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1.  Effects of developmental manganese, stress, and the combination of both on monoamines, growth, and corticosterone 
Toxicology reports  2014;1:1046-1061.
Developmental exposure to manganese (Mn) or stress can each be detrimental to brain development. Here, Sprague-Dawley rats were exposed to two housing conditions and Mn from postnatal day (P)4–28. Within each litter two males and 2 females were assigned to the following groups: 0 (vehicle), 50, or 100 mg/kg Mn by oral gavage every other day. Half the litters were reared in cages with standard bedding and half with no bedding. One pair/group in each litter had an acute shallow water stressor before tissue collection (i.e., standing in shallow water). Separate litters were assessed at P11, 19, or 29. Mn-treated rats raised in standard cages showed no change in baseline corticosterone but following acute stress increased more than controls on P19; no Mn effects were seen on P11 or P29. Mn increased neostriatal dopamine in females at P19 and norepinephrine at P11 and P29. Mn increased hippocampal dopamine at P11 and P29 and 5-HT at P29 regardless of housing or sex. Mn had no effect on hypothalamic dopamine, but increased norepinephrine in males at P29 and 5-HT in males at all ages irrespective of rearing condition. Barren reared rats showed no or opposite effects of Mn, i.e., barren rearing + Mn attenuated corticosterone increases to acute stress. Barren rearing also altered the Mn-induced changes in dopamine and norepinephrine in the neostriatum, but not in the hippocampus. Barren rearing caused a Mn-associated increase in hypothalamic dopamine at P19 and P29 not seen in standard reared Mn-treated groups. Developmental Mn alters monoamines and corticosterone as a function of age, stress (acute and chronic), and sex.
PMCID: PMC4285371  PMID: 25574457
Manganese; barren cage rearing; corticosterone; dopamine; norepinephrine; serotonin; shallow water stress
2.  Neuronal reorganization in adult rats neonatally exposed to (±)-3,4-methylenedioxymethamphetamine 
Toxicology reports  2014;1:699-706.
The abuse of methylenedioxymethamphetamine (MDMA) during pregnancy is of concern. MDMA treatment of rats during a period of brain growth analogous to late human gestation leads to neurochemical and behavioral changes. MDMA from postnatal day (P)11–20 in rats produces reductions in serotonin and deficits in spatial and route-based navigation. In this experiment we examined the impact of MDMA from P11–20 (20 mg/kg twice daily, 8 h apart) on neuronal architecture. Golgi impregnated sections showed significant changes. In the nucleus accumbens, the dendrites were shorter with fewer spines, whereas in the dentate gyrus the dendritic length was decreased but with more spines, and for the entorhinal cortex, reductions in basilar and apical dendritic lengths in MDMA animals compared with saline animals were seen. The data show that neuronal cytoarchitectural changes are long-lasting following developmental MDMA exposure and are in regions consistent with the learning and memory deficits observed in such animals.
PMCID: PMC4235131  PMID: 25419515
development; Golgi-Cox staining; dentate gyrus; entorhinal cortex
3.  Assessing Spatial Learning and Memory in Rodents 
ILAR Journal  2014;55(2):310-332.
Maneuvering safely through the environment is central to survival of almost all species. The ability to do this depends on learning and remembering locations. This capacity is encoded in the brain by two systems: one using cues outside the organism (distal cues), allocentric navigation, and one using self-movement, internal cues and nearby proximal cues, egocentric navigation. Allocentric navigation involves the hippocampus, entorhinal cortex, and surrounding structures; in humans this system encodes allocentric, semantic, and episodic memory. This form of memory is assessed in laboratory animals in many ways, but the dominant form of assessment is the Morris water maze (MWM). Egocentric navigation involves the dorsal striatum and connected structures; in humans this system encodes routes and integrated paths and, when overlearned, becomes procedural memory. In this article, several allocentric assessment methods for rodents are reviewed and compared with the MWM. MWM advantages (little training required, no food deprivation, ease of testing, rapid and reliable learning, insensitivity to differences in body weight and appetite, absence of nonperformers, control methods for proximal cue learning, and performance effects) and disadvantages (concern about stress, perhaps not as sensitive for working memory) are discussed. Evidence-based design improvements and testing methods are reviewed for both rats and mice. Experimental factors that apply generally to spatial navigation and to MWM specifically are considered. It is concluded that, on balance, the MWM has more advantages than disadvantages and compares favorably with other allocentric navigation tasks.
PMCID: PMC4240437  PMID: 25225309
4.  The GPCR repertoire in the demosponge Amphimedon queenslandica: insights into the GPCR system at the early divergence of animals 
BMC Evolutionary Biology  2014;14(1):270.
G protein-coupled receptors (GPCRs) play a central role in eukaryotic signal transduction. However, the GPCR component of this signalling system, at the early origins of metazoans is not fully understood. Here we aim to identify and classify GPCRs in Amphimedon queenslandica (sponge), a member of an earliest diverging metazoan lineage (Porifera). Furthermore, phylogenetic comparisons of sponge GPCRs with eumetazoan and bilaterian GPCRs will be essential to our understanding of the GPCR system at the roots of metazoan evolution.
