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1.  Immunogenicity, Safety, and Lot Consistency of a Novel Inactivated Enterovirus 71 Vaccine in Chinese Children Aged 6 to 59 Months 
Clinical and Vaccine Immunology : CVI  2013;20(12):1805-1811.
The determination of lot-to-lot consistency in the manufacturing process is a mandatory step in the clinical development of the novel enterovirus 71 (EV71) vaccine. A phase III, randomized, placebo-controlled, double-blind trial assessed the lot consistency, immunogenicity, and safety of the EV71 vaccine in children aged 6 to 59 months. Healthy children (n = 1,400) received one of three lots of the EV71 vaccine containing 400 U of EV71 antigen or a placebo at days 0 and 28. Blood samples were collected before dose 1 and at 28 days after dose 2 (day 56) for an anti-EV71 neutralizing antibody (NTAb) assay. The geometric mean titer (GMT) and the seropositivity rates (with titers of ≥1:8) were compared at day 56. After each dose, the solicited injection site and general adverse events (AEs) were recorded for 7 days, and unsolicited AEs were recorded for 28 days. At day 56, the seropositivity rates ranged from 99.7% to 100% for the vaccine groups. The NTAb GMTs for the vaccine were 140.3 (95% confidence interval [CI], 117.8 to 167.1), 141.5 (95% CI, 118.0 to 169.6), and 146.6 (95% CI, 122.5 to 175.3). The two-sided 95% CI of the log difference in GMTs between the pairs of lots were between −0.176 and 0.176, therefore meeting the predefined equivalence criteria. The percentages of subjects reporting any injection site AEs, general AEs, or serious AEs were similar across the four vaccination groups. In conclusion, the demonstration of consistency between the manufacturing lots confirms for the purposes of clinical development the reliability of the EV71 vaccine production process. (This study has been registered at under registration no. NCT01636245.)
PMCID: PMC3889509  PMID: 24108780
2.  The Cross-Neutralizing Activity of Enterovirus 71 Subgenotype C4 Vaccines in Healthy Chinese Infants and Children 
PLoS ONE  2013;8(11):e79599.
EV71 is one of major etiologic causes of hand-foot-mouth disease (HFMD) and leads to severe neurological complications in young children and infants. Recently inactivated EV71 vaccines have been developed by five manufactures and clinically show good safety and immunogenicity. However, the cross-neutralizing activity of these vaccines remains unclear, and is of particular interest because RNA recombination is seen more frequently in EV71 epidemics.
Methodology/Principal Findings
In this post-hoc study, sera from a subset of 119 infants and children in two clinical trials of EV71 subgenotype C4 vaccines ( Identifier: NCT01313715 and NCT01273246), were detected for neutralizing antibody (NTAb) titres with sera from infected patients as controls. Cytopathogenic effect method was employed to test NTAb against EV71 subgenotype B4, B5, C2, C4 and C5, which were prominent epidemic strains worldwide over the past decade. To validate the accuracy of the results, ELISpot assay was employed in parallel to detect NTAb in all the post-vaccine sera. After two-dose vaccination, 49 out of 53 participants in initially seronegative group and 52 out of 53 participants in initially seropositive group showed less than 4-fold differences in NTAb titers against five EV71 strains, whereas corresponding values among sera from pediatric patients recovering from EV71-induced HFMD and subclinically infected participants were 8/8 and 41/43, respectively. The geometric mean titers of participants against five subgenotypes EV71 all grew significantly after vaccinations, irrespective of the baseline NTAb titer. The relative fold increase in antibody titers (NTAb-FI) against B4, B5, C2, and C5 displayed a positive correlation to the NTAb-FI against C4.
The results demonstrated broad cross-neutralizing activity induced by two C4 EV71 vaccines in healthy Chinese infants and children. However, the degree of induced cross-protective immunity, and the potential escape evolution for EV71 still need to be monitored and researched in future for these new vaccines.
PMCID: PMC3834186  PMID: 24260259
3.  Characterizing Immunoglobulin Repertoire from Whole Blood by a Personal Genome Sequencer 
PLoS ONE  2013;8(9):e75294.
In human immune system, V(D)J recombination produces an enormously large repertoire of immunoglobulins (Ig) so that they can tackle different antigens from bacteria, viruses and tumor cells. Several studies have demonstrated the utility of next-generation sequencers such as Roche 454 and Illumina Genome Analyzer to characterize the repertoire of immunoglobulins. However, these techniques typically require separation of B cell population from whole blood and require a few weeks for running the sequencers, so it may not be practical to implement them in clinical settings. Recently, the Ion Torrent personal genome sequencer has emerged as a tabletop personal genome sequencer that can be operated in a time-efficient and cost-effective manner. In this study, we explored the technical feasibility to use multiplex PCR for amplifying V(D)J recombination for IgH, directly from whole blood, then sequence the amplicons by the Ion Torrent sequencer. The whole process including data generation and analysis can be completed in one day. We tested the method in a pilot study on patients with benign, atypical and malignant meningiomas. Despite the noisy data, we were able to compare the samples by their usage frequencies of the V segment, as well as their somatic hypermutation rates. In summary, our study suggested that it is technically feasible to perform clinical monitoring of V(D)J recombination within a day by personal genome sequencers.
