• Management of environmental streaming data to optimize Arctic shipping routes.

      Zhang, Zhihua; Crabbe, M. James C.; University of Bedfordshire; Shandong University (Springer Nature, 2021-07-20)
      Dynamic accurate predictions of Arctic sea ice, ocean, atmosphere, and ecosystem are necessary for safe and efficient Arctic maritime transportation; however a related technical roadmap has not yet been established. In this paper, we propose a management system for trans-Arctic maritime transportation supported by near real-time streaming data from air-space-ground-sea integrated monitoring networks and high spatio-temporal sea ice modeling. As the core algorithm of integrated monitoring networks, a long short-term memory (LSTM) neural network is embedded to improve Arctic sea ice mapping algorithms.Since the LSTM is localized in time and space, it can make full use of streaming data characteristics. The sea ice–related parameters from satellite remote sensing raw data are used as the input of the LSTM, while streaming data from shipborne radar networks and/or buoy measurements are used as training datasets to enhance the accuracy and resolution of environmental streaming data from outputs of LSTM. Due to large size of streaming data, the proposed management system of trans-Arctic shipping should be built on a cloud distribution platform using existing wireless communications networks among vessels and ports. Our management system will be used by the ongoing European Commission Horizon 2020 Programme “ePIcenter.”
    • Mastermind-Like 3 controls proliferation and differentiation in neuroblastoma

      Heynen, Guus J.J.E.; Nevedomskaya, Ekaterina; Palit, Sander; Basheer, Noorjahan Jagalur; Lieftink, Cor; Schlicker, Andreas; Zwart, Wilbert; Bernards, René; Bajpe, Prashanth Kumar; ; et al. (American Association for Cancer Research Inc., 2016-01-19)
      Neuroblastoma cell lines can differentiate upon treatment with retinoic acid (RA), a finding that provided the basis for the clinical use of RA to treat neuroblastoma. However, resistance to RA is often observed, which limits its clinical utility. Using a gain-of-function genetic screen, we identified an unexpected link between RA signaling and mastermindlike 3 (MAML3), a known transcriptional coactivator for NOTCH. Our findings indicate that MAML3 expression leads to the loss of activation of a subset of RA target genes, which hampers RA-induced differentiation and promotes resistance to RA. The regulatory DNA elements of this subset of RA target genes show overlap in binding of MAML3 and the RA receptor, suggesting a direct role for MAML3 in the regulation of these genes. In addition, MAML3 has RA-independent functions, including the activation of IGF1R and downstream AKT signaling via upregulation of IGF2, resulting in increased proliferation. These results demonstrate an important mechanistic role for MAML3 in proliferation and RA-mediated differentiation.
    • Measurement of specific radioactivity in proteins separated by two-dimensional gel electrophoresis

      Zhou, Shaobo; Mann, Christopher J.; Dunn, Michael J.; Preedy, Victor R.; Emery, Peter W.; (Wiley, 2006-03-07)
      We report a method to quantify the specific radioactivity of proteins that have been separated by 2-DE. Gels are stained with SyproRuby, and protein spots are excised. The SyproRuby dye is extracted from each spot using DMSO, and the fluorescence is quantified automatically using a plate reader. The extracted gel piece is then dissolved in hydrogen peroxide and radioactivity is quantified by liquid scintillation counting. Gentle agitation with DMSO for 24 h was found to extract all the SyproRuby dye from gel fragments. The fluorescence of the extract was linearly related to the amount of BSA loaded onto a series of 1-D gels. When rat muscle samples were run on 2-DE gels, the fluorescence extracted from 54 protein spots showed a good correlation (r = 0.79, p < 0.001) with the corresponding spot intensity measured by conventional scanning and image analysis. DMSO extraction was found not to affect the amount of radioactive protein left in the gel. When a series of BSA solutions of known specific radioactivity were run on 2-DE gels, the specific radioactivity measured by the new method showed a good correlation (r = 0.98, p < 0.01, n = 5) with the specific radioactivity measured directly before loading. Reproducibility of the method was measured in a series of 2-DE gels containing proteins from the livers of rats and mice that had been injected with [35S]methionine. Variability tended to increase when the amount of radioactivity in the protein spot was low, but for samples containing at least 10 dpm above background the CV was around 30%, which is comparable to that obtained when measuring protein expression by conventional image analysis of SyproRuby-stained 2-DE gels. Similar results were obtained whether spots were excised manually or using a spot excision robot. This method offers a high-throughput, cost-effective and reliable method of quantifying the specific radioactivity of proteins from metabolic labelling experiments carried out in vivo, so long as sufficient quantities of radioactive tracer are used.
    • MFG-E8 induced differences in proteomic profiles in mouse C2C12 cells and its effect on PI3K/Akt and ERK signal pathways

