Wendy Liu, MD, PhD

All Publications

• Differential expression of PIEZO1 and PIEZO2 mechanosensitive channels in ocular tissues implicates diverse functional roles. Experimental eye research Zhu, Y., Garcia-Sanchez, J., Dalal, R., Sun, Y., Kapiloff, M. S., Goldberg, J. L., Liu, W. W. 2023: 109675

  Abstract

  PIEZO1 and PIEZO2 are mechanosensitive ion channels that regulate many important physiological processes including vascular blood flow, touch, and proprioception. As the eye is subject to mechanical stress and is highly perfused, these channels may play important roles in ocular function and intraocular pressure regulation. PIEZO channel expression in the eye has not been well defined, in part due to difficulties in validating available antibodies against PIEZO1 and PIEZO2 in ocular tissues. It is also unclear if PIEZO1 and PIEZO2 are differentially expressed. To address these questions, we used single-molecule fluorescence in situ hybridization (smFISH) together with transgenic reporter mice expressing PIEZO fusion proteins under the control of their endogenous promoters to compare the expression and localization of PIEZO1 and PIEZO2 in mouse ocular tissues relevant to glaucoma. We detected both PIEZO1 and PIEZO2 expression in the trabecular meshwork, ciliary body, and in the ganglion cell layer (GCL) of the retina. Piezo1 mRNA was more abundantly expressed than Piezo2 mRNA in these ocular tissues. Piezo1 but not Piezo2 mRNA was detected in the inner nuclear layer and outer nuclear layer of the retina. Our results suggest that PIEZO1 and PIEZO2 are differentially expressed and may have distinct roles as mechanosensors in glaucoma-relevant ocular tissues.

  View details for DOI 10.1016/j.exer.2023.109675

  View details for PubMedID 37820892

• Mechanosensitive ion channel gene survey suggests potential roles in primary open angle glaucoma. Scientific reports Liu, W. W., Kinzy, T. G., Cooke Bailey, J. N., Xu, Z., Hysi, P., Wiggs, J. L. 2023; 13 (1): 15871

  Abstract

  Although glaucoma is a disease modulated by eye pressure, the mechanisms of pressure sensing in the eye are not well understood. Here, we investigated associations between mechanosensitive ion channel gene variants and primary open-angle glaucoma (POAG). Common (minor allele frequency > 5%) single nucleotide polymorphisms located within the genomic regions of 20 mechanosensitive ion channel genes in the K2P, TMEM63, PIEZO and TRP channel families were assessed using genotype data from the NEIGHBORHOOD consortium of 3853 cases and 33,480 controls. Rare (minor allele frequency < 1%) coding variants were assessed using exome array genotyping data for 2606 cases and 2606 controls. Association with POAG was analyzed using logistic regression adjusting for age and sex. Two rare PIEZO1 coding variants with protective effects were identified in the NEIGHBOR dataset: R1527H, (OR 0.17, P = 0.0018) and a variant that alters a canonical splice donor site, g.16-88737727-C-G Hg38 (OR 0.38, P = 0.02). Both variants showed similar effects in the UK Biobank and the R1527H also in the FinnGen database. Several common variants also reached study-specific thresholds for association in the NEIGHBORHOOD dataset. These results identify novel variants in several mechanosensitive channel genes that show associations with POAG, suggesting that these channels may be potential therapeutic targets.

  View details for DOI 10.1038/s41598-023-43072-3

  View details for PubMedID 37741866

  View details for PubMedCentralID PMC10517927

• Managing primary open-angle glaucoma in the setting of suboptimal surgical outcomes in the fellow eye. Journal of cataract and refractive surgery Huang, M. J., Samuelson, T. W., De Francesco, T., Levin, A., Sieck, E., Gazzard, G., Porter, M., Gallardo, M., Chang, R. T., Liu, W. W., Chaya, C., Gulati, S., Shah, M. 2023; 49 (7): 764

