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1.  Beta2-agonists for exercise-induced asthma 
Paediatrics & Child Health  2014;19(7):355-356.
PMCID: PMC4173903  PMID: 25332673
2.  Light-Dependent Phosphorylation of Bardet Biedl Syndrome 5 in Photoreceptor Cells Modulates its Interaction with Arrestin1 
Cellular and molecular life sciences : CMLS  2013;70(23):10.1007/s00018-013-1403-4.
Arrestins are dynamic proteins which move between cell compartments triggered by stimulation of G-protein-coupled receptors. Even more dynamically in vertebrate photoreceptors, arrestin1 (Arr1) moves between the inner and outer segments according to the lighting conditions. Previous studies have shown that the light-driven translocation of Arr1 in rod photoreceptors is initiated by rhodopsin through a phospholipase C/protein kinase C (PKC) signaling cascade. The purpose of this study is to identify the PKC substrate that regulates the translocation of Arr1.
Mass spectrometry was used to identify the primary phosphorylated proteins in extracts prepared from PKC-stimulated mouse eye cups, confirming the finding with in vitro phosphorylation assays. Our results show that BBS5 is the principal protein phosphorylated either by phorbol ester stimulation or by light stimulation of PKC. Via immunoprecipitation of BBS5 in rod outer segments, Arr1 was pulled down; phosphorylation of BBS5 reduced this co-precipitation of Arr1. Immunofluorescence and immunoelectron microscopy showed that BBS5 principally localizes along the axonemes of rods and cones, but also in photoreceptor inner segments, and synaptic regions.
Our principal findings in this study are three-fold. First, we demonstrate that BBS5 is post-translationally regulated by phosphorylation via PKC, an event that is triggered by light in photoreceptor cells. Second, we find a direct interaction between BBS5 and Arr1, an interaction that is modulated by phosphorylation of BBS5. Finally, we show that BBS5 is distributed along the photoreceptor axoneme, co-localizing with Arr1 in the dark. These findings suggest a role for BBS5 in regulating light-dependent translocation of Arr1 and a model describing its role in Arr1 translocation is proposed.
PMCID: PMC3819411  PMID: 23817741
arrestin; cilia; BBS5; BBSome; translocation
Molecular genetics and metabolism  2013;110(4):446-453.
Phenylacetic acid (PAA) is the active moiety in sodium phenylbutyrate (NaPBA) and glycerol phenylbutyrate (GPB, HPN-100), both are approved for treatment of urea cycle disorders (UCDs) - rare genetic disorders characterized by hyperammonemia. PAA is conjugated with glutamine in the liver to form phenylacetyleglutamine (PAGN), which is excreted in urine. PAA plasma levels ≥500 μg/dL have been reported to be associated with reversible neurological adverse events (AEs) in cancer patients receiving PAA intravenously. Therefore, we have investigated the relationship between PAA levels and neurological AEs in patients treated with these PAA pro-drugs as well as approaches to identifying patients most likely to experience high PAA levels.
The relationship between nervous system AEs, PAA levels and the ratio of plasma PAA to PAGN were examined in 4683 blood samples taken serially from: [1] healthy adults [2], UCD patients ≥2 months of age, and [3] patients with cirrhosis and hepatic encephalopathy (HE). The plasma ratio of PAA to PAGN was analyzed with respect to its utility in identifying patients at risk of high PAA values.
Only 0.2% (11) of 4683 samples exceeded 500 ug/ml. There was no relationship between neurological AEs and PAA levels in UCD or HE patients, but transient AEs including headache and nausea that correlated with PAA levels were observed in healthy adults. Irrespective of population, a curvilinear relationship was observed between PAA levels and the plasma PAA:PAGN ratio, and a ratio > 2.5 (both in μg/mL) in a random blood draw identified patients at risk for PAA levels > 500 μg/ml.
The presence of a relationship between PAA levels and reversible AEs in healthy adults but not in UCD or HE patients may reflect intrinsic differences among the populations and/or metabolic adaptation with continued dosing. The plasma PAA:PAGN ratio is a functional measure of the rate of PAA metabolism and represents a useful dosing biomarker.
PMCID: PMC4108288  PMID: 24144944
BUPHENYL; glycerol phenylbutyrate; HPN-100; neurological adverse events; pharmacokinetics; RAVICTI; sodium phenylbutyrate
5.  Exploring Natural Product Chemistry and Biology with Multicomponent Reactions. 5. Discovery of a Novel Tubulin-Targeting Scaffold Derived from the Rigidin Family of Marine Alkaloids 
Journal of medicinal chemistry  2013;56(17):6886-6900.
