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1.  Role of Ubiquitin Ligases and the Proteasome in Oncogenesis: Novel Targets for Anticancer Therapies 
Journal of Clinical Oncology  2013;31(9):1231-1238.
The ubiquitin proteasome system (UPS) regulates the ubiquitination, and thus degradation and turnover, of many proteins vital to cellular regulation and function. The UPS comprises a sequential series of enzymatic processes using four key enzyme families: E1 (ubiquitin-activating enzymes), E2 (ubiquitin-carrier proteins), E3 (ubiquitin-protein ligases), and E4 (ubiquitin chain assembly factors). Because the UPS is a crucial regulator of the cell cycle, and abnormal cell-cycle control can lead to oncogenesis, aberrancies within the UPS pathway can result in a malignant cellular phenotype and thus has become an attractive target for novel anticancer agents. This article will provide an overall review of the mechanics of the UPS, describe aberrancies leading to cancer, and give an overview of current drug therapies selectively targeting the UPS.
doi:10.1200/JCO.2012.44.0958
PMCID: PMC3807137  PMID: 23358974
2.  Artificial Neural Network Based Analysis of High Throughput Screening Data for Improved Prediction of Active Compounds 
Journal of biomolecular screening  2009;14(10):1236-1244.
Artificial Neural Networks (ANNs) are trained using High Throughput Screening (HTS) data to recover active compounds from a large data set. Improved classification performance was obtained on combining predictions made by multiple ANNs. The HTS data, acquired from a Methionine Aminopeptidases Inhibition study, consisted of a library of 43,347 compounds, and the ratio of active to non-active compounds, RA/N, was 0.0321. Back-propagation ANNs were trained and validated using Principal Components derived from the physico-chemical features of the compounds. On selecting the training parameters carefully, an ANN recovers one-third of all active compounds from the validation set with a three-fold gain in RA/N value. Further gains in RA/N values were obtained upon combining the predictions made by a number of ANNs. The generalization property of the back-propagation ANNs was utilized to train those ANNs with the same training samples, after being initialized with different sets of random weights. As a result, only 10% of all available compounds were needed for training and validation, and the rest of the data set was screened with more than a ten-fold gain of the original RA/N value. Thus, ANNs trained with limited HTS data might become useful in recovering active compounds from large data sets.
doi:10.1177/1087057109351312
PMCID: PMC4080791  PMID: 19940083
pattern classification; neural networks; generalization property
3.  Nerve-Langerhans cell interactions in diabetes and aging 
Histology and histopathology  2012;27(12):1589-1598.
Summary
Cutaneous infections are a leading cause of hospitalization of diabetic patients. Langerhans cells (LCs) are antigen-presenting cutaneous dendritic cells that protect against infections, and effects of diabetes and aging on these cells are unclear. We examined LCs in footpads of rats with streptozotocin-induced diabetes at 3 months of age following 4 weeks of diabetes, and at 6 months following 16 weeks of diabetes. Immunostaining of LCs using the selective marker protein langerin showed cutaneous LC composition increased between 3 and 6 months of age owing to increased LC numbers and size in control rats. In diabetic rats, LC numbers increased with age but, unlike 6 month old controls, cell size did not, suggesting that diabetes impairs the increase in cell size that is a hallmark of LC maturation. Diabetes reduced LC numbers after 4 weeks and numbers and sizes following 16 weeks. We examined the relation between LC and innervation and found that, while axon density decreased with aging, it was not affected by 16 weeks of diabetes. However, LCs expressing the neuronal marker PGP9.5 represented a source of error in axonal counts. These findings support the hypothesis that diabetes substantially impacts LC proliferation and maturation independent of effects on cutaneous innervation. Accordingly, the interactions of diabetes and aging on LCs may be important factors in predisposing diabetic patients to cutaneous ulcers and infections.
PMCID: PMC3714397  PMID: 23059889
Langerhans cell; Diabetes; Intraepidermal nerve fibers; Aging; Langerin
4.  Adaptive Plasticity of Vaginal Innervation in Term Pregnant Rats 
Reproductive Sciences  2011;18(12):1237-1245.
Changes in reproductive status place varied functional demands on the vagina. These include receptivity to male intromission and sperm transport in estrus, barrier functions during early pregnancy, and providing a conduit for fetal passage at parturition. Peripheral innervation regulates vaginal function, which in turn may be influenced by circulating reproductive hormones. We assessed vaginal innervation in diestrus and estrus (before and after the estrous cycle surge in estrogen), and in the early (low estrogen) and late (high estrogen) stages in pregnancy. In vaginal sections from cycling rats, axons immunoreactive for the pan-neuronal marker protein gene product 9.5 (PGP 9.5) showed a small reduction at estrus relative to diestrus, but this difference did not persist after correcting for changes in target size. No changes were detected in axons immunoreactive for tyrosine hydroxylase (sympathetic), vesicular acetylcholine transporter (parasympathetic), or calcitonin gene-related peptide and transient receptor potential vanilloid type 1 (TRPV-1; sensory nociceptors). In rats at 10 days of pregnancy, innervation was similar to that observed in cycling rats. However, at 21 days of pregnancy, axons immunoreactive for PGP 9.5 and each of the subpopulation-selective markers were significantly reduced both when expressed as percentage of sectional area or after correcting for changes in target size. Because peripheral nerves regulate vaginal smooth muscle tone, blood flow, and pain sensitivity, reductions in innervation may represent important adaptive mechanisms facilitating parturition.
doi:10.1177/1933719111410706
PMCID: PMC3343098  PMID: 21666101
autonomic innervation; estrous cycle; parturition; sensory innervation
5.  Guidance on the evaluation of Plasmodium vivax vaccines in populations exposed to natural infection 
Vaccine  2009;27(41):10.1016/j.vaccine.2009.07.018.
