• Peptide-specific, TCR-alpha-driven, coreceptor-independent negative selection in TCR alpha-chain transgenic mice

      Furmanski, Anna L.; Bartok, Istvan; Chai, Jian-Guo; Singh, Yogesh; Ferreira, Cristina; Scott, Diane; Holland, Stephen J.; Bourdeaux, Christophe; Crompton, Tessa; Dyson, Julian (American Association of Immunologists, 2010-01-06)
      As thymocytes differentiate, Ag sensitivity declines, with immature CD4-CD8- double-negative (DN) cells being most susceptible to TCRsignaling events. We show that expression of alphabetaTCR from the DN3 stage lowers the threshold for activation, allowing recognition of MHC peptides independently of the TCR beta-chain and without either T cell coreceptor. The MHC class I-restricted C6 TCR recognizes the Y-chromosome-derived Ag HYK(k)Smcy. Positive selection in C6 alphabetaTCR females is skewed to the CD8 compartment, whereas transgenic male mice exhibit early clonal deletion of thymocytes. We investigated the effect of the HYK(k)Smcy complex on developing thymocytes expressing the C6 TCR alpha-chain on a TCR-alpha(-/-) background. On the original selecting haplotype, the skew to the CD8 lineage is preserved. This is MHC dependent, as the normal bias to the CD4 subset is seen on an H2b background. In male H2k C6 alpha-only mice, the presence of the HYK(k)Smcy complex leads to a substantial deletion of thymocytes from the DN subset. This phenotype is replicated in H2k C6 alpha-only female mice expressing an Smcy transgene. Deletion is not dependent on the beta variable segment of the C6 TCR or on a restricted TCR-beta repertoire. In contrast, binding of HYK(k)Smcy and Ag-specific activation of mature CD8+ T cells is strictly dependent on the original C6 beta-chain. These data demonstrate that, in comparison with mature T cells, alphabetaTCR+ immature thymocytes can recognize and transduce signals in response to specific MHC-peptide complexes with relaxed binding requirements.
    • Petal, sepal, or tepal? B-genes and monocot flowers

      Dodsworth, Steven; Royal Botanic Gardens, Kew (Elsevier Ltd, 2016-11-25)
      In petaloid monocots expansion of B-gene expression into whorl 1 of the flower results in two whorls of petaloid organs (tepals), as opposed to sepals in whorl 1 of typical eudicot flowers. Recently, new gene-silencing technologies have provided the first functional data to support this, in the genus Tricyrtis (Liliaceae).
    • Pharmacokinetics of radioimmunotherapeutic agent of direct labeling mAb 188Re-HAb18

      Lou, Chao; Chen, Zhi-Nan; Bian, Hui-Jie; Li, Jie; Zhou, Shaobo; (Baishideng Publishing Group, 2002-02-15)
      AIM:To label anti-hepatoma monoclonal antibody (mAb) fragment HAb18 F(ab')2 was labeled with 188Re for the pharmacokinetic model of 188Re-HAb18 F(ab')2 and to evaluate its pharmacokinetic parameters in hepatoma-bearing nude mice. METHODS:HAb18 F(ab')2 was directly labeled with 188Re using 2-mercaptoethanol (2-ME) as reducing agents. Labeling efficiency and immunoreactivity of 188Re-HAb18 F(ab')2 were evaluated by Whatman 3MM paper chromatography and live cell assay, respectively. Biodistribution analysis was also conducted in nude mice bearing human hepatoma in which animals were sacrificed at different time points (1, 4, 18, 24 and 24h) after 188Re-HAb18 F(ab')2 was injected through tail-vein into hepatoma-bearing nude mice. The blood and radioactivity of organs and mass were measured. The concentrations of (188)Re-HAb18 F(ab')2 were evaluated with a pharmacokinetic 3P97 software. RESULTS:The optimum labeling efficiency and immunoreactive fraction were 91.7% and 0.78% respectively. The parameters of 188Re-HAb18 F(ab')2 were: T (1/2),2.29 h;Vd,1.49 x 10(-9)L x Bq(-1);AUC, 20.49 x 10(9)Bq x h x L(-1);CL, 0.45 x 10(-3)L x h(-1). 188Re-HAb18 F(ab')2 could locate specially in hepatoma with high selective reactivity of HAb18 F(ab')2. 188Re-HAb18 F(ab')2 was mainly eliminated by kidney. The maximal tumor to blood ratio was at 48 h,and maximal tumor to liver ratio was at 18 h. CONCLUSION:The pharmacokinetics of 188Re-HAb18 F(ab')2 fit a l-compartment model. 188Re-HAb18 F(ab')2 can be uptaken selectively at the hepatoma site.
    • Phenotype of striatofugal medium spiny neurons in parkinsonian and dyskinetic nonhuman primates: a call for a reappraisal of the functional organization of the basal ganglia