We present a curated list of 220 GPCRs in the sponge genome after excluding incomplete sequences and false positives from our initial dataset of 282 predicted GPCR sequences obtained using Pfam search. Phylogenetic analysis reveals that the sponge genome contains members belonging to four of the five major GRAFS families including Glutamate (33), Rhodopsin (126), Adhesion (40) and Frizzled (3). Interestingly, the sponge Rhodopsin family sequences lack orthologous relationships with those found in eumetazoan and bilaterian lineages, since they clustered separately to form sponge specific groups in the phylogenetic analysis. This suggests that sponge Rhodopsins diverged considerably from that found in other basal metazoans. A few sponge Adhesions clustered basal to Adhesion subfamilies commonly found in most vertebrates, suggesting some Adhesion subfamilies may have diverged prior to the emergence of Bilateria. Furthermore, at least eight of the sponge Adhesion members have a hormone binding motif (HRM domain) in their N-termini, although hormones have yet to be identified in sponges. We also phylogenetically clarified that sponge has homologs of metabotropic glutamate (mGluRs) and GABA receptors.
Our phylogenetic comparisons of sponge GPCRs with other metazoan genomes suggest that sponge contains a significantly diversified set of GPCRs. This is evident at the family/subfamily level comparisons for most GPCR families, in particular for the Rhodopsin family of GPCRs. In summary, this study provides a framework to perform future experimental and comparative studies to further verify and understand the roles of GPCRs that predates the divergence of bilaterian and eumetazoan lineages.
Electronic supplementary material
The online version of this article (doi:10.1186/s12862-014-0270-4) contains supplementary material, which is available to authorized users.
PMCID: PMC4302439  PMID: 25528161
Neurotransmitters; G protein-coupled receptors; Adhesion; Signal transduction; Porifera; Eumetazoa
5.  Plasma heat shock protein 27 is associated with coronary artery disease, abdominal aortic aneurysm and peripheral artery disease 
SpringerPlus  2014;3:635.
Low protein levels of Hsp27 have been reported in atherosclerotic plaques. In addition, human studies have indicated that circulating Hsp27 levels are lower in coronary artery disease patients compared with controls. It remains, however, unclear whether this applies to other forms of atherosclerotic disease.
Plasma Hsp27 from 280 subjects was examined by ELISA. The cohort included 80 coronary artery disease (CAD), 40 peripheral artery disease (PAD) and 80 abdominal aortic aneurysm (AAA) patients. Eighty elderly subjects, without any clinical history of vascular diseases, were used as a control group. Receiver operating curve (ROC) and logistic regression model analysis were performed to evaluate the potential value of Hsp27 as a circulating biomarker.
Patients with atherosclerotic vascular diseases had significantly lower levels of Hsp27 than control subjects (p < 0.001). Moreover, Hsp27 was significantly lower in CAD patients than other atherosclerotic vascular disease groups (p < 0.001). There was no difference in Hsp27 levels between the AAA and PAD groups. Using the ROC-generated optimal cut-off values for Hsp27, logistic regression modeling indicated that low plasma Hsp27 was independently associated with the presence of multiple forms of atherosclerotic disease.
In conclusion, circulating Hsp27 is significantly lower in patients with multiple forms of atherosclerotic arterial disease.
PMCID: PMC4218927  PMID: 25392804
6.  Diagnosis and management of idiopathic normal-pressure hydrocephalus 
Neurology: Clinical Practice  2013;3(5):375-385.
The diagnosis and management of idiopathic normal-pressure hydrocephalus (iNPH), a disorder of gait impairment, incontinence, and dementia that affects elderly patients, incorporates an organized approach using familiar principles for neurologists. The starting point is a comprehensive history and neurologic examination, review of neuroimaging, and evaluation of the differential diagnosis. Coexisting disorders should be treated before specific iNPH testing is performed. Specific iNPH testing includes assessing patient response to temporary CSF removal and testing CSF hydrodynamics. In properly selected patients, all iNPH symptoms, including dementia, can improve after shunt surgery. The longitudinal care of iNPH patients with shunts includes evaluation of the differential diagnosis of worsening iNPH symptoms and treatment of coexisting disorders. Evaluation of shunt obstruction is often indicated, and if it is found, surgical correction is likely to result in symptomatic improvement.
PMCID: PMC3806933  PMID: 24175154
7.  A computerized neuropsychological test battery designed for idiopathic normal pressure hydrocephalus 
A tool for standardized and repeated neuropsychological assessments in patients with idiopathic normal pressure hydrocephalus (INPH) is needed. The objective of this study was to develop a computerized neuropsychological test battery designed for INPH and to evaluate its reliability, validity and patient’s ability to complete the tests.
Based on a structured review of the literature on neuropsychological testing in INPH, the eight tests most sensitive to the INPH cognitive profile were implemented in a computerized format. The Geriatric Depression Scale (GDS) was also included. Tests were presented on a touch-screen monitor, with animated instructions and speaker sound. The battery was evaluated with the following cohorts: A. Test-retest reliability, 44 healthy elderly; B. Validity against standard pen and pencil testing, 28 patients with various cognitive impairments; C. Ability to complete test battery, defined as completion of at least seven of the eight tests, 40 investigated for INPH.
A. All except the figure copy test showed good test-retest reliability, r = 0.67-0.90; B. A high correlation was seen between conventional and computerized tests (r = 0.66-0.85) except for delayed recognition and figure copy task; C. Seventy-eight percent completed the computerized battery; Patients diagnosed with INPH (n = 26) performed worse on all tests, including depression score, compared to healthy controls.
A new computerized neuropsychological test battery designed for patients with communicating hydrocephalus and INPH was introduced. Its reliability, validity for general cognitive impairment and completion rate for INPH was promising. After exclusion of the figure copy task, the battery is ready for clinical evaluation and as a next step we suggest validation for INPH and a comparison before and after shunt surgery.
