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1.  An Integrated Approach to the Taxonomic Identification of Prehistoric Shell Ornaments 
PLoS ONE  2014;9(6):e99839.
Shell beads appear to have been one of the earliest examples of personal adornments. Marine shells identified far from the shore evidence long-distance transport and imply networks of exchange and negotiation. However, worked beads lose taxonomic clues to identification, and this may be compounded by taphonomic alteration. Consequently, the significance of this key early artefact may be underestimated. We report the use of bulk amino acid composition of the stable intra-crystalline proteins preserved in shell biominerals and the application of pattern recognition methods to a large dataset (777 samples) to demonstrate that taxonomic identification can be achieved at genus level. Amino acid analyses are fast (<2 hours per sample) and micro-destructive (sample size <2 mg). Their integration with non-destructive techniques provides a valuable and affordable tool, which can be used by archaeologists and museum curators to gain insight into early exploitation of natural resources by humans. Here we combine amino acid analyses, macro- and microstructural observations (by light microscopy and scanning electron microscopy) and Raman spectroscopy to try to identify the raw material used for beads discovered at the Early Bronze Age site of Great Cornard (UK). Our results show that at least two shell taxa were used and we hypothesise that these were sourced locally.
PMCID: PMC4061022  PMID: 24936797
2.  DUSP1 Is a Novel Target for Enhancing Pancreatic Cancer Cell Sensitivity to Gemcitabine 
PLoS ONE  2014;9(1):e84982.
Pancreatic ductal adenocarcinoma (PDAC) is a deadly cancer with a poor prognosis that is characterized by excessive mitogenic pathway activation and marked chemoresistance to a broad spectrum of chemotherapeutic drugs. Dual specificity protein phosphatase 1 (DUSP1) is a key negative regulator of mitogen activated protein kinases (MAPKs). Yet, DUSP1 is overexpressed in pancreatic cancer cells (PCCs) in PDAC where it paradoxically enhances colony formation in soft agar and promotes in vivo tumorigenicity. However, it is not known whether DUSP1 overexpression contributes to PDAC chemoresistance. Using BxPC3 and COLO-357 human PCCs, we show that gemcitabine activates c-JUN N-terminal kinase (JNK) and p38 mitogen activated protein kinase (p38 MAPK), key kinases in two major stress-activated signaling pathways. Gemcitabine-induced JNK and p38 MAPK activation mediates increased apoptosis, but also transcriptionally upregulates DUSP1, as evidenced by increased DUSP1 mRNA levels and RNA polymerase II loading at DUSP1 gene body. Conversely, shRNA-mediated inhibition of DUSP1 enhances JNK and p38 MAPK activation and gemcitabine chemosensitivity. Using doxycycline-inducible knockdown of DUSP1 in established orthotopic pancreatic tumors, we found that combining gemcitabine with DUSP1 inhibition improves animal survival, attenuates angiogenesis, and enhances apoptotic cell death, as compared with gemcitabine alone. Taken together, these results suggest that gemcitabine-mediated upregulation of DUSP1 contributes to a negative feedback loop that attenuates its beneficial actions on stress pathways and apoptosis, raising the possibility that targeting DUSP1 in PDAC may have the advantage of enhancing gemcitabine chemosensitivity while suppressing angiogenesis.
PMCID: PMC3883684  PMID: 24409315
3.  Genetic Background Alters the Severity and Onset of Neuromuscular Disease Caused by the Loss of Ubiquitin-Specific Protease 14 (Usp14) 
PLoS ONE  2013;8(12):e84042.
