A new plasmid display technology for the in vitro selection of functional phenotype-genotype linked proteins

Background Display technologies which allow peptides or proteins to be physically associated with the encoding DNA are central to procedures which involve screening of protein libraries in vitro for new or altered function. Here we describe a new system designed specifically for the display of libraries of diverse, functional proteins which utilises the DNA binding protein nuclear factor κB (NF-κB) p50 to establish a phenotype-genotype link between the displayed protein and the encoding gene. Results A range of model fusion proteins to either the amino- or carboxy-terminus of NF-κB p50 have been constructed and shown to retain the picomolar affinity and DNA specificity of wild-type NF-κB p50. Through use of an optimal combination of binding buffer and DNA target sequence, the half-life of p50-DNA complexes could be increased to over 47 h, enabling the competitive selection of a variety of protein-plasmid complexes with enrichment factors of up to 6000-fold per round. The p50-based plasmid display system was used to enrich a maltose binding protein complex to homogeneity in only three rounds from a binary mixture with a starting ratio of 1:108 and to enrich to near homogeneity a single functional protein from a phenotype-genotype linked Escherichia coli genomic library using in vitro functional selections. Conclusions A new display technology is described which addresses the challenge of functional protein display. The results demonstrate that plasmid display is sufficiently sensitive to select a functional protein from large libraries and that it therefore represents a useful addition to the repertoire of display technologies.

Pituitary tumours : all silent on the epigenetics front

Investigation of the epigenome of sporadic pituitary tumours is providing a more detailed understanding of aberrations that characterise this tumour type. Early studies, in this and other tumour types adopted candidate-gene approaches to characterise CpG island methylation as a mechanism responsible for or associated with gene silencing. However, more recently, investigators have adopted approaches that do not require a priori knowledge of the gene and transcript, as example differential display techniques, and also genome-wide, array-based approaches, to 'uncover' or 'unmask' silenced genes. Furthermore, through use of chromatin immunoprecipitation as a selective enrichment technique; we are now beginning to identify modifications that target the underlying histones themselves and that have roles in gene-silencing events. Collectively, these studies provided convincing evidence that change to the tumour epigenome are not simply epiphenomena but have functional consequences in the context of pituitary tumour evolution. Our ability to perform these types of studies has been and is increasingly reliant upon technological advances in the genomics and epigenomics arena. In this context, other more recent advances and developing technologies, and, in particular, next generation or flow cell re-sequencing techniques offer exciting opportunities for our future studies of this tumour type.

Differential gene expression in breast cancer cell lines and stroma-tumor differences in microdissected breast cancer biopsies revealed by display array analysis

To examine gene-expression patterning in late-stage breast cancer biopsies, we used a microdissection technique to separate tumor from the surrounding breast tissue or stroma. A DD-PCR protocol was then used to amplify expressed products, which were resolved using PAGE and used as probe to hybridize with representative human arrays and cDNA libraries. The probe derived from the tumor–stroma comparison was hybridized with a gene array and an arrayed cDNA library derived from a GCT of bone; 21 known genes or expressed sequence tags were detected, of which 17 showed differential expression. These included factors associated with epithelial to mesenchymal transition (vimentin), the cargo selection protein (TIP47) and the signal transducer and activator of transcription (STAT3). Northern blot analysis was used to confirm those genes also expressed by representative breast cancer cell lines. Notably, 6 genes of unknown function were restricted to tumor while the majority of stroma-associated genes were known. When applied to transformed breast cancer cell lines (MDA-MB-435 and T47D) that are known to have different metastatic potential, DD array analysis revealed a further 20 genes; 17 of these genes showed differential expression. Use of microdissection and the DD-PCR array protocol allowed us to identify factors whose localized expression within the breast may play a role in abnormal breast development or breast carcinogenesis.