In this paper we give guidance for the design and conduct of vaccine trials against Plasmodium vivax malaria. The paper supplements earlier guidelines on the planning of vaccine trials against Plasmodium falciparum malaria [WHO. Guidelines for the evaluation of Plasmodium falciparum vaccines in populations exposed to natural infections. Geneva: World Health Organization; 1997, http://www.who.int/vaccine_research/feuill_1_4-2.pdf], with further considerations in two later documents [Moorthy VS, Reed Z, Smith PG. Measurement of malaria vaccine efficacy in phase III trials: report of a WHO consultation. Vaccine 2007 July 9;25(28):5115-23; Moorthy V, Reed Z, Smith P. MALVAC 2008: measures of efficacy of malaria vaccines in phase 2b and phase 3 trials – scientific, regulatory and public health perspectives. Vaccine 2009 January 29;27(5):624-8]. We deal specifically with study design and methodological issues for the assessment of pre-erythrocytic and blood-stage vaccines against P. vivax. The role of vaccines in blocking transmission of P. vivax is not considered as the methodological issues are similar to those for P. falciparum, though longer follow-up would be required because of the potential for relapse discussed below. In this paper we discuss the rationale and background to trials of P. vivax vaccines, requirements for Phase IIb and Phase III field trials, implementation of clinical trials, methods of measurement and analysis, and ethical aspects.
doi:10.1016/j.vaccine.2009.07.018
PMCID: PMC3844894  PMID: 19638270
Plasmodium vivax malaria; Vaccine
6.  Vitamin D Receptor and Enzyme Expression in Dorsal Root Ganglia of Adult Female Rats: Modulation by Ovarian Hormones 
Vitamin D insufficiency impacts sensory processes including pain and proprioception, but little is known regarding vitamin D signaling in adult sensory neurons. We analyzed female rat dorsal root ganglia (DRG) for vitamin receptor (VDR) and the vitamin D metabolizing enzymes CYP27B1 and CYP24. Western blots and immunofluorescence revealed the presence of these proteins in sensory neurons. Nuclear VDR immunoreactivity was present within nearly all neurons, while cytoplasmic VDR was found preferentially in unmyelinated calcitonin gene-related peptide (CGRP)-positive neurons, colocalizing with CYP27B1 and CYP24. These data suggest that 1,25(OH)2D3 may affect sensory neurons through nuclear or extranuclear signaling pathways. In addition, local vitamin D metabolite concentrations in unmyelinated sensory neurons may be controlled through expression of CYP27B1 and CYP24. Because vitamin D deficiency appears to exacerbate some peri-menopausal pain syndromes, we assessed the effect of ovariectomy on vitamin D-related proteins. Two weeks following ovariectomy, total VDR expression in DRG dropped significantly, owing to a slight decrease in the percentage of total neurons expressing nuclear VDR and a large drop in unmyelinated CGRP-positive neurons expressing cytoplasmic VDR. Total CYP27B1 expression dropped significantly, predominantly due to decreased expression within unmyelinated CGRP-positive neurons. CYP24 expression remained unchanged. Therefore, unmyelinated CGRP-positive neurons appear to have a distinct vitamin D phenotype with hormonally-regulated ligand and receptor levels. These findings imply that vitamin D signaling may play a specialized role in a neural cell population that is primarily nociceptive.
doi:10.1016/j.jchemneu.2010.10.001
PMCID: PMC3005085  PMID: 20969950
1,25-dihydroxyvitamin D3; reproductive hormones; pain; sensory; CYP27B1; CYP24
7.  Molecular and cellular effects of NEDD8 activating enzyme (NAE) inhibition in myeloma 
Molecular cancer therapeutics  2012;11(4):942-951.
The NEDD8 activating enzyme (NAE) is upstream of the 20S proteasome in the ubiquitin/proteasome pathway and catalyzes the first step in the neddylation pathway. NEDD8 modification of cullins is required for ubiquitination of cullin-ring ligases (CRLs), which regulate degradation of a distinct subset of proteins. The more targeted impact of NAE on protein degradation prompted us to study MLN4924, an investigational NAE inhibitor, in preclinical multiple myeloma (MM) models. In vitro treatment with MLN4924 led to dose-dependent decrease of viability (EC50=25–150nM) in a panel of human MM cell lines. MLN4924 was similarly active against a bortezomib-resistant ANBL-6 subline and its bortezomib-sensitive parental cells. MLN4924 had sub-μM activity (EC50 values <500nM) against primary CD138+ MM patient cells and exhibited at least additive effect when combined with dexamethasone, doxorubicin and bortezomib against MM.1S cells. The bortezomib-induced compensatory up-regulation of transcripts for ubiquitin/proteasome was not observed with MLN4924 treatment, suggesting distinct functional roles of NAE vs 20S proteasome. MLN4924 was well tolerated at doses up to 60mg/kg 2x daily and significantly reduced tumor burden in both a subcutaneous and an orthotopic mouse model of MM. These studies provide the framework for the clinical investigation of MLN4924 in MM.