Trial registration NCT01265251.
PMCID: PMC4181752  PMID: 25279138
Dementia; Hydrocephalus, Normal pressure; Neuropsychological tests; Neuropsychology; Reliability and validity; Software
8.  Genome-wide association study identifies a sequence variant within the DAB2IP gene conferring susceptibility to abdominal aortic aneurysm 
Gretarsdottir, Solveig | Baas, Annette F | Thorleifsson, Gudmar | Holm, Hilma | den Heijer, Martin | de Vries, Jean-Paul P M | Kranendonk, Steef E | Zeebregts, Clark J A M | van Sterkenburg, Steven M | Geelkerken, Robert H | van Rij, Andre M | Williams, Michael J A | Boll, Albert P M | Kostic, Jelena P | Jonasdottir, Adalbjorg | Jonasdottir, Aslaug | Walters, G Bragi | Masson, Gisli | Sulem, Patrick | Saemundsdottir, Jona | Mouy, Magali | Magnusson, Kristinn P | Tromp, Gerard | Elmore, James R | Sakalihasan, Natzi | Limet, Raymond | Defraigne, Jean-Olivier | Ferrell, Robert E | Ronkainen, Antti | Ruigrok, Ynte M | Wijmenga, Cisca | Grobbee, Diederick E | Shah, Svati H | Granger, Christopher B | Quyyumi, Arshed A | Vaccarino, Viola | Patel, Riyaz S | Zafari, A Maziar | Levey, Allan I | Austin, Harland | Girelli, Domenico | Pignatti, Pier Franco | Olivieri, Oliviero | Martinelli, Nicola | Malerba, Giovanni | Trabetti, Elisabetta | Becker, Lewis C | Becker, Diane M | Reilly, Muredach P | Rader, Daniel J | Mueller, Thomas | Dieplinger, Benjamin | Haltmayer, Meinhard | Urbonavicius, Sigitas | Lindblad, Bengt | Gottsäter, Anders | Gaetani, Eleonora | Pola, Roberto | Wells, Philip | Rodger, Marc | Forgie, Melissa | Langlois, Nicole | Corral, Javier | Vicente, Vicente | Fontcuberta, Jordi | España, Francisco | Grarup, Niels | Jørgensen, Torben | Witte, Daniel R | Hansen, Torben | Pedersen, Oluf | Aben, Katja K | de Graaf, Jacqueline | Holewijn, Suzanne | Folkersen, Lasse | Franco-Cereceda, Anders | Eriksson, Per | Collier, David A | Stefansson, Hreinn | Steinthorsdottir, Valgerdur | Rafnar, Thorunn | Valdimarsson, Einar M | Magnadottir, Hulda B | Sveinbjornsdottir, Sigurlaug | Olafsson, Isleifur | Magnusson, Magnus Karl | Palmason, Robert | Haraldsdottir, Vilhelmina | Andersen, Karl | Onundarson, Pall T | Thorgeirsson, Gudmundur | Kiemeney, Lambertus A | Powell, Janet T | Carey, David J | Kuivaniemi, Helena | Lindholt, Jes S | Jones, Gregory T | Kong, Augustine | Blankensteijn, Jan D | Matthiasson, Stefan E | Thorsteinsdottir, Unnur | Stefansson, Kari
Nature genetics  2010;42(8):692-697.
We performed a genome-wide association study on 1,292 individuals with abdominal aortic aneurysms (AAAs) and 30,503 controls from Iceland and The Netherlands, with a follow-up of top markers in up to 3,267 individuals with AAAs and 7,451 controls. The A allele of rs7025486 on 9q33 was found to associate with AAA, with an odds ratio (OR) of 1.21 and P = 4.6 × 10−10. In tests for association with other vascular diseases, we found that rs7025486[A] is associated with early onset myocardial infarction (OR = 1.18, P = 3.1 × 10−5), peripheral arterial disease (OR = 1.14, P = 3.9 × 10−5) and pulmonary embolism (OR = 1.20, P = 0.00030), but not with intracranial aneurysm or ischemic stroke. No association was observed between rs7025486[A] and common risk factors for arterial and venous diseases—that is, smoking, lipid levels, obesity, type 2 diabetes and hypertension. Rs7025486 is located within DAB2IP, which encodes an inhibitor of cell growth and survival.
PMCID: PMC4157066  PMID: 20622881
9.  Obesity-Linked Homologues TfAP-2 and Twz Establish Meal Frequency in Drosophila melanogaster 
PLoS Genetics  2014;10(9):e1004499.
In all animals managing the size of individual meals and frequency of feeding is crucial for metabolic homeostasis. In the current study we demonstrate that the noradrenalin analogue octopamine and the cholecystokinin (CCK) homologue Drosulfakinin (Dsk) function downstream of TfAP-2 and Tiwaz (Twz) to control the number of meals in adult flies. Loss of TfAP-2 or Twz in octopaminergic neurons increased the size of individual meals, while overexpression of TfAP-2 significantly decreased meal size and increased feeding frequency. Of note, our study reveals that TfAP-2 and Twz regulate octopamine signaling to initiate feeding; then octopamine, in a negative feedback loop, induces expression of Dsk to inhibit consummatory behavior. Intriguingly, we found that the mouse TfAP-2 and Twz homologues, AP-2β and Kctd15, co-localize in areas of the brain known to regulate feeding behavior and reward, and a proximity ligation assay (PLA) demonstrated that AP-2β and Kctd15 interact directly in a mouse hypothalamus-derived cell line. Finally, we show that in this mouse hypothalamic cell line AP-2β and Kctd15 directly interact with Ube2i, a mouse sumoylation enzyme, and that AP-2β may itself be sumoylated. Our study reveals how two obesity-linked homologues regulate metabolic homeostasis by modulating consummatory behavior.