In this study, we identified and characterized an N-ethyl-N-nitrosourea (ENU) induced mutation in Usp14 (nmf375) that leads to adult-onset neurological disease. The nmf375 mutation causes aberrant splicing of Usp14 mRNA, resulting in a 95% reduction in USP14. We previously showed that loss of USP14 in ataxia (axJ) mice results in reduced ubiquitin levels, motor endplate disease, Purkinje cell axonal dystrophy and decreased hippocampal paired pulse facilitation (PPF) during the first 4-6 weeks of life, and early postnatal lethality by two months of age. Although the loss of USP14 is comparable between the nmf375 and axJ mice, the nmf375 mice did not exhibit these axJ developmental abnormalities. However, by 12 weeks of age the nmf375 mutants present with ubiquitin depletion and motor endplate disease, indicating a continual role for USP14-mediated regulation of ubiquitin pools and neuromuscular junction (NMJ) structure in adult mice. The observation that motor endplate disease was only seen after ubiquitin depletion suggests that the preservation of NMJ structure requires the stable maintenance of synaptic ubiquitin pools. Differences in genetic background were shown to affect ubiquitin expression and dramatically alter the phenotypes caused by USP14 deficiency.
PMCID: PMC3865287  PMID: 24358326
4.  Effects of First-Grade Number Knowledge Tutoring With Contrasting Forms of Practice 
The purpose of this study was to investigate the effects of 1st-grade number knowledge tutoring with contrasting forms of practice. Tutoring occurred 3 times per week for 16 weeks. In each 30-min session, the major emphasis (25 min) was number knowledge; the other 5 min provided practice in 1 of 2 forms. Nonspeeded practice reinforced relations and principles addressed in number knowledge tutoring. Speeded practice promoted quick responding and use of efficient counting procedures to generate many correct responses. At-risk students were randomly assigned to number knowledge tutoring with speeded practice (n = 195), number knowledge tutoring with nonspeeded practice (n = 190), and control (no tutoring, n = 206). Each tutoring condition produced stronger learning than control on all 4 mathematics outcomes. Speeded practice produced stronger learning than nonspeeded practice on arithmetic and 2-digit calculations, but effects were comparable on number knowledge and word problems. Effects of both practice conditions on arithmetic were partially mediated by increased reliance on retrieval, but only speeded practice helped at-risk children compensate for weak reasoning ability.
PMCID: PMC3779611  PMID: 24065865
mathematics; practice; fluency; arithmetic; word problems
5.  Aboriginal birth cohort (ABC): a prospective cohort study of early life determinants of adiposity and associated risk factors among Aboriginal people in Canada 
BMC Public Health  2013;13:608.
Aboriginal people living in Canada have a high prevalence of obesity, type 2 diabetes, and cardiovascular disease (CVD). To better understand the pre and postnatal influences on the development of adiposity and related cardio-metabolic factors in adult Aboriginal people, we will recruit and follow prospectively Aboriginal pregnant mothers and their children – the Aboriginal Birth Cohort (ABC) study.
We aim to recruit 300 Aboriginal pregnant mothers and their newborns from the Six Nations Reserve, and follow them prospectively to age 3 years. Key details of environment and health including maternal nutrition, glucose tolerance, physical activity, and weight gain will be collected. At birth, cord blood and placenta samples will be collected, as well as newborn anthropometric measurements. Mothers and offspring will be followed annually with serial measurements of diet and physical activity, growth trajectory, and adiposity.
There is an urgent need to understand maternal and child factors that underlie the early development of adiposity and type 2 diabetes in Aboriginal people. The information generated from this cohort will assist the Six Nations community in developing interventions to prevent early adiposity in Aboriginal children.
PMCID: PMC3702421  PMID: 23800270
Aboriginal; Birth cohort; Early origins; Adiposity
6.  Usp14 Deficiency Increases Tau Phosphorylation without Altering Tau Degradation or Causing Tau-Dependent Deficits 
PLoS ONE  2012;7(10):e47884.