A transcription factor map as revealed by a genome-wide gene expression analysis of whole-blood mRNA transcriptome in multiple sclerosis

Background Several lines of evidence suggests that transcription factors are involved in the pathogenesis of Multiple Sclerosis (MS) but a complete mapping the whole network has been elusive. One of the reasons is that there are several clinical subtypes of MS and transcription factors which may be involved in one subtype may not be in others. We investigated the possibility that this network could be mapped using microarray technologies and modern bioinformatics methods on a dataset from whole blood in 99 untreated MS patients (36 Relapse Remitting MS, 43 Primary Progressive MS, and 20 Secondary Progressive MS) and 45 age-matched healthy controls, Methodology/Principal Findings We have used two different analytical methodologies: a differential expression analysis and a differential co-expression analysis, which have converged on a significant number of regulatory motifs that seem to be statistically overrepresented in genes which are either differentially expressed (or differentially co-expressed) in cases and controls (e.g. V$KROX_Q6, p-value < 3.31E-6; V$CREBP1_Q2, p-value < 9.93E-6, V$YY1_02, p-value < 1.65E-5). Conclusions/significance: Our analysis uncovered a network of transcription factors that potentially dysregulate several genes in MS or one or more of its disease subtypes. Analysing the published literature we have found that these transcription factors are involved in the early T-lymphocyte specification and commitment as well as in oligodendrocytes dedifferentiation and development. The most significant transcription factors motifs were for the Early Growth response EGR/KROX family, ATF2, YY1 (Yin and Yang 1), E2F-1/DP-1 and E2F-4/DP-2 heterodimers, SOX5, and CREB and ATF families.

Offspring of mothers fed a high fat diet display hepatic cell cycle inhibition and associated changes in gene expression and DNA methylation

The association between an adverse early life environment and increased susceptibility to later-life metabolic disorders such as obesity, type 2 diabetes and cardiovascular disease is described by the developmental origins of health and disease hypothesis. Employing a rat model of maternal high fat (MHF) nutrition, we recently reported that offspring born to MHF mothers are small at birth and develop a postnatal phenotype that closely resembles that of the human metabolic syndrome. Livers of offspring born to MHF mothers also display a fatty phenotype reflecting hepatic steatosis and characteristics of non-alcoholic fatty liver disease. In the present study we hypothesised that a MHF diet leads to altered regulation of liver development in offspring; a derangement that may be detectable during early postnatal life. Livers were collected at postnatal days 2 (P2) and 27 (P27) from male offspring of control and MHF mothers (n = 8 per group). Cell cycle dynamics, measured by flow cytometry, revealed significant G0/G1 arrest in the livers of P2 offspring born to MHF mothers, associated with an increased expression of the hepatic cell cycle inhibitor Cdkn1a. In P2 livers, Cdkn1a was hypomethylated at specific CpG dinucleotides and first exon in offspring of MHF mothers and was shown to correlate with a demonstrable increase in mRNA expression levels. These modifications at P2 preceded observable reductions in liver weight and liver:brain weight ratio at P27, but there were no persistent changes in cell cycle dynamics or DNA methylation in MHF offspring at this time. Since Cdkn1a up-regulation has been associated with hepatocyte growth in pathologic states, our data may be suggestive of early hepatic dysfunction in neonates born to high fat fed mothers. It is likely that these offspring are predisposed to long-term hepatic dysfunction.

An analysis of DNA methylation in human adipose tissue reveals differential modification of obesity genes before and after gastric bypass and weight loss

Background Environmental factors can influence obesity by epigenetic mechanisms. Adipose tissue plays a key role in obesity-related metabolic dysfunction, and gastric bypass provides a model to investigate obesity and weight loss in humans. Results Here, we investigate DNA methylation in adipose tissue from obese women before and after gastric bypass and significant weight loss. In total, 485,577 CpG sites were profiled in matched, before and after weight loss, subcutaneous and omental adipose tissue. A paired analysis revealed significant differential methylation in omental and subcutaneous adipose tissue. A greater proportion of CpGs are hypermethylated before weight loss and increased methylation is observed in the 3′ untranslated region and gene bodies relative to promoter regions. Differential methylation is found within genes associated with obesity, epigenetic regulation and development, such as CETP, FOXP2, HDAC4, DNMT3B, KCNQ1 and HOX clusters. We identify robust correlations between changes in methylation and clinical trait, including associations between fasting glucose and HDAC4, SLC37A3 and DENND1C in subcutaneous adipose. Genes investigated with differential promoter methylation all show significantly different levels of mRNA before and after gastric bypass. Conclusions This is the first study reporting global DNA methylation profiling of adipose tissue before and after gastric bypass and associated weight loss. It provides a strong basis for future work and offers additional evidence for the role of DNA methylation of adipose tissue in obesity.

