Adult human biliary tree stem cells differentiate to β-pancreatic islet cells by treatment with a recombinant human Pdx1 peptide

Generation of β-pancreatic cells represents a major goal in research. The aim of this study was to explore a protein-based strategy to induce differentiation of human biliary tree stem cells (hBTSCs) towards β-pancreatic cells. A plasmid containing the sequence of the human pancreatic and duodenal homeobox 1 (PDX1) has been expressed in E. coli. Epithelial-Cell-Adhesion-Molecule positive hBTSCs or mature human hepatocyte cell line, HepG2, were grown in medium to which Pdx1 peptide was added. Differentiation toward pancreatic islet cells were evaluated by the expression of the β-cell transcription factors, Pdx1 and musculoapo-neurotic fibrosarcoma oncogene homolog A, and of the pancreatic hormones, insulin, glucagon, and somatostatin, investigated by real time polymerase chain reaction, western blot, light microscopy and immunofluorescence. C-peptide secretion in response to high glucose was also measured. Results indicated how purified Pdx1 protein corresponding to the primary structure of the human Pdx1 by mass spectroscopy was efficiently produced in bacteria, and transduced into hBTSCs. Pdx1 exposure triggered the expression of both intermediate and mature stage β-cell differentiation markers only in hBTSCs but not in HepG2 cell line. Furthermore, hBTSCs exposed to Pdx1 showed up-regulation of insulin, glucagon and somatostatin genes and formation of 3-dimensional islet-like structures intensely positive for insulin and glucagon. Finally, Pdx1-induced islet-like structures exhibited glucose-regulated C-peptide secretion. In conclusion, the human Pdx1 is highly effective in triggering hBTSC differentiation toward functional β-pancreatic cells

Proteomic Approaches within the NCI Early Detection Research Network for the Discovery and Identification of Cancer Biomarkers

In the postgenome era, proteomics provides a powerful approach for the analysis of normal and transformed cell functions, for the identification of disease-specific targets, and for uncovering novel endpoints for the evaluation of chemoprevention agents and drug toxicity. Unfortunately, the genomic information that has greatly expounded the genetic basis of cancer does not allow an accurate prediction of what is actually occurring at the protein level within a given cell type at any given time. The gene expression program of a given cell is affected by numerous factors in the in vivo environment resulting from tissue complexity and organ system orchestration, with cells acting in concert with each other and responding to changes in their microenvironment. Repositories of genomic information can be considered master “inventory lists” of genes and their maps, which need to be supplemented with protein-derived information. The National Cancer Institute's Early Detection Research Network is employing proteomics, or “protein walking”, in the discovery and evaluation of biomarkers for cancer detection and for the identification of high-risk subjects. Armed with microdissection techniques, including the use of Laser Capture Microdissection (LCM) to procure pure populations of cells directly from human tissue, the Network is facilitating the development of technologies that can overcome the problem of tissue heterogeneity and address the need to identify markers in easily accessible biological fluids. Proteomic approaches complement plasma-based assays of circulating DNA for cancer detection and risk assessment. LCM, coupled with downstream proteomics applications, such as two-dimensional polyacrylamide gel electrophoresis and SELDI (surface enhanced laser desorption ionization) separation followed by mass spectrometry (MS) analysis, may greatly facilitate the characterization and identification of protein expression changes that track normal and disease phenotypes. We highlight recent work from Network investigators to demonstrate the potential of proteomics to identify proteins present in cancer tissues and body fluids that are relevant for cancer screening.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/73353/1/j.1749-6632.2001.tb03870.x.pd

Making open data work for plant scientists

Despite the clear demand for open data sharing, its implementation within plant science is still limited. This is, at least in part, because open data-sharing raises several unanswered questions and challenges to current research practices. In this commentary, some of the challenges encountered by plant researchers at the bench when generating, interpreting, and attempting to disseminate their data have been highlighted. The difficulties involved in sharing sequencing, transcriptomics, proteomics, and metabolomics data are reviewed. The benefits and drawbacks of three data-sharing venues currently available to plant scientists are identified and assessed: (i) journal publication; (ii) university repositories; and (iii) community and project-specific databases. It is concluded that community and project-specific databases are the most useful to researchers interested in effective data sharing, since these databases are explicitly created to meet the researchers’ needs, support extensive curation, and embody a heightened awareness of what it takes to make data reuseable by others. Such bottom-up and community-driven approaches need to be valued by the research community, supported by publishers, and provided with long-term sustainable support by funding bodies and government. At the same time, these databases need to be linked to generic databases where possible, in order to be discoverable to the majority of researchers and thus promote effective and efficient data sharing. As we look forward to a future that embraces open access to data and publications, it is essential that data policies, data curation, data integration, data infrastructure, and data funding are linked together so as to foster data access and research productivity