PMCID: PMC3772839  PMID: 24058670
4.  Ionotropic glutamate receptors mediate OFF responses in light-adapted ON bipolar cells 
Vision research  2012;68:48-58.
Previous studies have suggested that photoreceptor synaptic inputs to depolarizing bipolar cells (DBCs or ON bipolar cells) are mediated by mGluR6 receptors and those to hyperpolarizing bipolar cells (HBCs or OFF bipolar cells) are mediated by AMPA/kainate receptors. Here we show that in addition to mGluR6 receptors which mediate the sign-inverting, depolarizing light responses, subpopulations of cone-dominated and rod/cone mixed DBCs use GluR4 AMPA receptors to generate a transient sign-preserving OFF response under light adapted conditions. These AMPA receptors are located at the basal junctions postsynaptic to rods and they are silent under dark-adapted conditions, as tonic glutamate release in darkness desensitizes these receptors. Light adaptation enhances rod-cone coupling and thus allows cone photocurrents with an abrupt OFF depolarization to enter the rods. The abrupt rod depolarization triggers glutamate activation of unoccupied AMPA receptors, resulting in a transient OFF response in DBCs. It has been widely accepted that the DNQX-sensitive, OFF transient responses in retinal amacrine cells and ganglion cells are mediated exclusively by HBCs. Our results suggests that this view needs revision as AMPA receptors in subpopulations of DBCs are likely to significantly contribute to the DNQX-sensitive OFF transient responses in light-adapted third- and higher-order visual neurons.
PMCID: PMC3733217  PMID: 22842089
Depolarizing bipolar cells; transient OFF responses; GluR4 receptors; mGluR6 receptors; 6,7-dinitroquinoxaline-2,3-dione (DNQX); cyclothiazide; dark and light adaptation
5.  Progress on the research and development of inactivated EV71 whole-virus vaccines 
Human Vaccines & Immunotherapeutics  2013;9(8):1701-1705.
The prevalence of diseases caused by EV71 infection has become a serious public health problem in the Western Pacific region. Due to a lack of effective treatment options, controlling EV71 epidemics has mainly focused on the research and development (R&D) of EV71 vaccines. Thus far, five organizations have completed pre-clinical studies focused on the development of inactivated EV71 whole-virus vaccines, including vaccine strain screening, process optimization, safety and immunogenicity evaluation, and are in different stages of clinical trials. Among these organizations, three companies in Mainland China [Beijing Vigoo Biological Co., Ltd. (Vigoo), Sinovac Biotech Ltd. (Sinovac) and Institute of Medical Biology, Chinese Academy of Medical Science (CAMS)] have recently completed Phase III trials for the vaccines they developed. In addition, the other two vaccines, developed by National Health Research Institutes (NHRI) of Taiwan and Inviragen Pte., Ltd (Inviragen), of Singapore, have also completed Phase I clinical trials. Published clinical trial results indicate that the inactivated EV71 vaccines have good safety and immunogenicity in the target population (infants) and confer a relatively high rate of protection against EV71 infection-related diseases. The results of clinical trials suggest a promising future for the clinical use of EV71 vaccines. Here, we review and highlight the recent progress on the R&D of inactivated EV71 whole-virus vaccines.
PMCID: PMC3906269  PMID: 23744508
enterovirus 71; hand-foot-mouth diseases (HFMD); inactivated whole-virus vaccine; immunogenicity; protective effect; standard
6.  Development and Evaluation of a Pseudovirus-Luciferase Assay for Rapid and Quantitative Detection of Neutralizing Antibodies against Enterovirus 71 
PLoS ONE  2013;8(6):e64116.
The level of neutralizing antibodies (NtAb) induced by vaccine inoculation is an important endpoint to evaluate the efficacy of EV71 vaccine. In order to evaluate the efficacy of EV71 vaccine, here, we reported the development of a novel pseudovirus system expression firefly luciferase (PVLA) for the quantitative measurement of NtAb. We first evaluated and validated the sensitivity and specificity of the PVLA method. A total of 326 serum samples from an epidemiological survey and 144 serum specimens from 3 clinical trials of EV71 vaccines were used, and the level of each specimen's neutralizing antibodies (NtAb) was measured in parallel using both the conventional CPE-based and PVLA-based assay. Against the standard neutralization assay based on the inhibition of the cytopathic effect (CPE), the sensitivity and specificity of the PVLA method are 98% and 96%, respectively. Then, we tested the potential interference of NtAb against hepatitis A virus, Polio-I, Polio-II, and Polio-III standard antisera (WHO) and goat anti-G10/CA16 serum, the PVLA based assay showed no cross-reactivity with NtAb against other specific sera. Importantly, unlike CPE based method, no live replication-competent EV71 is used during the measurement. Taken together, PVLA is a rapid and specific assay with higher sensitivity and accuracy. It could serve as a valuable tool in assessing the efficacy of EV71 vaccines in clinical trials and disease surveillance in epidemiology studies.