We developed synthetic chemistry to access the marine alkaloid rigidins and over forty synthetic analogues based on the 7-deazaxanthine, 7-deazaadenine, 7-deazapurine and 7-deazahypoxanthine skeletons. Analogues based on the 7-deazahypoxanthine skeleton exhibited nanomolar potencies against cell lines representing cancers with dismal prognoses, tumor metastases and multidrug resistant cells. Studies aimed at elucidating the mode(s) of action of the 7-deazahypoxanthines in cancer cells revealed that they inhibited in vitro tubulin polymerization and disorganized microtubules in live HeLa cells. Experiments evaluating the effects of the 7-deazahypoxanthines on the binding of [3H]colchicine to tubulin identified the colchicine site on tubulin as the most likely target for these compounds in cancer cells. Because many microtubule-targeting compounds are successfully used to fight cancer in the clinic, we believe the new chemical class of antitubulin agents represented by the 7-deazahypoxanthine rigidin analogues have significant potential as new anticancer agents.
PMCID: PMC3915529  PMID: 23927793
6.  Ammonia Control in Children Ages 2 Months through 5 Years with Urea Cycle Disorders: Comparison of Sodium Phenylbutyrate and Glycerol Phenylbutyrate 
The Journal of pediatrics  2013;162(6):1228-1234.e1.
To examine ammonia levels, pharmacokinetics (PK), and safety of glycerol phenylbutyrate (GPB, HPN-100) and sodium phenylbutyrate (NaPBA) in young children with urea cycle disorders (UCDs).
Study design
This open label switch-over study enrolled patients ages 29 days to under 6 years taking NaPBA. Patients underwent 24-hr blood and urine sampling on NaPBA and again on a PBA-equimolar dose of GPB and completed questionnaires regarding signs and symptoms associated with NaPBA and/or their UCD.
15 patients (8 ASL, 3 ASS, 3 OTC, 1 ARG) ages 2 months through 5 years enrolled in and completed the study. Daily ammonia exposure (24-hour AUC) was lower on GPB and met predefined non-inferiority criteria (ratio of means 0.79; 95% CI 0.593–1.055; p = 0.03 Wilcoxon; 0.07 t-test). Six patients experienced mild AEs on GPB; there were no SAEs or significant lab changes. Liver tests and ASA levels among patients with ASL were unchanged or improved on GPB. Eleven of 15 patients reported 35 symptoms on Day 1; 23 of these 35 symptoms improved or resolved on GPB. Mean systemic exposure to PBA, PAA and PAGN were similar and PAA exposure tended to be higher in the youngest children on both drugs. Urinary PAGN concentration was greater on morning voids and varied less over 24 hours on GPB versus NaPBA.
GPB results in more evenly distributed urinary output of PAGN over 24 hours, was associated with fewer symptoms and offers ammonia control comparable with that observed with NaPBA in young children with UCDs.
PMCID: PMC4017326  PMID: 23324524
clinical trial; pharmacokinetics; phenylacetic acid; phenylacetylglutamine; urinary PAGN
7.  Functional analysis of Normalized Difference Vegetation Index curves reveals overwinter mule deer survival is driven by both spring and autumn phenology 
Large herbivore populations respond strongly to remotely sensed measures of primary productivity. Whereas most studies in seasonal environments have focused on the effects of spring plant phenology on juvenile survival, recent studies demonstrated that autumn nutrition also plays a crucial role. We tested for both direct and indirect (through body mass) effects of spring and autumn phenology on winter survival of 2315 mule deer fawns across a wide range of environmental conditions in Idaho, USA. We first performed a functional analysis that identified spring and autumn as the key periods for structuring the among-population and among-year variation of primary production (approximated from 1 km Advanced Very High Resolution Radiometer Normalized Difference Vegetation Index (NDVI)) along the growing season. A path analysis showed that early winter precipitation and direct and indirect effects of spring and autumn NDVI functional components accounted for 45% of observed variation in overwinter survival. The effect size of autumn phenology on body mass was about twice that of spring phenology, while direct effects of phenology on survival were similar between spring and autumn. We demonstrate that the effects of plant phenology vary across ecosystems, and that in semi-arid systems, autumn may be more important than spring for overwinter survival.
PMCID: PMC3983931  PMID: 24733951
demography; Normalized Difference Vegetation Index; phenology curve; population dynamics; ungulate; winter severity
8.  Two waves of colonization straddling the K–Pg boundary formed the modern reef fish fauna 
Living reef fishes are one of the most diverse vertebrate assemblages on Earth. Despite its prominence and ecological importance, the origins and assembly of the reef fish fauna is poorly described. A patchy fossil record suggests that the major colonization of reef habitats must have occurred in the Late Cretaceous and early Palaeogene, with the earliest known modern fossil coral reef fish assemblage dated to 50 Ma. Using a phylogenetic approach, we analysed the early evolutionary dynamics of modern reef fishes. We find that reef lineages successively colonized reef habitats throughout the Late Cretaceous and early Palaeogene. Two waves of invasion were accompanied by increasing morphological convergence: one in the Late Cretaceous from 90 to 72 Ma and the other immediately following the end-Cretaceous mass extinction. The surge in reef invasions after the Cretaceous–Palaeogene boundary continued for 10 Myr, after which the pace of transitions to reef habitats slowed. Combined, these patterns match a classic niche-filling scenario: early transitions to reefs were made rapidly by morphologically distinct lineages and were followed by a decrease in the rate of invasions and eventual saturation of morphospace. Major alterations in reef composition, distribution and abundance, along with shifts in climate and oceanic currents, occurred during the Late Cretaceous and early Palaeogene interval. A causal mechanism between these changes and concurrent episodes of reef invasion remains obscure, but what is clear is that the broad framework of the modern reef fish fauna was in place within 10 Myr of the end-Cretaceous extinction.