      Nadjar, Agnes; Brotchie, Jonathan M.; Guigoni, Celine; Li, Qin; Zhou, Shaobo; Wang, Guijie; Ravenscroft, Paula; Georges, François; Crossman, Alan R.; Bezard, Erwan; et al. (Society for Neuroscience, 2006-08-23)
      The classic view of anatomofunctional organization of the basal ganglia is that striatopallidal neurons of the "indirect" pathway express D2 dopamine receptors and corelease enkephalin with GABA, whereas striatopallidal neurons of the "direct" pathway bear D1 dopamine receptors and corelease dynorphin and substance P with GABA. Although many studies have investigated the pathophysiology of the basal ganglia after dopamine denervation and subsequent chronic levodopa (L-dopa) treatment, none has ever considered the possibility of plastic changes leading to profound reorganization and/or biochemical phenotype modifications of medium spiny neurons. Therefore, we studied the phenotype of striatal neurons in four groups of nonhuman primates, including the following: normal, parkinsonian, parkinsonian chronically treated with L-dopa without exhibiting dyskinesia, and parkinsonian chronically treated with L-dopa exhibiting overt dyskinesia. To identify striatal cells projecting to external (indirect) or internal (direct) segments of the globus pallidus, the retrograde tracer cholera toxin subunit B (CTb) was injected stereotaxically into the terminal areas. Using immunohistochemistry techniques, brain sections were double labeled for CTb and dopamine receptors, opioid peptides, or the substance P receptor (NK1). We also used HPLC-RIA to assess opioid levels throughout structures of the basal ganglia. Our results suggest that medium spiny neurons retain their phenotype because no variations were observed in any experimental condition. Therefore, it appears unlikely that dyskinesia is related to a phenotype modification of the striatal neurons. However, this study supports the concept of axonal collateralization of striatofugal cells that project to both globus pallidus pars externa and globus pallidus pars interna. Striatofugal pathways are not as segregated in the primate as previously considered.
    • Phenotype of striatofugal medium spiny neurons in parkinsonian and dyskinetic nonhuman primates: a call for a reappraisal of the functional organization of the basal ganglia

      Nadjar, Agnes; Brotchie, Jonathan M.; Guigoni, Celine; Li, Qin; Zhou, Shaobo; Wang, Guijie; Ravenscroft, Paula; Georges, François; Crossman, Alan R.; Bezard, Erwan; et al. (Society for Neuroscience, 2006-08-23)
      The classic view of anatomofunctional organization of the basal ganglia is that striatopallidal neurons of the “indirect” pathway express D2 dopamine receptors and corelease enkephalin with GABA, whereas striatopallidal neurons of the “direct” pathway bear D1 dopamine receptors and corelease dynorphin and substance P with GABA. Although many studies have investigated the pathophysiology of the basal ganglia after dopamine denervation and subsequent chronic levodopa (l-dopa) treatment, none has ever considered the possibility of plastic changes leading to profound reorganization and/or biochemical phenotype modifications of medium spiny neurons. Therefore, we studied the phenotype of striatal neurons in four groups of nonhuman primates, including the following: normal, parkinsonian, parkinsonian chronically treated with l-dopa without exhibiting dyskinesia, and parkinsonian chronically treated with l-dopa exhibiting overt dyskinesia. To identify striatal cells projecting to external (indirect) or internal (direct) segments of the globus pallidus, the retrograde tracer cholera toxin subunit B (CTb) was injected stereotaxically into the terminal areas. Using immunohistochemistry techniques, brain sections were double labeled for CTb and dopamine receptors, opioid peptides, or the substance P receptor (NK1). We also used HPLC-RIA to assess opioid levels throughout structures of the basal ganglia. Our results suggest that medium spiny neurons retain their phenotype because no variations were observed in any experimental condition. Therefore, it appears unlikely that dyskinesia is related to a phenotype modification of the striatal neurons. However, this study supports the concept of axonal collateralization of striatofugal cells that project to both globus pallidus pars externa and globus pallidus pars interna. Striatofugal pathways are not as segregated in the primate as previously considered.
    • Phylogenetic signal from genomic repeat abundances of hominid primates