PMCID: PMC3673970  PMID: 23755115
7.  Investigation of sit-to-stand and stand-to-sit in an above knee amputee 
the objective of this pilot study is twofold: 1) to extract key factors/features in sit-to-stand and stand-to-sit (STS) performed by an above knee (AK) amputee; 2) to propose a convenient way to quantify symmetry. One male unilateral transfemoral amputee participated in the pilot study. The subject was instructed to rise in a comfortable and natural manner and conduct a series of sit-to-stand, stand-to-sit. We simultaneously measured kinematics, kinetics and muscle activities. Principal component analysis (PCA) was used to reduce the dimension and identify modes and a convenient index of STS symmetry (slope of the major axis of the error ellipse) is proposed using the insole pressure sensors. Based on the preliminary results it is recommended that kinematics and kinetics in both the sagittal and frontal planes be considered for an AK amputee performing STS. The information might be useful for further research on amputee STS.
PMCID: PMC3667594  PMID: 22256034
8.  Comparative analysis of 4C-Seq data generated from enzyme-based and sonication-based methods 
BMC Genomics  2013;14:345.
Circular chromosome conformation capture, when coupled with next-generation sequencing (4C-Seq), can be used to identify genome-wide interaction of a given locus (a “bait” sequence) with all of its interacting partners. Conventional 4C approaches used restriction enzyme digestion to fragment chromatin, and recently sonication approach was also applied for this purpose. However, bioinformatics pipelines for analyzing sonication-based 4C-Seq data are not well developed. In addition, data consistency as well as similarity between the two methods has not been explored previously. Here we present a comparative analysis of 4C-Seq data generated by both methods, using an enhancer element of Pou5f1 gene in mouse embryonic stem (ES) cells.
From biological replicates, we found good correlation (r>0.6) for inter-chromosomal interactions identified in either enzyme or sonication method. Compared to enzyme approach, sonication method generated less distal intra-chromosomal interactions, possibly due to the difference in chromatin fragmentation. From all mapped interactions, we further applied statistical models to identify enriched interacting regions. Interestingly, data generated from the two methods showed 30% overlap of the reproducible interacting regions. The interacting sites in the reproducible regions from both methods are similarly enriched with active histone marks. In addition, the interacting sites identified from sonication-based data are enriched with ChIP-Seq signals of transcription factors Oct4, Klf4, Esrrb, Tcfcp2i1, and Zfx that are critical for reprogramming and pluripotency.
Both enzyme-based and sonication-based 4C-Seq methods are valuable tools to explore long-range chromosomal interactions. Due to the nature of sonication-based method, correlation analysis of the 4C interactions with transcription factor binding should be more straightforward.
PMCID: PMC3679908  PMID: 23705979
Circular chromosome conformation capture; Sonication; 4C-Seq; Bioinformatics; Next-generation sequencing
9.  Use of Multidimensional Fluorescence Resonance Energy Transfer To Establish the Orientation of Cholecystokinin Docked at the Type A Cholecystokinin Receptor† 
Biochemistry  2008;47(36):9574-9581.
Fluorescence resonance energy transfer (FRET) represents a powerful tool to establish relative distances between donor and acceptor fluorophores. By utilizing several donors situated in distinct positions within a docked full agonist ligand and several acceptors distributed at distinct sites within its receptor, multiple interdependent dimensions can be determined. These can provide a unique method to establish or confirm three-dimensional structure of the molecular complex. In this work, we have utilized full agonist analogues of cholecystokinin (CCK) with Aladan distributed throughout the pharmacophore in positions 24, 29, and 33, along with receptor constructs derivatized with Alexa546 at positions 94, 102, 204, and 341 in the helical bundle and first, second, and third extracellular loops, respectively. These provided 12 FRET distances to overlay on working models of the CCK-occupied receptor. These established that the carboxyl terminus of CCK resides at the external surface of the lipid bilayer, adjacent to the receptor amino-terminal tail, rather than being inserted into the helical bundle. They also provide important experimentally derived constraints for understanding spatial relationships between the docked ligand and the flexible extracellular loop regions. Multidimensional FRET provides a new independent method to establish and refine structural insights into ligand–receptor complexes.
PMCID: PMC3648886  PMID: 18700727
10.  The interactomes of POU5F1 and SOX2 enhancers in human embryonic stem cells 
Scientific Reports  2013;3:1588.
