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1.  An investigation of the effects of antiretroviral CNS penetration effectiveness on procedural learning in HIV+ drug users 
Journal of clinical and experimental neuropsychology  2013;35(9):10.1080/13803395.2013.838939.
Treatment with combination antiretroviral therapy (cART) regimens with a high capacity to penetrate the blood-brain barrier has been associated with lower levels of human immunodeficiency virus (HIV) in the central nervous system (CNS). This study examined neurocognitive performance among a sample of 118 HIV+ substance dependent individuals (SDIs) and 310 HIV− SDIs. HIV+ participants were prescribed cART regimens with varying capacity to penetrate the CNS as indexed by the revised CNS penetration effectiveness (CPE) scale. Participants completed the Rotary Pursuit Task (RPT) and the Weather Prediction Task (WPT)--two measures of procedural learning (PL) with known sensitivity to HIV infection--and a control task of sustained attention. HIV+ SDIs prescribed cART with relatively high CNS penetrance performed significantly more poorly on both tasks than HIV− controls. Task performance of HIV+ SDIs prescribed cART with relatively low CNS penetrance did not differ significantly from either HIV− controls or the HIV+/High CPE group, although a trend towards lower RPT performance relative to HIV− participants was observed. Between-group differences were not seen on a control task of motor impulsivity (Immediate Memory Task), indicating that the observed deficits among HIV+/High CPE SDIs may have some specificity.
doi:10.1080/13803395.2013.838939
PMCID: PMC3856247  PMID: 24079384
HIV; neurocognition; substance abuse; antiretroviral therapy; addiction; procedural learning; CPE
2.  The Oncogenic Response to MiR-335 Is Associated with Cell Surface Expression of Membrane-Type 1 Matrix Metalloproteinase (MT1-MMP) Activity 
PLoS ONE  2015;10(7):e0132026.
MicroRNA miR-335 has been reported to have both tumor suppressor and oncogenic activities. In order to determine possible tissue and cell type differences in response to miR-335, we examined the effect of miR-335 on cell expression of MT1-MMP, a proteinase commonly expressed in tumors and associated with cell proliferation and migration. miR-335 increased cell surface expression of MT1-MMP in fibrosarcoma HT-1080 and benign prostate BPH-1 cells, but not in prostate LNCaP or breast MCF-7 tumor cells. miR-335 stimulated proliferation and cell migration in a wound healing in vitro assay in HT-1080, BPH-1, and U87 glioblastoma cells, cells which demonstrated significant cell surface expression of MT1-MMP. In contrast, miR-335 did not affect proliferation or migration in cells without a prominent plasma membrane associated MT1-MMP activity. Our data suggest that differences in response to miR-335 by tumor cells may lie in part in the mechanism of regulation of MT1-MMP production.
doi:10.1371/journal.pone.0132026
PMCID: PMC4512721  PMID: 26204513
3.  Interactions of Disulfide-deficient Selenocysteine Analogs of μ-Conotoxin BuIIIB with the Voltage-Gated Sodium Channel NaV1.3 
The FEBS journal  2014;281(13):2885-2898.
Inhibitors of the neuronal voltage-gated sodium channel subtype NaV1.3 are of interest as pharmacological tools for the study of neuropathic pain associated with spinal cord injury and have potential therapeutic applications. The recently described μ-conotoxin BuIIIB from Conus bullatus (μ-BuIIIB) was shown to block NaV1.3 with sub-micromolar potency (Kd = 0.2 μM), making it one of the most potent peptidic inhibitors of this subtype described to date. However, oxidative folding of μ-BuIIIB results in numerous folding isoforms, making it difficult to obtain sufficient quantities of the active form of the peptide for detailed structure-activity studies. Here we report the synthesis and characterization of μ-BuIIIB analogs incorporating a disulfide-deficient, diselenide-containing scaffold designed to simplify synthesis and facilitate structure-activity studies directed at identifying amino acid residues involved in NaV1.3 blockade. Our results indicate that, like other μ-conotoxins, the C-terminal residues (Trp16, Arg18 and His20) are most crucial for NaV1 block. At the N-terminus, replacement of Glu3 by Ala resulted in an analog with increased potency for NaV1.3 (Kd = 0.07 μM), implicating this position as a potential site for modification for increased potency and/or selectivity. Further examination of this position showed that increased negative charge, through γ-carboxyglutamate replacement, decreased potency (Kd = 0.33 μM), while replacement with positively-charged 2,4-diamonobutyric acid increased potency (Kd = 0.036 μM). These results provide a foundation for the design and synthesis of μ-BuIIIB-based analogs with increased potency against NaV1.3.
doi:10.1111/febs.12835
PMCID: PMC4160447  PMID: 24814369
Conotoxin; disulfide; neuropathic pain; selenocysteine; voltage-gated sodium channel
4.  miR-520c and miR-373 target mTOR and SIRT1, activate the Ras/Raf/MEK/Erk pathway and NF-κB, with up-regulation of MMP9 in human fibrosarcoma cells 
Journal of cellular physiology  2012;227(2):867-876.
