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1.  Magnitude Effects for Experienced Rewards at Short Delays in the Escalating Interest Task 
Psychonomic bulletin & review  2013;20(2):302-309.
A first-person shooter video game was adapted for the study of choice between smaller sooner and larger later rewards. Participants chose when to fire a weapon that increased in damage potential over a short interval. When the delay to maximum damage was shorter (5 – 8 s), people showed greater sensitivity to the consequences of their choices than when the delay was longer (17 – 20 s). Participants also evidenced a magnitude effect by waiting proportionally longer when the damage magnitudes were doubled for all rewards. The experiment replicated the standard magnitude effect with this new video game preparation over time scales similar to those typically used in nonhuman animal studies and without complications due to satiation or cost.
PMCID: PMC3594385  PMID: 23188742
2.  Multiphoton Imaging with a Direct-diode Pumped Femtosecond Ti:sapphire Laser 
Journal of microscopy  2012;249(2):83-86.
A direct-diode pumped Ti:sapphire femtosecond oscillator is used to perform multiphoton imaging for the first time.
PMCID: PMC3553270  PMID: 23189919
3.  Use of a Synthetic Xeno-Free Culture Substrate for Induced Pluripotent Stem Cell Induction and Retinal Differentiation 
The purpose of this study was to determine whether a proprietary xeno-free synthetic culture surface could be used to aid in the production and subsequent retinal-specific differentiation of clinical-grade induced pluripotent stem cells (iPSCs). It was found that Synthemax cell culture surfaces provide an ideal surface for the xeno-free production, culture, and differentiation of adult somatic cell-derived iPSCs. These findings demonstrate the potential utility of these surfaces for the production of clinical-grade retinal neurons for transplantation and induction of retinal regeneration.
The purpose of this study was to determine whether a proprietary xeno-free synthetic culture surface could be used to aid in the production and subsequent retinal-specific differentiation of clinical-grade induced pluripotent stem cells (iPSCs). iPSCs were generated using adult somatic cells via infection with either a single cre-excisable lentiviral vector or four separate nonintegrating Sendai viruses driving expression of the transcription factors OCT4, SOX2, KLF4, and c-MYC. Retinal precursor cells were derived via targeted differentiation of iPSCs with exogenous delivery of dkk-1, noggin, insulin-like growth factor-1, basic fibroblast growth factor, acidic fibroblast growth factor, and DAPT. Phase contrast microscopy, immunocytochemistry, hematoxylin and eosin staining, and reverse transcription-polymerase chain reaction were used to determine reprogramming efficiency, pluripotency, and fate of undifferentiated and differentiated iPSCs. Following viral transduction, cells underwent prototypical morphological changes resulting in the formation of iPSC colonies large enough for manual isolation/passage at 3–4 weeks postinfection. Both normal and disease-specific iPSCs expressed markers of pluripotency and, following transplantation into immune-compromised mice, formed teratomas containing tissue comprising all three germ layers. When subjected to our established retinal differentiation protocol, a significant proportion of the xeno-free substrate-derived cells expressed retinal cell markers, the number of which did not significantly differ from that derived on traditional extracellular matrix-coated dishes. Synthetic cell culture substrates provide a useful surface for the xeno-free production, culture, and differentiation of adult somatic cell-derived iPSCs. These findings demonstrate the potential utility of these surfaces for the production of clinical-grade retinal neurons for transplantation and induction of retinal regeneration.
PMCID: PMC3659741  PMID: 23283489
Induced pluripotent stem cells; Stem cell culture; Retina; Reprogramming; Pluripotent stem cells; Clinical translation
4.  Defining mental illnesses: can values and objectivity get along? 
BMC Psychiatry  2013;13:346.
The creation of each edition of the Diagnostic and Statistical Manual (DSM) of psychiatry has proven enormously controversial. The current effort to revise the ‘bible’ of disorder definitions for the field of mental health is no exception. The controversy around DSM-5 reached a crescendo with the announcement from National Institute of Mental Health (NIMH) that the institute would focus efforts on the development of their own psychiatric nosology, the Research Domain Criteria (RDoC) (NIMH, 2013).