The genes POU5F1 and SOX2 are critical for pluripotency and reprogramming, yet the chromosomal organization around these genes remains poorly understood. We assayed long-range chromosomal interactions on putative enhancers of POU5F1 and SOX2 genes in human embryonic stem cells (hESCs) using 4C-Seq technique. We discovered that their frequent interacting regions mainly overlap with early DNA replication domains. The interactomes are associated with active histone marks and enriched with 5-hydroxymethylcytosine sites. In hESCs, genes within the interactomes have elevated expression. Additionally, some genes associated with the POU5F1 enhancer contribute to pluripotency. Binding sites for multiple DNA binding proteins, including ATF3, CTCF, GABPA, JUND, NANOG, RAD21 and YY1, are enriched in both interactomes. The RARG locus, frequently interacting with the POU5F1 locus, has abundant RAD21 binding sites co-localized with other protein binding sites. Thus the interactomes of these two pluripotency genes could be an important part of the regulatory network in hESCs.
PMCID: PMC4070749  PMID: 23549118
11.  DNA Methylation in the Malignant Transformation of Meningiomas 
PLoS ONE  2013;8(1):e54114.
Meningiomas are central nervous system tumors that originate from the meningeal coverings of the brain and spinal cord. Most meningiomas are pathologically benign or atypical, but 3–5% display malignant features. Despite previous studies on benign and atypical meningiomas, the key molecular pathways involved in malignant transformation remain to be determined, as does the extent of epigenetic alteration in malignant meningiomas. In this study, we explored the landscape of DNA methylation in ten benign, five atypical and four malignant meningiomas. Compared to the benign tumors, the atypical and malignant meningiomas demonstrate increased global DNA hypomethylation. Clustering analysis readily separates malignant from atypical and benign tumors, implicating that DNA methylation patterns may serve as diagnostic biomarkers for malignancy. Genes with hypermethylated CpG islands in malignant meningiomas (such as HOXA6 and HOXA9) tend to coincide with the binding sites of polycomb repressive complexes (PRC) in early developmental stages. Most genes with hypermethylated CpG islands at promoters are suppressed in malignant and benign meningiomas, suggesting the switching of gene silencing machinery from PRC binding to DNA methylation in malignant meningiomas. One exception is the MAL2 gene that is highly expressed in benign group and silenced in malignant group, representing de novo gene silencing induced by DNA methylation. In summary, our results suggest that malignant meningiomas have distinct DNA methylation patterns compared to their benign and atypical counterparts, and that the differentially methylated genes may serve as diagnostic biomarkers or candidate causal genes for malignant transformation.
PMCID: PMC3551961  PMID: 23349797
12.  Recent Structural and Mechanistic Insights into Endplate Acetylcholine Receptors 
Voluntary movement mediated by skeletal muscle relies on endplate acetylcholine receptors (AChR) to detect nerve-released ACh and depolarize themuscle fiber. Recent structural and mechanistic studies of the endplate AChR have catalyzed a leap in our understanding of the molecular steps in this chemical-to-electrical transduction process. Studies of acetylcholine binding protein (AChBP) give insight into ACh recognition, the first step in activation of the AChR. An atomic structural model of the Torpedo AChR at a resolution of 0.4 nm, together with single-ion channel recording methods, allow tracing of the link between the agonist binding event and gating of the ion channel, as well as determination of how the channel moves when it opens to allow flow of cations. Structural models of the human AChR enable precise mapping of disease-causing mutations, while studies of the speed with which single AChR channels open and close cast light on pathogenic mechanisms.
PMCID: PMC3478106  PMID: 18567853
acetylcholine receptor; acetylcholine binding protein; agonist recognition; binding-gating coupling mechanism; congenital myasthenic syndrome
13.  Comparative Analysis of the Immunogenicity and Protective Effects of Inactivated EV71 Vaccines in Mice 
PLoS ONE  2012;7(9):e46043.
Enterovirus 71 (EV71) is the major causative agent of hand, foot, and mouth disease (HFMD). Three inactivated EV71 whole-virus vaccines of different strains developed by different manufacturers in mainland China have recently entered clinical trials. Although several studies on these vaccines have been published, a study directly comparing the immunogenicity and protective effects among them has not been carried out, which makes evaluating their relative effectiveness difficult. Thus, properly comparing newly developed vaccines has become a priority, especially in China.
Methods and Findings
This comparative immunogenicity study was carried out on vaccine strains (both live and inactivated), final container products (FCPs) without adjuvant, and corresponding FCPs containing adjuvant (FCP-As) produced by three manufacturers. These vaccines were evaluated by neutralizing antibody (NAb) responses induced by the same or different dosages at one or multiple time points post-immunization. The protective efficacy of the three vaccines was also determined in one-day-old ICR mice born to immunized female mice. Survival rates were observed in these suckling mice after challenge with 20 LD50 of EV71/048M3C2. Three FCP-As, in a dose of 200 U, generated nearly 100% NAb positivity rates and similar geometric mean titers (GMTs), especially at 14–21 days post-inoculation. However, the dynamic NAb responses were different among three vaccine strains or three FCPs. The FCP-As at the lowest dose used in clinical trials (162 U) showed good protective effects in suckling mice against lethal challenge (90–100% survival), while the ED50 of NAb responses and protective effects varied among three FCP-As.