PMCID: PMC3996619  PMID: 24695431
macroevolution; reef fishes; Cretaceous–Palaeogene mass extinction; niche-filling models
9.  Species-specific bioluminescence facilitates speciation in the deep sea 
Marine Biology  2014;161(5):1139-1148.
The vast darkness of the deep sea is an environment with few obvious genetic isolating barriers, and little is known regarding the macroevolutionary processes that have shaped present-day biodiversity in this habitat. Bioluminescence, the production and emission of light from a living organism through a chemical reaction, is thought to occur in approximately 80 % of the eukaryotic life that inhabits the deep sea (water depth greater than 200 m). In this study, we show, for the first time, that deep-sea fishes that possess species-specific bioluminescent structures (e.g., lanternfishes, dragonfishes) are diversifying into new species at a more rapid rate than deep-sea fishes that utilize bioluminescence in ways that would not promote isolation of populations (e.g., camouflage, predation). This work adds to our understanding of how life thrives and evolution shaped present-day biodiversity in the deep sea, the largest and arguably least explored habitat on earth.
Electronic supplementary material
The online version of this article (doi:10.1007/s00227-014-2406-x) contains supplementary material, which is available to authorized users.
PMCID: PMC3996283  PMID: 24771948
10.  Influence of eye size and beam entry angle on dose to non-targeted tissues of the eye during stereotactic x-ray radiosurgery of AMD 
Physics in medicine and biology  2013;58(19):6887-6896.
Age-related macular degeneration is a leading cause of vision loss for the elderly population of industrialized nations. The IRay® Radiotherapy System, developed by Oraya® Therapeutics, Inc., is a stereotactic low-voltage irradiation system designed to treat the wet form of the disease. The IRay System uses three robotically positioned 100 kVp collimated photon beams to deliver an absorbed dose of up to 24 Gy to the macula. The present study uses the Monte Carlo radiation transport code MCNPX to assess absorbed dose to six non-targeted tissues within the eye - total lens, radiosensitive tissues of the lens, optic nerve, distal tip of the central retinal artery, non-targeted portion of the retina, and the ciliary body – all as a function of eye size and beam entry angle. The ocular axial length was ranged from 20 to 28 mm in 2 mm increments, with the polar entry angle of the delivery system varied from 18 to 34 degrees in 2 degree increments. The resulting data showed insignificant variations in dose for all eye sizes. Slight variations in the dose to the optic nerve and the distal tip of the central retinal artery were noted as the polar beam angle changed. An increase in non-targeted retinal dose was noted as the entry angle increased, while the dose to the lens, sensitive volume of the lens, and ciliary body decreased as the treatment polar angle increased. Polar angles of 26 degrees or greater resulted in no portion of the sensitive volume of the lens receiving an absorbed dose of 0.5 Gy or greater. All doses to non-targeted structures reported in this study were less than accepted thresholds for post-procedure complications.
PMCID: PMC3927464  PMID: 24025704
Age-related macular degeneration (AMD); stereotactic radiosurgery (SRS); non-targeted tissue complication probability (NTCP); dose volume histogram (DVH)
11.  Detection of Epidemic USA300 Community-Associated Methicillin-Resistant Staphylococcus aureus Strains by Use of a Single Allele-Specific PCR Assay Targeting a Novel Polymorphism of Staphylococcus aureus pbp3 
Journal of Clinical Microbiology  2013;51(8):2541-2550.
In recent years, the dramatic increase in community-associated methicillin-resistant Staphylococcus aureus (CA-MRSA) infections has become a significant health care challenge. Early detection of CA-MRSA is important because of its increased virulence associated with the arginine catabolic mobile element (ACME), Panton-Valentine leukocidin (PVL), and other toxins that may contribute to disease severity. In particular, the USA300 epidemic clone has emerged and now represents the cause of as much as 98% of CA-MRSA skin and soft tissue infections in the United States. Current diagnostic assays used to identify CA-MRSA strains are based on complex multiplex PCRs targeting the staphylococcal cassette chromosome mec (SCCmec) DNA junction, a multitude of genes, and noncoding DNA fragments or on a number of lengthy sequence-typing methods. Here, two nucleotide polymorphisms, G88A and G2047A, that were found to be in strict linkage disequilibrium in the S. aureus penicillin-binding protein 3 (pbp3) gene were also found to be highly associated with the USA300 clone of CA-MRSA. Clinical isolates that contained this pbp3 allele were also positive for the presence of SCCmec type IV, the ACME, and the PVL toxin gene and matched the t008 or t121 molecular spa types, which are associated specifically with the USA300 CA-MRSA clone. A single allele-specific PCR targeting the G88A polymorphism was developed and was found to be 100% sensitive and specific for the detection of USA300 CA-MRSA and 91.5% sensitive and 100% specific for the detection of all CA-MRSA isolates in this study.