Author Summary
The size of individual meals and feeding frequency are important for homeostatic control. Due to the complex neuroendocrine system regulating human food intake it is difficult to uncover the mechanisms underlying eating disorders. The genetically tractable model system Drosophila melanogaster has a comparatively simple brain; yet, similar to humans, its eating behavior can adapt to respond to nutritional needs. Our study describes how the obesity-linked homologues TfAP-2 (human TFAP2B) and Tiwaz (human KCTD15) regulate a unique feedback system involving noradrenalin-like octopamine and the CCK homolog Dsk, that exert positive and negative effects on Drosophila feeding behavior. Our findings provide insight into how two conserved obesity-linked genes regulate feeding behavior in order to maintain metabolic balance.
PMCID: PMC4154645  PMID: 25187989
Successful navigation requires interactions among multiple but overlapping neural pathways mediating distinct capabilities, including egocentric (self-oriented, route-based) and allocentric (spatial, map-based) learning. Route-based navigation has been shown to be impaired following acute exposure to the dopaminergic (DA) drugs (+)-methamphetamine and (+)-amphetamine, but not the serotoninergic (5-HT) drugs (±)-3,4-methylenedioxymethamphetamine or (±)-fenfluramine. The dopaminergic-rich neostriatum is involved in both allocentric and egocentric navigation. This experiment tested whether dorsal striatal DA loss using bilateral 6-hydroxydopamine (6-OHDA) injections impaired one or both types of navigation. Two weeks following 6-OHDA injections, rats began testing in the Cincinnati water maze (CWM) followed by the Morris water maze (MWM) for route-based and spatial navigation, respectively. 6-OHDA treatment significantly increased latency and errors in the CWM and path length, latency, and cumulative distance in the MWM with no difference on cued MWM trials. Neostriatal DA levels were reduced by 80% at 2 and 7 weeks post-treatment. In addition, 6-OHDA increased DA turnover and decreased norepinephrine (NE) levels. 6-OHDA injections did not alter monoamine levels in the prefrontal cortex. The data support that neostriatal DA modulates both types of navigation.
PMCID: PMC4131757  PMID: 22465436
Dopamine; Route-based learning; Spatial learning; 6-hydroxydopamine; Egocentric; Allocentric; Striatum
Study Design:
Single‐blind, randomized, clinical trial.
The effect of eccentric training for mid‐portion Achilles tendinopathy is well documented; however, its effect on insertional Achilles tendinopathy is inconclusive. The primary purpose of this study was to investigate the effect of eccentric training on pain and function for individuals with insertional Achilles tendinopathy.
All patients received a 12‐week conventional strengthening protocol. Patients who were randomly assigned to the experimental group received additional eccentric exercises. Patients completed the Short Form‐36 Health and Bodily Pain Surveys, the Foot and Ankle Outcomes Questionnaire, and the Visual Analog Scale at initial evaluation, after 6 weeks of therapy, and at 12 weeks after therapy.
Thirty‐six patients (20 control and 16 experimental; average age 54 years; 72% women) completed the study. Both groups experienced statistically significant decreases in pain and improvements in function. No statistically significant differences were noted between the groups for any of the outcome measures.
Conventional physical therapy consisting of gastrocnemius, soleus and hamstring stretches, ice massage on the Achilles tendon, and use of heel lifts and night splints with or without eccentric training is effective for treating insertional Achilles tendinopathy.
Level of Evidence:
Level 2
PMCID: PMC4127511  PMID: 25133077
Achilles tendinopathy; eccentric training; posterior heel pain
12.  Automated Synthesis of 18F-Fluoropropoxytryptophan for Amino Acid Transporter System Imaging 
BioMed Research International  2014;2014:492545.
Objective. This study was to develop a cGMP grade of [18F]fluoropropoxytryptophan (18F-FTP) to assess tryptophan transporters using an automated synthesizer. Methods. Tosylpropoxytryptophan (Ts-TP) was reacted with K18F/kryptofix complex. After column purification, solvent evaporation, and hydrolysis, the identity and purity of the product were validated by radio-TLC (1M-ammonium acetate : methanol = 4 : 1) and HPLC (C-18 column, methanol : water = 7 : 3) analyses. In vitro cellular uptake of 18F-FTP and 18F-FDG was performed in human prostate cancer cells. PET imaging studies were performed with 18F-FTP and 18F-FDG in prostate and small cell lung tumor-bearing mice (3.7 MBq/mouse, iv). Results. Radio-TLC and HPLC analyses of 18F-FTP showed that the Rf and Rt values were 0.9 and 9 min, respectively. Radiochemical purity was >99%. The radiochemical yield was 37.7% (EOS 90 min, decay corrected). Cellular uptake of 18F-FTP and 18F-FDG showed enhanced uptake as a function of incubation time. PET imaging studies showed that 18F-FTP had less tumor uptake than 18F-FDG in prostate cancer model. However, 18F-FTP had more uptake than 18F-FDG in small cell lung cancer model. Conclusion. 18F-FTP could be synthesized with high radiochemical yield. Assessment of upregulated transporters activity by 18F-FTP may provide potential applications in differential diagnosis and prediction of early treatment response.