These studies establish a standard method for measuring the immunogenicity of EV71 vaccines in mice. The data generated from our mouse model study indicated a clear dose-response relationship, which is important for vaccine quality control and assessment, especially for predicting protective efficacy in humans when combined with future clinical trial results.
PMCID: PMC3460965  PMID: 23029378
14.  Effects of repeated ankle stretching on calf muscle-tendon and ankle biomechanical properties in stroke survivors 
The objective of this study was to investigate changes in active and passive biomechanical properties of the calf muscle-tendon unit induced by controlled ankle stretching in stroke survivors.
Ten stroke survivors with ankle spasticity/contracture and ten healthy control subjects received intervention of 60-min ankle stretching. Joint biomechanical properties including resistance torque, stiffness and index of hysteresis were evaluated pre- and post-intervention. Achilles tendon length was measured using ultrasonography. The force output of the triceps surae muscles was characterized via the torque-angle relationship, by stimulating the calf muscles at a controlled intensity across different ankle positions.
Compared to healthy controls, the ankle position corresponding to the peak torque of the stroke survivors was shifted towards plantar flexion (P<0.001). Stroke survivors showed significantly higher resistance torques and joint stiffness (P<0.05), and these higher resistances were reduced significantly after the stretching intervention, especially in dorsiflexion (P = 0.013). Stretching significantly improved the force output of the impaired calf muscles in stroke survivors under matched stimulations (P<0.05). Ankle range of motion was also increased by stretching (P<0.001).
At the joint level, repeated stretching loosened the ankle joint with increased passive joint range of motion and decreased joint stiffness. At the muscle-tendon level, repeated stretching improved calf muscle force output, which might be associated with decreased muscle fascicle stiffness, increased fascicle length and shortening of the Achilles tendon. The study provided evidence of improvement in muscle tendon properties through stretching intervention.
PMCID: PMC3085098  PMID: 21211873
Stroke; Ultrasound; Muscle tendon; Spasticity; Triceps surae muscles
15.  Paclitaxel/sirolimus combination coated drug-eluting stent: In vitro and in vivo drug release studies 
Paclitaxel and sirolimus are the two major drugs for the treatment of coronary arterial disease in current drug eluting stents. The two drugs can effectively inhibit the in-stent restenosis through their independent pathways and show synergistic effect in preventing tumor tissue growth. We hypothesize that the combination of the two drugs in a drug eluting stent (DES) can also effectively suppress the neointima growth in the stented artery. The present work was focused on the investigation of paclitaxel/sirolimus combination release profiles from a novel biodegradable polymer (poly (D, L-lactide -co-glycolide) / amorphous calcium phosphate, PLGA/ACP) coated stent both in vitro and in vivo. For the in vitro, the drug releasing profiles were characterized by measuring the drug concentration in a drug release medium (Dulbecco’s phosphate buffered saline, DPBS, pH 7.4) at predetermined time points. For the in vivo, a rat aorta stenting model was employed. The results showed that both paclitaxel and sirolimus had a two-phase release profile both in vitro and in vivo, which is similar to the drug release profile of their individual coated DESs, and there is no evident of interference between two drugs. The data suggest that paclitaxel and sirolimus can be combined pharmacokinetically in a DES for the treatment of coronary arterial diseases.
PMCID: PMC3008332  PMID: 21126843
Amorphous Calcium Phosphate; Drug-Eluting Stent; Paclitaxel; Poly (D; L-lactide -co-glycolide); Sirolimus
16.  Enterovirus 71 viral capsid protein linear epitopes: Identification and characterization 
Virology Journal  2012;9:26.
To characterize the human humoral immune response against enterovirus 71 (EV71) infection and map human epitopes on the viral capsid proteins.
A series of 256 peptides spanning the capsid proteins (VP1, VP2, VP3) of BJ08 strain (genomic C4) were synthesized. An indirect enzyme-linked immunosorbent assay (ELISA) was carried out to detect anti-EV71 IgM and IgG in sera of infected children in acute or recovery phase. The partially overlapped peptides contained 12 amino acids and were coated in the plate as antigen (0.1 μg/μl). Sera from rabbits immunized with inactivated BJ08 virus were also used to screen the peptide panel.
A total of 10 human anti-EV71 IgM epitopes (vp1-14 in VP1; vp2-6, 21, 40 and 50 in VP2 and vp3-10, 12, 15, 24 and 75 in VP3) were identified in acute phase sera. In contrast, only one anti-EV71 IgG epitope in VP1 (vp1-15) was identified in sera of recovery stage. Four rabbit anti-EV71 IgG epitopes (vp1-14, 31, 54 and 71) were identified and mapped to VP1.
