles for CFH, ARMS 2, HTRA1, C3, or TLR3.
Conclusions
Approximately one fifth of CATT patients developed GA within 2 years of treatment. Independent baseline risk factors included poor VA, RAP, foveal intraretinal fluid, monthly dosing, and treatment with ranibizumab. Anti-vascular endothelial growth factor therapy may have a role in the development of GA.
doi:10.1016/j.ophtha.2013.08.015
PMCID: PMC3892560  PMID: 24084496
23.  Prevalence of anti-retinal autoantibodies in different stages of Age-related macular degeneration 
BMC Ophthalmology  2014;14:154.
Background
Age-related macular degeneration (AMD) is the leading cause of central vision loss in older adults. Anti-retinal autoantibodies (AAbs) have been found in individuals with AMD. The goal of the study was to determine the AAb specificity in different stages of AMD, and determine whether there is a prevalent AAb signature.
Methods
Sera of 134 participants in the Age-related Eye Disease Study were analyzed for anti-retinal AAbs by western blotting. The subjects were classified by diagnostic subgroups based upon their clinical classification: No AMD, Intermediate AMD, and Late AMD - geographic atrophy (GA) and Late AMD - neovascular (NV).
Results
The presence of anti-retinal AAb was detected in 58% patients with Intermediate and Late AMD, and 54% of those with no AMD. AAbs bound to fifteen different retinal antigens. Most individuals had 1 specific AAbs (67%), with the remainder having 2 to 4 different AAbs. Over 40% of patients with Intermediate AMD, and 46% of those with GA had anti-enolase AAbs, compared with 29% of individuals with NV and 29% with no AMD. Different AAbs signatures related to NV as compared to GA and/or Intermediate AMD were distinguished. Anti-40-kDa (10%) and 42-kDa (16%) autoantibodies were associated with Intermediate AMD, while anti-30-kDa AAbs (23%) were primarily present in GA. Anti-32-kDa (12%), 35-kDa (21%), and 60-kDa (8%) AAbs were more frequent in NV AMD.
Conclusions
A unique AAb pattern for each of the disease subgroups was present when AMD progressed from the intermediate to the late forms of severity. Differences in the frequency of specific AAbs between AMD subgroups suggested that they may participate in pathogenicity of AMD. Further studies are necessary to confirm these observations in the larger cohort and individual AMD patients over time.
doi:10.1186/1471-2415-14-154
PMCID: PMC4269864  PMID: 25488058
Age-related macular degeneration; AREDS; Autoantibodies; Enolase; Antibody signature; Biomarker; Retina; Macula; Smoking; Arthritis
24.  Computer Simulations of Voltage-Gated Cation Channels 
The relentless growth in computational power has seen increasing applications of molecular dynamics (MD) simulation to the study of membrane proteins in realistic membrane environments, which include explicit membrane lipids, water and ions. The concomitant increasing availability of membrane protein structures for ion channels, and transporters -- to name just two examples -- has stimulated many of these MD studies. In the case of voltage-gated cation channels (VGCCs) recent computational works have focused on ion-conduction and gating mechanisms, along with their regulation by agonist/antagonist ligands. The information garnered from these computational studies is largely inaccessible to experiment and is crucial for understanding the interplay between the structure and function as well as providing new directions for experiments. This article highlights recent advances in probing the structure and function of potassium channels and offers a perspective on the challenges likely to arise in making analogous progress in characterizing sodium channels.
doi:10.1021/jz300089g
PMCID: PMC3328965  PMID: 22523619
Ion Channels; Ionic Conduction; Gating; S4 helix; Regulation; Molecular Dynamics Simulations; Structure and Function
25.  TCR Triggering by pMHC Ligands Tethered on Surfaces via Poly(Ethylene Glycol) Depends on Polymer Length 
PLoS ONE  2014;9(11):e112292.
Antigen recognition by T cells relies on the interaction between T cell receptor (TCR) and peptide-major histocompatibility complex (pMHC) at the interface between the T cell and the antigen presenting cell (APC). The pMHC-TCR interaction is two-dimensional (2D), in that both the ligand and receptor are membrane-anchored and their movement is limited to 2D diffusion. The 2D nature of the interaction is critical for the ability of pMHC ligands to trigger TCR. The exact properties of the 2D pMHC-TCR interaction that enable TCR triggering, however, are not fully understood. Here, we altered the 2D pMHC-TCR interaction by tethering pMHC ligands to a rigid plastic surface with flexible poly(ethylene glycol) (PEG) polymers of different lengths, thereby gradually increasing the ligands’ range of motion in the third dimension. We found that pMHC ligands tethered by PEG linkers with long contour length were capable of activating T cells. Shorter PEG linkers, however, triggered TCR more efficiently. Molecular dynamics simulation suggested that shorter PEGs exhibit faster TCR binding on-rates and off-rates. Our findings indicate that TCR signaling can be triggered by surface-tethered pMHC ligands within a defined 3D range of motion, and that fast binding rates lead to higher TCR triggering efficiency. These observations are consistent with a model of TCR triggering that incorporates the dynamic interaction between T cell and antigen-presenting cell.
doi:10.1371/journal.pone.0112292
PMCID: PMC4226474  PMID: 25383949

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