  Abstract

  A 62-year-old woman with mild myopia presented to her local optometrist for a routine examination and was found to have intraocular pressure (IOP) of 30 mm Hg in both eyes and cupped nerves. She had a family history of glaucoma in her father. She was started on latanoprost in both eyes and was referred for a glaucoma evaluation. On initial evaluation, her IOP was 25 mm Hg in the right eye and 26 mm Hg in the left eye. Central corneal thickness measured 592 µm in the right eye and 581 µm in the left eye. Her angles were open to gonioscopy without any peripheral anterior synechia. She had 1+ nuclear sclerosis with a corrected distance visual acuity (CDVA) of 20/25 in the right eye and 20/30- in the left eye and uncorrected near visual acuity of J1+ in each eye. Her nerves were 0.85 mm in the right eye and 0.75 mm in the left eye. Optical coherence tomography (OCT) showed retinal nerve fiber layer thinning and a dense superior arcuate scotoma into fixation in her right eye, and superior and inferior arcuate scotomas in her left eye (Figures 1 and 2JOURNAL/jcrs/04.03/02158034-202307000-00019/figure1/v/2023-06-26T195222Z/r/image-tiffJOURNAL/jcrs/04.03/02158034-202307000-00019/figure2/v/2023-06-26T195222Z/r/image-tiff, Supplemental Figures 1 and 2, available at http://links.lww.com/JRS/A882 and http://links.lww.com/JRS/A883). She was successively trialed on fixed combination brimonidine-timolol, dorzolamide, and netarsudil, in addition to her latanoprost, but her IOP remained in the mid- to upper 20s in both eyes. The addition of acetazolamide lowered the pressure to 19 mm Hg in both eyes, but she tolerated it poorly. Methazolamide was also attempted with similar side effects. We elected to perform left eye cataract surgery combined with 360-degree viscocanaloplasty and insertion of a Hydrus microstent (Alcon Laboratories, Inc.). Surgery was uncomplicated with IOP of 16 mm Hg on postoperative day 1 with no glaucoma medications. However, by postoperative week 3, IOP returned to 27 mm Hg, and despite restarting latanoprost-netarsudil and finishing her steroid taper, IOP remained at 27 mm Hg by postoperative week 6. Brimonidine-timolol was added back to her left eye regimen and at postoperative week 8, IOP had elevated to 45 mm Hg. Maximizing her therapy with the addition of topical dorzolamide and oral methazolamide brought her IOP back down to 30 mm Hg. At that point, the decision was made to proceed with trabeculectomy of the left eye. The trabeculectomy was uneventful. However, postoperative attempts to augment filtration were rendered less successful by extremely thick Tenon layer. At her most recent follow-up the pressure in the left eye was mid-teens with brimonidine-timolol and dorzolamide. Her right eye IOP is in the upper 20s on maximum topical therapy. Knowing her postoperative course in the left eye, how would you manage the right eye? In addition to currently available options, would you consider a supraciliary shunt such as the MINIject (iSTAR) if such a device were U.S. Food and Drug Administration (FDA)-approved?

  View details for DOI 10.1097/j.jcrs.0000000000001221

  View details for PubMedID 37390324

• Correlation between central visual field defects and stereopsis in patients with early to moderate visual field loss. Ophthalmology. Glaucoma Liu, W. W., Shalaby, W. S., Shiuey, E. J., Raghu, R., Petkovsek, D., Myers, J. S., Wizov, S. S., Spaeth, G. L., Shukla, A. G. 2023

  Abstract

  To investigate an association between stereoacuity and the presence of central visual field defects (CVFDs) due to glaucoma.Prospective, cross-sectional cohort study.Participants with early to moderate glaucoma with visual acuity better than 20/40, less than 2-line difference in visual acuity between eyes, and 2 reliable Humphrey VFs (24-2 SITA standard) with mean deviation (MD) in the worse eye better than -12dB.Stereoacuity was measured using the Titmus stereo test. Participants with a significant field defect (P<0.5%) in any one of the central four points in the 24-2 SITA standard total deviation map in either eye were classified as having a CVFD. Vision-related quality of life (VR-QOL) was measured by the 25-item National Eye Institute Visual Function Questionnaire (NEI-VFQ-25) scores. Logistic regression was used to determine the association between level of stereoacuity and age, gender, race, glaucoma type, presence of CVFDs, visual acuity, contrast sensitivity, and visual field MD.Stereoacuity in the CVFD and no CVFD groups.Sixty-five participants met the inclusion criteria. The mean age was 64.3 ± 8.0 years and 64.6% were women. Median stereoacuity was 60 arc sec (inter-quartile range 40-120). Forty-two (65%) patients had CVFDs and 23 (35%) patients did not. Median stereoacuity for the CVFD group was worse than the non-CVFD group (60 arc sec (IQR 50-140) vs. 40 arc sec (IQR 40-80); p=0.001), respectively. The non-CVFD group had a higher percentage of participants with normal stereopsis compared to the non-CVFD group (61% vs 21%, p=0.001). Multivariable analysis found that the presence of CVFDs was associated with worse stereopsis level (odds ratio 4.49, p=0.021). The CVFD group had a lower VFQ-25 composite score (84.0 vs 91.4, p=0.004), and lower VFQ-25 sub-scale scores for general vision, near activities, and mental health (P<0.05).CVFDs were associated with increased odds of poor stereoacuity in patients with early to moderate glaucomatous VF loss. Specifically, patients without CVFDs are more likely to have normal stereopsis and higher VR-QOL than those with CVFDs. Patients with CVFDs should be counseled regarding how depth perception difficulties may affect daily living.