      Martín-Peciña, María; Ruiz-Ruano, Francisco J.; Camacho, Juan Pedro M.; Dodsworth, Steven (2018-11-25)
    • Phylogenetic signal of genomic repeat abundances can be distorted by random homoplasy: a case study from hominid primates

      Martín-Peciña, Maria; Ruiz-Ruano, Francisco J.; Camacho, Juan Pedro M.; Dodsworth, Steven (Oxford University Press (OUP), 2019-02-11)
      The genomic abundance of different types of repetitive DNA elements contains a phylogenetic signal useful for inferring the evolutionary history of different groups of organisms. Here we test the reliability of this approach using the Hominidae family of primates, whose consensus phylogeny is well accepted. We used the software RepeatExplorer to identify the different repetitive DNA clusters and quantify their abundances. With these data, we performed phylogenetic analyses by maximum parsimony, including one, two or three individuals per species, technical replicates, and including or discarding two clusters of repetitive elements (i.e. a satellite DNA and an endogenous retrovirus) that generated random homoplasy, because they were abundant in Pan and Gorilla but almost absent in Homo and Pongo. The only phylogenetic tree congruent with the accepted topology for hominids, thus coinciding with that obtained from the mitogenomes of the same individuals, was the one built after filtering out the libraries for the two homoplasious clusters and using three individuals per species. Our results suggest some caution in the use of repeat abundance for phylogenetic studies, because some element abundances are homoplasious, which severely distorts the phylogenetic signal owing to their differential amplification among evolutionary lineages.
    • Plastid phylogenomics resolves ambiguous relationships within the orchid family and provides a solid timeframe for biogeography and macroevolution

      Serna-Sánchez, Maria Alejandra; Pérez-Escobar, Oscar A.; Bogarín, Diego; Torres-Jimenez, María Fernanda; Alvarez-Yela, Astrid Catalina; Arcila-Galvis, Juliana E.; Hall, Climbie F.; de Barros, Fábio; Pinheiro, Fábio; Dodsworth, Steven; et al. (SpringerNature, 2021-03-25)
      Recent phylogenomic analyses based on the maternally inherited plastid organelle have enlightened evolutionary relationships between the subfamilies of Orchidaceae and most of the tribes. However, uncertainty remains within several subtribes and genera for which phylogenetic relationships have not ever been tested in a phylogenomic context. To address these knowledge-gaps, we here provide the most extensively sampled analysis of the orchid family to date, based on 78 plastid coding genes representing 264 species, 117 genera, 18 tribes and 28 subtribes. Divergence times are also provided as inferred from strict and relaxed molecular clocks and birth-death tree models. Our taxon sampling includes 51 newly sequenced plastid genomes produced by a genome skimming approach. We focus our sampling efforts on previously unplaced clades within tribes Cymbidieae and Epidendreae. Our results confirmed phylogenetic relationships in Orchidaceae as recovered in previous studies, most of which were recovered with maximum support (209 of the 262 tree branches). We provide for the first time a clear phylogenetic placement for Codonorchideae within subfamily Orchidoideae, and Podochilieae and Collabieae within subfamily Epidendroideae. We also identify relationships that have been persistently problematic across multiple studies, regardless of the different details of sampling and genomic datasets used for phylogenetic reconstructions. Our study provides an expanded, robust temporal phylogenomic framework of the Orchidaceae that paves the way for biogeographical and macroevolutionary studies.
    • The polyprotein and FAR lipid binding proteins of nematodes: shape and monomer/dimer states in ligand-free and bound forms