MicroRNA 520c and 373 (miR-520c and miR-373) have been characterized as oncogenes and play critical roles in cancer cell metastasis. However, the relationship between these two microRNAs and matrix metalloproteins (MMPs), which are important in cancer cell metastasis, remains unknown. Here we report new evidence in which miR-520c and miR-373 effects in human fibrosarcoma HT1080 cells are associated with MMP9 activity, and this up-regulation of MMP9 is not only at the activity and protein levels, but also at that of its mRNA. Our experimental data demonstrate that these effects occur not by direct binding to the MMP9 promoter, but by miR-520c and miR-373 directly targeting the 3′UTRs of mRNAs of mTOR and SIRT1 (negative regulators of expression of MMP9 via inactivating the Ras/Raf/MEK/Erk signaling pathway and transcription factor NF-κB activity); and thus suppressing translation levels of SIRT1 and mTOR. Moreover, inhibition of key kinases of the Ras/Raf/MEK/Erk signaling pathway and western-blots for selected proteins further identified miR-520c and miR-373 as activating this signaling pathway and NF-κB. In conclusion, miR-520c and miR-373 increased the expression of MMP9 by directly targeting the 3′UTRs of mRNAs of mTOR and SIRT1 and suppressing their translation; resulting in activation of the Ras/Raf/MEK/Erk signaling pathway and NF-κB; and finally increasing the mRNA, protein, and activity of MMP9 and enhancing cell migration and cell growth in 3-D type I collagen gels.
doi:10.1002/jcp.22993
PMCID: PMC3225649  PMID: 21898400
5.  Pharmacological fractionation of tetrodotoxin-sensitive sodium currents in rat dorsal root ganglion neurons by μ-conotoxins 
British Journal of Pharmacology  2013;169(1):102-114.
Background and Purpose
Adult rat dorsal root ganglion (DRG) neurons normally express transcripts for five isoforms of the α-subunit of voltage-gated sodium channels: NaV1.1, 1.6, 1.7, 1.8 and 1.9. Tetrodotoxin (TTX) readily blocks all but NaV1.8 and 1.9, and pharmacological agents that discriminate among the TTX-sensitive NaV1-isoforms are scarce. Recently, we used the activity profile of a panel of μ-conotoxins in blocking cloned rodent NaV1-isoforms expressed in Xenopus laevis oocytes to conclude that action potentials of A- and C-fibres in rat sciatic nerve were, respectively, mediated primarily by NaV1.6 and NaV1.7.
Experimental Approach
We used three μ-conotoxins, μ-TIIIA, μ-PIIIA and μ-SmIIIA, applied individually and in combinations, to pharmacologically differentiate the TTX-sensitive INa of voltage-clamped neurons acutely dissociated from adult rat DRG. We examined only small and large neurons whose respective INa were >50% and >80% TTX-sensitive.
Key Results
In both small and large neurons, the ability of the toxins to block TTX-sensitive INa was μ-TIIIA < μ-PIIIA < μ-SmIIIA, with the latter blocking ≳90%. Comparison of the toxin-susceptibility profiles of the neuronal INa with recently acquired profiles of rat NaV1-isoforms, co-expressed with various NaVβ-subunits in X. laevis oocytes, were consistent: NaV1.1, 1.6 and 1.7 could account for all of the TTX-sensitive INa, with NaV1.1 < NaV1.6 < NaV1.7 for small neurons and NaV1.7 < NaV1.1 < NaV1.6 for large neurons.
Conclusions and Implications
Combinations of μ-conotoxins can be used to determine the probable NaV1-isoforms underlying the INa in DRG neurons. Preliminary experiments with sympathetic neurons suggest that this approach is extendable to other neurons.
doi:10.1111/bph.12119
PMCID: PMC3632242  PMID: 23351163
μ-conotoxin PIIIA; μ-conotoxin SmIIIA; μ-conotoxin TIIIA; dorsal root ganglion; superior cervical ganglion; tetrodotoxin; voltage-gated sodium channel; whole-cell patch clamp
6.  Co-expression of NaVβ subunits alters the kinetics of inhibition of voltage-gated sodium channels by pore-blocking μ-conotoxins 
British Journal of Pharmacology  2013;168(7):1597-1610.