The RDoC seem to be structured around the concern that the only way to find objectivity in the classification of diseases or disorders in psychiatry is to begin with biology and work back to symptoms. Values infuse medical categories in various ways and drive practical considerations about where and how to divide up constellations of already agreed upon symptoms.
We briefly argue that all nosologies are infused with values and, while we should continue to sharpen the psychiatric nosology, normativity will permeate even the strictest biologically based taxonomy; this need not be a bad thing.
PMCID: PMC3877989  PMID: 24365131
5.  Cyclic Amp-Dependent Resuscitation of Dormant Mycobacteria by Exogenous Free Fatty Acids 
PLoS ONE  2013;8(12):e82914.
One third of the world population carries a latent tuberculosis (TB) infection, which may reactivate leading to active disease. Although TB latency has been known for many years it remains poorly understood. In particular, substances of host origin, which may induce the resuscitation of dormant mycobacteria, have not yet been described. In vitro models of dormant (“non-culturable”) cells of Mycobacterium smegmatis (mc2155) and Mycobacterium tuberculosis H37Rv were used. We found that the resuscitation of dormant M. smegmatis and M. tuberculosis cells in liquid medium was stimulated by adding free unsaturated fatty acids (FA), including arachidonic acid, at concentrations of 1.6–10 µM. FA addition enhanced cAMP levels in reactivating M. smegmatis cells and exogenously added cAMP (3–10 mM) or dibutyryl-cAMP (0.5–1 mM) substituted for FA, causing resuscitation of M. smegmatis and M. tuberculosis dormant cells. A M. smegmatis null-mutant lacking MSMEG_4279, which encodes a FA-activated adenylyl cyclase (AC), could not be resuscitated by FA but it was resuscitated by cAMP. M. smegmatis and M. tuberculosis cells hyper-expressing AC were unable to form non-culturable cells and a specific inhibitor of AC (8-bromo-cAMP) prevented FA-dependent resuscitation. RT-PCR analysis revealed that rpfA (coding for resuscitation promoting factor A) is up-regulated in M. smegmatis in the beginning of exponential growth following the cAMP increase in lag phase caused by FA-induced cell activation. A specific Rpf inhibitor (4-benzoyl-2-nitrophenylthiocyanate) suppressed FA-induced resuscitation. We propose a novel pathway for the resuscitation of dormant mycobacteria involving the activation of adenylyl cyclase MSMEG_4279 by FAs resulted in activation of cellular metabolism followed later by increase of RpfA activity which stimulates cell multiplication in exponential phase. The study reveals a probable role for lipids of host origin in the resuscitation of dormant mycobacteria, which may function during the reactivation of latent TB.
PMCID: PMC3871856  PMID: 24376605
6.  The Spectral Properties of (-)-Epigallocatechin 3-O-Gallate (EGCG) Fluorescence in Different Solvents: Dependence on Solvent Polarity 
PLoS ONE  2013;8(11):e79834.
(-)-Epigallocatechin 3-O-gallate (EGCG) a molecule found in green tea and known for a plethora of bioactive properties is an inhibitor of heat shock protein 90 (HSP90), a protein of interest as a target for cancer and neuroprotection. Determination of the spectral properties of EGCG fluorescence in environments similar to those of binding sites found in proteins provides an important tool to directly study protein-EGCG interactions. The goal of this study is to examine the spectral properties of EGCG fluorescence in an aqueous buffer (AB) at pH=7.0, acetonitrile (AN) (a polar aprotic solvent), dimethylsulfoxide (DMSO) (a polar aprotic solvent), and ethanol (EtOH) (a polar protic solvent). We demonstrate that EGCG is a highly fluorescent molecule when excited at approximately 275 nm with emission maxima between 350 and 400 nm depending on solvent. Another smaller excitation peak was found when EGCG is excited at approximately 235 nm with maximum emission between 340 and 400 nm. We found that the fluorescence intensity (FI) of EGCG in AB at pH=7.0 is significantly quenched, and that it is about 85 times higher in an aprotic solvent DMSO. The Stokes shifts of EGCG fluorescence were determined by solvent polarity. In addition, while the emission maxima of EGCG fluorescence in AB, DMSO, and EtOH follow the Lippert-Mataga equation, its fluorescence in AN points to non-specific solvent effects on EGCG fluorescence. We conclude that significant solvent-dependent changes in both fluorescence intensity and fluorescence emission shifts can be effectively used to distinguish EGCG in aqueous solutions from EGCG in environments of different polarity, and, thus, can be used to study specific EGCG binding to protein binding sites where the environment is often different from aqueous in terms of polarity.