      Li, He; Guan, Kaifang; Li, Xu; Ma, Ying; Zhou, Shaobo; Harbin Institute of Technology; Jilin University; University of Bedfordshire (Elsevier, 2018-11-28)
      Milk fat globule-EGF factor 8 (MFG-E8) is one of the major proteins in milk fat globule membrane. In this study, mouse-derived C2C12 myoblast cells were served as an experimentally tractable model system for investigating the molecular basis of skeletal muscle cell specification and development. To examine the biochemical adaptations associated with myocytes formation comprehensively, a liquid chromatography coupled with tandem mass spectrometry label-free semi-quantitative  approach was used to analyse the myogenic C2C12 proliferation program. Over 1987  proteins were identified in C2C12 cells. The MFG-E8 (200 mg/mL) and MFG-E8 (500 26 mg/mL) with significant differences were compared based on the relative abundance. The result profiles of regulation of MFG-E8 to the expression of proteins in C2C12 cells revealed that differential waves of expression of proteins linked to intracellular signaling, transcription, cytoarchitecture, adhesion, metabolism, and muscle contraction across during the C2C12 cell proliferation process. Based on the analysis of  KEGG and STRING database, further to verification the expression of PI3K and ERK phosphorylation levels by Western blot. This study found that the data of proteomic was complementary to recent MFG-E8 studies of protein expression patterns in developing myotubes and provided a holistic framework for understanding how diverse biochemical processes are coordinated at the cellular level during skeletal muscle development.
    • MicroRNAs for virus pathogenicity and host responses, identified in SARS-CoV-2 genomes, may play roles in viral-host co-evolution in putative zoonotic host species

      Lange, Sigrun; Arisan, Elif Damla; Grant, Guy H.; Uysal-Onganer, Pinar; University of Westminster; Gebze Technical University; University of Bedfordshire (MDPI, 2021-01-16)
      Our recent study identified seven key microRNAs (miR-8066, 5197, 3611, 3934-3p, 1307-3p, 3691-3p, 1468-5p) similar between SARS-CoV-2 and the human genome, pointing at miR-related mechanisms in viral entry and the regulatory effects on host immunity. To identify the putative roles of these miRs in zoonosis, we assessed their conservation, compared with humans, in some key wild and domestic animal carriers of zoonotic viruses, including bat, pangolin, pig, cow, rat, and chicken. Out of the seven miRs under study, miR-3611 was the most strongly conserved across all species; miR-5197 was the most conserved in pangolin, pig, cow, bat, and rat; miR-1307 was most strongly conserved in pangolin, pig, cow, bat, and human; miR-3691-3p in pangolin, cow, and human; miR-3934-3p in pig and cow, followed by pangolin and bat; miR-1468 was most conserved in pangolin, pig, and bat; while miR-8066 was most conserved in pangolin and pig. In humans, miR-3611 and miR-1307 were most conserved, while miR-8066, miR-5197, miR-3334-3p and miR-1468 were least conserved, compared with pangolin, pig, cow, and bat. Furthermore, we identified that changes in the miR-5197 nucleotides between pangolin and human can generate three new miRs, with differing tissue distribution in the brain, lung, intestines, lymph nodes, and muscle, and with different downstream regulatory effects on KEGG pathways. This may be of considerable importance as miR-5197 is localized in the spike protein transcript area of the SARS-CoV-2 genome. Our findings may indicate roles for these miRs in viral-host co-evolution in zoonotic hosts, particularly highlighting pangolin, bat, cow, and pig as putative zoonotic carriers, while highlighting the miRs' roles in KEGG pathways linked to viral pathogenicity and host responses in humans. This in silico study paves the way for investigations into the roles of miRs in zoonotic disease.
    • Migration of BEAS-2B cells enhanced by H1299 cell derived-exosomes