The role of microcomputer-based laboratory display in supporting the construction of new understanding in kinematics

Teachers' failure to utilise MBL activities more widely may be due to not recognising their capacity to transform the nature of laboratory activities to be more consistent with contemporary constructivist theories of learning. This research aimed to increase understanding of how MBL activities specifically designed to be consistent with a constructivist theory of learning support or constrain student construction of understanding. The first author conducted the research with his Year 11 physics class of 29 students. Dyads completed nine tasks relating to kinematics using a Predict-Observe-Explain format. Data sources included video and audio recordings of students and teacher during four 70-minute sessions, students' display graphs and written notes, semi-structured student interviews, and the teacher's journal. The study identifies the actors and describes the patterns of interactions in the MBL. Analysis of students' discourse and actions identified many instances where students' initial understanding of kinematics were mediated in multiple ways. Students invented numerous techniques for manipulating data in the service of their emerging understanding. The findings are presented as eight assertions. Recommendations are made for developing pedagogical strategies incorporating MBL activities which will likely catalyse student construction of understanding.

Comparative Electrochemical Study of Unsubstituted and Substituted Bis(phthalocyaninato) Rare Earth(III) Complexes

The electrochemistry of homoleptic substituted phthalocyaninato rare earth double-decker complexes M(TBPc)2 and M(OOPc)2 [M = Y, La...Lu except Pm; H2TBPc = 3(4),12(13),21(22),30(31)-tetra-tert-butylphthalocyanine, H2OOPc = 3,4,12,13,21,22,30,31-octakis(octyloxy)phthalocyanine] has been comparatively studied by cyclic voltammetry (CV) and differential pulse voltammetry (DPV) in CH2Cl2 containing 0.1 M tetra-n-butylammonium perchlorate (TBAP). Two quasi-reversible one-electron oxidations and three or four quasi-reversible one-electron reductions have been revealed for these neutral double-deckers of two series of substituted complexes, respectively. For comparison, unsubstituted bis(phthalocyaninato) rare earth analogues M(Pc)2 (M = Y, La...Lu except Pm; H2Pc = phthalocyanine) have also been electrochemically investigated. Two quasi-reversible one-electron oxidations and up to five quasi-reversible one-electron reductions have been revealed for these neutral double-decker compounds. The three bis(phthalocyaninato)cerium compounds display one cerium-centered redox wave between the first ligand-based oxidation and reduction. The half-wave potentials of the first and second oxidations and first reduction for double-deckers of the tervalent rare earths depend on the size of the metal center. The difference between the redox potentials of the second and third reductions for MIII(Pc)2, which represents the potential difference between the first oxidation and first reduction of [MIII(Pc)2]−, lies in the range 1.08−1.37 V and also gradually diminishes along with the lanthanide contraction, indicating enhanced π−π interactions in the double-deckers connected by the smaller, lanthanides. This corresponds well with the red-shift of the lowest energy band observed in the electronic absorption spectra of reduced double-decker [MIII(Pc′)2]− (Pc′ = Pc, TBPc, OOPc).

A Petrov-Galerkin method for a singularly perturbed ordinary differential equation with non-smooth data

In this paper, a singularly perturbed ordinary differential equation with non-smooth data is considered. The numerical method is generated by means of a Petrov-Galerkin finite element method with the piecewise-exponential test function and the piecewise-linear trial function. At the discontinuous point of the coefficient, a special technique is used. The method is shown to be first-order accurate and singular perturbation parameter uniform convergence. Finally, numerical results are presented, which are in agreement with theoretical results.