Regulated protein degradation by the proteasome plays an essential role in the enhancement and suppression of signaling pathways in the nervous system. Proteasome-associated factors are pivotal in ensuring appropriate protein degradation, and we have previously demonstrated that alterations in one of these factors, the proteasomal deubiquitinating enzyme ubiquitin-specific protease 14 (Usp14), can lead to proteasome dysfunction and neurological disease. Recent studies in cell culture have shown that Usp14 can also stabilize the expression of over-expressed, disease-associated proteins such as tau and ataxin-3. Using Usp14-deficient axJ mice, we investigated if loss of Usp14 results in decreased levels of endogenous tau and ataxin-3 in the nervous system of mice. Although loss of Usp14 did not alter the overall neuronal levels of tau and ataxin-3, we found increased levels of phosphorylated tau that correlated with the onset of axonal varicosities in the Usp14-deficient mice. These changes in tau phosphorylation were accompanied by increased levels of activated phospho-Akt, phosphorylated MAPKs, and inactivated phospho-GSK3β. However, genetic ablation of tau did not alter any of the neurological deficits in the Usp14-deficient mice, demonstrating that increased levels of phosphorylated tau do not necessarily lead to neurological disease. Due to the widespread activation of intracellular signaling pathways induced by the loss of Usp14, a better understanding of the cellular pathways regulated by the proteasome is required before effective proteasomal-based therapies can be used to treat chronic neurological diseases.
PMCID: PMC3483306  PMID: 23144711
7.  Ubiquitin homeostasis is critical for synaptic development and function 
The Journal of Neuroscience  2011;31(48):17505-17513.
The ubiquitin-proteasome system (UPS) controls protein abundance and is essential for many aspects of neuronal function. In ataxia (axJ) mice, profound neurological and synaptic defects result from a loss-of-function mutation in the proteasome-associated deubiquitinating enzyme Usp14, which is required for recycling ubiquitin from proteasomal substrates. Here, we show that transgenic complementation of axJ mice with neuronally-expressed ubiquitin prevents early postnatal lethality, restores muscle mass, and corrects developmental and functional deficits resulting from the loss of Usp14, demonstrating that ubiquitin deficiency is a major cause of the neurological defects observed in the axJ mice. We also show that proteasome components are normally induced during the first two weeks of postnatal development, which coincides with dramatic alterations in polyubiquitin chain formation. These data demonstrate a critical role for ubiquitin homeostasis in synaptic development and function, and show that ubiquitin deficiency may contribute to diseases characterized by synaptic dysfunction.
PMCID: PMC3253363  PMID: 22131412
8.  Acute Pharmacological Modulation of mGluR8 reduces measures of anxiety 
Behavioural brain research  2010;212(2):168-173.
Metabotropic glutamate receptors (mGluRs), which are coupled to second messenger pathways via G proteins, modulate glutamatergic and GABAergic neurotransmission. Because of their role in modulating neurotransmission, mGluRs are attractive therapeutic targets for anxiety disorders. Previously we showed that mGluR8−/− male mice showed higher measures of anxiety in the open field and elevated plus maze than age-matched wild-type mice. In this study, we assessed the potential effects of acute pharmacological modulation of mGluR8 on measures of avoidable and unavoidable anxiety. In addition to wild-type mice, we also tested apolipoprotein E-deficient (Apoe−/−) mice, as these mice show increased levels of anxiety-like behaviors and therefore might show an altered sensitivity to mGluR8 stimulation. mGluR8 stimulation with the specific agonist DCPG, or modulation with AZ12216052, a new, positive allosteric modulator of mGluR8 reduced measures of anxiety in both wild-type mice. The effects of mGluR8 positive allosteric modulators, which only affect neurotransmission in the presence of extracellular glutamate, seem particularly promising for patients with anxiety disorders showing benzodiazepine insensitivity.
PMCID: PMC2892883  PMID: 20385173
metabotropic; group-III mGluR; allosteric modulator; behavior
9.  The proteasome-associated deubiquitinating enzyme Usp14 is essential for the maintenance of synaptic ubiquitin levels and the development of neuromuscular junctions 
Dysfunction of the ubiquitin proteasome system (UPS) has been implicated in the pathogenesis of many neurological diseases, including Alzheimer's, spinocerebellar ataxia, and several motor neuron diseases. Recent research indicates that changes in synaptic transmission may play a critical role in the progression of neurological disease; however, the mechanisms by which the UPS regulates synaptic structure and function have not been well characterized. In this report, we show that Usp14 is indispensable for synaptic development and function at neuromuscular junctions (NMJs). Usp14-deficient axJ mice display a resting tremor, a reduction in muscle mass, and notable hind-limb rigidity without any detectable loss of motor neurons. Instead, loss of Usp14 causes developmental defects at motor neuron endplates. Presynaptic defects include phosphorylated neurofilament accumulations, nerve terminal sprouting and poor arborization of the motor nerve terminals, while postsynaptic acetylcholine receptors display immature plaque-like morphology. These structural changes in the NMJ correlated with ubiquitin loss in the spinal cord and sciatic nerve. Further studies demonstrated that the greatest loss of ubiquitin was found in synaptosomal fractions, suggesting that the endplate swellings may be caused by decreased protein turnover at the synapse. Transgenic restoration of Usp14 in the nervous system corrected the levels of monomeric ubiquitin in the motor neuron circuit and the defects that were observed in the motor endplates and muscles of the axJ mice. These data define a critical role for Usp14 at mammalian synapses and suggest a requirement for local ubiquitin recycling by the proteasome to control the development and function of NMJs.