      Wang, Shuwei; Ju, Tuoyu; Wang, Jiajia; Yang, Fan; Qu, Kaige; Liu, Wei; Wang, Zuobin; Jilin University; Changchun University of Science and Technology; University of Bedfordshire (Elsevier Ltd, 2021-01-12)
      Previous studies reported that exosomes (Exos) secreted by tumor cells could affect the tumor cells themselves and normal cells. However, the effects of exosomes derived from tumor cells on normal cells’ migration and mechanical characteristics are rarely reported. This work explores the effects of H1299 cell-derived exosomes (H1299-Exos) on the migration of BEAS-2B cells, and analyzes possible mechanical mechanisms. In the experiments, exosomes were isolated from the culture supernatants of H1299 cells by ultracentrifugation. The H1299-Exos were confirmed by scanning electron microscope (SEM) and western blotting (WB). The BEAS-2B cell migration was assessed using scratch assays. Cytoskeletal structure changes were detected by immunofluorescence. Surface morphology and mechanical properties were measured by atomic force microscopy (AFM). After incubation with H1299-Exos for 48 h, BEAS-2B cells enhanced migration ability, with increased filopodia and cytoskeletal rearrangements. The changes in the morphology and mechanical properties of the cells caused by H1299-Exos were detected using AFM, including the increase in cell length and the decrease in cell height, Young's modulus and adhesion. In short, H1299-Exos promoted the BEAS-2B cell migrations. It indicates that the morphological and mechanical properties can be used as a means to assess normal cell alterations induced by tumor cell derived-exosomes. This provides a method for studying the effects of exosomes secreted by tumor cells on normal cells and the changes in their physical properties.
    • Milk fat globule membrane protein promotes C2C12 cell proliferation through the PI3K/Akt signaling pathway

      Li, He; Xu, Weili; Ma, Ying; Zhou, Shaobo; Xiao, Ran; Harbin Institute of Technology; University of Bedfordshire (Elsevier, 2018-04-07)
      Milk fat globule membrane (MFGM) protein is known to have several health benefits, including an anti-sarcopenia effect; however, its mechanism is unclear. The aim of this study was to investigate the potential mechanism of action of the MFGM protein. The MFGM protein was extracted and separated into 4 fractions, and Fraction 2 (57 % of total MFGM) demonstrated the greatest effect on C2C12 cell proliferation. Milk fat globule-EGF factor 8 (MFG-E8) accounted for 82.35 % of the MFGM protein. The effects of whole Fraction 2 (100 μg/mL, 200 μg/mL and 300 μg/mL) on cell proliferation and morphology were measured. Using qRT-PCR or a Western blot assay, several regulatory factors, e.g., PI3K P85α, p-pI3K p85α (Tyr 508), Akt, p-Akt (Ser 473), mTOR and p-mTOR (Ser 2448), were measured in cells incubated with 200 μg/mL of Fraction 2 with or without wortmannin. The results demonstrated that Fraction 2 induced C2C12 cell proliferation in a dose-dependent manner, upregulated the mRNA expression of mTOR and p70S6K, and activated PI3K, Akt, mTOR and P70S6K phosphorylation; however, Fraction 2 inhibited FOXO3a and 4E-BP. The results demonstrate that the MFGM protein, predominantly MFG-E8, promotes cell proliferation through the PI3K/Akt/mTOR signaling pathway. This study elucidated the molecular mechanism of the MFGM protein, primarily MFG-E8, in promoting C2C12 cell proliferation via the PI3K/Akt/mTOR/P70S6K signal pathway.
    • Mining threatens Colombian ecosystems