  View details for DOI 10.1016/j.ogla.2023.04.003

  View details for PubMedID 37080537

• 4-year Surgical Outcomes of Gonioscopy-assisted Transluminal Trabeculotomy (GATT) in Patients with Open-angle Glaucoma. Ophthalmology. Glaucoma Liu, W. W., Petkovsek, D., Shalaby, W. S., Arbabi, A., Moster, M. R. 2023

  Abstract

  To provide 4-year data on the efficacy and safety of gonioscopy-assisted transluminal trabeculotomy (GATT) in patients with open-angle glaucoma.Retrospective case series.Eyes of patients >18 years of age who underwent GATT by a single surgeon at Wills Eye Hospital with at least 36 months follow-up.Postoperative changes in outcome measures including intraocular pressure (IOP), medication use and visual acuity were recorded. Failure was defined as IOP > 21mmHg or less than 20% reduction below baseline at any postoperative visit after three months, or need for further glaucoma surgery.Main outcome measures were failure rate, IOP, number of glaucoma medications, and visual acuity at 4 years.59 patients (74 eyes), age 57.1±18.5 years (37.8% female) underwent the GATT procedure. Average follow-up was 47.0±6.7 months (range 35.6 to 76.5 months). Mean IOP was 27.0±10.0 mmHg preoperatively and 14.8±6.5 mmHg at 4 years (45% IOP decrease; P<0.01). Mean number of medications decreased from 3.2±1.0 preoperatively to 2.3±1.0 at 4 years (P< 0.01). The cumulative failure rate at 4 years was 53.9%, and the cumulative reoperation rate was 42.0%. No significant differences between patients with primary open-angle glaucoma and other types of glaucoma were found.GATT can be a safe and effective conjunctival-sparing surgery for in treating various forms of open-angle glaucoma at 4 years.

  View details for DOI 10.1016/j.ogla.2023.01.005

  View details for PubMedID 36702382

• Epigenetics in glaucoma: a link between histone methylation and neurodegeneration. The Journal of clinical investigation Liu, W. W., Sun, Y. 2023; 133 (15)

  View details for DOI 10.1172/JCI173784

  View details for PubMedID 37526085

• Epigenetics in glaucoma: a link between DNA methylation and neurodegeneration. The Journal of clinical investigation Liu, W. W., Sun, Y. 2022; 132 (21)

  Abstract

  Normal-tension glaucoma is a form of optic nerve degeneration that is characterized by loss of retinal ganglion cells independent of eye pressure elevation. In this issue of the JCI, Pan et al. report the discovery in a Japanese family of a mutation in the METTL23 gene, which encodes a DNA methyltransferase that causes normal-pressure glaucoma in haploinsufficiency. Inherited as an autosomal dominant condition, METTL23 deficiency revealed an important function in the regulation of pS2 and the downstream NF-kappaB signaling pathway, which has previously been linked to glaucomatous optic nerve degeneration. These findings are the first direct link between defective epigenetic regulatory machinery and genetic forms of optic nerve degeneration.