PMCID: PMC4127279  PMID: 25136592
13.  A sequence variant associated with sortilin-1 (SORT1) on 1p13.3 is independently associated with abdominal aortic aneurysm 
Human Molecular Genetics  2013;22(14):2941-2947.
Abdominal aortic aneurysm (AAA) is a common human disease with a high estimated heritability (0.7); however, only a small number of associated genetic loci have been reported to date. In contrast, over 100 loci have now been reproducibly associated with either blood lipid profile and/or coronary artery disease (CAD) (both risk factors for AAA) in large-scale meta-analyses. This study employed a staged design to investigate whether the loci for these two phenotypes are also associated with AAA. Validated CAD and dyslipidaemia loci underwent screening using the Otago AAA genome-wide association data set. Putative associations underwent staged secondary validation in 10 additional cohorts. A novel association between the SORT1 (1p13.3) locus and AAA was identified. The rs599839 G allele, which has been previously associated with both dyslipidaemia and CAD, reached genome-wide significance in 11 combined independent cohorts (meta-analysis with 7048 AAA cases and 75 976 controls: G allele OR 0.81, 95% CI 0.76–0.85, P = 7.2 × 10−14). Modelling for confounding interactions of concurrent dyslipidaemia, heart disease and other risk factors suggested that this marker is an independent predictor of AAA susceptibility. In conclusion, a genetic marker associated with cardiovascular risk factors, and in particular concurrent vascular disease, appeared to independently contribute to susceptibility for AAA. Given the potential genetic overlap between risk factor and disease phenotypes, the use of well-characterized case–control cohorts allowing for modelling of cardiovascular disease risk confounders will be an important component in the future discovery of genetic markers for conditions such as AAA.
PMCID: PMC3690970  PMID: 23535823
14.  Probiotics as Therapy in Gastroenterology 
The objective of this study was to determine how gastroenterologists perceive and use probiotic-based therapies in practice.
In the United States, there has been a recent increase in research investigating the therapeutic capacities of probiotics in human disease and an accompanying increase in product availability and marketing. How medical care providers have interpreted the available literature and incorporated it into their practice has not been earlier assessed.
A 16-question survey (see Survey, Supplemental Digital Content 1, was distributed to practicing gastroenterologists and physicians with a specific interest in GI disorders within a large metropolitan area.
All physicians responded that they believed probiotics to be safe for most patients and 98% responded that probiotics have a role in treating gastrointestinal illnesses or symptoms. Currently 93% of physicians have patients taking probiotics most often for irritable bowel syndrome. Commonly used probiotics included yogurt-based products, Bifidobacterium infantis 35624 (Align), and VSL#3. Most surveyed physicians recommended probiotics for irritable bowel syndrome, antibiotic, and Clostridium difficile-associated diarrhea because they believed that the literature supports their usage for these conditions. However, physician practice patterns did not consistently correlate with published, expert-panel-generated recommendations for evidence-based probiotic use.
This study suggests most gastrointestinal disease specialists recognize a role for and have used probiotics as part of their therapeutic armamentarium; however, the effective implementation of this practice will benefit from additional supporting studies and the eventual development of clinical practice guidelines supported by the major gastroenterology societies.
PMCID: PMC4094138  PMID: 20216432
probiotic; gastroenterology; opinion; recommendation; survey
15.  MHC class-I associated phosphopeptides are the targets of memory-like immunity in leukemia 
Science translational medicine  2013;5(203):203ra125.
Deregulation of signaling pathways involving phosphorylation is a hallmark of malignant transformation. Degradation of phosphoproteins generates cancer-specific phosphopeptides that are associated with MHC-I and II molecules and recognized by T-cells. We identified 95 phosphopeptides presented on the surface of primary hematological tumors and normal tissues, including 61 that were tumor-specific. Phosphopeptides were more prevalent on more aggressive and malignant samples. CD8 T-cell lines specific for these phosphopeptides recognized and killed both leukemia cell lines and HLA-matched primary leukemia cells ex vivo. Healthy individuals showed surprisingly high levels of CD8 T-cell responses against many of these phosphopeptides within the circulating memory compartment. This immunity was significantly reduced or absent in some leukemia patients, which correlated with clinical outcome, and was restored following allogeneic stem cell transplantation. These results suggest that phosphopeptides may be targets of cancer immune surveillance in humans, and point to their importance for development of vaccine-based and T-cell adoptive transfer immunotherapies..
PMCID: PMC4071620  PMID: 24048523
16.  Tissue-specific effects of saposin A and saposin B on glycosphingolipid degradation in mutant mice 
Human Molecular Genetics  2013;22(12):2435-2450.
Individual saposin A (A−/−) and saposin B (B−/−)-deficient mice show unique phenotypes caused by insufficient degradation of myelin-related glycosphingolipids (GSLs): galactosylceramide and galactosylsphingosine and sulfatide, respectively. To gain insight into the interrelated functions of saposins A and B, combined saposin AB-deficient mice (AB−/−) were created by knock-in point mutations into the saposins A and B domains on the prosaposin locus. Saposin A and B proteins were undetectable in AB−/− mice, whereas prosaposin, saposin C and saposin D were expressed near wild-type (WT) levels. AB−/− mice developed neuromotor deterioration at >61 days and exhibited abnormal locomotor activity and enhanced tremor. AB−/− mice (∼96 days) lived longer than A−/− mice (∼85 days), but shorter than B−/− mice (∼644 days). Storage materials were observed in Schwann cells and neuronal processes by electron microscopy. Accumulation of p62 and increased levels of LC3-II were detected in the brainstem suggesting altered autophagy. GSL analyses by (liquid chromatography) LC/MS identified substantial increases in lactosylceramide in AB−/− mouse livers. Sulfatide accumulated, but galactosylceramide remained at WT levels, in the AB−/− mouse brains and kidneys. Brain galactosylsphingosine in AB−/− mice was ∼68% of that in A−/− mice. These findings indicate that combined saposins A and B deficiencies attenuated GalCer-β-galactosylceramidase and GM1-β-galactosidase functions in the degradation of lactosylceramide preferentially in the liver. Blocking sulfatide degradation from the saposin B deficiency diminished galactosylceramide accumulation in the brain and kidney and galctosylsphingosine in the brain. These analyses of AB−/− mice continue to delineate the tissue differential interactions of saposins in GSL metabolism.