PMCID: PMC3838354  PMID: 24278192
7.  PD5: A General Purpose Library for Primer Design Software 
PLoS ONE  2013;8(11):e80156.
Complex PCR applications for large genome-scale projects require fast, reliable and often highly sophisticated primer design software applications. Presently, such applications use pipelining methods to utilise many third party applications and this involves file parsing, interfacing and data conversion, which is slow and prone to error. A fully integrated suite of software tools for primer design would considerably improve the development time, the processing speed, and the reliability of bespoke primer design software applications.
The PD5 software library is an open-source collection of classes and utilities, providing a complete collection of software building blocks for primer design and analysis. It is written in object-oriented C++ with an emphasis on classes suitable for efficient and rapid development of bespoke primer design programs. The modular design of the software library simplifies the development of specific applications and also integration with existing third party software where necessary. We demonstrate several applications created using this software library that have already proved to be effective, but we view the project as a dynamic environment for building primer design software and it is open for future development by the bioinformatics community. Therefore, the PD5 software library is published under the terms of the GNU General Public License, which guarantee access to source-code and allow redistribution and modification.
The PD5 software library is downloadable from Google Code and the accompanying Wiki includes instructions and examples:
PMCID: PMC3836914  PMID: 24278254
9.  The intramembrane protease Sppl2a is required for B cell and DC development and survival via cleavage of the invariant chain 
The protease Sppl2a cleaves the N-terminal fragment of invariant chain (CD74) and is required for efficient B cell development and function.
B cell development requires tight regulation to allow for the generation of a diverse repertoire while preventing the development of autoreactive cells. We report, using N-ethyl-N-nitrosourea (ENU)–induced mutagenesis, the identification of a mutant mouse (chompB) with a block in early B cell development. The blockade occurs after the transitional 1 (T1) stage and leads to a decrease in mature B cell subsets and deficits in T cell–dependent antibody responses. Additionally, chompB mice have decreases in myeloid dendritic cells (DCs). The mutation was mapped to the intramembrane protease signal peptide peptidase-like 2a (Sppl2a), a gene not previously implicated in immune cell development. Proteomic analysis identified the invariant chain (CD74) as a key substrate of Sppl2a and suggests that regulated intramembrane proteolysis of CD74 by Sppl2a contributes to B cell and DC survival. Moreover, these data suggest that modulation of Sppl2a may be a useful therapeutic strategy for treatment of B cell dependent autoimmune disorders.
PMCID: PMC3549714  PMID: 23267013
10.  Updated structure of Drosophila cryptochrome 
Nature  2013;495(7441):E3-E4.
PMCID: PMC3694752  PMID: 23518567
11.  Eliminating the scattering ambiguity in multifocal, multimodal multiphoton imaging systems 
Journal of biophotonics  2012;5(0):425-436.
Four images of Drosophila Melanogaster antennal lobe structure labeled with red fluorescent protein. The images are separated axially by 7 μm in depth, and were all acquired simultaneously from a single-element detector.
PMCID: PMC3670971  PMID: 22461190
12.  Comparing Retrospective Reports to Real-Time/Real-Place Mobile Assessments in Individuals With Schizophrenia and a Nonclinical Comparison Group 
Schizophrenia Bulletin  2012;38(3):396-404.