  View details for DOI 10.1172/JCI163670

  View details for PubMedID 36317630

• Differences in primary cilia amongst retinal ganglion cell subtypes Kowal, T., Dhande, O., Ning, K., Wang, B., Wang, Q., Liu, W., Berbari, N., Hu, Y., Sun, Y. ASSOC RESEARCH VISION OPHTHALMOLOGY INC. 2022

  View details for Web of Science ID 000844401302301

• Distribution of prototypical primary cilia markers in subtypes of retinal ganglion cells. The Journal of comparative neurology Kowal, T. J., Dhande, O. S., Wang, B., Wang, Q., Ning, K., Liu, W., Berbari, N. F., Hu, Y., Sun, Y. 2022

  Abstract

  Loss of retinal ganglion cells (RGCs) underlies several forms of retinal disease including glaucomatous optic neuropathy, a leading cause of irreversible blindness. Several rare genetic disorders associated with cilia dysfunction have retinal degeneration as a clinical hallmark. Much of the focus of ciliopathy associated blindness is on the connecting cilium of photoreceptors; however, RGCs also possess primary cilia. It is unclear what roles RGC cilia play, what proteins and signaling machinery localize to RGC cilia, or how RGC cilia are differentiated across the subtypes of RGCs. To better understand these questions, we assessed the presence or absence of a prototypical cilia marker Arl13b and a widely distributed neuronal cilia marker AC3 in different subtypes of mouse RGCs. Interestingly, not all RGC subtype cilia are the same and there are significant differences even among these standard cilia markers. Alpha-RGCs positive for osteopontin, calretinin, and SMI32 primarily possess AC3-positive cilia. Directionally selective RGCs that are CART positive or Trhr positive localize either Arl13b or AC3, respectively, in cilia. Intrinsically photosensitive RGCs differentially localize Arl13b and AC3 based on melanopsin expression. Taken together, we characterized the localization of gold standard cilia markers in different subtypes of RGCs and conclude that cilia within RGC subtypes may be differentially organized. Future studies aimed at understanding RGC cilia function will require a fundamental ability to observe the cilia across subtypes as their signaling protein composition is elucidated. A comprehensive understanding of RGC cilia may reveal opportunities to understanding how their dysfunction leads to retinal degeneration.

  View details for DOI 10.1002/cne.25326

  View details for PubMedID 35434813

• Repair of Tube Erosion by Modifying the Tube Extender JOURNAL OF GLAUCOMA Liu, W. W., Werner, A., Chen, T. C. 2020; 29 (7): 604-606

  Abstract

  We describe here a case report of a novel technique for tube erosion repair, which modifies and utilizes the commercially available tube extender (Model TE). The modification of the tube extender makes the commercially available tube extender more compact and is useful in cases where conjunctival mobility and space are limited. This debulking of the tube extender may reduce the risk of future tube exposure and dellen formation.

  View details for DOI 10.1097/IJG.0000000000001505

  View details for Web of Science ID 000559090500025

  View details for PubMedID 32251073

  View details for PubMedCentralID PMC7337120

• Three-dimensional Neuroretinal Rim Thickness and Visual Fields in Glaucoma: A Broken-stick Model. Journal of glaucoma Liu, W. W., McClurkin, M., Tsikata, E., Hui, P. C., Elze, T., Celebi, A. R., Khoueir, Z., Lee, R., Shieh, E., Simavli, H., Que, C., Guo, R., de Boer, J., Chen, T. C. 2020; 29 (10): 952-963

  Abstract

  In open-angle glaucoma, when neuroretinal rim tissue measured by volumetric optical coherence tomography (OCT) scans is below a third of the normal value, visual field (VF) damage becomes detectable.To determine the amount of neuroretinal rim tissue thickness below which VF damage becomes detectable.In a retrospective cross-sectional study, 1 eye per subject (of 57 healthy and 100 open-angle glaucoma patients) at an academic institution had eye examinations, VF testing, spectral-domain OCT retinal nerve fiber layer (RNFL) thickness measurements, and optic nerve volumetric scans. Using custom algorithms, the minimum distance band (MDB) neuroretinal rim thickness was calculated from optic nerve scans. "Broken-stick" regression was performed for estimating both the MDB and RNFL thickness tipping-point thresholds, below which were associated with initial VF defects in the decibel scale. The slopes for the structure-function relationship above and below the thresholds were computed. Smoothing curves of the MDB and RNFL thickness covariates were evaluated to examine the consistency of the independently identified tipping-point pairs.Plots of VF total deviation against MDB thickness revealed plateaus of VF total deviation unrelated to MDB thickness. Below the thresholds, VF total deviation decreased with MDB thickness, with the associated slopes significantly greater than those above the thresholds (P<0.014). Below 31% of global MDB thickness, and 36.8% and 43.6% of superior and inferior MDB thickness, VF damage becomes detectable. The MDB and RNFL tipping points were in good accordance with the correlation of the MDB and RNFL thickness covariates.When neuroretinal rim tissue, characterized by MDB thickness in OCT, is below a third of the normal value, VF damage in the decibel scale becomes detectable.