These data suggested that human IgM epitopes were mainly mapped to VP2 and VP3 with multi-epitope responses occurred at acute infection, while the only IgG epitope located on protein VP1 was activated in recovery phase sera. The dynamic changes of humoral immune response at different stages of infection may have public health significance in evaluation of EV71 vaccine immunogenicity and the clinical application of diagnostic reagents.
PMCID: PMC3292509  PMID: 22264266
EV71; Capsid protein; Epitopes; Humoral immune response
17.  Curariform Antagonists Bind in Different Orientations to Acetylcholine-binding Protein* 
The Journal of biological chemistry  2003;278(25):23020-23026.
Acetylcholine-binding protein (AChBP) recently emerged as a prototype for relating structure to function of the ligand binding domain of nicotinic acetylcholine receptors (AChRs). To understand interactions of competitive antagonists at the atomic structural level, we studied binding of the curare derivatives d-tubocurarine (d-TC) and metocurine to AChBP using computational methods, mutagenesis, and ligand binding measurements. To account for protein flexibility, we used a 2-ns molecular dynamics simulation of AChBP to generate multiple snapshots of the equilibrated dynamic structure to which optimal docking orientations were determined. Our results predict a predominant docking orientation for both d-TC and metocurine, but unexpectedly, the bound orientations differ fundamentally for each ligand. At one subunit interface of AChBP, the side chain of Tyr-89 closely approaches a positively charged nitrogen in d-TC but is farther away from the equivalent nitrogen in metocurine, whereas, at the opposing interface, side chains of Trp-53 and Gln-55 closely approach the metocurine scaffold but not that of d-TC. The different orientations correspond to ~170° rotation and ~30° degree tilt of the curare scaffold within the binding pocket. Mutagenesis of binding site residues in AChBP, combined with measurements of ligand binding, confirms the different docking orientations. Thus structurally similar ligands can adopt distinct orientations at receptor binding sites, posing challenges for interpreting structure-activity relationships for many drugs.
PMCID: PMC3191914  PMID: 12682067
18.  Pathway-Wide Association Study Implicates Multiple Sterol Transport and Metabolism Genes in HDL Cholesterol Regulation 
Pathway-based association methods have been proposed to be an effective approach in identifying disease genes, when single-marker association tests do not have sufficient power. The analysis of quantitative traits may be benefited from these approaches, by sampling from two extreme tails of the distribution. Here we tested a pathway association approach on a small genome-wide association study (GWAS) on 653 subjects with extremely high high-density lipoprotein cholesterol (HDL-C) levels and 784 subjects with low HDL-C levels. We identified 102 genes in the sterol transport and metabolism pathways that collectively associate with HDL-C levels, and replicated these association signals in an independent GWAS. Interestingly, the pathways include 18 genes implicated in previous GWAS on lipid traits, suggesting that genuine HDL-C genes are highly enriched in these pathways. Additionally, multiple biologically relevant loci in the pathways were not detected by previous GWAS, including genes implicated in previous candidate gene association studies (such as LEPR, APOA2, HDLBP, SOAT2), genes that cause Mendelian forms of lipid disorders (such as DHCR24), and genes expressing dyslipidemia phenotypes in knockout mice (such as SOAT1, PON1). Our study suggests that sampling from two extreme tails of a quantitative trait and examining genetic pathways may yield biological insights from smaller samples than are generally required using single-marker analysis in large-scale GWAS. Our results also implicate that functionally related genes work together to regulate complex quantitative traits, and that future large-scale studies may benefit from pathway-association approaches to identify novel pathways regulating HDL-C levels.
PMCID: PMC3268595  PMID: 22303337
GWAS; lipid; HDL-C; pathway analysis; cholesterol; sterol transport; sterol metabolism; genetic association
19.  Light responses and morphology of bNOS-immunoreactive neurons in the mouse retina 
The Journal of comparative neurology  2010;518(13):2456-2474.
Nitric oxide (NO), produced by NO synthase (NOS), modulates the function of all retinal neurons and ocular blood vessels and participates in the pathogenesis of ocular diseases. To further understand the regulation of ocular NO release, we systematically studied the morphology, topography and light responses of NOS-containing amacrine cells (NOACs) in dark-adapted mouse retina. Immunohistological staining for neuronal NOS (bNOS), combined with retrograde labeling of ganglion cells (GCs) with Neurobiotin (NB, a gap junction permeable dye) and Lucifer yellow (LY, a less permeable dye), was used to identify NOACs. The light responses of ACs were recorded under whole-cell voltage clamp conditions and cell morphology was examined with a confocal microscope. We found that in dark-adapted conditions bNOS-immunoreactivity (IR) was present primarily in the inner nuclear layer and the ganglion cell layer. bNOS-IR somas were negative for LY, thus they were identified as ACs; nearly 6 % of the cells were labeled by NB but not by LY, indicating that they were dye-coupled with GCs. Three morphological subtypes of NOACs (NI, NII and displaced) were identified. The cell density, inter-cellular distance and the distribution of NOACs were studied in whole retinas. Light evoked depolarizing highly sensitive ON-OFF responses in NI cells and less sensitive OFF responses in NII cells. Frequent (1 to 2 Hz) or abrupt change of light-intensity evoked larger peak responses. The possibility for light to modify NO release from NOACs is discussed.