Background and Purpose
Voltage-gated sodium channels (VGSCs) are assembled from two classes of subunits, a pore-bearing α-subunit (NaV1) and one or two accessory β-subunits (NaVβs). Neurons in mammals can express one or more of seven isoforms of NaV1 and one or more of four isoforms of NaVβ. The peptide μ-conotoxins, like the guanidinium alkaloids tetrodotoxin (TTX) and saxitoxin (STX), inhibit VGSCs by blocking the pore in NaV1. Hitherto, the effects of NaVβ-subunit co-expression on the activity of these toxins have not been comprehensively assessed.
Experimental Approach
Four μ-conotoxins (μ-TIIIA, μ-PIIIA, μ-SmIIIA and μ-KIIIA), TTX and STX were tested against NaV1.1, 1.2, 1.6 or 1.7, each co-expressed in Xenopus laevis oocytes with one of NaVβ1, β2, β3 or β4 and, for NaV1.7, binary combinations of thereof.
Key Results
Co-expression of NaVβ-subunits modifies the block by μ-conotoxins: in general, NaVβ1 or β3 co-expression tended to increase kon (in the most extreme instance by ninefold), whereas NaVβ2 or β4 co-expression decreased kon (in the most extreme instance by 240-fold). In contrast, the block by TTX and STX was only minimally, if at all, affected by NaVβ-subunit co-expression. Tests of NaVβ1 : β2 chimeras co-expressed with NaV1.7 suggest that the extracellular portion of the NaVβ subunit is largely responsible for altering μ-conotoxin kinetics.
Conclusions and Implications
These results are the first indication that NaVβ subunit co-expression can markedly influence μ-conotoxin binding and, by extension, the outer vestibule of the pore of VGSCs. μ-Conotoxins could, in principle, be used to pharmacologically probe the NaVβ subunit composition of endogenously expressed VGSCs.
doi:10.1111/bph.12051
PMCID: PMC3605869  PMID: 23146020
μ-conotoxin KIIIA; μ-conotoxin PIIIA; μ-conotoxin SmIIIA; μ-conotoxin TIIIA; NaVβ-subunit; saxitoxin; site 1; tetrodotoxin; voltage-gated sodium channel; Xenopus oocytes
7.  One-step zero-background IgG reformatting of phage-displayed antibody fragments enabling rapid and high-throughput lead identification 
Nucleic Acids Research  2013;42(4):e26.
We describe a novel cloning method, referred to as insert-tagged (InTag) positive selection, for the rapid one-step reformatting of phage-displayed antibody fragments to full-length immunoglobulin Gs (IgGs). InTag positive selection enables recombinant clones of interest to be directly selected without cloning background, bypassing the laborious process of plating out cultures and colony screening and enabling the cloning procedure to be automated and performed in a high-throughput format. This removes a significant bottleneck in the functional screening of phage-derived antibody candidates and enables a large number of clones to be directly reformatted into IgG without the intermediate step of Escherichia coli expression and testing of soluble antibody fragments. The use of InTag positive selection with the Dyax Fab-on-phage antibody library is demonstrated, and optimized methods for the small-scale transient expression of IgGs at high levels are described. InTag positive selection cloning has the potential for wide application in high-throughput DNA cloning involving multiple inserts, markedly improving the speed and quality of selections from protein libraries.
doi:10.1093/nar/gkt1142
PMCID: PMC3936716  PMID: 24253301
8.  Lactam-stabilized helical analogues of the analgesic μ-conotoxin KIIIA 
Journal of medicinal chemistry  2011;54(21):7558-7566.
μ-Conotoxin KIIIA (μ-KIIIA) blocks mammalian voltage-gated sodium channels (VGSCs) and is a potent analgesic following systemic administration in mice. Previous structure-activity studies of μ-KIIIA identified a helical pharmacophore for VGSC blockade. This suggested a route for designing truncated analogues of μ-KIIIA by incorporating the key residues into an α-helical scaffold. As (i, i+4) lactam bridges constitute a proven approach for stabilizing α-helices, we designed and synthesized six truncated analogues of μ-KIIIA containing single lactam bridges at various locations. The helicity of these lactam analogues was analysed by NMR spectroscopy, and their activities were tested against mammalian VGSC subtypes NaV1.1 through 1.7. Two of the analogues, Ac-cyclo9/13[Asp9,Lys13]KIIIA7–14 and Ac-cyclo9/13[Lys9,Asp13]KIIIA7–14, displayed µM activity against VGSC subtypes NaV1.2 and NaV1.6; importantly, the subtype selectivity profile for these peptides matched that of μ-KIIIA. Our study highlights structure-activity relationships within these helical mimetics and provides a basis for the design of additional truncated peptides as potential analgesics.
doi:10.1021/jm200839a
PMCID: PMC3228837  PMID: 21962108
9.  Temporal Variation and Host Association in the Campylobacter Population in a Longitudinal Ruminant Farm Study▿ 
Applied and Environmental Microbiology  2011;77(18):6579-6586.