Multivariate Analysis in Metabolomics

Metabolomics aims to provide a global snapshot of all small-molecule metabolites in cells and biological fluids, free of observational biases inherent to more focused studies of metabolism. However, the staggeringly high information content of such global analyses introduces a challenge of its own; efficiently forming biologically relevant conclusions from any given metabolomics dataset indeed requires specialized forms of data analysis. One approach to finding meaning in metabolomics datasets involves multivariate analysis (MVA) methods such as principal component analysis (PCA) and partial least squares projection to latent structures (PLS), where spectral features contributing most to variation or separation are identified for further analysis. However, as with any mathematical treatment, these methods are not a panacea; this review discusses the use of multivariate analysis for metabolomics, as well as common pitfalls and misconceptions

Multivariate Analysis in Metabolomics

Metabolomics aims to provide a global snapshot of all small-molecule metabolites in cells and biological fluids, free of observational biases inherent to more focused studies of metabolism. However, the staggeringly high information content of such global analyses introduces a challenge of its own; efficiently forming biologically relevant conclusions from any given metabolomics dataset indeed requires specialized forms of data analysis. One approach to finding meaning in metabolomics datasets involves multivariate analysis (MVA) methods such as principal component analysis (PCA) and partial least squares projection to latent structures (PLS), where spectral features contributing most to variation or separation are identified for further analysis. However, as with any mathematical treatment, these methods are not a panacea; this review discusses the use of multivariate analysis for metabolomics, as well as common pitfalls and misconceptions

What NMR can do in the biopharmaceutical industry

LBK

Fingerprinting the Substrate Specificity of M1 and M17 Aminopeptidases of Human Malaria, Plasmodium falciparum

Plasmodium falciparum, the causative agent of human malaria, expresses two aminopeptidases, PfM1AAP and PfM17LAP, critical to generating a free amino acid pool used by the intraerythrocytic stage of the parasite for proteins synthesis, growth and development. These exopeptidases are potential targets for the development of a new class of anti-malaria drugs.To define the substrate specificity of recombinant forms of these two malaria aminopeptidases we used a new library consisting of 61 fluorogenic substrates derived both from natural and unnatural amino acids. We obtained a detailed substrate fingerprint for recombinant forms of the enzymes revealing that PfM1AAP exhibits a very broad substrate tolerance, capable of efficiently hydrolyzing neutral and basic amino acids, while PfM17LAP has narrower substrate specificity and preferentially cleaves bulky, hydrophobic amino acids. The substrate library was also exploited to profile the activity of the native aminopeptidases in soluble cell lysates of P. falciparum malaria.This data showed that PfM1AAP and PfM17LAP are responsible for majority of the aminopeptidase activity in these extracts. These studies provide specific substrate and mechanistic information important for understanding the function of these aminopeptidases and could be exploited in the design of new inhibitors to specifically target these for anti-malaria treatment