      Pérez-Escobar, Oscar Alejandro; Cámara-Leret, Rodrigo; Antonelli, Alexandre; Bateman, Richard M.; Bellot, Sidonie; Chomicki, Guillaume; Cleef, Antoine; Diazgranados, Mauricio; Dodsworth, Steven; Jaramillo, Carlos; et al. (American Association for the Advancement of Science, 2018-03-30)
    • Modelling, inference and big data in biophysics

      Ho, Joshua W.K.; Grant, Guy H.; Victor Chang Cardiac Research Institute; University of New South Wales; University of Bedfordshire (Springer, 2017-08-31)
      In recognition of the increasing importance of big data in biophysics, a new session called 'Modelling, inference, big data' is incorporated into the IUPAB/EBSA Congress on 18 July 2017 at Edinburgh, UK.
    • Mutagenic and chemical modification of the ABA-1 allergen of the nematode Ascaris: consequences for structure and lipid binding properties

      McDermott, Lindsay C.; Moore, Joyce; Brass, Andrew; Price, Nicholas C.; Kelly, Sharon M.; Cooper, Alan; Kennedy, Malcolm W.; University of Glasgow; University of Manchester (American Chemical Society, 2001-08-21)
      The polyprotein allergens/antigens of nematodes (NPAs) are the only lipid binding proteins known to be produced as polyproteins. Cleavage of the large polyprotein precursors at regularly spaced proteinase cleavage sites produces 10 or 11 individual protein units of approximately 15 kDa. The sequences of these units are highly diverse within and between species, but there are five absolutely or strongly conserved amino acid positions (Trp15, Gln20, Leu42, Cys64, and Cys120). We have tested the role of these signature amino acids by mutational or chemical alteration of the ABA-1 protein of Ascaris, and examined the resulting modified proteins for perturbations of their lipid binding activities and structural integrity. Substitution of Trp15 and Gln20 both affect the stability of the protein in terms of resistance to thermal or chemical denaturation, but the ligand binding function is unaffected. Mutation of Leu42, however, disrupts both the protein's structural stability and functional integrity, as does chemical disruption of the disulfide bridge formed between Cys64 and Cys120. We also find that the C-terminal, but not the N-terminal, half of the protein binds fatty acids, indicating that the binding site may be confined to this part of the protein. This also supports the idea that the NPA units are themselves derived from an ancient duplication event, and that they may comprise two functionally distinct domains.
    • Negative regulation of autophagy in activating nucleotide-binding oligomerization domain-like receptor family pyrin domain-containing 3 inflammasomes in the hippocampus of an epilepsy rat model