Campylobacter jejuni and C. coli were quantified and typed, using multilocus sequence typing (MLST), from fecal samples collected from a mixed cattle and sheep farm during summer. Cattle had a significantly higher prevalence than sheep (21.9% [74/338] and 14.0% [30/214], respectively), but both decreased over time. There were no differences in the average Campylobacter concentrations shed by cattle (600 CFU g−1) and sheep (820 CFU g−1), although sheep did show a significant temporal reduction in the number of Campylobacter organisms shed in their feces. A total of 21 different sequence types (STs) (97.7% C. jejuni, 2.3% C. coli) were isolated from cattle, and 9 different STs were isolated from sheep (40.6% C. jejuni, 59.4% C. coli). The Campylobacter population in cattle was relatively stable, and the frequencies of genotypes isolated showed little temporal variation. However, the composition of subtypes isolated from sheep did show significant temporal differences. The cattle and sheep consistently showed significant differences in their carriage of Campylobacter species, STs, and CCs despite the fact that both were exposed to the same farming environment. This work has highlighted the patterns of a Campylobacter population on a ruminant farm by identifying the existence of both temporal and between-host variations.
doi:10.1128/AEM.00428-11
PMCID: PMC3187171  PMID: 21784915
10.  Regulation of MT1-MMP activity by β-catenin in MDCK non-cancer and HT1080 cancer cells 
Journal of cellular physiology  2010;225(3):810-821.
Past studies on β-catenin in cancer cells focused on nuclear localized β-catenin and its involvement in the Wnt pathway. Our goal here was to investigate the function of β-catenin in both the cytoplasm and nucleus on the regulation of MT1-MMP expression and activity. We found that β-catenin in MDCK non-cancer cells inhibited the cell surface localization of MT1-MMP, and thus its proteolytic activity on pro-MMP2 activation, via direct interaction with the 18-amino acid cytoplasmic tail of MT1-MMP in the cytoplasm. In contrast, β-catenin in HT1080 cancer cells enhanced the activity of MT1-MMP by entering the nucleus and activating transcription factor Tcf-4/Lef, and elevating the level of MT1-MMP protein. We also found that enhancement of cell growth in 3-D/2-D type I collagen gels and of cell migration by MT1-MMP were inhibited by β-catenin in MDCK cells, whereas these functions were enhanced in HT1080 cells. In addition, regulation of MT1-MMP by β-catenin involved E-cadherin in MDCK cells and Wnt-3a in HT1080 cells. Taken together, our results present a differential effect of cytoplasmic and nuclear β-catenin on MT1-MMP activity in non-cancer cells versus cancer cells. These differences were most probably due to different subcellular locations and different involved pathways of β-catenin in these cells.
doi:10.1002/jcp.22292
PMCID: PMC2939945  PMID: 20589835
β-catenin; MT1-MMP; interacting/associating; cytoplasmic tail of MT1-MMP
11.  Sleep Deprivation Differentially Impairs Cognitive Performance in Abstinent Methylenedioxymethamphetamine (“Ecstasy”) Users 
Methylenedioxymethamphetamine (MDMA; “Ecstasy”) is a popular recreational drug and brain serotonin (5-HT) neurotoxin. Neuroimaging data indicate that some human MDMA users develop persistent deficits in brain 5-HT neuronal markers. Although the consequences of MDMA-induced 5-HT neurotoxicity are not fully understood, abstinent MDMA users have been found to have subtle cognitive deficits and altered sleep architecture. The present study sought to test the hypothesis that sleep disturbance plays a role in cognitive deficits in MDMA users. Nineteen abstinent MDMA users and 21 control subjects participated in a 5 d inpatient study in a clinical research unit. Baseline sleep quality was measured using the Pittsburgh Sleep Quality Inventory. Cognitive performance was tested three times daily using a computerized cognitive battery. On the third day of admission, subjects began a 40 h sleep deprivation period and continued cognitive testing using the same daily schedule. At baseline, MDMA users performed less accurately than controls on a task of working memory and more impulsively on four of the seven computerized tests. During sleep deprivation, MDMA users, but not controls, became increasingly impulsive, performing more rapidly at the expense of accuracy on tasks of working and short-term memory. Tests of mediation implicated baseline sleep disturbance in the cognitive decline seen during sleep deprivation. These findings are the first to demonstrate that memory problems in MDMA users may be related, at least in part, to sleep disturbance and suggest that cognitive deficits in MDMA users may become more prominent in situations associated with sleep deprivation.