doi:10.1158/1535-7163.MCT-11-0563
PMCID: PMC3755358  PMID: 22246439
myeloma; cancer; NEDD8; NAE; microenvironment
8.  Bone Morphogenetic Protein 4 Mediates Estrogen-Regulated Sensory Axon Plasticity in the Adult Female Reproductive Tract 
Peripheral axons are structurally plastic even in the adult, and altered axon density is implicated in many disorders and pain syndromes. However, mechanisms responsible for peripheral axon remodeling are poorly understood. Physiological plasticity is characteristic of the female reproductive tract: vaginal sensory innervation density is low under high estrogen conditions such as term pregnancy, while density is high in low-estrogen conditions such as menopause. We exploited this system in rats to identify factors responsible for adult peripheral neuroplasticity. Calcitonin gene-related peptide-immunoreactive sensory innervation is distributed primarily within the vaginal submucosa. Submucosal smooth muscle cells express Bone Morphogenetic Protein 4 (BMP4). With low estrogen, BMP4 expression was elevated indicating negative regulation by this hormone. Vaginal smooth muscle cells induced robust neurite outgrowth by co-cultured dorsal root ganglion neurons, which was prevented by neutralizing BMP4 with noggin or anti-BMP4. Estrogen also prevented axon outgrowth, and this was reversed by exogenous BMP4. Nuclear accumulation of phosphorylated Smad1, a primary transcription factor for BMP4 signaling, was high in vagina-projecting sensory neurons following ovariectomy, and reduced by estrogen. BMP4 regulation of innervation was confirmed in vivo using lentiviral transduction to overexpress BMP4 in an estrogen-independent manner. Submucosal regions with high virally induced BMP4 expression had high innervation density despite elevated estrogen. These findings show that BMP4, an important factor in early nervous system development and regeneration after injury, is a critical mediator of adult physiological plasticity as well. Altered BMP4 expression may therefore contribute to sensory hyperinnervation, a hallmark of several pain disorders including vulvodynia.
doi:10.1523/JNEUROSCI.1704-12.2013
PMCID: PMC3581866  PMID: 23325243
9.  β-adrenoceptor blockers increase cardiac sympathetic innervation by inhibiting autoreceptor suppression of axon growth 
Beta-adrenoceptor antagonists are used widely to reduce cardiovascular sympathetic tone, but withdrawal is accompanied by sympathetic hyperactivity. Receptor supersensitivity accounts for some but not all aspects of this withdrawal syndrome. Therefore, we investigated effects of β-blockers on sympathetic innervation. Rats received infusions of adrenergic receptor blockers or saline for one week. The nonselective β-blocker propranolol and the β1-antagonist metoprolol both increased myocardial sympathetic axon density. At 2 days following propranolol discontinuation, β-receptor sensitivity and responsiveness to isoproterenol were similar to controls. However, tyramine-induced mobilization of norepinephrine stores produced elevated ventricular contractility consistent with enhanced sympathetic neuroeffector properties. In addition, rats undergoing discontinuation showed exaggerated increases in mean arterial pressure in response to air puff or noise startle. In sympathetic neuronal cell cultures, both propranolol and metoprolol increased axon outgrowth but the β2-blocker ICI 118,551did not. Norepinephrine synthesis suppression by α-methyl p-tyrosine also increased sprouting and concurrent dobutamine administration reduced it, confirming that locally synthesized norepinephrine inhibits outgrowth via β1 adrenoceptors. Immunohistochemistry revealed β1 adrenoceptor protein on sympathetic axon terminations. In rats with coronary artery ligation, propranolol reversed heart failure-induced ventricular myocardial sympathetic axon depletion, but did not affect infarct-associated sympathetic hyperinnervation. We conclude that sympathetic neurons possess β1 autoreceptors that negatively regulate axon outgrowth. Chronic β-adrenoceptor blockade disrupts this feedback system, leading to ventricular sympathetic axon proliferation and increased neuroeffector gain, which are likely to contribute to β-blocker withdrawal syndrome.
doi:10.1523/JNEUROSCI.1667-10.2010
PMCID: PMC2952276  PMID: 20844139
Beta-blocker; outgrowth; adrenergic receptor; cardiovascular; autoreceptors; hyperinnervation
10.  Systemic and Topical Hormone Therapies Reduce Vaginal Innervation Density in Post-Menopausal Women 
Menopause (New York, N.y.)  2012;19(6):630-635.
Objective
Menopause is often accompanied by vaginal discomfort including burning, itching, dryness and spontaneous or provoked pain. While direct effects of estrogen withdrawal on vaginal cells are implicated, surgical menopause in rodents causes autonomic and sensory nerves to proliferate, suggesting that indirect effects mediated by changes in vaginal innervation may contribute. We assessed whether post-menopausal women display hormone-dependent changes in vaginal innervation.