PMCID: PMC2766780  PMID: 19726649
proteasome; ubiquitin; neuromuscular junction; Usp14; synapse; Motoneuron
10.  Induction of Hematopoietic Differentiation of Mouse Embryonic Stem Cells by an AGM-Derived Stromal Cell Line is Not Further Enhanced by Overexpression of HOXB4 
Stem Cells and Development  2010;19(11):1687-1698.
Hematopoietic differentiation of embryonic stem (ES) cells can be enhanced by co-culture with stromal cells derived from hematopoietic tissues and by overexpression of the transcription factor HOXB4. In this study, we compare the hematopoietic inductive effects of stromal cell lines derived from different subregions of the embryonic aorta-gonad-mesonephros tissue with the commonly used OP9 stromal cell line and with HOXB4 activation. We show that stromal cell lines derived from the aorta and surrounding mesenchyme (AM) act at an earlier stage of the differentiation process compared with the commonly used OP9 stromal cells. AM stromal cells were able to promote the further differentiation of isolated brachyury-GFP+ mesodermal cells into hematopoietic progenitors, whereas the OP9 stromal cells could not support the differentiation of these cells. Co-culture and analyses of individual embryoid bodies support the hypothesis that the AM stromal cell lines could enhance the de novo production of hematopoietic progenitors, lending support to the idea that AM stromal cells might act on prehematopoietic mesoderm. The induction level observed for AM stromal cells was comparable to HOXB4 activation, but no additive effect was observed when these 2 inductive strategies were combined. Addition of a γ-secretase inhibitor reduced the inductive effects of both the stromal cell line and HOXB4, providing clues to possible shared molecular mechanisms.
PMCID: PMC3128308  PMID: 20184433
11.  Transgenic rescue of ataxia mice reveals a male-specific sterility defect 
Developmental biology  2008;325(1):33-42.
Homozygous ataxia (axJ) mice have reduced expression of ubiquitin-specific protease 14 (Usp14), resulting in severe neuromuscular defects and death by 2 months of age. Transgenic expression of Usp14 exclusively in the nervous system of axJ mice (axJ-Tg) prevents early lethality and restores motor system function to the axJ mice, enabling an analysis of the reproductive capabilities of Usp14-deficient mice. Although female axJ-Tg mice had a 75% reduction of Usp14 in the ovaries, they were able to produce normal litters. Ovary transfer experiments also demonstrated that the ovaries of axJ mice were capable of producing viable pups. In contrast, male axJ and axJ-Tg mice displayed a 50% reduction in testicular Usp14 levels and were infertile, indicating that Usp14 is required for development and function of the male reproductive system. Immunohistochemistry experiments showed that Usp14 is found in the redundant nuclear envelope and cytoplasmic droplet of epididymal spermatozoa. Analysis of axJ testes demonstrated a 50% reduction in testis weight, a 100-fold reduction in sperm number and the presence of abnormal spermatozoa in the epididymis. Histological examination of the Usp14-deficient testes revealed abnormal spermatogenesis and the presence of degenerating germ cells, indicating that Usp14 and the ubiquitin proteasome system are required for spermatid differentiation during spermiogenesis.