  View details for DOI 10.1097/IJG.0000000000001604

  View details for PubMedID 32925518

  View details for PubMedCentralID PMC7541591

• Imaging Retinal Ganglion Cell Death and Dysfunction in Glaucoma. International ophthalmology clinics Liu, W. W., Margeta, M. A. 2019; 59 (4): 41-54

  View details for DOI 10.1097/IIO.0000000000000285

  View details for PubMedID 31569133

• Diagnosing Myasthenia Gravis with an Ice Pack NEW ENGLAND JOURNAL OF MEDICINE Liu, W. W., Chen, A. 2016; 375 (19): E39

  View details for DOI 10.1056/NEJMicm1509523

  View details for Web of Science ID 000387534200001

  View details for PubMedID 27959645

• Thermosensory processing in the Drosophila brain NATURE Liu, W. W., Mazor, O., Wilson, R. I. 2015; 519 (7543): 353-+

  Abstract

  In Drosophila, just as in vertebrates, changes in external temperature are encoded by bidirectional opponent thermoreceptor cells: some cells are excited by warming and inhibited by cooling, whereas others are excited by cooling and inhibited by warming. The central circuits that process these signals are not understood. In Drosophila, a specific brain region receives input from thermoreceptor cells. Here we show that distinct genetically identified projection neurons (PNs) in this brain region are excited by cooling, warming, or both. The PNs excited by cooling receive mainly feed-forward excitation from cool thermoreceptors. In contrast, the PNs excited by warming ('warm-PNs') receive both excitation from warm thermoreceptors and crossover inhibition from cool thermoreceptors through inhibitory interneurons. Notably, this crossover inhibition elicits warming-evoked excitation, because warming suppresses tonic activity in cool thermoreceptors. This in turn disinhibits warm-PNs and sums with feed-forward excitation evoked by warming. Crossover inhibition could cancel non-thermal activity (noise) that is positively correlated among warm and cool thermoreceptor cells, while reinforcing thermal activity which is anti-correlated. Our results show how central circuits can combine signals from bidirectional opponent neurons to construct sensitive and robust neural codes.

  View details for DOI 10.1038/nature14170

  View details for Web of Science ID 000351171900041

  View details for PubMedID 25739502

  View details for PubMedCentralID PMC5488797

• Transient and Specific Inactivation of Drosophila Neurons In Vivo Using a Native Ligand-Gated Ion Channel CURRENT BIOLOGY Liu, W. W., Wilson, R. I. 2013; 23 (13): 1202-1208

  Abstract

  A key tool in neuroscience is the ability to transiently inactivate specific neurons on timescales of milliseconds to minutes. In Drosophila, there are two available techniques for accomplishing this (shibire(ts) and halorhodopsin [1-3]), but both have shortcomings [4-9]. Here we describe a complementary technique using a native histamine-gated chloride channel (Ort). Ort is the receptor at the first synapse in the visual system. It forms large-conductance homomeric channels that desensitize only modestly in response to ligand [10]. Many regions of the CNS are devoid of histaminergic neurons [11, 12], raising the possibility that Ort could be used to artificially inactivate specific neurons in these regions. To test this idea, we performed in vivo whole-cell recordings from antennal lobe neurons misexpressing Ort. In these neurons, histamine produced a rapid and reversible drop in input resistance, clamping the membrane potential below spike threshold and virtually abolishing spontaneous and odor-evoked activity. Every neuron type in this brain region could be inactivated in this manner. Neurons that did not misexpress Ort showed negligible responses to histamine. Ort also performed favorably in comparison to the available alternative effector transgenes. Thus, Ort misexpression is a useful tool for probing functional connectivity among Drosophila neurons.