Methods
Vaginal biopsies from 20 postmenopausal women undergoing surgery for stress urinary incontinence and pelvic organ prolapse were fixed and immunostained for the pan-neuronal marker, PGP9.5, the sympathetic marker tyrosine hydroxylase, the parasympathetic marker vasoactive intestinal polypeptide, and the sensory nociceptor marker calcitonin gene-related peptide. Innervation density was measured as apparent percentage of section area occupied by immunofluorescent axons. Specimens were grouped according to whether participants received systemic hormone therapy (HT), topical (vaginal) HT, or no HT.
Results
Women not receiving HT showed relatively high levels of total innervation, with most axons expressing tyrosine hydroxylase or vasoactive intestinal polypeptide immunoreactivity. In patients receiving systemic HT, overall innervation was reduced, as were presumptive parasympathetic, sympathetic and sensory axon populations. Topical HT elicited more dramatic reductions in innervation than systemic HT.
Conclusions
Hormone therapy reduces autonomic and sensory vaginal innervation density, which may in part contribute to relief from vaginal discomfort. Moreover, topical therapy is more effective than systemic therapy, which may help explain the greater improvement reported with topical as compared to systemic HT.
doi:10.1097/gme.0b013e31823b8983
PMCID: PMC3319450  PMID: 22205148
Estrogen; menopause; vagina; autonomic; sensory; dyspareunia
11.  Estrogen Promotes Sympathetic Nerve Regeneration in the Rat Proximal Urethra 
Urology  2009;73(6):1392-1396.
Objectives:
Sympathetic innervation mediates tonic contraction of proximal urethral smooth muscle, thus contributing to urinary continence. Urethral innervation is particularly susceptible to damage during vaginal delivery, a time characterized by decreasing estrogen levels. Because regeneration of other nerves types can be influenced by estrogen, the present study was conducted to assess whether sympathetic reinnervation of the rat proximal urethra is affected by differences in estrogen levels.
Methods:
Adult female rats were ovariectomized and implanted with pellets containing vehicle or estrogen to achieve serum levels similar to rodent pregnancy. Rats were injected intravenously with vehicle or the selective sympathetic neurotoxin 6-hydroxydopamine, which produces uniform and complete destruction of terminal sympathetic axons. At 1, 4, 12 and 25 days, tyrosine hydroxylase-immunoreactive sympathetic innervation of the proximal urethral smooth muscle was assessed quantitatively.
Results:
In rats with intact innervation, the proximal urethra is densely innervated, and nerve density is comparable irrespective of estrogen status. 6-Hydroxydopamine induced marked sympathetic axon disruption by 1 day and complete denervation by 4 days post-injection in ovariectomized rats receiving vehicle or estrogen. In vehicle-treated rats, few nerves were present at 12 days post-sympathectomy, and innervation remained substantially below normal levels at 25 days. In estrogen-treated rats, sympathetic reinnervation was 2-fold greater at 12 days, and by 25 days was comparable to controls.
Conclusion:
Estrogen improves sympathetic reinnervation of the proximal urethra. Estrogen titers in individuals with urethral sympathetic nerve damage may therefore influence the rate and extent of urethral smooth muscle reinnervation.
doi:10.1016/j.urology.2008.11.052
PMCID: PMC2734410  PMID: 19362354
Noradrenergic; urethral sphincter; regeneration; 17β-estradiol
12.  Inhibition of the Nedd8 system sensitizes cells to DNA Inter-strand crosslinking agents 
Molecular Cancer Research  2012;10(3):369-377.
The Fanconi Anemia (FA) pathway is required for repair of DNA interstrand crosslinks (ICLs). FA pathway-deficient cells are hypersensitive to DNA ICL-inducing drugs such as Cisplatin. Conversely, hyperactivation of the FA pathway is a mechanism that may underlie cellular resistance to DNA ICL agents. Modulating FANCD2 monoubiquitination, a key step in the FA pathway, may be an effective therapeutic approach to conferring cellular sensitivity to ICL agents. Here, we show that inhibition of the Nedd8 conjugation system increases cellular sensitivity to DNA ICL-inducing agents. Mechanistically, the Nedd8 inhibition, either by siRNA-mediated knockdown of Nedd8 conjugating enzymes or treatment with a Nedd8 activating enzyme inhibitor MLN4924, suppressed DNA damage-induced FANCD2 monoubiquitination and CHK1 phosphorylation. Our data indicate that inhibition of the FA pathway is largely responsible for the heightened cellular sensitivity to DNA ICLs upon Nedd8 inhibition. These results suggest that a combination of Nedd8 inhibition with ICL-inducing agents may be an effective strategy for sensitizing a subset of drug-resistant cancer cells.
doi:10.1158/1541-7786.MCR-11-0497
PMCID: PMC3307881  PMID: 22219386
Fanconi Anemia; Nedd8; Chemosensitization
13.  Radiosensitization of human pancreatic cancer cells by MLN4924, an investigational NEDD8-activating enzyme inhibitor 
Cancer research  2011;72(1):282-293.