PMCID: PMC3708521  PMID: 23446636
17.  Drosophila Insulin-Producing Cells Are Differentially Modulated by Serotonin and Octopamine Receptors and Affect Social Behavior 
PLoS ONE  2014;9(6):e99732.
A set of 14 insulin-producing cells (IPCs) in the Drosophila brain produces three insulin-like peptides (DILP2, 3 and 5). Activity in IPCs and release of DILPs is nutrient dependent and controlled by multiple factors such as fat body-derived proteins, neurotransmitters, and neuropeptides. Two monoamine receptors, the octopamine receptor OAMB and the serotonin receptor 5-HT1A, are expressed by the IPCs. These receptors may act antagonistically on adenylate cyclase. Here we investigate the action of the two receptors on activity in and output from the IPCs. Knockdown of OAMB by targeted RNAi led to elevated Dilp3 transcript levels in the brain, whereas 5-HT1A knockdown resulted in increases of Dilp2 and 5. OAMB-RNAi in IPCs leads to extended survival of starved flies and increased food intake, whereas 5-HT1A-RNAi produces the opposite phenotypes. However, knockdown of either OAMB or 5-HT1A in IPCs both lead to increased resistance to oxidative stress. In assays of carbohydrate levels we found that 5-HT1A knockdown in IPCs resulted in elevated hemolymph glucose, body glycogen and body trehalose levels, while no effects were seen after OAMB knockdown. We also found that manipulations of the two receptors in IPCs affected male aggressive behavior in different ways and 5-HT1A-RNAi reduced courtship latency. Our observations suggest that activation of 5-HT1A and OAMB signaling in IPCs generates differential effects on Dilp transcription, fly physiology, metabolism and social interactions. However the findings do not support an antagonistic action of the two monoamines and their receptors in this particular system.
PMCID: PMC4055686  PMID: 24923784
18.  Effect of Implantation on engineered skeletal muscle constructs 
The development of engineered skeletal muscle would provide a viable tissue for replacement and repair of muscle damaged by disease or injury. Current tissue engineering methods result in three-dimensional (3-D) muscle constructs that generate tension, but do not advance phenotypically beyond neonatal characteristics (Larkin et al., 2006). To develop to an adult phenotype, innervation and vascularization of the construct must occur. In this study, 3-D muscle constructs were implanted into the hindlimb of a rat along the sciatic nerve with the sural nerve isolated, transected and sutured to the construct to encourage innervation. Aortic ring anchors were sutured to the tendons of the biceps femoris muscle so that the construct would move dynamically with the endogenous muscle. After 1 week in vivo, constructs were explanted, evaluated for force production, and stained for muscle, nerve, and collagen markers. Implanted muscle constructs showed a developing capillary system, an epimysium-like outer layer of connective tissue, and an increase in myofiber content. The beginning of alpha-bungarotoxin clustering suggests that neuromuscular junctions (NMJ) could form on the implanted muscle given more time in vivo. Additionally, the constructs increased maximum isometric force from 192±41μN to 549±103μN (245% increase) compared to in vitro controls that increased from 276±23μN to 329±27μN (25% increase). These findings suggest that engineered muscle tissue survives 1 week implantation and begins to develop the necessary interfaces needed to advance the phenotype toward adult muscle. However, in terms of force production, the muscle constructs need longer implantation times to fully develop an adult phenotype.
PMCID: PMC3355234  PMID: 22328229
tissue engineering; implantation; skeletal muscle; innervation; vascularization
19.  Cerebral blood flow and cerebrovascular reactivity at rest and during sub-maximal exercise: Effect of age and 12-week exercise training 
Age  2012;35(3):905-920.
Chronic reductions in cerebral blood flow (CBF) and cerebrovascular reactivity to CO2 are risk factors for cerebrovascular disease. Higher aerobic fitness is associated with higher CBF at any age; however, whether CBF or reactivity can be elevated following an exercise training intervention in healthy individuals is unknown. The aim of this study was to assess the effect of exercise training on CBF and cerebrovascular reactivity at rest and during exercise in young and older individuals. Ten young (23 ± 5 years; body mass index (BMI), 26 ± 3 kg m−2; \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$ {\mathop{V}\limits^{ \cdot }{_{\text{O2}}}}\max $$\end{document}, 35 ± 5 ml kg−1 min−1) and 10 older (63 ± 5 years; BMI, 25 ± 3.0 kg m−2; \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$ {\mathop{V}\limits^{ \cdot }{_{\text{O2}}}}\max $$\end{document}, 26 ± 4 ml kg-1 min−1) previously sedentary individuals breathed 5 % CO2 for 3 min at rest and during steady-state cycling exercise (30 and 70 % heart rate range (HRR)) prior to and following a 12-week aerobic exercise intervention. Effects of training on middle cerebral artery blood velocity (MCAv) at rest were unclear in both age groups. The absolute MCAv response to exercise was greater in the young (9 and 9 cm s−1 (30 and 70 % HRR, respectively) vs. 5 and 4 cm s−1 (older), P < 0.05) and was similar following training. Cerebrovascular reactivity was elevated following the 12-week training at rest (2.87 ± 0.76 vs. 2.54 ± 1.12 cm s−1 mm Hg−1, P = 0.01) and during exercise, irrespective of age. The finding of a training-induced elevation in cerebrovascular reactivity provides further support for exercise as a preventative tool in cerebrovascular and neurological disease with ageing.