Retrospective reports are often used as the primary source of information for important diagnostic decisions, treatment, and clinical research. Whether such reports accurately represent individuals’ past experiences in the context of a serious mental illness such as schizophrenia is unclear. In the current study, 24 individuals with schizophrenia and 26 nonclinical participants used a mobile device to complete multiple real-time/real-place assessments daily, over 7 consecutive days. At the end of the week, participants were also asked to provide a retrospective report summarizing the same period. Comparison of the data captured by the 2 methods showed that participants from both groups retrospectively overestimated the intensity of negative and positive daily experiences. In the clinical group, overestimations for affect were greater than for psychotic symptoms, which were relatively comparable to their retrospective reports. In both samples, retrospective reports were more closely associated with the week’s average than the most intense or most recent ratings captured with a mobile device. Multilevel modeling revealed that much of the variability in weekly assessments was not explained by between-person differences and could not be captured by a single retrospective estimate. Based on the findings of this study, clinicians and researchers should be aware that while retrospective summary reports of the severity of certain symptoms compare relatively well with average momentary ratings, they are limited in their ability to capture variability in one’s affective or psychotic experiences over time.
PMCID: PMC3329976  PMID: 22302902
experience sampling method (ESM); ecological momentary assessment (EMA); mobile interventions; mHealth; delusions; hallucinations
13.  Control of Sleep by Cyclin A and its Regulator 
Science (New York, N.y.)  2012;335(6076):1617-1621.
How and why the brain reversibly switches from a waking to a sleep state remain among the most intriguing questions in biology. We show that CyclinA (CycA) and Regulator of Cyclin A1 (Rca1), essential cell cycle factors, function in post-mitotic neurons to promote sleep in Drosophila melanogaster. Reducing the abundance of CycA in neurons delayed the wake-sleep transition, caused multiple arousals from sleep and reduced the homeostatic response to sleep deprivation. CycA is expressed in ~40–50 neurons in the adult brain, most of which are intermingled with circadian clock neurons, suggesting functional interactions among neurons controlling sleep and circadian behavior.
PMCID: PMC3380085  PMID: 22461610
14.  Robust Detection of Rare Species Using Environmental DNA: The Importance of Primer Specificity 
PLoS ONE  2013;8(3):e59520.
Environmental DNA (eDNA) is being rapidly adopted as a tool to detect rare animals. Quantitative PCR (qPCR) using probe-based chemistries may represent a particularly powerful tool because of the method’s sensitivity, specificity, and potential to quantify target DNA. However, there has been little work understanding the performance of these assays in the presence of closely related, sympatric taxa. If related species cause any cross-amplification or interference, false positives and negatives may be generated. These errors can be disastrous if false positives lead to overestimate the abundance of an endangered species or if false negatives prevent detection of an invasive species. In this study we test factors that influence the specificity and sensitivity of TaqMan MGB assays using co-occurring, closely related brook trout (Salvelinus fontinalis) and bull trout (S. confluentus) as a case study. We found qPCR to be substantially more sensitive than traditional PCR, with a high probability of detection at concentrations as low as 0.5 target copies/µl. We also found that number and placement of base pair mismatches between the Taqman MGB assay and non-target templates was important to target specificity, and that specificity was most influenced by base pair mismatches in the primers, rather than in the probe. We found that insufficient specificity can result in both false positive and false negative results, particularly in the presence of abundant related species. Our results highlight the utility of qPCR as a highly sensitive eDNA tool, and underscore the importance of careful assay design.
PMCID: PMC3608683  PMID: 23555689
15.  Mast Cell Dependent Vascular Changes Associated with an Acute Response to Cold Immersion in Primary Contact Urticaria 
PLoS ONE  2013;8(2):e56773.
While a number of the consequences of mast cell degranulation within tissues have been documented including tissue-specific changes such as bronchospasm and the subsequent cellular infiltrate, there is little known about the immediate effects of mast cell degranulation on the associated vasculature, critical to understanding the evolution of mast cell dependent inflammation.
To characterize the microcirculatory events that follow mast cell degranulation.
Methodology/Principal Findings
Perturbations in dermal blood flow, temperature and skin color were analyzed using laser-speckle contrast imaging, infrared and polarized-light colorimetry following cold-hand immersion (CHI) challenge in patients with cold-induced urticaria compared to the response in healthy controls. Evidence for mast cell degranulation was established by documentation of serum histamine levels and the localized release of tryptase in post-challenge urticarial biopsies. Laser-speckle contrast imaging quantified the attenuated response to cold challenge in patients on cetirizine. We found that the histamine-associated vascular response accompanying mast cell degranulation is rapid and extensive. At the tissue level, it is characterized by a uniform pattern of increased blood flow, thermal warming, vasodilation, and recruitment of collateral circulation. These vascular responses are modified by the administration of an antihistamine.