PMCID: PMC3719641  PMID: 23698534
12.  The Covert World of Fish Biofluorescence: A Phylogenetically Widespread and Phenotypically Variable Phenomenon 
PLoS ONE  2014;9(1):e83259.
The discovery of fluorescent proteins has revolutionized experimental biology. Whereas the majority of fluorescent proteins have been identified from cnidarians, recently several fluorescent proteins have been isolated across the animal tree of life. Here we show that biofluorescence is not only phylogenetically widespread, but is also phenotypically variable across both cartilaginous and bony fishes, highlighting its evolutionary history and the possibility for discovery of numerous novel fluorescent proteins. Fish biofluorescence is especially common and morphologically variable in cryptically patterned coral-reef lineages. We identified 16 orders, 50 families, 105 genera, and more than 180 species of biofluorescent fishes. We have also reconstructed our current understanding of the phylogenetic distribution of biofluorescence for ray-finned fishes. The presence of yellow long-pass intraocular filters in many biofluorescent fish lineages and the substantive color vision capabilities of coral-reef fishes suggest that they are capable of detecting fluoresced light. We present species-specific emission patterns among closely related species, indicating that biofluorescence potentially functions in intraspecific communication and evidence that fluorescence can be used for camouflage. This research provides insight into the distribution, evolution, and phenotypic variability of biofluorescence in marine lineages and examines the role this variation may play.
PMCID: PMC3885428  PMID: 24421880
13.  Activation of Phospholipase C Mimics the Phase Shifting Effects of Light on Melatonin Rhythms in Retinal Photoreceptors 
PLoS ONE  2013;8(12):e83378.
Many aspects of retinal photoreceptor function and physiology are regulated by the circadian clocks in these cells. It is well established that light is the primary stimulus that entrains these clocks; yet, the biochemical cascade(s) mediating light’s effects on these clocks remains unknown. This deficiency represents a significant gap in our fundamental understanding of photoreceptor signaling cascades and their functions. In this study, we utilized re-aggregated spheroid cultures prepared from embryonic chick retina to determine if activation of phospholipase C in photoreceptors in the absence of light can phase shift the melatonin secretion rhythms of these cells in a manner similar to that induced by light. We show that spheroid cultures rhythmically secrete melatonin and that these melatonin rhythms can be dynamically phase shifted by exposing the cultures to an appropriately timed light pulse. Importantly, we show that activation of phospholipase C using m-3M3FBS in the absence of light induces a phase delay in photoreceptor melatonin rhythms that mirrors that induced by light. The implication of this finding is that the light signaling cascade that entrains photoreceptor melatonin rhythms involves activation of phospholipase C.
PMCID: PMC3873303  PMID: 24386190
14.  Urinary Phenylacetylglutamine as Dosing Biomarker for Patients with Urea Cycle Disorders 
Molecular genetics and metabolism  2012;107(3):308-314.
We have analyzed pharmacokinetic data for glycerol phenylbutyrate (also GT4P or HPN-100) and sodium phenylbutyrate with respect to possible dosing biomarkers in patients with urea cycle disorders (UCD).
Study Design
These analyses are based on over 3000 urine and plasma data points from 54 adult and 11 pediatric UCD patients (ages 6–17) who participated in three clinical studies comparing ammonia control and pharmacokinetics during steady state treatment with glycerol phenylbutyrate or sodium phenylbutyrate. All patients received phenylbutyric acid equivalent doses of glycerol phenylbutyrate or sodium phenylbutyrate in a cross over fashion and underwent 24-hour blood samples and urine sampling for phenylbutyric acid, phenylacetic acid and phenylacetylglutamine.