PMCID: PMC3636405  PMID: 22669592
Ageing; Exercise training; Fitness; Cerebral blood flow; CO2 reactivity
20.  Biogeography and body size shuffling of aquatic salamander communities on a shifting refuge 
Freshwater habitats of coastal plains are refugia for many divergent vertebrate lineages, yet these environments are highly vulnerable to sea-level fluctuations, which suggest that resident communities have endured dynamic histories. Using the fossil record and a multi-locus nuclear phylogeny, we examine divergence times, biogeography, body size evolution and patterns of community assembly of aquatic salamanders from North American coastal plains since the Late Cretaceous. At least five salamander families occurred on the extensive Western Interior Coastal Plain (WICP), which existed from the Late Cretaceous through the Eocene. Four of these families subsequently colonized the emergent Southeastern Coastal Plain (SECP) by the Early Oligocene to Late Miocene. Three families ultimately survived and underwent extensive body size evolution in situ on the SECP. This included at least two major size reversals in recent taxa that are convergent with confamilial WICP ancestors. Dynamics of the coastal plain, major lineage extinctions and frequent extreme changes in body size have resulted in significant shuffling of the size structure of aquatic salamander communities on this shifting refuge since the Cretaceous.
PMCID: PMC3619470  PMID: 23466988
Amphibia; Caudata; coastal plain; body size evolution; community evolution; incumbency
21.  Class III correction using an inter-arch spring-loaded module 
Progress in Orthodontics  2014;15(1):32.
A retrospective study was conducted to determine the cephalometric changes in a group of Class III patients treated with the inter-arch spring-loaded module (CS2000®, Dynaflex, St. Ann, MO, USA).
Thirty Caucasian patients (15 males, 15 females) with an average pre-treatment age of 9.6 years were treated consecutively with this appliance and compared with a control group of subjects from the Bolton-Brush Study who were matched in age, gender, and craniofacial morphology to the treatment group. Lateral cephalograms were taken before treatment and after removal of the CS2000® appliance. The treatment effects of the CS2000® appliance were calculated by subtracting the changes due to growth (control group) from the treatment changes.
All patients were improved to a Class I dental arch relationship with a positive overjet. Significant sagittal, vertical, and angular changes were found between the pre- and post-treatment radiographs. With an average treatment time of 1.3 years, the maxillary base moved forward by 0.8 mm, while the mandibular base moved backward by 2.8 mm together with improvements in the ANB and Wits measurements. The maxillary incisor moved forward by 1.3 mm and the mandibular incisor moved forward by 1.0 mm. The maxillary molar moved forward by 1.0 mm while the mandibular molar moved backward by 0.6 mm. The average overjet correction was 3.9 mm and 92% of the correction was due to skeletal contribution and 8% was due to dental contribution. The average molar correction was 5.2 mm and 69% of the correction was due to skeletal contribution and 31% was due to dental contribution.
Mild to moderate Class III malocclusion can be corrected using the inter-arch spring-loaded appliance with minimal patient compliance. The overjet correction was contributed by forward movement of the maxilla, backward and downward movement of the mandible, and proclination of the maxillary incisors. The molar relationship was corrected by mesialization of the maxillary molars, distalization of the mandibular molars together with a rotation of the occlusal plane.
PMCID: PMC4047765  PMID: 24934153
22.  Impaired relaxation despite upregulated calcium-handling protein atrial myocardium from type 2 diabetic patients with preserved ejection fraction 
Diastolic dysfunction is a key factor in the development and pathology of cardiac dysfunction in diabetes, however the exact underlying mechanism remains unknown, especially in humans. We aimed to measure contraction, relaxation, expression of calcium-handling proteins and fibrosis in myocardium of diabetic patients with preserved systolic function.
Right atrial appendages from patients with type 2 diabetes mellitus (DM, n = 20) and non-diabetic patients (non-DM, n = 36), all with preserved ejection fraction and undergoing coronary artery bypass grafting (CABG), were collected. From appendages, small cardiac muscles, trabeculae, were isolated to measure basal and β-adrenergic stimulated myocardial function. Expression levels of calcium-handling proteins, sarcoplasmic reticulum Ca2+ ATPase (SERCA2a) and phospholamban (PLB), and of β1-adrenoreceptors were determined in tissue samples by Western blot. Collagen deposition was determined by picro-sirius red staining.
In trabeculae from diabetic samples, contractile function was preserved, but relaxation was prolonged (Tau: 74 ± 13 ms vs. 93 ± 16 ms, non-DM vs. DM, p = 0.03). The expression of SERCA2a was increased in diabetic myocardial tissue (0.75 ± 0.09 vs. 1.23 ± 0.15, non-DM vs. DM, p = 0.007), whereas its endogenous inhibitor PLB was reduced (2.21 ± 0.45 vs. 0.42 ± 0.11, non-DM vs. DM, p = 0.01). Collagen deposition was increased in diabetic samples. Moreover, trabeculae from diabetic patients were unresponsive to β-adrenergic stimulation, despite no change in β1-adrenoreceptor expression levels.