      Wu, Jia-Mei; Chen, Liqiang; Wang, Shuo; Li, Yingyu; Liu, Lei; Chen, Guang; Wang, Shu-Qiu; Zhou, Shaobo (American Scientific Publishers, 2019-06-25)
      Epilepsy, characterized by unpredictable and periodic seizures, is associated with chronic hippocampal inflammation and autophagy. Moreover, a molecular relationship between autophagy and inflammation in neurodegenerative disorders has been reported, highlighting the role of autophagy in the regulation of inflammation. To the best of our knowledge, there is no previous evidence of an association between nucleotide-binding oligomerization domain-like receptor family pyrin domaincontaining 3 (NLRP3) inflammasome and autophagy in epilepsy. Hence, we in this study aimed at investigating the possible association between NLRP3 inflammasome activation and autophagy in the development of epilepsy. A rat model of temporal lobe epilepsy was induced with lithiumpilocarpine. Five groups, i.e., control (n=20), status epilepticus (SE, n=30), SE+control (siRNA; n=15), SE+NLRP3 siRNA (n=30), and SE+wortmannin (n=30), were investigated. Real-time quantitative reverse transcription polymerase chain reaction, Western blotting, and quantum dotbased immunohistochemistry were used to detect the mRNA/protein expression levels of NLRP3, caspase-1, interleukin (IL)-1, IL-18, Beclin-1, and microtubule-associated protein light chain 3 (LC3) in the hippocampus. In addition, transmission electron microscopy was utilized to investigate autophagosome in the hippocampus of SE rats. We found that mRNA and protein expressions of NLRP3, caspase-1, IL-1, IL-18, LC3, and Beclin-1 were activated in the hippocampus. Gene silencing of NLRP3 suppressed caspase-1, IL-1, and IL-18 release and significantly ameliorated hippocampal damage. Furthermore, the LC3 and Beclin-1 expression levels decreased significantly after treatment with wortmannin. Importantly, NLRP3 inflammasome activation and IL-1/IL-18 releases were significantly enhanced after treatment with wortmannin, which implied a negative association between autophagy inhibition and NLRP3 inflammasome activation. Our study provides the first evidence that autophagy plays an important role in NLRP3 inflammasome activation in the development of epilepsy. These findings suggest that regulation of autophagy may be a promising potential strategy for treating patients with epilepsy.
    • A new family of periplasmic-binding proteins that sense arsenic oxyanions

      Badilla, Consuelo; Osborne, Thomas H.; Cole, Ambrose; Watson, Cameron; Djordjevic, Snezana; Santini, Joanne M. (SpringerNature, 2018-04-19)
      Arsenic contamination of drinking water affects more than 140 million people worldwide. While toxic to humans, inorganic forms of arsenic (arsenite and arsenate), can be used as energy sources for microbial respiration. AioX and its orthologues (ArxX and ArrX) represent the first members of a new sub-family of periplasmic-binding proteins that serve as the first component of a signal transduction system, that’s role is to positively regulate expression of arsenic metabolism enzymes. As determined by X-ray crystallography for AioX, arsenite binding only requires subtle conformational changes in protein structure, providing insights into protein-ligand interactions. The binding pocket of all orthologues is conserved but this alone is not sufficient for oxyanion selectivity, with proteins selectively binding either arsenite or arsenate. Phylogenetic evidence, clearly demonstrates that the regulatory proteins evolved together early in prokaryotic evolution and had a separate origin from the metabolic enzymes whose expression they regulate.
    • Non-destructive genome skimming for aquatic copepods

      Vakati, Vinod; Dodsworth, Steven; Neijiang Normal University; Hanyang University; University of Bedfordshire (Springer, 2020-01-21)
      Copepods are important ecologically and represent a large amount of aquatic biomass in both freshwater and marine systems. Despite this, the taxonomy of copepods and other meiofauna is not well understood, hampered by tiny sizes, cryptic taxa, intraspecific polymorphisms and total specimen destruction where DNA methods are employed. In this article we highlight these issues and propose a more up-to-date approach for dealing with them. Namely, we recommend non-destructive DNA extraction methods, coupled with high-throughput sequencing (HTS). Whilst DNA yields may be low, they should still be sufficient for HTS library preparation and DNA sequencing. At the same time morphological specimens can be preserved and the crucial link between morphology and DNA sequence is maintained. This is critical for an integrative taxonomy and a fuller understanding of biodiversity patterns as well as evolutionary processes in meiofauna.
    • Non-redundant role for the transcription factor Gli1 at multiple stages of thymocyte development