Radiotherapy is used in locally advanced pancreatic cancers where it can improve survival in combination with gemcitabine. However, prognosis is still poor in this setting where more effective therapies remain needed. MLN4924 is an investigational small molecule currently in Phase I clinical trials. MLN4924 inhibits NAE (NEDD8 Activating Enzyme), a pivotal regulator of the E3 ubiquitin ligase SCF (SKP1, Cullins, and F-box protein), that has been implicated recently in DNA repair. In this study, we provide evidence that MLN4924 can be used as an effective radiosensitizer in pancreatic cancer. Specifically, MLN4924 (20–100 nM) effectively inhibited cullin neddylation and sensitized pancreatic cancer cells to ionizing radiation in vitro with a sensitivity enhancement ratio (SER) of ~1.5. Mechanistically, MLN4924 treatment stimulated an accumulation of several SCF substrates, including CDT1, WEE1 and NOXA, in parallel with an enhancement of radiation-induced DNA damage, aneuploidy, G2/M phase cell cycle arrest and apoptosis. RNAi-mediated knockdown of CDT1 and WEE1 partially abrogated MLN4924-induced aneuploidy, G2/M arrest, and radiosensization, indicating a causal effect. Further, MLN4924 was an effective radiosensitizer in mouse xenograft models of human pancreatic cancer. Our findings offer proof of concept for use of MLN4924 as a novel class of radiosensitizer for the treatment of pancreatic cancer.
doi:10.1158/0008-5472.CAN-11-2866
PMCID: PMC3251739  PMID: 22072567
NAE inhibitor; MLN4924; CRL/SCF E3 ubiquitin ligase; radiosensitization; DNA damage; pancreatic cancer cells
14.  Hypersensitivity and Hyperinnervation of the Rat Hind Paw Following Carrageenan-induced Inflammation 
Neuroscience letters  2011;495(1):67-71.
Studies of human tissue show that many chronic pain syndromes are accompanied by abnormal increases in numbers of peripheral sensory nerve fibers. It is not known if sensory nerve sprouting occurs as a result of inflammation present in these conditions, or other factors such as infection or extensive tissue damage. In the present study, we used a well established model of inflammation to examine cutaneous innervation density in relation to mechanical and thermal hypersensitivity. Adult female rats were ovariectomized to eliminate fluctuations in female reproductive hormones and one week later, a hind paw was injected with carrageenan or saline vehicle. Behavioral testing showed that saline vehicle injection did not alter thermal or mechanical thresholds compared to pre-injection baselines. Carrageenan injections resulted in markedly reduced paw withdrawal thresholds at 24 and 72 h after injection; this was accompanied by increased mechanical sensitivity of the contralateral paw at 72h. Analysis of innervation density using PGP9.5 as a pan-neuronal marker at 72h showed that inflammation resulted in a 2-fold increase in cutaneous innervation density. We conclude that inflammation alone is sufficient to induce sprouting of sensory cutaneous axon endings leading local tissue hyperinnervation, which may contribute to hypersensitivity that occurs in painful inflammatory conditions.
doi:10.1016/j.neulet.2011.03.043
PMCID: PMC3119508  PMID: 21439352
Pain; sensory nerves; axon sprouting; skin
15.  Vitamin D Deficiency Promotes Skeletal Muscle Hypersensitivity and Sensory Hyperinnervation 
The Journal of Neuroscience  2011;31(39):13728-13738.
Musculoskeletal pain affects nearly half of all adults, most of whom are vitamin D deficient. Previous findings demonstrated that putative nociceptors (“pain-sensing” nerves) express vitamin D receptors (VDRs), suggesting responsiveness to 1,25-dihydroxyvitamin D. In the present study, rats receiving vitamin D-deficient diets for 2– 4 weeks showed mechanical deep muscle hypersensitivity, but not cutaneous hypersensitivity. Muscle hypersensitivity was accompanied by balance deficits and occurred before onset of overt muscle or bone pathology. Hypersensitivity was not due to hypocalcemia and was actually accelerated by increased dietary calcium. Morphometry of skeletal muscle innervation showed increased numbers of presumptive nociceptor axons (peripherin-positive axons containing calcitonin gene-related peptide), without changes in sympathetic or skeletal muscle motor innervation. Similarly, there was no change in epidermal innervation. In culture, sensory neurons displayed enriched VDR expression in growth cones, and sprouting was regulated by VDR-mediated rapid response signaling pathways, while sympathetic outgrowth was not affected by different concentrations of 1,25-dihydroxyvitamin D. These findings indicate that vitamin D deficiency can lead to selective alterations in target innervation, resulting in presumptive nociceptor hyperinnervation of skeletal muscle, which in turn is likely to contribute to muscular hypersensitivity and pain.
doi:10.1523/JNEUROSCI.3637-11.2011
PMCID: PMC3319727  PMID: 21957236
16.  Characterization of the role of COP9 signalosome in regulating cullin E3 ubiquitin ligase activity 
Molecular Biology of the Cell  2011;22(24):4706-4715.
Cullin RING E3 ligases require covalent modification with Nedd8 for activity. Neddylation is reversed by the COP9 signalosome (CSN). We characterize the role of CSN-dependent deneddylation in vivo and propose a model in which CSN binds to cullin ligases in their active conformation and functions to recruit important regulatory factors.