Patients received phenylbutyric acid equivalent doses of glycerol phenylbutyrate ranging from 1.5–31.8 g/day and of sodium phenylbutyrate ranging from 1.3–31.7 g/day. Plasma metabolite levels varied widely, with average fluctuation indices ranging from 1979% –5690% for phenylbutyric acid, 843% to 3931% for phenylacetic acid, and 881% -to 1434% for phenylacetylglutamine. Mean percent recovery of phenylbutyric acid as urinary phenylacetylglutamine was 66.4 and 69.0 for pediatric patients and 68.7 and 71.4 for adult patients on glycerol phenylbutyrate and sodium phenylbutyrate, respectively. The correlation with dose was strongest for urinary phenylacetylglutamine excretion, either as morning spot urine (r=0.730, p<0.001) or as total 24-hour excretion (r=0.791 p<0.001), followed by plasma phenylacetylglutamine AUC24-hour, plasma phenylacetic acid AUC24-hour and phenylbutyric acid AUC24-hour. Plasma phenylacetic acid levels in adult and pediatric patients did not show a consistent relationship with either urinary phenylacetylglutamine or ammonia control.
The findings are collectively consistent with substantial yet variable pre-systemic (1st pass) conversion of phenylbutyric acid to phenylacetic acid and/or phenylacetylglutamine. The variability of blood metabolite levels during the day, their weaker correlation with dose, the need for multiple blood samples to capture trough and peak, and the inconsistency between phenylacetic acid and urinary phenylacetylglutamine as a marker of waste nitrogen scavenging limit the utility of plasma levels for therapeutic monitoring. By contrast, 24-hour urinary phenylacetylglutamine and morning spot urine phenylacetylglutamine correlate strongly with dose and appear to be clinically useful non-invasive biomarkers for compliance and therapeutic monitoring.
PMCID: PMC3608516  PMID: 22958974
15.  Estimated prevalence and patterns of presumed osteoporosis among older Americans based on Medicare data 
Estimates of osteoporosis (OP) prevalence based on bone mineral density testing and fracture occurrence may be imprecise for small demographic groups. Medicare data are a useful supplemental source of information on OP.
We studied people ages ≥65 years covered by Medicare 2005. Cases of presumed OP were beneficiaries with physician services or inpatient claims for OP or for an associated fracture (hip, distal forearm, spine) in 1999–2005.
Among 911,327 beneficiaries with 6 or 7 years of Medicare coverage, the overall prevalence of OP and associated fractures was 29.7%. Prevalence was four times higher for women than men, increased with age, and was two times higher for whites, Hispanic Americans, and Asian Americans than African Americans. Among people with OP-associated fracture claims, the proportion with an OP diagnosis was 49.7% overall (women, 57.1%; men, 21.9%) and was lower for men than women and for African Americans than other ethnic groups.
The low proportion of beneficiaries who had an OP-associated fracture and also had an OP diagnosis, particularly among men and African American women, suggests suboptimal recognition and management of OP. Study limitations included lack of validation of our definition of OP and potential misclassification of race/ethnicity.
PMCID: PMC3767011  PMID: 19189165
Epidemiology; Fracture; Osteoporosis; Prevalence
16.  Prevalence of hypothalamic-pituitary-adrenal axis suppression in children treated for asthma with inhaled corticosteroid 
Paediatrics & Child Health  2012;17(5):e34-e39.
To determine the prevalence of hypothalamic-pituitary-adrenal (HPA) axis suppression in asthmatic children on inhaled corticosteroids (ICS).
Clinical and demographic variables were recorded on preconstructed, standardized forms. HPA axis suppression was measured by morning serum cortisol levels and confirmed by low-dose adrenocorticotropic hormone stimulation testing.
In total, 214 children participated. Twenty children (9.3%, 95% CI 5.3% to 13.4%) had HPA axis suppression. Odds of HPA axis suppression increased with ICS dose (OR 1.005, 95% CI 1.003 to 1.009, P<0.001). All children with HPA axis suppression were on a medium or lower dose of ICS for their age (200 μg/day to 500 μg/day). HPA axis suppression was not predicted by drug type, dose duration, concomitant use of long-acting beta-agonist or nasal steroid, or clinical features.
Laboratory evidence of HPA axis suppression exists in children taking ICS for asthma. Children should be regularly screened for the presence of HPA axis suppression when treated with high-dose ICS (>500 μg/day). Consideration should be given to screening children on medium-dose ICS.
PMCID: PMC3381924  PMID: 23633903
Asthma; Hypothalamic-pituitary-adrenal axis; Inhaled corticosteroid
17.  Endovascular Mechanical Thrombectomy for the Treatment of Acute Ischemic Stroke Due to Arterial Dissection 
Interventional Neuroradiology  2012;18(1):74-79.
Arterial dissections account for 2% of strokes in all age groups, and up to 25% in patients aged 45 years or younger. The safety of endovascular intervention in this patient population is not well characterized.
We identified all patients in the Merci registry – a prospective, multi-center post-market database enrolling patients treated with the Merci Retriever thrombectomy device – with arterial dissection as the most likely stroke etiology. Stroke presentation and procedural details were obtained prospectively; data regarding procedural complications, intracerebral hemorrhage (ICH), and the use of stenting of the dissected artery were obtained retrospectively.