      Drakopoulou, Ekati; Outram, Susan V.; Rowbotham, Nicola J.; Ross, Susan; Furmanski, Anna L.; Saldana, Jose Ignacio; Hager-Theodorides, Ariadne L.; Crompton, Tessa (Taylor & Francis, 2010-10-15)
      The Hedgehog (Hh) signaling pathway influences multiple stages of murine T-cell development. Hh signaling mediates transcriptional changes by the activity of the Gli family of transcription factors, Gli1, Gli2 and Gli3. Both Gli2 and Gli3 are essential for mouse developmentand can be processed to function as transcriptional repressors or transcriptional activators, whereas Gli1, itself a transcriptional target of Hh pathway activation, can only function as a transcriptional activator and is not essential for mouse development. Gli1-deficient mice are healthy and appear normal and nonredundant functions for Gli1 have been difficult to identify. Here we show that Gli1 is non-redundant in the regulation of T-cell development in the thymus, at multiple developmental stages. Analysis of Gli1-deficient embryonic mouse thymus shows a role for Gli1 to promote the differentiation of CD4⁻CD8⁻ double negative (DN) thymocytes before pre- TCR signal transduction, and a negative regulatory function after pre-TCR signaling. In addition, introduction of a Class I-restricted transgenic TCR into the adult Gli1-deficient and embryonic Gli2-deficient thymus showed that both Gli1 and Gli2 influence its selection to the CD8 lineage.
    • A novel 3D in vitro model of glioblastoma reveals resistance to temozolomide which was potentiated by hypoxia

      Musah-Eroje, Ahmed; Watson, Sue; University of Nottingham; University of Bedfordshire (Springer, 2019-01-29)
      Glioblastoma (GBM) is the most common invasive malignant brain tumour in adults. It is traditionally investigated in vitro by culturing cells as a monolayer (2D culture) or as neurospheres (clusters enriched in cancer stem cells) but neither system accurately reflects the complexity of the three-dimensional (3D) chemoresistant microenvironment of GBM. Using three GBM cell-lines (U87, U251, and SNB19), the effect of culturing cells in a Cultrex-based basement membrane extract (BME) [3D Tumour Growth Assay (TGA)] on morphology, gene expression, metabolism, and temozolomide chemoresistance was investigated. Cells were easily harvested from the 3D model and cultured as a monolayer (2D) and neurospheres. Indeed, the SNB19 cells formed neurospheres only after they were first cultured in the 3D model. The expression of CD133 and OCT4 was upregulated in the neurosphere and 3D assays respectively. Compared with cells cultured in the 2D model, cells were more resistant to temozolomide in the 3D model and this resistance was potentiated by hypoxia. Taken together, these results suggest that micro-environmental factors influence GBM sensitivity to temozolomide. Knowledge of the mechanisms involved in temozolomide resistance in this 3D model might lead to the identification of new strategies that enable the more effective use of the current standard of care agents. PURPOSE MATERIALS AND METHODS RESULTS CONCLUSION
    • A novel approach to oral iron delivery using ferrous sulphate loaded solid lipid nanoparticles