PMCID: PMC2651617  PMID: 18926813
ubiquitin; proteasome; testis; Usp14; fertility
12.  Protective Cytotoxic T-Cell Responses Induced by Venezuelan Equine Encephalitis Virus Replicons Expressing Ebola Virus Proteins 
Journal of Virology  2005;79(22):14189-14196.
Infection with Ebola virus causes a severe disease accompanied by high mortality rates, and there are no licensed vaccines or therapies available for human use. Filovirus vaccine research efforts still need to determine the roles of humoral and cell-mediated immune responses in protection from Ebola virus infection. Previous studies indicated that exposure to Ebola virus proteins expressed from packaged Venezuelan equine encephalitis virus replicons elicited protective immunity in mice and that antibody-mediated protection could only be demonstrated after vaccination against the glycoprotein. In this study, the murine CD8+ T-cell responses to six Ebola virus proteins were examined. CD8+ T cells specific for Ebola virus glycoprotein, nucleoprotein, and viral proteins (VP24, VP30, VP35, and VP40) were identified by intracellular cytokine assays using splenocytes from vaccinated mice. The cells were expanded by restimulation with peptides and demonstrated cytolytic activity. Adoptive transfer of the CD8+ cytotoxic T cells protected filovirus naïve mice from challenge with Ebola virus. These data support a role for CD8+ cytotoxic T cells as part of a protective mechanism induced by vaccination against six Ebola virus proteins and provide additional evidence that cytotoxic T-cell responses can contribute to protection from filovirus infections.
PMCID: PMC1280180  PMID: 16254354
13.  Protection from Ebola Virus Mediated by Cytotoxic T Lymphocytes Specific for the Viral Nucleoprotein 
Journal of Virology  2001;75(6):2660-2664.
Cytotoxic T lymphocytes (CTLs) are proposed to be critical for protection from intracellular pathogens such as Ebola virus. However, there have been no demonstrations that protection against Ebola virus is mediated by Ebola virus-specific CTLs. Here, we report that C57BL/6 mice vaccinated with Venezuelan equine encephalitis virus replicons encoding the Ebola virus nucleoprotein (NP) survived lethal challenge with Ebola virus. Vaccination induced both antibodies to the NP and a major histocompatibility complex class I-restricted CTL response to an 11-amino-acid sequence in the amino-terminal portion of the Ebola virus NP. Passive transfer of polyclonal NP-specific antiserum did not protect recipient mice. In contrast, adoptive transfer of CTLs specific for the Ebola virus NP protected unvaccinated mice from lethal Ebola virus challenge. The protective CTLs were CD8+, restricted to the Db class I molecule, and recognized an epitope within amino acids 43 to 53 (VYQVNNLEEIC) in the Ebola virus NP. The demonstration that CTLs can prevent lethal Ebola virus infection affects vaccine development in that protective cellular immune responses may be required for optimal protection from Ebola virus.
PMCID: PMC115890  PMID: 11222689
14.  The Epstein-Barr Virus Immediate-Early Gene Product, BRLF1, Interacts with the Retinoblastoma Protein during the Viral Lytic Cycle 
Journal of Virology  1998;72(10):8043-8051.
Retinoblastoma protein (Rb) is a key regulator of cellular proliferation, controlling entry into G1/S in the cell cycle, largely through its action in binding the cellular transcription factor E2F, which activates genes important in DNA synthesis. Small DNA tumor viruses encode gene products which can functionally inactivate Rb, promoting cellular proliferation and viral DNA synthesis. In this study, the Epstein-Barr virus (EBV) immediate-early lytic gene product, BRLF1 (R), is shown to bind Rb in vivo, shortly after induction of the viral lytic cycle in EBV-infected Akata cells. Furthermore, the temporal kinetics of R-Rb interaction correlate with displacement of E2F1 from Rb. Mapping of the domains required for the interaction of R and Rb proteins reveals that R binds specifically to the N terminus of Rb, outside the Rb pocket, and that the first 200 amino acids of R are required for this interaction. The interaction of R and Rb may initiate cell cycle progression and facilitate viral DNA synthesis during lytic replication.
PMCID: PMC110141  PMID: 9733844

Results 1-14 (14)