Cullin RING ligases (CRLs) are the largest family of cellular E3 ubiquitin ligases and mediate polyubiquitination of a number of cellular substrates. CRLs are activated via the covalent modification of the cullin protein with the ubiquitin-like protein Nedd8. This results in a conformational change in the cullin carboxy terminus that facilitates the ubiquitin transfer onto the substrate. COP9 signalosome (CSN)-mediated cullin deneddylation is essential for CRL activity in vivo. However, the mechanism through which CSN promotes CRL activity in vivo is currently unclear. In this paper, we provide evidence that cullin deneddylation is not intrinsically coupled to substrate polyubiquitination as part of the CRL activation cycle. Furthermore, inhibiting substrate-receptor autoubiquitination is unlikely to account for the major mechanism through which CSN regulates CRL activity. CSN also did not affect recruitment of the substrate-receptor SPOP to Cul3, suggesting it may not function to facilitate the exchange of Cul3 substrate receptors. Our results indicate that CSN binds preferentially to CRLs in the neddylation-induced, active conformation. Binding of the CSN complex to active CRLs may recruit CSN-associated proteins important for CRL regulation. The deneddylating activity of CSN would subsequently promote its own dissociation to allow progression through the CRL activation cycle.
doi:10.1091/mbc.E11-03-0251
PMCID: PMC3237615  PMID: 22013077
17.  NEDD8 targeting drug MLN4924 elicits DNA re-replication by stabilizing Cdt1 in S Phase, triggering checkpoint activation, apoptosis and senescence in cancer cells 
Cancer research  2010;70(24):10310-10320.
MLN4924 is a first-in-class experimental cancer drug that inhibits the NEDD8-activating enzyme, thereby inhibiting cullin-RING E3 ubiquitin ligases and stabilizing many cullin substrates. The mechanism by which MLN4924 inhibits cancer cell proliferation has not been defined, although it is accompanied by DNA re-replication and attendant DNA damage. Here we show that stabilization of the DNA replication factor Cdt1, a substrate of Cullins 1 and 4, is critical for MLN4924 to trigger DNA re-replication and inhibit cell proliferation. Even only one hour of exposure to MLN4924, which was sufficient to elevate Cdt1 for 4–5 hours, was found to be sufficient to induce DNA re-replication and to activate apoptosis and senescence pathways. Cells in S phase were most susceptible, suggesting that MLN4924 will be most toxic on highly proliferating cancers. Although MLN4924-induced cell senescence appears to be dependent on induction of p53 and its downstream effector p21Waf1, we found that p53−/−and p21−/− cells were even more susceptible than wild-type cells to MLN4924. Our results suggested that apoptosis, not senescence, may be more important for the anti-proliferative effect of MLN4924. Further, our findings show that transient exposure to this new investigational drug should be useful for controlling p53-negative cancer cells, which often pose significant clinical challenge.
doi:10.1158/0008-5472.CAN-10-2062
PMCID: PMC3059213  PMID: 21159650
MLN4924; re-replication; senescence
18.  Selection and quantification of infection endpoints for trials of vaccines against intestinal helminths 
Vaccine  2011;29(20):3686-3694.
Vaccines against human helminths are being developed but the choice of optimal parasitological endpoints and effect measures to assess their efficacy has received little attention. Assuming negative binomial distributions for the parasite counts, we rank the statistical power of three measures of efficacy: ratio of mean parasite intensity at the end of the trial, the odds ratio of infection at the end of the trial, and the rate ratio of incidence of infection during the trial. We also use a modelling approach to estimate the likely impact of trial interventions on the force of infection, and hence statistical power. We conclude that (1) final mean parasite intensity is a suitable endpoint for later phase vaccine trials, and (2) mass effects of trial interventions are unlikely to appreciably reduce the force of infection in the community – and hence statistical power – unless there is a combination of high vaccine efficacy and a large proportion of the population enrolled.
doi:10.1016/j.vaccine.2011.03.026
PMCID: PMC3093614  PMID: 21435404
Helminth vaccines; Parasitological endpoints; Mathematical model
19.  Single-dose liposomal amphotericin B (AmBisome®) for the treatment of Visceral Leishmaniasis in East Africa: study protocol for a randomized controlled trial 
Trials  2011;12:66.
Background
AmBisome® is an efficacious, safe anti-leishmanial treatment. There is growing interest in its use, either as a single dose or in combination treatments. In East Africa, the minimum optimal single-dosage has not been identified.
Methods/Design
An open-label, 2-arm, non-inferiority, multi-centre randomised controlled trial is being conducted to determine the optimal single-dose treatment with AmBisome®.
Patients in the single-dose arm will receive one infusion on day 1, at a dose depending on body weight. For the first group of patients entered to the trial, the dose will be 7.5 mg/kg, but if this dose is found to be ineffective then in subsequent patient series the dose will be escalated progressively to 10, 12.5 and 15 mg/kg. Patients in the reference arm will receive a multi-dose regimen of AmBisome® (3 mg/kg/day on days 1-5, 14 and 21: total dose 21 mg/kg). Patients will be hospitalised for approximately one month after the start of treatment and then followed up at three and six months. The primary endpoint is the status of patients six months after treatment. A secondary endpoint is assessment at day 30. Treatment success is determined as the absence of parasites on microscopy samples taken from bone marrow, lymph node or splenic aspirates. Interim analyses to assess the comparative efficacy of the single dose are planned after recruitment of 20 and 40 patients per arm. The final non-inferiority analysis will include 120 patients per arm, to determine if the single-dose efficacy 6 months after treatment is not more than 10% inferior to the multi-dose.