      Solovyova, Alexandra S.; Meenan, Nicola; McDermott, Lindsay C.; Garofalo, Antonio; Bradley, Jannette E.; Kennedy, Malcolm W.; Byron, Olwyn; University of Glasgow; National Academy of Science of the Ukraine; University of Nottingham (Springer, 2003-08-31)
      Nematodes produce two classes of small, helix-rich fatty acid- and retinol-binding proteins whose structures and in vivo functions remain to be elucidated. These are the polyprotein allergens (NPA) and the FAR proteins. The solution properties of recombinant forms of these proteins from parasitic [Ascaris suum (As) and Onchocerca volvulus (Ov)] and free-living [Caenorhabditis elegans (Ce)] nematodes have been examined. Analytical ultracentrifugation (AUC) showed that, contrary to previous findings, the rAs-NPA-1A polyprotein unit (approximately 15 kDa) is a monomer, and this stoichiometry is unaltered by ligand (oleic acid) binding. The rOv-FAR-1 and rCe-FAR-5 proteins differ in that the former forms a tight dimer and the latter a monomer, and these oligomeric states are also unaffected by ligand binding or protein concentration. Sedimentation equilibrium experiments showed that the partial specific volume v of the unliganded proteins agree well with the value calculated from amino acid composition extrapolated to experimental temperature, and was unaffected upon ligand binding. Data from small-angle X-ray scattering (SAXS) indicated that both of the monomeric proteins rAs-NPA-1A and rCe-FAR-5 are globular, although slightly elongated and flattened. These data are in good agreement with shapes predicted from sedimentation velocity experiments and hydrodynamic bead modelling. On the basis of functional and secondary structural homology with the ligand-binding domain of the retinoic acid receptor RXRalpha, de novo atomic resolution structures for rAs-NPA-1A and rCe-FAR-5 have been constructed which are consistent with the SAXS and hydrodynamic data.
    • Potential of herbariomics for studying repetitive DNA in angiosperms

      Dodsworth, Steven; Guignard, Maite S.; Christenhusz, Maarten J.M.; Cowan, Robyn S.; Knapp, Sandra; Maurin, Olivier; Struebig, Monika; Leitch, Andrew R.; Chase, Mark W.; Forest, Felix; et al. (Frontiers Media, 2018-10-29)
      Repetitive DNA has an important role in angiosperm genomes and is relevant to our understanding of genome size variation, polyploidisation and genome dynamics more broadly. Much recent work has harnessed the power of high-throughput sequencing (HTS) technologies to advance the study of repetitive DNA in flowering plants. Herbarium collections provide a useful historical perspective on genome diversity through time, but their value for the study of repetitive DNA has not yet been explored. We propose that herbarium DNA may prove as useful for studies of repetitive DNA content as it has for reconstructed organellar genomes and low-copy nuclear sequence data. Here we present a case study in the tobacco genus (Nicotiana; Solanaceae), showing that herbarium specimens can provide accurate estimates of the repetitive content of angiosperm genomes by direct comparison with recently-collected material. We show a strong correlation between the abundance of repeat clusters, e.g., different types of transposable elements and satellite DNA, in herbarium collections versus recent material for four sets of Nicotiana taxa. These results suggest that herbarium specimen genome sequencing (herbariomics) holds promise for both repeat discovery and analyses that aim to investigate the role of repetitive DNAs in genomic evolution, particularly genome size evolution and/or contributions of repeats to the regulation of gene space.
    • The prediction of miRNAs in SARS-CoV-2 genomes: hsa-miR databases identify 7 key miRs linked to host responses and virus pathogenicity-related KEGG pathways significant for comorbidities