Human type 2 diabetic atrial myocardium showed increased fibrosis without systolic dysfunction but with impaired relaxation, especially during β-adrenergic challenge. Interestingly, changes in calcium-handling protein expression suggests accelerated active calcium re-uptake, thus improved relaxation, indicating a compensatory calcium-handling mechanism in diabetes in an attempt to maintain diastolic function at rest despite impaired relaxation in the diabetic fibrotic atrial myocardium. Our study addresses important aspects of the underlying mechanisms of diabetes-associated diastolic dysfunction, which is crucial to developing new therapeutic treatments.
PMCID: PMC3997226  PMID: 24708792
Diastolic dysfunction; Human myocardium; Type 2 diabetes; Relaxation; Contraction; Adrenergic regulation; Calcium signaling; Myocardial fibrosis
23.  The Epilepsy Phenome/Genome Project (EPGP) informatics platform 
The Epilepsy Phenome/Genome Project (EPGP) is a large-scale, multi-institutional, collaborative network of 27 epilepsy centers throughout the U.S., Australia, and Argentina, with the objective of collecting detailed phenotypic and genetic data on a large number of epilepsy participants. The goals of EPGP are (1) to perform detailed phenotyping on 3750 participants with specific forms of non-acquired epilepsy and 1500 parents without epilepsy, (2) to obtain DNA samples on these individuals, and (3) to ultimately genotype the samples in order to discover novel genes that cause epilepsy. To carry out the project, a reliable and robust informatics platform was needed for standardized electronic data collection and storage, data quality review, and phenotypic analysis involving cases from multiple sites.
EPGP developed its own suite of web-based informatics applications for participant tracking, electronic data collection (using electronic case report forms/surveys), data management, phenotypic data review and validation, specimen tracking, electroencephalograph and neuroimaging storage, and issue tracking. We implemented procedures to train and support end-users at each clinical site.
Thus far, 3780 study participants have been enrolled and 20,957 web-based study activities have been completed using this informatics platform. Over 95% of respondents to an end-user satisfaction survey felt that the informatics platform was successful almost always or most of the time.
The EPGP informatics platform has successfully and effectively allowed study management and efficient and reliable collection of phenotypic data. Our novel informatics platform met the requirements of a large, multicenter research project. The platform has had a high level of end-user acceptance by principal investigators and study coordinators, and can serve as a model for new tools to support future large scale, collaborative research projects collecting extensive phenotypic data.
PMCID: PMC3655424  PMID: 22579394
EPGP; Informatics; Epilepsy; Research; Internet; Electronic data collection; Phenotyping
24.  Rapid onset of cardiomyopathy in STZ-induced female diabetic mice involves the downregulation of pro-survival Pim-1 
Diabetic women are five times more likely to develop congestive heart failure compared with two fold for men. The underlying mechanism for this gender difference is not known. Here we investigate the molecular mechanisms responsible for this female disadvantage and attempt safeguarding cardiomyocytes viability and function through restoration of pro-survival Pim-1.
Methods and Results
Diabetes was induced by injection of streptozotocin in CD1 mice of both genders. Functional and dimensional parameters measurement using echocardiography revealed diastolic dysfunction in female diabetic mice within 8 weeks after STZ-induced diabetes. This was associated with significant downregulation of pro-survival Pim-1 and upregulation of pro-apoptotic Caspase-3, microRNA-1 and microRNA-208a. Male diabetic mice did not show any significant changes at this time point (P < 0.05 vs. female diabetic). Further, the onset of ventricular remodelling was quicker in female diabetic mice showing marked left ventricular dilation, reduced ejection fraction and poor contractility (P < 0.05 vs. male diabetic at 12 and 16 weeks of STZ-induced diabetes). Molecular analysis of samples from human diabetic hearts confirmed the results of pre-clinical studies, showing marked downregulation of Pim-1 in the female diabetic heart (P < 0.05 vs. male diabetic). Finally, in vitro restoration of Pim-1 reversed the female disadvantage in diabetic cardiomyocytes.
We provide novel insights into the molecular mechanisms behind the rapid onset of cardiomyopathy in female diabetics. These results suggest the requirement for the development of gender-specific treatments for diabetic cardiomyopathy.
PMCID: PMC4073808  PMID: 24685144
Diabetes; Cardiomyopathy; Gender difference; Cardiac dysfunction; Apoptosis
25.  Molecular programming of B cell memory 
Nature reviews. Immunology  2011;12(1):24-34.
The development of high-affinity B cell memory is regulated through three separable phases, each involving antigen recognition by specific B cells and cognate T helper cells. Initially, antigen-primed B cells require cognate T cell help to gain entry into the germinal centre pathway to memory. Once in the germinal centre, B cells with variant B cell receptors must access antigens and present them to germinal centre T helper cells to enter long-lived memory B cell compartments. Following antigen recall, memory B cells require T cell help to proliferate and differentiate into plasma cells. A recent surge of information — resulting from dynamic B cell imaging in vivo and the elucidation of T follicular helper cell programmes — has reshaped the conceptual landscape surrounding the generation of memory B cells. In this Review, we integrate this new information about each phase of antigen-specific B cell development to describe the newly unravelled molecular dynamics of memory B cell programming.
PMCID: PMC3947622  PMID: 22158414

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