Of 980 patients in the registry, ten were identified with arterial dissection (8/10 ICA; 2/10 vertebrobasilar). The median age was 48 years with a baseline NIH stroke scale score of 16 and median time to treatment of 4.9 h. The procedure resulted in thrombolysis in cerebral ischemia (TICI) scores of 2a or better in eight out of ten and TICI 2b or better in six out of ten patients. Stenting of the dissection was performed in four of nine (44%). The single complication (1/9; 11%) – extension of a dissected carotid artery – was treated effectively with stenting. No symptomatic ICH or stroke in a previously unaffected territory occurred. A favorable functional outcome was observed in eight out of ten patients.
Despite severe strokes on presentation, high rates of recanalization (8/10) and favorable functional outcomes (8/10) were observed. These results suggest that mechanical thrombectomy in patients with acute stroke resulting from arterial dissection is feasible, safe, and may be associated with favorable functional outcomes.
PMCID: PMC3312093  PMID: 22440604
stroke, acute stroke, arterial dissection, mechanical thrombectomy, endovascular, carotid dissection, vertebral dissection, stenting, interventional
18.  Effective Arrestin–Specific Immunotherapy of Experimental Autoimmune Uveitis with RTL: A Prospect for Treatment of Human Uveitis 
To evaluate the immunotherapeutic efficacy of recombinant T cell receptor ligands (RTLs) specific for arrestin immunity in treatment of experimental autoimmune uveitis (EAU) in humanized leukocyte antigen (HLA-DR3) transgenic (Tg) mice.
We generated de novo recombinant human DR3-derived RTLs bearing covalently tethered arrestin peptides 291–310 (RTL351) or 305–324 (RTL352). EAU was induced by immunization of HLA-DR3 mice with arrestin or arrestin peptide and treated with RTLs by subcutaneous delivery. T cell proliferation and cytokine expression was measured in RTL-treated and control mice.
RTL351 prevented the migration of cells outside of the spleen and the recruitment of inflammatory cells into the eye, and provided full protection against inflammation from EAU induced with arrestin or arrestin peptides. RTL351 significantly inhibited T cell proliferation and secretion of inflammatory cytokines interleukin 2 (IL-2), interferon γ (IFN-γ), IL-6, and IL-17 and chemokines (macrophage inflammatory proteins [MIP-1a] and regulated and normal T cell expressed and secreted [RANTES]), which is in agreement with the suppression of intraocular inflammation. RTL350 (“empty,” no peptide) and RTL352 were not effective.
Immunotherapy with a single RTL351 successfully prevented and treated arrestin-induced EAU in HLA-DR3 mice and provided proof of concept for therapy of autoimmune uveitis in human patients. The beneficial effects of RTL351 should be attributed to a significant decrease in Th1/Th17 mediated inflammation.
Translational Relevance:
Successful therapies for autoimmune uveitis must specifically inhibit pathogenic inflammation without inducing generalized immunosuppression. RTLs can offer such an option. The single retina-specific RTLs may have a value as potential immunotherapeutic drug for human autoimmune uveitis because they effectively prevent disease induced by multiple T cell specificities.
PMCID: PMC3763891  PMID: 24049712
experimental autoimmune uveitis; immunotherapy; inflammation
20.  Review: The history and role of naturally occurring mouse models with Pde6b mutations 
Molecular Vision  2013;19:2579-2589.
Mouse models are useful tools for developing potential therapies for human inherited retinal diseases, such as retinitis pigmentosa (RP), since more strains are being identified with the same mutant genes and phenotypes as humans with corresponding retinal degenerative diseases. Mutations in the beta subunit of the human rod phosphodiesterase (PDE6B) gene are a common cause of autosomal recessive RP (arRP). This article focuses on two well-established naturally occurring mouse models of arRP caused by spontaneous mutations in Pde6b, their discovery, phenotype, mechanism of degeneration, strengths and limitations, and therapeutic approaches to restore vision and delay disease progression. Viral vector, especially adeno-associated viral vector (AAV) -mediated gene replacement therapy, pharmacological treatment, cell-based therapy and other approaches that extend the therapeutic window of treatment, is a potentially promising strategy for improving photoreceptor function and significantly slowing the process of retinal degeneration.
PMCID: PMC3869645  PMID: 24367157
21.  Hypoxia Imaging with [F-18] FMISO-PET in Head and Neck Cancer: Potential for Guiding Intensity Modulated Radiation Therapy in Overcoming Hypoxia-Induced Treatment Resistance 
Background and Purpose
Positron emission tomography (PET) imaging with [F-18] fluoromisonidazole (FMISO) has been validated as a hypoxic tracer [1, 2]. Head and neck cancer exhibits hypoxia, inducing aggressive biologic traits that impart resistance to treatment. Delivery of modestly higher radiation doses to tumors with stable areas of chronic hypoxia can improve tumor control [3]. Advanced radiation treatment planning (RTP) and delivery techniques such as Intensity Modulated Radiation Therapy (IMRT) can deliver higher doses to a small volume without increasing morbidity. We investigated the utility of co-registered FMISO-PET and CT images to develop clinically feasible RTPs with higher tumor control probabilities (TCP).