doi:10.1523/JNEUROSCI.4654-09.2009
PMCID: PMC3047479  PMID: 19890014
12.  Slugs: Potential Novel Vectors of Escherichia coli O157 
Field and laboratory studies were performed to determine whether slugs could act as novel vectors for pathogen (e.g., Escherichia coli O157) transfer from animal feces to salad vegetables. Escherichia coli O157 was isolated from 0.21% of field slugs from an Aberdeenshire sheep farm. These isolates carried the verocytotoxin genes (vt1 and vt2) and the attaching and effacing gene (eae), suggesting that they are potentially pathogenic to humans. Strain typing using multilocus variable number tandem repeats analysis showed that slug and sheep isolates were indistinguishable. Laboratory experiments using an E. coli mutant resistant to nalidixic acid showed that the ubiquitous slug species Deroceras reticulatum could carry viable E. coli on its external surface for up to 14 days. Slugs that had been fed E. coli shed viable bacteria in their feces with numbers showing a short but statistically significant linear log decline. Further, it was found that E. coli persisted for up to 3 weeks in excreted slug feces, and hence, we conclude that slugs have the potential to act as novel vectors of E. coli O157.
doi:10.1128/AEM.72.1.144-149.2006
PMCID: PMC1352200  PMID: 16391036
13.  Modelling of oedemous limbs and venous ulcers using partial differential equations 
Background
Oedema, commonly known as tissue swelling, occurs mainly on the leg and the arm. The condition may be associated with a range of causes such as venous diseases, trauma, infection, joint disease and orthopaedic surgery. Oedema is caused by both lymphatic and chronic venous insufficiency, which leads to pooling of blood and fluid in the extremities. This results in swelling, mild redness and scaling of the skin, all of which can culminate in ulceration.
Methods
We present a method to model a wide variety of geometries of limbs affected by oedema and venous ulcers. The shape modelling is based on the PDE method where a set of boundary curves are extracted from 3D scan data and are utilised as boundary conditions to solve a PDE, which provides the geometry of an affected limb. For this work we utilise a mixture of fourth order and sixth order PDEs, the solutions of which enable us to obtain a good representative shape of the limb and associated ulcers in question.
Results
A series of examples are discussed demonstrating the capability of the method to produce good representative shapes of limbs by utilising a series of curves extracted from the scan data. In particular we show how the method could be used to model the shape of an arm and a leg with an associated ulcer.
Conclusion
We show how PDE based shape modelling techniques can be utilised to generate a variety of limb shapes and associated ulcers by means of a series of curves extracted from scan data. We also discuss how the method could be used to manipulate a generic shape of a limb and an associated wound so that the model could be fine-tuned for a particular patient.
doi:10.1186/1742-4682-2-28
PMCID: PMC1198260  PMID: 16078992
14.  DNA Methylation Maintains Allele-specific KIR Gene Expression in Human Natural Killer Cells 
Killer immunoglobulin-like receptors (KIR) bind self–major histocompatibility complex class I molecules, allowing natural killer (NK) cells to recognize aberrant cells that have down-regulated class I. NK cells express variable numbers and combinations of highly homologous clonally restricted KIR genes, but uniformly express KIR2DL4. We show that NK clones express both 2DL4 alleles and either one or both alleles of the clonally restricted KIR 3DL1 and 3DL2 genes. Despite allele-independent expression, 3DL1 alleles differed in the core promoter by only one or two nucleotides. Allele-specific 3DL1 gene expression correlated with promoter and 5′ gene DNA hypomethylation in NK cells in vitro and in vivo. The DNA methylase inhibitor, 5-aza-2′-deoxycytidine, induced KIR DNA hypomethylation and heterogeneous expression of multiple KIR genes. Thus, NK cells use DNA methylation to maintain clonally restricted expression of highly homologous KIR genes and alleles.
doi:10.1084/jem.20021127
PMCID: PMC2193817  PMID: 12538663
killer cells, natural; killer inhibitory receptor; alleles; DNA methylation; gene expression regulation

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