Monitoring the hemodynamic responses within tissues that are associated with mast cell degranulation provides additional insight into the evolution of the acute inflammatory response and offers a unique approach to assess the effectiveness of treatment intervention.
PMCID: PMC3579929  PMID: 23451084
16.  The cost of lower respiratory tract infections hospital admissions in the Canadian Arctic 
International Journal of Circumpolar Health  2013;72:10.3402/ijch.v72i0.21595.
Inuit infants who reside in the Nunavut (NU) regions of Arctic Canada have extremely high rates of lower respiratory tract infections (LRTIs) associated with significant health expenditures, but the costs in other regions of Arctic Canada have not been documented.
This prospective surveillance compares, across most of Arctic Canada, the rates and costs associated with LRTI admissions in infants less than 1 year of age, and the days of hospitalization and costs adjusted per live birth.
This was a hospital-based surveillance of LRTI admissions of infants less than 1 year of age, residing in Northwest Territories (NT), the 3 regions of Nunavut (NU); [Kitikmeot (KT), Kivalliq (KQ) and Qikiqtani (QI)] and Nunavik (NK) from 1 January 2009 to 30 June 2010. Costs were obtained from the territorial or regional governments and hospitals, and included transportation, hospital stay, physician fees and accommodation costs. The rates of LRTI hospitalizations, days of hospitalization and associated costs were calculated per live birth in each of the 5 regions.
There were 513 LRTI admissions during the study period. For NT, KT, KQ, QI and NK, the rates of LRTI hospitalization per 1000 live births were 38, 389, 230, 202 and 445, respectively. The total days of LRTI admission per live birth were 0.25, 3.3, 2.6, 1.7 and 3 for the above regions. The average cost per live birth for LRTI admission for these regions was $1,412, $22,375, $14,608, $8,254 and $10,333. The total cost for LRTI was $1,498,232 in NT, $15,662,968 in NU and $3,874,881 in NK. Medical transportation contributed to a significant proportion of the costs.
LRTI admission rates in NU and Nunavik are much higher than that in NT and remain among the highest rates globally. The costs of these admissions are exceptionally high due to the combination of very high rates of admission, very expensive medical evacuations and prolonged hospitalizations. Decreasing the rates of LRTI in this population could result in substantial health savings.
PMCID: PMC3748437  PMID: 23967411
Lower Respiratory tract infections (or LRTI); costs; Canadian Arctic; Inuit
17.  insomniac and Cullin-3 Regulate Sleep and Wakefulness in Drosophila 
Neuron  2011;72(6):964-976.
In a forward genetic screen in Drosophila, we have isolated insomniac, a mutant that severely reduces the duration and consolidation of sleep. Anatomically-restricted genetic manipulations indicate that insomniac functions within neurons to regulate sleep. insomniac expression does not oscillate in a circadian manner, and conversely, the circadian clock is intact in insomniac mutants, suggesting that insomniac regulates sleep by pathways distinct from the circadian clock. The protein encoded by insomniac is a member of the BTB/POZ superfamily, which includes many proteins that function as adaptors for the Cullin-3 (Cul3) ubiquitin ligase complex. We show that Insomniac can physically associate with Cul3, and that reduction of Cul3 activity in neurons recapitulates the insomniac phenotype. The extensive evolutionary conservation of insomniac and Cul3 suggests that protein degradation pathways may have a general role in governing the sleep and wakefulness of animals.
PMCID: PMC3244879  PMID: 22196332
18.  Deciding when to “cash in” when outcomes are continuously improving: An escalating interest task 
Behavioural processes  2011;88(2):101-110.