      Arisan, Elif Damla; Dart, Alwyn; Grant, Guy H.; Arisan, Serdar; Cuhadaroglu, Songul; Lange, Sigrun; Uysal-Onganer, Pinar; ; Gebze Technical University; St George’s University of London; et al. (MDPI, 2020-06-04)
      Severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) is a member of the betacoronavirus family, which causes COVID-19 disease. SARS-CoV-2 pathogenicity in humans leads to increased mortality rates due to alterations of significant pathways, including some resulting in exacerbated inflammatory responses linked to the "cytokine storm" and extensive lung pathology, as well as being linked to a number of comorbidities. Our current study compared five SARS-CoV-2 sequences from different geographical regions to those from SARS, MERS and two cold viruses, OC43 and 229E, to identify the presence of miR-like sequences. We identified seven key miRs, which highlight considerable differences between the SARS-CoV-2 sequences, compared with the other viruses. The level of conservation between the five SARS-CoV-2 sequences was identical but poor compared with the other sequences, with SARS showing the highest degree of conservation. This decrease in similarity could result in reduced levels of transcriptional control, as well as a change in the physiological effect of the virus and associated host-pathogen responses. MERS and the milder symptom viruses showed greater differences and even significant sequence gaps. This divergence away from the SARS-CoV-2 sequences broadly mirrors the phylogenetic relationships obtained from the whole-genome alignments. Therefore, patterns of mutation, occurring during sequence divergence from the longer established human viruses to the more recent ones, may have led to the emergence of sequence motifs that can be related directly to the pathogenicity of SARS-CoV-2. Importantly, we identified 7 key-microRNAs (miRs 8066, 5197, 3611, 3934-3p, 1307-3p, 3691-3p, 1468-5p) with significant links to KEGG pathways linked to viral pathogenicity and host responses. According to Bioproject data (PRJNA615032), SARS-CoV-2 mediated transcriptomic alterations were similar to the target pathways of the selected 7 miRs identified in our study. This mechanism could have considerable significance in determining the symptom spectrum of future potential pandemics. KEGG pathway analysis revealed a number of critical pathways linked to the seven identified miRs that may provide insight into the interplay between the virus and comorbidities. Based on our reported findings, miRNAs may constitute potential and effective therapeutic approaches in COVID-19 and its pathological consequences.
    • Proteomic analysis of age-related changes in ovine cerebrospinal fluid

      Chen, Carl P.C.; Preston, Jane E.; Zhou, Shaobo; Fuller, Heidi R.; Morgan, David G.A.; Chen, Ruoli; King’s College London; Chang Gung University; University of Bedfordshire; RJAH Orthopaedic Hospital; et al. (Elsevier, 2018-04-21)
      Cerebrospinal fluid (CSF) circulates through the brain and has a unique composition reflecting the biological processes of the brain. Identifying ageing CSF biomarkers can aid in understanding the ageing process and interpreting CSF protein changes in neurodegenerative diseases. In this study, ovine CSF proteins from young (1-2 year old), middle aged (3-6 year old) and old (7-10 year old) sheep were systemically studied. CSF proteins were labelled with iTRAQ tagging reagents and fractionated by 2-dimensional high performance, liquid chromatography. Tryptic peptides were identified using MS/MS fragmentation ions for sequencing and quantified from iTRAQ reporter ion intensities at m/z 114, 115, 116 and 117. Two hundred thirty one peptides were detected, from which 143 proteins were identified. There were 52 proteins with >25% increase in concentrations in the old sheep compared to the young. 33 of them increased >25% but <50%, 13 increased >50% but <1 fold, 6 increased >1 fold [i.e. haptoglobin (Hp), haemoglobin, neuroendocrine protein 7B2, IgM, fibrous sheath interacting protein 1, vimentin]. There were 18 proteins with >25% decrease in concentrations in the old sheep compared to the young. 17 of them decreased >25% but <50%, and histone deacetylase 7 (HDAC7) was gradually decreased for over 80%. Glutathione S-transferase was decreased in middle aged CSF compared to both young and old CSF. The differential expressions of 3 proteins (Hp, neuroendocrine protein 7B2, IgM) were confirmed by immunoassays. These data expand our current knowledge regarding ovine CSF proteins, supply the necessary information to understand the ageing process in the brain and provide a basis for diagnosis of neurodegenerative diseases.
    • Public T cell receptor beta-chains are not advantaged during positive selection