Discussion
An effective, safe single-dose treatment would reduce hospitalization and treatment costs. Results will inform the design of combination treatment studies.
Trial Registration
ClinicalTrials.gov NCT00832208
doi:10.1186/1745-6215-12-66
PMCID: PMC3061925  PMID: 21375777
20.  GOAPhAR: An Integrative Discovery Tool for Annotation, Pathway Analysis 
We have developed the web based tool GOAPhAR (Gene Ontology, Annotations and Pathways for Array Research), that integrates information from disparate sources regarding gene annotations, protein annotations, identifiers associated with probe sets, functional pathways, protein interactions, Gene Ontology, publicly available microarray datasets and tools for statistically validating clusters in microarray data. Genes of interest can be input as Affymetrix probe identifiers, Genbank, or Unigene identifiers for human, mouse or rat genomes. Results are provided in a user friendly interface with hyperlinks to the sources of information.
PMCID: PMC2995274  PMID: 21132056
GOAPhAR: Gene ontology, Annotations and pathways for array research
21.  The NEDD8 Conjugation Pathway and Its Relevance in Cancer Biology and Therapy 
Genes & Cancer  2010;1(7):708-716.
Cancer cells depend on signals that promote cell cycle progression and prevent programmed cell death that would otherwise result from cumulative, aberrant stress. These activities require the temporally controlled destruction of specific intracellular proteins by the ubiquitin-proteasome system (UPS). To a large extent, the control points in this process include a family of E3 ubiquitin ligases called cullin-RING ligases (CRLs). The ligase activity of these multicomponent complexes requires modification of the cullin protein situated at their core with a ubiquitin-like protein called NEDD8. Neddylation results in conformational rearrangements within the CRL, which are necessary for ubiquitin transfer to a substrate. The NEDD8 pathway thus has a critical role in mediating the ubiquitination of numerous CRL substrate proteins involved in cell cycle progression and survival including the DNA replication licensing factor Cdt-1, the NF-κB transcription factor inhibitor pIκBα, and the cell cycle regulators cyclin E and p27. The initial step required for attachment of NEDD8 to a cullin is catalyzed by the E1, NEDD8-activating enzyme (NAE). The first-in-class inhibitor of NAE, MLN4924, has been shown to block the activity of NAE and prevent the subsequent neddylation of cullins. Preclinical studies have demonstrated antitumor activity in various solid tumors and hematological malignancies, and preliminary clinical data have shown the anticipated pharmacodynamic effects in humans. Here, we review the NEDD8 pathway, its importance in cancer, and the therapeutic potential of NAE inhibition.
doi:10.1177/1947601910382898
PMCID: PMC3092238  PMID: 21779466
NEDD8; ubiquitin; cullin-RING ligase; DNA rereplication
22.  Rate and Amplification of Drug Resistance among Previously-Treated Patients with Tuberculosis in Kampala, Uganda 
Background
Drug-resistant Mycobacterium tuberculosis has emerged as a global threat. In resource-constrained settings, patients with a history of tuberculosis (TB) treatment may have drug-resistant disease and may experience poor outcomes. There is a need to measure the extent of and risk factors for drug resistance in such patients.
Methods
From July 2003 through November 2006, we enrolled 410 previously treated patients with TB in Kampala, Uganda. We measured the prevalence of resistance to first- and second-line drugs and analyzed risk factors associated with baseline and acquired drug resistance.
Results
The prevalence of multidrug-resistant TB was 12.7% (95% confidence interval [95% CI], 9.6%–16.3%). Resistance to second-line drugs was low. Factors associated with multidrug-resistant TB at enrollment included a history of treatment failure (odds ratio, 23.6; 95% CI, 7.7–72.4), multiple previous TB episodes (odds ratio, 15.6; 95% CI, 5.0–49.1), and cavities present on chest radiograph (odds ratio, 5.9; 95% CI, 1.2–29.5). Among a cohort of 250 patients, 5.2% (95% CI, 2.8%–8.7%) were infected with M. tuberculosis that developed additional drug resistance. Amplification of drug resistance was associated with existing drug resistance at baseline (P<.01) and delayed sputum culture conversion (P<.01).
Conclusions
The burden of drug resistance in previously treated patients with TB in Uganda is sizeable, and the risk of generating additional drug resistance is significant. There is an urgent need to improve the treatment for such patients in low-income countries.
doi:10.1086/592252
PMCID: PMC2883442  PMID: 18808360
23.  Sustainable development of a GCP-compliant clinical trials platform in Africa: the Malaria Clinical Trials Alliance perspective 
Malaria Journal  2010;9:103.
Background
The Malaria Clinical Trials Alliance (MCTA), a programme of INDEPTH network of demographic surveillance centres, was launched in 2006 with two broad objectives: to facilitate the timely development of a network of centres in Africa with the capacity to conduct clinical trials of malaria vaccines and drugs under conditions of good clinical practice (GCP); and to support, strengthen and mentor the centres in the network to facilitate their progression towards self-sustaining clinical research centres.