A first-person shooter video game was adapted for the study of choice between smaller sooner and larger later outcomes. Participants chose when to fire a weapon that increased in damage potential over a 10 s interval, an escalating interest situation. Across two experiments, participants demonstrated sensitivity to the nature of the mathematical function that defined the relationship between waiting and damage potential. In Experiment 1, people tended to wait longer when doing so allowed them to eliminate targets more quickly. In Experiment 2, people tended to wait longer to increase the probability of a constant magnitude outcome than to increase the magnitude of a 100% certain outcome that was matched for the same expected value (i.e., probability times magnitude). The two experiments demonstrated sensitivity to the way in which an outcome improves when the outcome is continuously available. The results also demonstrate that this new video game task is useful for generating sensitivity to delay to reinforcement over time scales that are typically used in nonhuman animal studies.
PMCID: PMC3523357  PMID: 21871951
Impulsivity; Delay discounting; Video game; Probability; Magnitude
19.  Complications Associated with the Administration of Dantrolene 1987 to 2006: A Report from the North American Malignant Hyperthermia Registry of the Malignant Hyperthermia Association of the United States 
Anesthesia and analgesia  2011;112(5):1115-1123.
Dantrolene is the only specific treatment for malignant hyperthermia (MH), a genetic disorder in which life-threatening temperature increase has been induced by inhaled anesthetics and succinylcholine. Because MH presents with nonspecific signs and delay of treatment can be fatal, dantrolene may be given as soon as MH is suspected. We report the complications associated with dantrolene administration as documented in AMRA (adverse metabolic/musculoskeletal reaction to anesthesia) reports submitted to the North American Malignant Hyperthermia Registry.
AMRA reports were analyzed for differences between subjects with and without complications attributed to dantrolene. Documentation of dantrolene dose and subject weight were inclusion criteria. Because some reported complications were likely due to factors other than dantrolene, a reduced set of cases was also defined. Chi-square and Mann-Whitney tests were used. Logistic regression was applied to describe factors associated with increased risk of complications.
In the full dataset of 368 subjects, the most frequent complications associated with dantrolene were muscle weakness (21.7%), phlebitis (9%), gastrointestinal upset (4.1%) and respiratory failure (3.8%). Logistic regression described a 29% increase in risk of any complication when the total dantrolene dose was doubled, a 144% increase in risk when fluid administration was part of treatment, an 83% decrease in risk in the presence of neurosurgery and a 74% decrease in risk in the presence of oral surgery.
In the dataset reduced by removal ofsome serious complications that were judged likely to have been due to preexisting disease or the MH event, there were 349 subjects. The most frequent complications associated with dantrolene were muscle weakness (14.6%), phlebitis (9.2%) and gastrointestinal upset (4.3%). In this reduced dataset, logistic regression described a 25% increase in risk of any complication when the total dantrolene dose was doubled, a 572% increase in risk in the presence of obstetric or gynecologic surgery, a 56% decrease in risk if furosemide was given and no relationship with fluid administration or other types of surgery.
Complications after dantrolene are common, but rarely life threatening. Unidentified factors in the surgical environment are associated with changes in the risk of complications. Fluid management, as part of the treatment of MH, has an important association with the risk of complications after dantrolene administration and should be monitored closely.
PMCID: PMC3498049  PMID: 21372281
20.  Structure of an Enclosed Dimer Formed by The Drosophila Period Protein 
Journal of molecular biology  2011;413(3):561-572.
Period (PER) is the major transcription inhibitor in metazoan circadian clocks and lies at the center of several feedback loops that regulate gene expression. Dimerization of Drosophila PER influences nuclear translocation, repressor activity and behavioral rhythms. The structure of a central, 346-residue PER fragment reveals two associated Per-Arnt-Sim (PAS) domains followed by a protruding α-helical extension (αF). A closed, pseudo-symmetric dimer forms from a cross handshake interaction of the N-terminal PAS domain with αF of the opposing subunit. Strikingly, a shift of αF against the PAS β-sheet generates two alternative subunit interfaces in the dimer. Together with a previously reported PER structure in which αF extends, these data indicate that αF unlatches to switch association of PER with itself to its partner Timeless. The variable positions of the αF helix provide snapshots of a helix dissociation mechanism that has relevance to other PAS protein systems. Conservation of PER interaction residues among a family of PAS-AB containing transcription factors suggests that contacts mediating closed PAS-AB dimers serve a general function.