Vibrational Spectroscopy of Glycans in Helium Nanodroplets

A central theme among the glycosciences is the Janus-faced nature ofglycans. Their tremendous structural diversity enables a myriad ofbiological functions ranging from energy storage to molecular recogni-tion processes. But at the same time, this structural diversity poses aformidable challenge for glycan analysis that impedes the full develop-ment of structural glycobiology. In contrast to genomics and proteomics,glycomics lacks generic sequencing methods that allow reliable, high-throughput analyses with low sample consumption. Instead, a variety ofsophisticated methods is used for glycan analysis, including mass spec-trometry. A general challenge using mass spectrometry alone, however,is the unambiguous identification of isomeric glycans. Therefore, it isoften coupled to orthogonal techniques, such as liquid chromatography.In the last two decades, the combination of mass spectrometry andgas-phase action spectroscopy emerged for glycan analysis. Variouschallenges, however, limited gas-phase spectroscopy to smaller glycans.In particular, the thermal activation of ions during the measurement ininfrared multiple-photon dissociation spectroscopy leads to significantline-broadening, which limits the amount of structural information thatcan be obtained by this method.This work overcomes these limitations by combining mass spectrom-etry and cryogenic vibrational spectroscopy using superfluid helium nanodroplets. The unique low-temperature environment of heliumdroplets allows the acquisition of vibrational spectra in the absenceof significant thermal contributions. A systematic study of isomericglycans demonstrates the outstanding resolving power that provides avariety of well-resolved absorption bands that are unique to each isomer:a true spectral fingerprint. The unique optical signatures allow theresolution of even minute structural details, such as the stereochemicalorientation of a single hydroxy group.The exceptional ability of this method to resolve structural detailswas used to investigate an elusive rearrangement reaction, called fu-cose migration, which frequently leads to the detection of misleadingfragment ions and erroneous sequence assignments in tandem massspectrometry. Because fucose migration was only observed in fragmentions, it was strictly associated with the fragmentation process. In thiswork, cryogenic vibrational spectroscopy reveals that fucose migrationis not restricted to fragment ions, and instead occurs in intact ions aswell. These results generalize fucose migration to a universal issue inmass spectrometry as a whole.In another study, the combination of cryogenic vibrational spec-troscopy and first-principles theory was used to unravel the structureof glycosyl cations, the key intermediates during chemical glycosyla-tion reactions. Various reaction pathways were postulated many yearsago, but the exact structure of glycosyl cations remained obscure dueto their short-lived and reactive nature in the condensed phase. Inthis work, the fragmentation of precursor ions was used to generateglycosyl cations in the gas phase, which provides a unique clean-roomenvironment that stabilizes these transient intermediates. The highlyresolved vibrational spectra obtained for various glycosyl cations allowan in-depth structural analysis that reveals detailed insights into the two fundamental structural motifs that enable stereoselective glycosylationreactions: neighboring group participation and remote participation.These results facilitate the mechanistic understanding of glycosylationreactions and will eventually lead to a more rational design of buildingblocks that is based on structural rather than anecdotal evidence.Een centraal thema in de glyco-wetenschappen is het dubbele karaktervan glycanen. Enerzijds maakt de enorme structurele diversiteit vanglycanen een groot aantal biologische functies mogelijk, variërend vanenergieopslag tot moleculaire herkenningsprocessen. Maar tegelijkertijdvormt deze structurele diversiteit een enorme uitdaging voor de ana-lyse van glycanen, wat de ontwikkeling van structurele glycobiologiebelemmert. In tegenstelling tot genomics en proteomics, ontbreekthet glycomics aan een generieke sequentiemethode die betrouwbare,high-throughput analyses van kleine hoeveelheden glycanen mogelijkmaakt. In plaats daarvan wordt een verscheidenheid aan geavanceerdemethoden gebruikt, waaronder massaspectrometrie. Met uitsluitendmassa-spectrometrische methoden, is een eenduidige identificatie vanverschillende isomeren van glycanen echter slechts beperkt mogelijk.Daarom wordt massaspectrometrie vaak gekoppeld aan orthogonale tech-nieken, zoals vloeistofchromatografie. In de laatste twee decennia is ookde combinatie van massaspectrometrie met gasfase-actiespectroscopieopgekomen voor glycaan analyse. Deze combinatie is tot dusver metname gebruikt voor de analyse van kleinere glycanen. In het bijzonderleidt de thermische activering van ionen gedurende infrarood multifotondissociatie spectroscopie tot een significante lijnverbreding, hetgeen de hoeveelheid structurele informatie beperkt die met deze methodeverkregen kan worden.Het onderzoek beschreven in dit proefschrift lost deze beperkingenop door massaspectrometrie te combineren met cryogene vibratiespec-troscopie, gebruikmakend van superfluïde helium nanodruppels. Delage temperatuur in de heliumdruppels maakt de opname van vibratie-spectra mogelijk in afwezigheid van significante thermische bijdragen.Een systematische studie van isomere glycanen toont het uitstekendeoplossend vermogen van deze techniek. De spectra bevatten volledigopgeloste absorptiebanden die uniek zijn voor een specifiek isomeer:een echte spectrale vingerafdruk. Deze spectra maken een identificatiemogelijk van zeer subtiele structurele details, zoals de stereochemie vaneen enkele hydroxygroep.Het uitzonderlijke vermogen van deze methode om structurele de-tails te onderscheiden is gebruikt om een lastige migratie-reactie, dezogenaamde fucose migratie, te onderzoeken. Deze migratie-reactie leidtvaak tot de detectie van misleidende fragmentionen en foutieve sequen-tietoewijzingen in tandem massaspectrometrie. Omdat fucose migratiealleen werd waargenomen in fragmentionen, werd het enkel geassoci-eerd met het fragmentatieproces. Zoals beschreven in dit proefschrift,onthult cryogene vibratiespectroscopie dat fucose migratie niet beperktis tot fragmentionen maar dat dit ook voorkomt in intacte ionen. Dezeresultaten maken duidelijk dat fucose migratie een universele kwestie isin de massaspectrometrie.In een andere studie is de combinatie van cryogene vibratiespectro-scopie en ab initio theorie gebruikt om de structuur van glycosylkationen,de belangrijkste tussenproducten tijdens chemische glycosyleringsreac-ties, te ontrafelen. Verschillende reactiepaden zijn vele jaren geledengepostuleerd, maar de exacte structuur van glycosylkationen bleef onduidelijk vanwege hun kortstondige en reactieve aard in de gecondenseerdefase. In dit werk is de fragmentatie van precursor-ionen gebruikt omglycosylkationen te genereren in de gasfase. Dit creëert een unieke“clean-room” omgeving die deze tijdelijke tussenproducten stabiliseert.De vibratiespectra verkregen voor verschillende glycosylkationen makeneen diepgaande structurele analyse mogelijk. Dit onthult gedetailleerdeinzichten in de twee fundamentele structurele motieven die stereoselec-tieve glycosyleringsreacties mogelijk maken: participatie van naburigegroepen en participatie op afstand. Deze resultaten maken een mecha-nistisch begrip van glycosyleringsreacties mogelijk en zullen uiteindelijkleiden tot een rationeler ontwerp van de benodigde bouwstenen, geba-seerd op structureel in plaats van anekdotisch bewijs