Case description
Sixteen research centres in 10 African malaria-endemic countries were selected that were already working with the Malaria Vaccine Initiative (MVI) or the Medicines for Malaria Venture (MMV). All centres were visited to assess their requirements for research capacity development through infrastructure strengthening and training. Support provided by MCTA included: laboratory and facility refurbishment; workshops on GCP, malaria diagnosis, strategic management and media training; and training to support staff to undertake accreditation examinations of the Association of Clinical Research Professionals (ACRP). Short attachments to other network centres were also supported to facilitate sharing practices within the Alliance. MCTA also played a key role in the creation of the African Media & Malaria Research Network (AMMREN), which aims to promote interaction between researchers and the media for appropriate publicity and media reporting of research and developments on malaria, including drug and vaccine trials.
Conclusion
In three years, MCTA strengthened 13 centres to perform GCP-compliant drug and vaccine trials, including 11 centres that form the backbone of a large phase III malaria vaccine trial. MCTA activities have demonstrated that centres can be brought up to GCP compliance on this time scale, but the costs are substantial and there is a need for further support of other centres to meet the growing demand for clinical trial capacity. The MCTA experience also indicates that capacity development in clinical trials is best carried out in the context of preparation for specific trials. In this regard MCTA centres involved in the phase III malaria vaccine trial were, on average, more successful at consolidating the training and infrastructure support than those centres focussing only on drug trials.
doi:10.1186/1475-2875-9-103
PMCID: PMC2873521  PMID: 20406478
24.  Modulation of rat parasympathetic cardiac ganglion phenotype and NGF synthesis by adrenergic nerves 
Cardiac function is regulated by interactions among intrinsic and extrinsic autonomic neurons, and the mechanisms responsible for organizing these circuits are poorly understood. Parasympathetic neurons elsewhere synthesize the neurotrophin NGF, which may promote postganglionic axonal associations where parasympathetic axons inhibit sympathetic transmitter release. Previous studies have shown that parasympathetic NGF content and neurochemical phenotype are regulated by sympathetic innervation. In this study we assessed contributions of sympathetic input on cardiac ganglion neuronal phenotype and NGF expression. Because cardiac ganglia are reported to contain putative noradrenergic neurons, we eliminated sympathetic input both surgically (extrinsic) and chemically (extrinsic plus intrinsic). In controls, most cardiac ganglion neurons expressed vesicular acetylcholine transporter, frequently colocalized with vesicular monoamine transporter, but lacked catecholamine histofluorescence. Most cardiac ganglion neurons expressed NGF transcripts, and 40% contained mature and 47% proNGF immunoreactivity. Guanethidine treatment for 7 days decreased numbers of neurons expressing vesicular acetylcholine transporter, NGF transcripts and NGF immunoreactivity, but did not affect proNGF or vesicular monoamine transporter immunoreactivity. Stellate ganglionectomy had comparable effects on neurochemical phenotype and mature NGF immunoreactivity, but proNGF expression was additionally reduced.
These findings show that individual cardiac ganglion neurons display markers of both cholinergic and noradrenergic transmission. Sympathetic noradrenergic innervation maintains levels of cholinergic but not noradrenergic marker protein. Sympathetic innervation also promotes cardiac ganglion neuronal NGF synthesis. Because chemical blockade of all noradrenergic transmission is no more effective than extrinsic sympathectomy, local intrinsic noradrenergic transmission is not a factor in regulating ganglion neuron phenotype.
doi:10.1016/j.autneu.2008.10.012
PMCID: PMC2650853  PMID: 19019738
Nerve Growth Factor; ProNGF; Stellate Ganglionectomy; Guanethidine; Parasympathetic Nervous System
25.  Neurotrimin is an estrogen-regulated determinant of peripheral sympathetic innervation 
Journal of neuroscience research  2008;86(14):3086-3095.
Mechanisms underlying axon degeneration in peripheral neuropathies and during normal remodeling are poorly understood. Because estrogen induces widespread sympathetic axon degeneration within female reproductive tract smooth muscle, we surveyed estrogen-regulated genes in rat myometrium. Microarray analysis revealed that the neural cell adhesion protein neurotrimin (Ntm) was markedly upregulated at 6h and down-regulated by 24h after injection of 17β-estradiol and real time RT-PCR confirmed this pattern of expression. Protein analysis by western blotting showed that uterine Ntm protein is also upregulated in vivo at 6−24h following estrogen injection, and that Ntm protein is increased selectively in the myometrium during the high-estrogen phase of the estrous cycle. Cultured myometrial smooth muscle cells display peri-nuclear accumulations of Ntm protein, and 17β-Estradiol also increases intracellular levels of Ntm and its secretion into the culture medium. To determine if neurotrimin is required for estrogen-induced sympathetic pruning, sympathetic neurons were co-cultured with uterine smooth muscle cells transfected with siRNA directed against Ntm. While estrogen inhibited neurite outgrowth in non-transfected co-cultures, estrogen's ability to reduce sympathetic outgrowth was impaired substantially following Ntm downregulation. This supports a role for neurotrimin in mediating estrogen-induced sympathetic pruning in some peripheral targets. Together with earlier studies, these findings support the idea that physiological sympathetic axon degeneration is a multifactorial process requiring dynamic regulation of multiple repellant proteins.
doi:10.1002/jnr.21768
PMCID: PMC2577172  PMID: 18627025
neurodegeneration; estrogen; sympathetic; IgLON

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