PMCID: PMC3252215  PMID: 21907720
Circadian; helix dissociation; PER; PAS dimer; transcriptional regulation
21.  Retinal Pigment Epithelium and Müller Progenitor Cell Interaction Increase Müller Progenitor Cell Expression of PDGFRα and Ability to Induce Proliferative Vitreoretinopathy in a Rabbit Model 
Stem Cells International  2012;2012:106486.
Purpose. Proliferative vitreoretinopathy (PVR) is a complication of retinal detachment characterized by redetachment of the retina as a result of membrane formation and contraction. A variety of retinal cells, including retinal pigment epithelial (RPE) and Müller glia, and growth factors may be responsible. Platelet-derived growth factor receptor alpha (PDGFRα) is found in large quantities in PVR membranes, and is intrinsic to the development of PVR in rabbit models. This study explores the expression of PDGFR in cocultures of RPE and Müller cells over time to examine how these two cell types may collaborate in the development of PVR. We also examine how changes in PDGFRα expression alter Müller cell pathogenicity. Methods. Human MIO-M1 Müller progenitor (MPC) and ARPE19 cells were studied in a transmembrane coculture system. Immunocytochemistry and Western blot were used to look at PDGFRα, PDGFRβ, and GFAP expression. A transfected MPC line cell line expressing the PDGFRα (MIO-M1α) was generated, and tested in a rabbit model for its ability to induce PVR. Results. The expression of PDGFRα and PDGFRβ was upregulated in MIO-M1 MPCs cocultured with ARPE19 cells; GFAP was slightly decreased. Increased expression of PDGFRα in the MIO-M1 cell line resulted in increased pathogenicity and enhanced ability to induce PVR in a rabbit model. Conclusions. Müller and RPE cell interaction can lead to upregulation of PDGFRα and increased Müller cell pathogenicity. Müller cells may play a more active role than previously thought in the development of PVR membranes, particularly when stimulated by an RPE-cell-rich environment. Additional studies of human samples and in animal models are warranted.
PMCID: PMC3432558  PMID: 22966235
22.  A Study of the Use of Impulse Oscillometry in the Evaluation of Children With Asthma: Analysis of Lung Parameters, Order Effect, and Utility Compared With Spirometry 
Pediatric pulmonology  2011;47(1):18-26.
The ability to objectively measure lung function in children is critical in the assessment and treatment of asthma in this age group. We thus determined the effectiveness of impulse oscillometry (IOS) as a non-invasive technique to assess lung function in children and in comparison to spirometry for sensitivity and specificity, testing variability, and the order effect of sequential testing of IOS and spirometry.
One hundred seventeen children sequentially evaluated in a pediatric clinic and under medical care for disease, were asked to perform IOS and spirometry. The utility of IOS and spirometry in differentiating children that had asthma versus those children who did not was then analyzed.
In the primary analysis (n = 117), bronchodilator response using IOS distinguished asthmatics from non-asthmatics, P = 0.0008 for R10. Receiver–operator characteristic curve (ROC) analysis of R10 bronchodilator response at the best cut-off (–8.6% change) correctly identified 77% of patients with asthma and excluded 76% of non-asthmatics. Amongst those children able to perform spirometry (asthmatics, n = 66; non-asthmatics, n = 16), FEV1 did not reveal a difference between these two groups, while area of reactance (AX) did distinguish these groups (P = 0.0092). Sequential testing of IOS and then spirometry (n = 47) showed a significant decrement in lung function as determined by IOS following performance of spirometry (P = 0.0309).
In the diagnosis and management of children with lung disease, IOS is a non-invasive approach that easily and objectively measures lung impedance and should be considered as both an adjunct, and in some situations, an alternative to standard spirometry.
PMCID: PMC3423092  PMID: 22170806
pediatric asthma; lung function; bronchodilator response; spirometry; impulse oscillometry
23.  Structure of Full-length Drosophila Cryptochrome 
Nature  2011;480(7377):396-399.