Molecular biology techniques as a tool for detection and characterisation of Mycobacterium avium subsp. paratuberculosis

Mycobacterium avium subsp. paratuberculosis (M. paratuberculosis) is the causative agent of paratuberculosis, also known as Johne’s disease, a chronic intestinal infection in cattle and other ruminants. Paratuberculosis is characterised by diarrhea and weight loss that occurs after a period of a few months up to several years without any clinical signs. The considerable economic losses to dairy and beef cattle producers are caused by reduced milk production and poor reproduction performance in subclinically infected animals. Early diagnosis of infected cattle is essential to prevent the spread of the disease. Efforts have been made to eradicate paratuberculosis by using a detection and cull strategy, but eradication is hampered by the lack of suitable and sensitive diagnostic methods. This thesis, based on five scientific investigations, describes the development of different DNA amplification strategies for detection and characterisation of M. paratuberculosis. Various ways to pre-treat bacterial cultures, tissue specimens and fecal samples prior to PCR analysis were investigated. Internal positive PCR control molecules were developed and used in PCR analyses to improve the reliability and to facilitate the interpretation of the results. The sensitivity of the ultimate methods was found to be approximate that of culture and allowed detection of low numbers of M. paratuberculosis expected to be found in subclinically infected animals. Genomic DNA of a Swedish mycobacterial isolate, incorrectly identified by PCR as M. paratuberculosis was characterised. The isolate was closely related to M. cookii and harboured one copy of a DNA segment with 94% similarity to IS900, the target sequence used in diagnostic PCR for detection of M. paratuberculosis. This finding highlighted the urgency of developing or evaluating PCR systems based on genes other than IS900. A PCR-based fingerprinting method using primers targeting the enterobacterial intergenic consensus sequence (ERIC) and the IS900 sequence was developed and successfully used to distinguish M. paratuberculosis from closely related mycobacteria, including the above mentioned mycobacterial isolate. In conclusion, the molecular biology techniques developed in these studies have proved useful for accelerating the diagnostic detection and characterisation of M. paratuberculosis