      Furmanski, Anna L.; Ferreira, Cristina; Bartok, Istvan; Dimakou, Sofia; Rice, Jason; Stevenson, Freda; Millrain, Maggie M.; Simpson, Elizabeth; Dyson, Julian (American Association of Immunologists, 2008-01-04)
      Studies of human and murine T cells have shown that public TCR beta-chain rearrangements can dominate the Ag-specific and naive repertoires of distinct individuals. We show that mouse T cells responding to the minor histocompatibility Ag HYDbSmcy share an invariant Vbeta8.2-Jbeta2.3 TCR gene rearrangement. The dominance of this rearrangement shows that it successfully negotiated thymic selection and was highly favored during clonal expansion in all animals examined. We hypothesized that such beta-chains are advantaged during thymic and/or peripheral selection and, as a result, may be over-represented in the naive repertoire. A sequencing study was undertaken to examine the diversity of Vbeta8.2-Jbeta2.3 CDR3 loops from naive T cell repertoires of multiple mice. Public TCR beta-chain sequences were identified across different repertoires and MHC haplotypes. To determine whether such public beta-chains are advantaged during thymic selection, individual chains were followed through T cell development in a series of novel bone marrow competition chimeras. We demonstrate that beta-chains were positively selected with similar efficiency regardless of CDR3 loop sequence. Therefore, the establishment and maintenance of public beta-chains in the periphery is predominantly controlled by post-thymic events through modification of the primary, thymus-derived TCR repertoire.
    • Purification and identification of novel xanthine oxidase inhibitory peptides derived from round scad (Decapterus maruadsi) protein hydrolysates

      Hu, Xiao; Zhou, Ya; Zhou, Shaobo; Chen, Shengjun; Wu, Yanyan; Li, Laihao; Yang, Xianqing; Chinese Academy of Fishery Sciences; Jiangsu Ocean University; Shanghai Ocean University; et al. (MDPI, 2021-09-24)
      The objective of the present study was to investigate the xanthine oxidase (XO) inhibitory effects of peptides purified and identified from round scad (Decapterus maruadsi) hydrolysates (RSHs). In this study, RSHs were obtained by using three proteases (neutrase, protamex and alcalase). Among them, the RSHs of 6-h hydrolysis by neutrase displayed the strongest XO inhibitory activity and had an abundance of small peptides (&lt;500 Da). Four novel peptides were purified by immobilized metal affinity chromatography and identified by nano-high-performance liquid chromatography mass/mass spectrometry. Their amino acid sequences were KGFP (447.53 Da), FPSV (448.51 Da), FPFP (506.59 Da) and WPDGR (629.66 Da), respectively. Then the peptides were synthesized to evaluate their XO inhibitory activity. The results indicated that the peptides of both FPSV (5 mM) and FPFP (5 mM) exhibited higher XO inhibitory activity (22.61 +- 1.81% and 20.09 +- 2.41% respectively). Fluorescence spectra assay demonstrated that the fluorescence quenching mechanism of XO by these inhibitors (FPSV and FPFP) was a static quenching procedure. The study of inhibition kinetics suggested that the inhibition of both FPSV and FPFP was reversible, and the type of their inhibition was a mixed one. Molecular docking revealed the importance of π-π stacking between Phe residue (contained in peptides) and Phe914 (contained in the XO) in the XO inhibitory activity of the peptides.
    • QSAR and molecular docking for the search of AOX inhibitors: a rational drug discovery approach