The Cryptochrome/Photolyase (CRY/PL) family of photoreceptors mediates adaptive responses to UV and blue light exposure in all kingdoms of life 1; 2; 3; 4; 5. Whereas PLs function predominantly in DNA repair of cyclobutane pyrimidine dimers (CPDs)and 6-4 photolesions caused by UV radiation, CRYs transduce signals important for growth, development, magnetosensitivity and circadian clocks1; 2; 3; 4; 5. Despite these diverse functions, PLs/CRYs preserve a common structural fold, a dependence on flavin adenine dinucleotide (FAD) and an internal photoactivation mechanism3; 6. However, members of the CRY/PL family differ in the substrates recognized (protein or DNA), photochemical reactions catalyzed and involvement of an antenna cofactor. It is largely unknown how the animal CRYs that regulate circadian rhythms act on their substrates. CRYs contain a variable C-terminal tail that appends the conserved PL homology domain (PHD) and is important for function 7; 8; 9; 10; 11; 12. Herein, we report a 2.3 Å resolution crystal structure of Drosophila CRY with an intact C-terminus. The C-terminal helix docks in the analogous groove that binds DNA substrates in PLs. Conserved Trp536 juts into the CRY catalytic center to mimic PL recognition of DNA photolesions. The FAD anionic semiquinone found in the crystals assumes a conformation to facilitate restructuring of the tail helix. These results help reconcile the diverse functions of the CRY/PL family by demonstrating how conserved protein architecture, and photochemistry can be elaborated into a range of light-driven functions.
PMCID: PMC3240699  PMID: 22080955
24.  Multiple orphan histidine kinases interact directly with Spo0A to control the initiation of endospore formation in Clostridium acetobutylicum 
Molecular microbiology  2011;80(3):641-654.
The phosphorylated Spo0A transcription factor controls the initiation of endospore formation in Clostridium acetobutylicum, but genes encoding key phosphorelay components, Spo0F and Spo0B, are missing in the genome. We hypothesized that the five orphan histidine kinases of C. acetobutylicum interact directly with Spo0A to control its phosphorylation state. Sequential targeted gene disruption and gene expression profiling provided evidence for two pathways for Spo0A activation, one dependent on a histidine kinase encoded by cac0323, the other on both histidine kinases encoded by cac0903 and cac3319. Purified Cac0903 and Cac3319 kinases autophosphorylated and transferred phosphoryl groups to Spo0A in vitro, confirming their role in Spo0A activation in vivo. A cac0437 mutant hyper-sporulated, suggesting that Cac0437 is a modulator that prevents sporulation and maintains cellular Spo0A~P homeostasis during growth. Accordingly, Cac0437 has apparently lost the ability to autophosphorylate in vitro; instead it catalyses the ATP-dependent dephosphorylation of Spo0A~P releasing inorganic phosphate. Direct phosphorylation of Spo0A by histidine kinases and dephosphorylation by kinase-like proteins may be a common feature of the clostridia that may represent the ancestral state before the great oxygen event some 2.4 billion years ago, after which additional phosphorelay proteins were recruited in the evolutionary lineage that led to the bacilli.
PMCID: PMC3097173  PMID: 21401736
sporulation; clostridia; autophosphorylation; phosphotransfer; phosphorelay
25.  Possible interactions between bacterial diversity, microbial activity and supraglacial hydrology of cryoconite holes in Svalbard 
The ISME journal  2010;5(1):150-160.
The diversity of highly active bacterial communities in cryoconite holes on three Arctic glaciers in Svalbard was investigated using terminal restriction fragment length polymorphism (T-RFLP) of the 16S rRNA locus. Construction and sequencing of clone libraries allowed several members of these communities to be identified, with Proteobacteria being the dominant one, followed by Cyanobacteria and Bacteroidetes. T-RFLP data revealed significantly different communities in holes on the (cold) valley glacier Austre Brøggerbreen relative to two adjacent (polythermal) valley glaciers, Midtre Lovénbreen and Vestre Brøggerbreen. These population compositions correlate with differences in organic matter content, temperature and the metabolic activity of microbial communities concerned. No within-glacier spatial patterns were observed in the communities identified over the 2-year period and with the 1 km-spaced sampling. We infer that surface hydrology is an important factor in the development of cryoconite bacterial communities.
PMCID: PMC3105670  PMID: 20664552
svalbard; bacterial diversity; biogeography; T-RFLP; evenness; spatial scaling

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