FMISO-PET images were used to determine hypoxic sub-volumes for boost planning. Example plans were generated for ten of the patients in the study who exhibited significant hypoxia. We created an IMRT plan for each patient with a simultaneous integrated boost (SIB) to the hypoxic sub-volumes. We also varied the boost for two patients.
A significant (mean 17%, median 15%) improvement in TCP is predicted when the modest additional boost dose to the hypoxic sub-volume is included.
Combined FMISO-PET imaging and IMRT planning permits delivery of higher doses to hypoxic regions, increasing the predicted TCP (mean 17%) without increasing expected complications.
PMCID: PMC3225491  PMID: 21872957
22.  Clinical spectrum of SIX3-associated mutations in holoprosencephaly: correlation between genotype, phenotype and function 
Journal of medical genetics  2009;46(6):389-398.
Holoprosencephaly (HPE) is the most common structural malformation of the human forebrain. There are several important HPE mutational target genes, including the transcription factor SIX3, which encodes an early regulator of Shh, Wnt, Bmp and Nodal signalling expressed in the developing forebrain and eyes of all vertebrates.
To characterise genetic and clinical findings in patients with SIX3 mutations.
Patients with HPE and their family members were tested for mutations in HPE-associated genes and the genetic and clinical findings, including those for additional cases found in the literature, were analysed. The results were correlated with a mutation-specific functional assay in zebrafish.
In a cohort of patients (n = 800) with HPE, SIX3 mutations were found in 4.7% of probands and additional cases were found through testing of relatives. In total, 138 cases of HPE were identified, 59 of whom had not previously been clinically presented. Mutations in SIX3 result in more severe HPE than in other cases of non-chromosomal, non-syndromic HPE. An over-representation of severe HPE was found in patients whose mutations confer greater loss of function, as measured by the functional zebrafish assay. The gender ratio in this combined set of patients was 1.5:1 (F:M) and maternal inheritance was almost twice as common as paternal. About 14% of SIX3 mutations in probands occur de novo. There is a wide intrafamilial clinical range of features and classical penetrance is estimated to be at least 62%.
Our data suggest that SIX3 mutations result in relatively severe HPE and that there is a genotype–phenotype correlation, as shown by functional studies using animal models.
PMCID: PMC3510661  PMID: 19346217
23.  A novel angiogenesis inhibitor impairs lovo cell survival via targeting against human VEGFR and its signaling pathway of phosphorylation 
Cell Death & Disease  2012;3(10):e406-.
Colorectal cancer represents the fourth commonest malignancy, and constitutes a major cause of significant morbidity and mortality among other diseases. However, the chemical therapy is still under development. Angiogenesis plays an important role in colon cancer development. We developed HMQ18–22 (a novel analog of taspine) with the aim to target angiogenesis. We found that HMQ18–22 significantly reduced angiogenesis of chicken chorioallantoic membrane (CAM) and mouse colon tissue, and inhibited cell migration and tube formation as well. Then, we verified the interaction between HMQ18–22 and VEGFR2 by AlphaScreen P-VEGFR assay, screened the targets on angiogenesis by VEGF Phospho Antibody Array, validated the target by western blot and RNAi in lovo cells. We found HMQ18–22 could decrease phosphorylation of VEGFR2(Tyr1214), VEGFR1(Tyr1333), Akt(Tyr326), protein kinase Cα (PKCα) (Tyr657) and phospholipase-Cγ-1 (PLCγ-1) (Tyr771). Most importantly, HMQ18–22 inhibited proliferation of lovo cell and tumor growth in a human colon tumor xenografted model of athymic mice. Compared with normal lovo cells proliferation, the inhibition on proliferation of knockdown cells (VEGFR2, VEGFR1, Akt, PKCα and PLCγ-1) by HMQ18–22 decreased. These results suggested that HMQ18–22 is a novel angiogenesis inhibitor and can be a useful therapeutic candidate for colon cancer intervention.
PMCID: PMC3481133  PMID: 23059825
HMQ18–22; angiogenesis; VEGFR; inhibition; signaling pathway
24.  Physical activity attitudes, intentions and behaviour among 18–25 year olds: A mixed method study 
BMC Public Health  2012;12:640.
Young people (18–25 years) during the adolescence/adulthood transition are vulnerable to weight gain and notoriously hard to reach. Despite increased levels of overweight/obesity in this age group, physical activity behaviour, a major contributor to obesity, is poorly understood. The purpose of this study was to explore physical activity (PA) behaviour among 18–25 year olds with influential factors including attitudes, motivators and barriers.