PMCID: PMC3000797  PMID: 20503422
retrograde labeling; patch clamp recording; dye coupling
20.  Synthesis and in vitro characterization of radioiodinatable benzodiazepines selective for type 1 and 2 cholecystokinin receptors 
Journal of medicinal chemistry  2009;52(7):2138-2147.
Radiolabeled antagonists of specific peptide receptors identify a higher number of receptor binding sites than agonists and may thus be preferable for in vivo tumor targeting. In this study, two novel radioiodinated 1,4-benzodiazepines, (S)-1-(3-iodophenyl)-3-(1-methyl-2-oxo-5-phenyl-2,3-dihydro-1H-benzo[e][1,4]diazepin-3-yl)urea (7) and (R)-1-(3-iodophenyl)-3-(1-methyl-2-oxo-5-phenyl-2,3-dihydro-1H-benzo[e][1,4]diazepin-3-yl)urea (9), were developed. They were characterized in vitro as high affinity selective antagonists at cholecystokinin type 1 and 2 (CCK1 and CCK2) receptors using receptor binding, calcium mobilization, and internalization studies. Their binding to human tumor tissues was assessed with in vitro receptor autoradiography and compared with an established peptidic CCK agonist radioligand. The 125I-labeled CCK1 receptor-selective compound 9 often revealed a substantially higher amount of CCK1 receptor binding sites in tumors than the agonist 125I-CCK. Conversely, the radioiodinated CCK2 receptor-selective compound 7 showed generally weaker tumor binding than 125I-CCK. In conclusion, compound 9 is an excellent radioiodinated non-peptidic antagonist ligand for direct and selective labeling of CCK1 receptors in vitro. Moreover, it represents a suitable candidate to test antagonist binding to CCK1 receptor-expressing tumors in vivo.
PMCID: PMC2666544  PMID: 19271701
21.  Internal forces during object manipulation 
Internal force is a set of contact forces that does not disturb object equilibrium. The elements of the internal force vector cancel each other and, hence, do not contribute to the resultant (manipulation) force acting on the object. The mathematical independence of the internal and manipulation forces allows for their independent (decoupled) control realized in robotic manipulators. To examine whether in humans internal force is coupled with the manipulation force and what grasping strategy the performers utilize, the subjects (n=6) were instructed to make cyclic arm movements with a customized handle. Six combinations of handle orientation and movement direction were tested. These involved: parallel manipulations (1) VV task (vertical orientation and vertical movement) and (2) HH task (horizontal orientation and horizontal movement); orthogonal manipulations (3) VH task (vertical orientation and horizontal movement) and (4) HV task (horizontal orientation and vertical movement); and diagonal manipulations (5) DV task (diagonal orientation and vertical movement) and (6) DH task (diagonal orientation and horizontal movement). Handle weight (from 3.8 to 13.8 N), and movement frequency (from 1 to 3 Hz) were systematically changed. The analysis was performed at the thumb-virtual finger level (VF, an imaginary finger that produces a wrench equal to the sum of wrenches produced by all the fingers). At this level, the forces of interest could be reduced to the internal force and internal moment. During the parallel manipulations, the internal (grip) force was coupled with the manipulation force (producing object acceleration) and the thumb-VF forces increased or decreased in phase: the thumb and VF worked in synchrony to grasp the object more strongly or more weakly. During the orthogonal manipulations, the thumb-VF forces changed out of phase: the plots of the internal force vs. object acceleration resembled an inverted letter V. The HV task was the only task where the relative phase (coupling) between the normal forces of the thumb and VF depended on oscillation frequency. During the diagonal manipulations, the coupling was different in the DV and DH tasks. A novel observation of substantial internal moments is described: the moments produced by the normal finger forces were counterbalanced by the moments produced by the tangential forces such that the resultant moments were close to zero. Implications of the findings for the notion of grasping synergies are discussed.