      Zariwala, Mohammed Gulrez; Elsaid, Naba; Jackson, Timothy L.; Corral López, Francisco; Farnaud, Sébastien; Somavarapu, Satyanarayana; Renshaw, Derek; University of Westminster; University College London; King's College Hospital; et al. (Elsevier, 2013-11-18)
      Iron (Fe) loaded solid lipid nanoparticles (SLN's) were formulated using stearic acid and iron absorption was evaluated in vitro using the cell line Caco-2 with intracellular ferritin formation as a marker of iron absorption. Iron loading was optimised at 1% Fe (w/w) lipid since an inverse relation was observed between initial iron concentration and SLN iron incorporation efficiency. Chitosan (Chi) was included to prepare chitosan coated SLN's. Particle size analysis revealed a sub-micron size range (300.3±31.75 nm to 495.1±80.42 nm), with chitosan containing particles having the largest dimensions. As expected, chitosan (0.1%, 0.2% and 0.4% w/v) conferred a net positive charge on the particle surface in a concentration dependent manner. For iron absorption experiments equal doses of Fe (20 μM) from selected formulations (SLN-FeA and SLN-Fe-ChiB) were added to Caco-2 cells and intracellular ferritin protein concentrations determined. Caco-2 iron absorption from SLN-FeA (583.98±40.83 ng/mg cell protein) and chitosan containing SLN-Fe-ChiB (642.77±29.37 ng/mg cell protein) were 13.42% and 24.9% greater than that from ferrous sulphate (FeSO4) reference (514.66±20.43 ng/mg cell protein) (p≤0.05). We demonstrate for the first time preparation, characterisation and superior iron absorption in vitro from SLN's, suggesting the potential of these formulations as a novel system for oral iron delivery.
    • Novel classes of fatty acid and retinol binding protein from nematodes

      McDermott, Lindsay C.; Cooper, Alan; Kennedy, Malcolm W.; University of Glasgow (Springer, 1999-02-28)
      Parasitic nematodes have recently been found to produce proteins which represent two new classes of fatty acid and retinoid binding protein. The first is the nematode polyprotein allergens/antigens (NPAs) which, as their name suggests, are synthesised as large polyproteins which are subsequently cleaved at regularly spaced sites to form multiple copies of a fatty acid binding protein of approximately 14.5 kDa. Binding studies using molecular environment-sensitive fluorescent ligands have shown that the binding site is highly unusual, producing blue-shifting in fluorescence to an unprecedented degree, suggesting a remarkably non-polar environment and isolation from solvent water. Computer-based structural predictions and biophysical observations have identified the NPAs as highly helical proteins which might form a four helix bundle, so constitute a new class of lipid binding protein from animals. The second class, like the NPAs, binds both fatty acids and retinol, but with a higher affinity for the latter. These are also highly helical but are structurally distinct from the NPAs. The biological function of these new classes of protein are discussed in the context of both the metabolic requirements of the parasites and the possible role of the proteins in control of the immune and inflammatory environment of the tissue sites parasitised.
    • A novel human neuronal cell model to study iron accumulation in Parkinson’s disease

      Mehta, Kosha; Ahmed, Bushra Y.; Farnaud, Sébastien; University of Bedfordshire (OMICS International, 2019-02-11)
      Objectives: With an estimated seven to ten million sufferers worldwide, Parkinson’s disease (PD) is the second most common age-related neurodegenerative disorder. Progress in elucidating its causes has been slow, partly due to the lack of human-relevant models. Similarly, while the contribution of iron is increasingly advocated, identifying its role in disease progression remains challenging mainly due to the lack of valid model. In this study, we created Parkinson-like conditions in a human neuron model and conducted preliminary studies on iron-related parameters to assess whether these cells replicated iron accumulation observed in Parkinsonism. Methods: ReNcell VM (human neural progenitor) were differentiated into dopaminergic neurons (dDCNs) and treated with neurotoxin 6-hydroxy dopamine (100 μM) to mimic Parkinsonism. Total intracellular, mitochondrial and cytoplasmic iron was measured by ferrozine assay. Expression of iron-related genes TFRC, SLC40A1, HAMP and SLC25A37 were assessed through real-time PCR. Results: Data showed that the treated dDCNs accumulated iron over time and exceeded levels measured in untreated dDCNs by 2.5-fold at 48 h (p<0.02). Following the treatment, the treated cells showed lower expression of TFRC (p<0.05), but substantially higher mRNA expressions of SLC40A1 (9-fold; p<0.02) and HAMP (5.7-fold; p<0.05), along with higher intracellular iron (p<0.05). Higher iron accumulation in the mitochondria than cytosol (p<0.05), was also observed with increased expression of the mitochondrial iron-importer SLC25A37 (p=0.08). Conclusion: Our Parkinsonian model demonstrates iron accumulation and elevated HAMP expression as previously described in PD phenotype. The observed mitochondrial iron shuttling, which is proposed to be one of the primary contributors of oxidative stress in PD, calls for further investigation. The differences observed in distribution of iron in our human model and with the expression of major iron-related proteins, indicate that our model reproduces the disease state successfully, and suggests that further study could help in advancing our understanding of PD.
    • A novel orvinol analog, BU08028, as a safe opioid analgesic without abuse liability in primates