An explanatory mixed method study design, based on health Behaviour Change Theories was used. Those at university/college and in the community, including those Not in Education, Employment or Training (NEET) were included. An initial self reported quantitative questionnaire survey underpinned by the Theory of Planned Behaviour and Social Cognitive Theory was conducted. 1313 questionnaires were analysed. Results from this were incorporated into a qualitative phase also grounded in these theories. Seven focus groups were conducted among similar young people, varying in education and socioeconomic status. Exploratory univariate analysis was followed by multi staged modelling to analyse the quantitative data. ‘Framework Analysis’ was used to analyse the focus groups.
Only 28% of 18–25 year olds achieved recommended levels of PA which decreased with age. Self-reported overweight/obesity prevalence was 22%, increasing with age, particularly in males. Based on the statistical modelling, positive attitudes toward PA were strong predictors of physical activity associated with being physically active and less sedentary. However, strong intentions to do exercise, was not associated with actual behaviour. Interactive discussions through focus groups unravelled attitudes and barriers influencing PA behaviour. Doing PA to feel good and to enjoy themselves was more important for young people than the common assumptions of ‘winning’ and ‘pleasing others’. Further this age group saw traditional health promotion messages as ‘empty’ and ‘fear of their future health’ was not a motivating factor to change current behaviour.
18–25 year olds are a difficult group to reach and have low levels of PA. Factors such as, ‘enjoyment’, ‘appearance ‘and ‘feeling good’ were deemed important by this specific age group. A targeted intervention incorporating these crucial elements should be developed to improve and sustain PA levels.
PMCID: PMC3490897  PMID: 22892291
Physical activity; Young people; 18–25 year olds; Mixed methods; Obesity; Transition
25.  Leprosy 
Clinical Evidence  2010;2010:0915.
The World Health Organization field leprosy classification is based on the number of skin lesions: paucibacillary leprosy (1–5 skin lesions), and multibacillary leprosy (more than 5 skin lesions). Worldwide, about 250,000 new cases of leprosy are reported each year, and about 2 million people have leprosy-related disabilities.
Methods and outcomes
We conducted a systematic review and aimed to answer the following clinical questions: What are the effects of interventions to prevent leprosy? What are the effects of treatments for leprosy? We searched: Medline, Embase, The Cochrane Library, and other important databases up to September 2009 (Clinical Evidence reviews are updated periodically, please check our website for the most up-to-date version of this review). We included harms alerts from relevant organisations such as the US Food and Drug Administration (FDA) and the UK Medicines and Healthcare products Regulatory Agency (MHRA).
We found 20 systematic reviews, RCTs, or observational studies that met our inclusion criteria. We performed a GRADE evaluation of the quality of evidence for interventions.
In this systematic review we present information relating to the effectiveness and safety of the following interventions: chemoprophylaxis with single-dose rifampicin, Bacillus Calmette–Guerin (BCG) plus killed Mycobacterium leprae vaccine, BCG vaccine, ICRC vaccine, multidrug treatment, multiple-dose treatment, Mycobacterium w vaccine, and single-dose treatment.
Key Points
Leprosy is a chronic granulomatous disease caused by Mycobacterium leprae, primarily affecting the peripheral nerves and skin. The WHO field leprosy classification is based on the number of skin lesions: paucibacillary leprosy (1–5 skin lesions), and multibacillary leprosy (more than 5 skin lesions).Worldwide, about 250,000 new cases of leprosy are reported each year, and about 2 million people have leprosy-related disabilities.
Chemoprophylaxis given to contacts of index cases is moderately effective in preventing leprosy. Chemoprophylaxis with single-dose rifampicin reduces the incidence of leprosy in contacts of new cases, although the effect is only seen in the first 2 years.
Vaccination is the most efficient method of preventing the contraction of leprosy. Vaccination with Bacillus Calmette–Guerin (BCG) vaccine, either alone or in combination with killed M leprae , reduces the incidence of leprosy. BCG and BCG plus killed M leprae seem to be as effective as each other at reducing the incidence of leprosy. ICRC vaccine prevents leprosy and produces few adverse effects, although its formulation is unclear and we only found evidence in one geographical area. Mycobacterium w vaccine reduces the incidence of leprosy compared with placebo.
Leprosy is generally treated with multidrug programmes. Despite sparse good RCT or cohort study evidence, there is consensus that multidrug treatment (rifampicin plus clofazimine plus dapsone) is highly effective for treating multibacillary leprosy. Placebo-controlled trials of multidrug treatment would now be considered unethical. Multidrug treatment with rifampicin plus dapsone is believed to improve skin lesions, nerve impairment, and relapse rates in people with paucibacillary leprosy, despite a lack of good evidence. Multiple-dose treatments with rifampicin monthly plus dapsone daily for 6 months are more effective than single-dose treatments with rifampicin plus minocycline plus ofloxacin for treating people with single skin lesions (although both achieve high cure rates).
PMCID: PMC3217821  PMID: 21418690

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