      Rosell-Hidalgo, Alicia; Young, Luke; Moore, Anthony L.; Ghafourian, Taravat; University of Sussex; University of Bedfordshire (Springer Science and Business Media Deutschland GmbH, 2020-12-08)
      The alternative oxidase (AOX) is a monotopic diiron carboxylate protein that catalyses the oxidation of ubiquinol and the reduction of oxygen to water. Although a number of AOX inhibitors have been discovered, little is still known about the ligand–protein interaction and essential chemical characteristics of compounds required for a potent inhibition. Furthermore, owing to the rapidly growing resistance to existing inhibitors, new compounds with improved potency and pharmacokinetic properties are urgently required. In this study we used two computational approaches, ligand–protein docking and Quantitative Structure–Activity Relationships (QSAR) to investigate binding of AOX inhibitors to the enzyme and the molecular characteristics required for inhibition. Docking studies followed by protein–ligand interaction fingerprint (PLIF) analysis using the AOX enzyme and the mutated analogues revealed the importance of the residues Leu 122, Arg 118 and Thr 219 within the hydrophobic cavity. QSAR analysis, using stepwise regression analysis with experimentally obtained IC50 values as the response variable, resulted in a multiple regression model with a good prediction accuracy. The model highlighted the importance of the presence of hydrogen bonding acceptor groups on specific positions of the aromatic ring of ascofuranone derivatives, acidity of the compounds, and a large linker group on the compounds on the inhibitory effect of AOX.
    • A quantitative investigation into the losses of proteins at different stages of a two-dimensional gel electrophoresis procedure

      Zhou, Shaobo; Bailey, Matthew J.; Dunn, Michael J.; Preedy, Victor R.; Emery, Peter W.; (Wiley, 2005-08-01)
      We report the results of a systematic investigation to quantify the losses of protein during a well-established two-dimensional polyacrylamide gel electrophoresis (2-DE) procedure. Radioactively labelled proteins ([(14)C]bovine serum albumin and a homogenate prepared from the liver of a rat that had been injected with [(35)S]methionine) were used, and recovery was quantified by digesting pieces of gel in H(2)O(2) and subjecting the digests to liquid scintillation counting. When samples were loaded onto the first dimension immobilised pH gradient strips by in-gel rehydration, recovery of protein from the strips was 44-80% of the amount of protein loaded, depending on the amount of protein in the sample. Most of the unrecovered protein appeared to have adhered to the reswelling tray. Losses during isoelectric focusing (IEF) were much smaller (7-14%), although approximately 2% of the protein appeared to migrate from sample strips to adjacent blank strips in the focussing apparatus. A further 17-24% of the proteins were lost into the buffers during equilibration prior to running in the second dimension. Losses during the second dimension run and subsequent staining with SYPRO Ruby amounted to less than 10%. The overall loss during 2-DE was reduced by approximately 25% when proteins were loaded onto the IEF strips using sample cups instead of by in-gel rehydration. These extensive and variable losses during the 2-DE procedure mean that spot intensities on 2-DE gels cannot be used to derive reliable, quantitative information on the amounts of proteins present in the original sample.
    • Reconstructing phylogenetic relationships based on repeat sequence similarities

      Vitales, Daniel; Garcia, Sonia; Dodsworth, Steven; Institut Botànic de Barcelona; Universitat de Barcelona; University of Bedfordshire (Elsevier, 2020-02-28)
      A recent phylogenetic method based on genome-wide abundance of different repeat types proved to be useful in reconstructing the evolutionary history of several plant and animal groups. Here, we demonstrate that an alternative information source from the repeatome can also be employed to infer phylogenetic relationships among taxa. Specifically, this novel approach makes use of the repeat sequence similarity matrices obtained from the comparative clustering analyses of RepeatExplorer 2, which are subsequently transformed to between-taxa distance matrices. These pairwise matrices are used to construct neighbour-joining trees for each of the top most-abundant clusters and they are finally summarized in a consensus network. This methodology was tested on three groups of angiosperms and one group of insects, resulting in congruent evolutionary hypotheses compared to more standard systematic analyses based on commonly used DNA markers. We propose that the combined application of these phylogenetic approaches based on repeat abundances and repeat sequence similarities could be helpful to understand mechanisms governing genome and repeatome evolution.
    • Regulation of fatty acid binding proteins by hypoxia inducible factors 1α and 2α in the placenta: Relevance to pre-eclampsia