      Ding, Huiping; Czoty, Paul W.; Kiguchi, Norikazu; Cami-Kobeci, Gerta; Sukhtankar, Devki D.; Nader, Michael A.; Husbands, Stephen M.; Ko, Mei-Chuan; ; Wake Forest University; et al. (National Academy of Sciences, 2016-08-29)
      Despite the critical need, no previous research has substantiated safe opioid analgesics without abuse liability in primates. Recent advances in medicinal chemistry have led to the development of ligands with mixed mu opioid peptide (MOP)/nociceptin-orphanin FQ peptide (NOP) receptor agonist activity to achieve this objective. BU08028 is a novel orvinol analog that displays a similar binding profile to buprenorphine with improved affinity and efficacy at NOP receptors. The aim of this preclinical study was to establish the functional profile of BU08028 in monkeys using clinically used MOP receptor agonists for side-by-side comparisons in various wellhoned behavioral and physiological assays. Systemic BU08028 (0.001-0.01 mg/kg) produced potent long-lasting (i.e., >24 h) antinociceptive and antiallodynic effects, which were blocked by MOP or NOP receptor antagonists. More importantly, the reinforcing strength of BU08028 was significantly lower than that of cocaine, remifentanil, or buprenorphine in monkeys responding under a progressive-ratio schedule of drug self-administration. Unlike MOP receptor agonists, BU08028 at antinociceptive doses and ?10-to 30-fold higher doses did not cause respiratory depression or cardiovascular adverse events as measured by telemetry devices. After repeated administration, the monkeys developed acute physical dependence on morphine, as manifested by precipitated withdrawal signs, such as increased respiratory rate, heart rate, and blood pressure. In contrast, monkeys did not show physical dependence on BU08028. These in vivo findings in primates not only document the efficacy and tolerability profile of bifunctional MOP/NOP receptor agonists, but also provide a means of translating such ligands into therapies as safe and potentially abusefree opioid analgesics.
    • A novel role for Hedgehog in T-cell receptor signaling: implications for development and immunity

      Rowbotham, Nicola J.; Hager-Theodorides, Ariadne L.; Furmanski, Anna L.; Crompton, Tessa (Taylor & Francis, 2007-09-07)
      The Hedgehog (Hh) signaling pathway is a key regulator of both embryonic development and homeostasis of adult tissues, including thymus and blood. In the thymus, Hh signals for differentiation, survival and proliferation in the early stages of T cell development, before TCR gene rearrangement. Our recent data has shown that Hh signaling also modulates T cell receptor (TCR) signal strength in more mature T lineage cells. We showed that constitutive activation of the Hh pathway in thymocytes (by transgenic expression of the transcriptional activator form of Gli2) decreased TCR signal strength with profound consequences for the thymus--allowing self-reactive T cells to escape deletion and altering T cell CD4/CD8 lineage decisions. In contrast, in the Sonic Hh deficient thymus, TCR signaling was increased, again influencing both TCR repertoire selection and CD4/8 lineage commitment. In peripheral T cells, the transcriptional changes induced by activation of the Hh signaling pathway lead to reduced T cell activation. Hh signaling also attenuated ERK phosphorylation and proliferation in mature T cells on TCR ligation. Modulation of TCR signal strength by Hh pathway activation has importance for immunity as the presence or absence of Hh in the environment in which a T cell is activated would shape the immune response.