  View details for DOI 10.1016/j.cub.2013.05.016

  View details for Web of Science ID 000321605600021

  View details for PubMedID 23770187

  View details for PubMedCentralID PMC3725270

• Glutamate is an inhibitory neurotransmitter in the Drosophila olfactory system PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Liu, W. W., Wilson, R. I. 2013; 110 (25): 10294-10299

  Abstract

  Glutamatergic neurons are abundant in the Drosophila central nervous system, but their physiological effects are largely unknown. In this study, we investigated the effects of glutamate in the Drosophila antennal lobe, the first relay in the olfactory system and a model circuit for understanding olfactory processing. In the antennal lobe, one-third of local neurons are glutamatergic. Using in vivo whole-cell patch clamp recordings, we found that many glutamatergic local neurons are broadly tuned to odors. Iontophoresed glutamate hyperpolarizes all major cell types in the antennal lobe, and this effect is blocked by picrotoxin or by transgenic RNAi-mediated knockdown of the GluClα gene, which encodes a glutamate-gated chloride channel. Moreover, antennal lobe neurons are inhibited by selective activation of glutamatergic local neurons using a nonnative genetically encoded cation channel. Finally, transgenic knockdown of GluClα in principal neurons disinhibits the odor responses of these neurons. Thus, glutamate acts as an inhibitory neurotransmitter in the antennal lobe, broadly similar to the role of GABA in this circuit. However, because glutamate release is concentrated between glomeruli, whereas GABA release is concentrated within glomeruli, these neurotransmitters may act on different spatial and temporal scales. Thus, the existence of two parallel inhibitory transmitter systems may increase the range and flexibility of synaptic inhibition.

  View details for DOI 10.1073/pnas.1220560110

  View details for Web of Science ID 000321500200061

  View details for PubMedID 23729809

  View details for PubMedCentralID PMC3690841

• Organic-inorganic interfaces and spiral growth in nacre JOURNAL OF THE ROYAL SOCIETY INTERFACE Yao, N., Epstein, A. K., Liu, W. W., Sauer, F., Yang, N. 2009; 6 (33): 367-376

  Abstract

  Nacre, the crown jewel of natural materials, has been extensively studied owing to its remarkable physical properties for over 160 years. Yet, the precise structural features governing its extraordinary strength and its growth mechanism remain elusive. In this paper, we present a series of observations pertaining to the red abalone (Haliotis rufescens) shell's organic-inorganic interface, organic interlayer morphology and properties, large-area crystal domain orientations and nacre growth. In particular, we describe unique lateral nano-growths and paired screw dislocations in the aragonite layers, and demonstrate that the organic material sandwiched between aragonite platelets consists of multiple organic layers of varying nano-mechanical resilience. Based on these novel observations and analysis, we propose a spiral growth model that accounts for both [001] vertical propagation via helices that surround numerous screw dislocation cores and simultaneous 010 lateral growth of aragonite sheet structure. These new findings may aid in creating novel organic-inorganic micro/nano composites through synthetic or biomineralization pathways.

  View details for DOI 10.1098/rsif.2008.0316

  View details for Web of Science ID 000264357800004

  View details for PubMedID 18753125

  View details for PubMedCentralID PMC2572677

• A Microfluidic Chamber for Analysis of Neuron-to-Cell Spread and Axonal Transport of an Alpha-Herpesvirus PLOS ONE Liu, W. W., Goodhouse, J., Jeon, N., Enquist, L. W. 2008; 3 (6): e2382

  Abstract

  Alpha-herpesviruses, including herpes simplex virus and pseudorabies virus (PRV), infect the peripheral nervous system (PNS) of their hosts. Here, we describe an in vitro method for studying neuron-to-cell spread of infection as well as viral transport in axons. The method centers on a novel microfluidic chamber system that directs growth of axons into a fluidically isolated environment. The system uses substantially smaller amounts of virus inoculum and media than previous chamber systems and yet offers the flexibility of applying multiple virology and cell biology assays including live-cell optical imaging. Using PRV infection of cultured PNS neurons, we demonstrate that the microfluidic chamber recapitulates all known facets of neuron-to-cell spread demonstrated in animals and other compartmented cell systems.

  View details for DOI 10.1371/journal.pone.0002382

  View details for Web of Science ID 000263280700003

  View details for PubMedID 18560518

  View details for PubMedCentralID PMC2426917