      Jadoon, Ayesha; Cunningham, Phil; McDermott, Lindsay C. (Elsevier, 2014-09-25)
      Pre-eclampsia is characterized by placental hypoxia and dyslipidemia. Arachidonic and docosahexanoic acids are essential maternal nutrients for fetal development. They are transported via placental trophoblast cells by membrane and cytosolic fatty acid binding proteins. Others report the expressions of these proteins which are increased in hypoxic trophoblasts. Using bioinformatics, BeWo cells, reporter assays, quantitative real-time PCR and immunoblotting we tested the hypothesis that hypoxia inducible factors 1α (HIF-1α) and/ or 2α (HIF-2α) regulate the expressions of FABP1, FABP3, FABP4 and FATP2 proteins. Three hypoxia responsive elements (HRE) were identified in FABP1 which cumulatively responded strongly to HIF-1α and weakly to HIF-2α. FABP3 expression partially responded to HIF-1α. Two putative HRE were validated in FABP4 both of which responded weakly toHIF-1α and HIF-2α. FATP2 protein expression reacted positively to hypoxia. Thus, fetal essential fatty acid supply via the placenta is protected under hypoxia. It will be interesting to determine if our findings are replicated in human pre-eclamptic placenta.
    • Regulation of murine normal and stress-induced erythropoiesis by Desert Hedgehog

      Lau, Ching-In; Outram, Susan V.; Saldaña, José Ignacio; Furmanski, Anna L.; Dessens, Johannes T.; Crompton, Tessa (American Society of Hematology, 2012-05-17)
      The function of Hedgehog signaling in hematopoiesis is controversial, with different experimental systems giving opposing results. Here we examined the role of Desert Hedgehog (Dhh) in the regulation of murine erythropoiesis. Dhh is one of 3 mammalian Hedgehog family proteins. Dhh is essential for testis development and Schwann cell function. We show, by analysis of Dhh-deficient mice, that Dhh negatively regulates multiple stages of erythrocyte differentiation. In Dhh-deficient bone marrow, the common myeloid progenitor (CMP) population was increased, but differentiation from CMP to granulocyte/macrophage progenitor was decreased, and the mature granulocyte population was decreased, compared with wild-type (WT). In contrast, differentiation from CMP to megakaryocyte/erythrocyte progenitor was increased, and the megakaryocyte/erythrocyte progenitor population was increased. In addition, we found that erythroblast populations were Dhh-responsive in vitro and ex vivo and that Dhh negatively regulated erythroblast differentiation. In Dhh-deficient spleen and bone marrow, BFU-Es and erythroblast populations were increased compared with WT. During recovery of hematopoiesis after irradiation, and under conditions of stress-induced erythropoiesis, erythrocyte differentiation was accelerated in both spleen and bone marrow of Dhh-deficient mice compared with WT.
    • Repeated parallel losses of inflexed stamens in Moraceae: phylogenomics and generic revision of the tribe Moreae and the reinstatement of the tribe Olmedieae (Moraceae)

      Gardner, Elliot M.; Garner, Mira; Cowan, Robyn S.; Dodsworth, Steven; Epitawalage, Niroshini; Arifiani, Deby; Baker, William J.; Forest, Felix; Maurin, Olivier; Zerega, Nyree JC; et al. (Wiley, 2021-07-06)
      We present a densely sampled phylogenomic study of the mulberry tribe (Moreae, Moraceae), an economically important clade with a global distribution, revealing multiple losses of inflexed stamens, a character traditionally used to circumscribe Moreae. Inflexed stamens facilitate ballistic pollen release and are associated with wind pollination, and the results presented here suggest that losses of this character state may have evolved repeatedly in Moraceae. Neither Moreae nor several of its major genera (Morus, Streblus, Trophis) were found to be monophyletic. A revised system for a monophyletic Moreae is presented, including the reinstatement of the genera Ampalis, Maillardia, Taxotrophis, and Paratrophis, and the recognition of the new genus Afromorus. Pseudostreblus is reinstated and transferred to the Parartocarpeae, and Sloetiopsis is reinstated and transferred to the Dorstenieae. The tribe Olmedieae is reinstated, replacing the Castilleae, owing to the reinstatement of the type Olmedia and its exclusion from Moreae. Streblus s.str. is excluded from Moreae and transferred to the Olmedieae, which is characterized primarily by involucrate inflorescences without regard to stamen position. Nine new combinations are made.