PMCID: PMC2847586  PMID: 15912369
Internal force; Manipulation force; Prehension; Prehension synergy; Grasping; Fingers
22.  Motor control goes beyond physics: differential effects of gravity and inertia on finger forces during manipulation of hand-held objects 
According to basic physics, the local effects induced by gravity and acceleration are identical and cannot be separated by any physical experiment. In contrast—as this study shows—people adjust the grip forces associated with gravitational and inertial forces differently. In the experiment, subjects oscillated a vertically-oriented handle loaded with five different weights (from 3.8 N to 13.8 N) at three different frequencies in the vertical plane: 1 Hz, 1.5 Hz and 2.0 Hz. Three contributions to the grip force—static, dynamic, and stato-dynamic fractions—were quantified. The static fraction reflects grip force related to holding a load statically. The stato-dynamic fraction reflects a steady change in the grip force when the same load is moved cyclically. The dynamic fraction is due to acceleration-related adjustments of the grip force during oscillation cycles. The slope of the relation between the grip force and the load force was steeper for the static fraction than for the dynamic fraction. The stato-dynamic fraction increased with the frequency and load. The slope of the dynamic grip force–load force relation decreased with frequency, and as a rule, increased with the load. Hence, when adjusting grip force to task requirements, the central controller takes into account not only the expected magnitude of the load force but also such factors as whether the force is gravitational or inertial and the contributions of the object mass and acceleration to the inertial force. As an auxiliary finding, a complex finger coordination pattern aimed at preserving the rotational equilibrium of the object during shaking movements was reported.
PMCID: PMC2845181  PMID: 15580485
Prehension; Grasping; Stato-dynamic relation; Grip force; Load force
23.  Finger force vectors in multi-finger prehension 
Journal of biomechanics  2003;36(11):1745-1749.
In a majority of studies on grasp, only normal forces were measured and only when a zero torque was exerted on a hand-held object. This study concerns finger force vectors during the torque production tasks. Subjects (n = 8) stabilized a handle with an attachment that allowed for change of external torque from −1.5 to 1.5Nm. Forces and moments exerted by the digit tips on the object were recorded. At the large (>−0.375Nm) supination torques the index/middle and ring/little pairs of fingers generated oppositely directed tangential forces. The index and middle finger produced forces in a downward direction and therefore did not support the load. At a zero torque and pronation torques, the middle, ring and little fingers produced forces along nearly the same direction. The vector of the index finger force was always directed differently from the vectors of other finger forces, the angles ranged from 19°30′ to 47°40′. The points of force application were systematically displaced with the torque, with the exception of the little finger. Tangential finger forces contributed substantially to the total torque exerted on the hand-held object.
PMCID: PMC2845161  PMID: 14522218
Finger forces; Prehension; Grasping
24.  The principle of superposition in human prehension 
Robotica  2004;22(2):231-234.
The experimental evidence supports the validity of the principle of superposition for multi-finger prehension in humans. Forces and moments of individual digits are defined by two independent commands: “Grasp the object stronger/weaker to prevent slipping” and “Maintain the rotational equilibrium of the object”. The effects of the two commands are summed up.
PMCID: PMC2827859  PMID: 20186284
Superposition principle; Human prehension; Robotics
25.  Prehension Stability: Experiments With Expanding and Contracting Handle 
Journal of neurophysiology  2005;95(4):2513-2529.
We studied adjustments in digit forces and moments during holding a vertically oriented handle under slow, externally imposed changes in the width of the grasp. Subjects (n = 8) grasped a customized motorized handle with five digits and held it statically in the air. The handle width either increased (expanded) or decreased (contracted) at a rate of 1.0, 1.5, or 2.0 mm/s, while the subjects were asked to ignore the handle width changes, and their attention was distracted. External torques of 0.0, 0.25, and 0.5 Nm were applied to the handle in two directions. Forces and moments at the digit tips were measured with six-component sensors. The analysis was performed at the virtual finger (VF) and individual finger (IF) levels (VF is an imagined finger that produces the same wrench, i.e., the force and moment, as several fingers combined). In all the tasks, the normal VF and thumb forces increased with the handle expansion and decreased with the handle contraction. Similar behavior was seen for the thumb tangential force. In contrast, the VF tangential force decreased with the handle expansion and increased with the handle contraction. The changes in the tangential forces assisted the perturbations in the tasks requiring exertion of the supination moments and annulled the perturbation in the pronation effort tasks. In the former tasks, the equilibrium was maintained by the changes of the moments of normal forces, whereas in the latter tasks, the equilibrium was maintained by the changes of the moments of the tangential forces. Analysis at the IF level has shown that the resultant force and moment exerted on the object could arise from dissimilar adjustments of individual fingers to the same handle width change. The complex adjustments of digit forces to handle width change may be viewed as coming from two sources. First, there are local spring-like adjustments of individual digit forces and moments caused by both mechanical properties of the digits and the action of spinal reflexes. These stiffness-like reactions mainly assist in perturbing the rotational equilibrium of the object rather than in maintaining it. Second, there are tilt-preventing adjustments defined by the common task constraints that unite the digits into a task-specific synergy. The “virtual springs theory” developed in robotics literature is insufficient for describing the phenomena observed in human grasping.
PMCID: PMC2827039  PMID: 16319210

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