Controlled gene expression using acute phase response elements

Previous work in our laboratory involved the creation of an inducible gene expression system based on the promoter of the major acute phase protein in humans, C-reactive protein (CRP). The aims of this project were to further characterise the CRP based expression vector and to modify it for use in pigs. The green fluorescent protein (GFP) was also evaluated as a reporter of transient inducible gene expression. The 30kb fragment containing the human CRP gene was sequenced and analysed for the presence of elements that may be responsible for the low basal levels of expression of the gene and for sequences that are responsible for the sexually dimorphic pattern of expression of the human CRP gene in transgenic mice. It was planned to use the information from these analyses to modify the expression vector for use in pigs. However, because it is not currently known how the CRP- based acute phase expression vector would behave in other species, the promoter of the major acute phase protein in pigs, ITIH4, was isolated. Due to the absence of homology between the pig ITIH4 promoter and the human CRP promoter, the pig ITIH4 promoter was further characterised. The investigations focused on the inducibility of the promoter in response to cytokine stimulation and the effect modification of the promoter would have on promoter activity and inducibility. It was found that the promoter was induced by IL-6 but not IL-1 (except at low concentrations). A combination of IL-1 and IL-6 was shown to result in a decrease in the inducibility of the construct by IL-6. Mutation of the promoter in order to decrease the basal level of expression and enhance inducibility was unsuccessful. In order to develop a system that will facilitate studies of inducible gene expression in vitro a destabilised variant of GFP was evaluated as a reporter gene. Although small increases in fluorescence intensity could be detected following stimulation, the analyses suggest that GFP is not as sensitive as other reporter genes for studying inducible gene expression

Heteronuclear bimetallic complexes with 3d and 4f elements

Three heteronuclear bimetallic complexes [Cu(MeOH)(L)Ln(NO3)3] ( 1-Ce ; Ln = Ce, 1-Pr ; Ln = Pr, and 1-Nd ; Ln = Nd) were prepared using H2L (1,3-bis[(3-methoxysalicylidene)amino]-2,2-dimethylpropane) in methanol, affording the complexes as green crystalline materials. These can be prepared in a one-pot synthesis from 2,2-dimethylpropan-1,3-diamine, o-vanillin, copper(II) nitrate, and Ln(III) nitrate (Ln = Ce, Pr, Nd). X-ray crystallography, high-resolution mass spectrometry, and UV-vis spectroscopy were used to characterize the bimetallic complexes. All three complexes showed the copper center adopting a five-coordinate square pyramidal geometry and the lanthanoid cation adopting a ten-coordinate geometry.Publisher PDFPeer reviewe

Two Distinct Coagulase-Dependent Barriers Protect Staphylococcus aureus from Neutrophils in a Three Dimensional in vitro Infection Model

Staphylococcus aureus is a pyogenic abscess-forming facultative pathogenic microorganism expressing a large set of virulence-associated factors. Among these, secreted proteins with binding capacity to plasma proteins (e.g. fibrinogen binding proteins Eap and Emp) and prothrombin activators such as Coagulase (Coa) and vWbp are involved in abscess formation. By using a three-dimensional collagen gel (3D-CoG) supplemented with fibrinogen (Fib) we studied the growth behavior of S. aureus strain Newman and a set of mutants as well as their interaction with mouse neutrophils by real-time confocal microscopy. In 3D-CoG/Fib, S. aureus forms microcolonies which are surrounded by an inner pseudocapsule and an extended outer dense microcolony-associated meshwork (MAM) containing fibrin. Coa is involved in formation of the pseudocapsule whereas MAM formation depends on vWbp. Moreover, agr-dependent dispersal of late stage microcolonies could be observed. Furthermore, we demonstrate that the pseudocapsule and the MAM act as mechanical barriers against neutrophils attracted to the microcolony. The thrombin inhibitor argatroban is able to prevent formation of both pseudocapsule and MAM and supports access of neutrophils to staphylococci. Taken together, this model can simulate specific stages of S. aureus abscess formation by temporal dissection of bacterial growth and recruitment of immune cells. It can complement established animal infection models in the development of new treatment options

Modulating Activity of Vancomycin and Daptomycin on the Expression of Autolysis Cell-Wall Turnover and Membrane Charge Genes in hVISA and VISA Strains

Glycopeptides are still the gold standard to treat MRSA (Methicillin Resistant Staphylococcus aureus) infections, but their widespread use has led to vancomycin-reduced susceptibility [heterogeneous Vancomycin-Intermediate-Staphylococcus aureus (hVISA) and Vancomycin-Intermediate-Staphylococcus aureus (VISA)], in which different genetic loci (regulatory, autolytic, cell-wall turnover and cell-envelope positive charge genes) are involved. In addition, reduced susceptibility to vancomycin can influence the development of resistance to daptomycin. Although the phenotypic and molecular changes of hVISA/VISA have been the focus of different papers, the molecular mechanisms responsible for these different phenotypes and for the vancomycin and daptomycin cross-resistance are not clearly understood. The aim of our study was to investigate, by real time RT-PCR, the relative quantitative expression of genes involved in autolysis (atl-lytM), cell-wall turnover (sceD), membrane charges (mprF-dltA) and regulatory mechanisms (agr-locus-graRS-walKR), in hVISA and VISA cultured with or without vancomycin and daptomycin, in order to better understand the molecular basis of vancomycin-reduced susceptibility and the modulating activity of vancomycin and daptomycin on the expression of genes implicated in their reduced susceptibility mechanisms. Our results show that hVISA and VISA present common features that distinguish them from Vancomycin-Susceptible Staphylococcus aureus (VSSA), responsible for the intermediate glycopeptide resistance i.e. an increased cell-wall turnover, an increased positive cell-wall charge responsible for a repulsion mechanism towards vancomycin and daptomycin, and reduced agr-functionality. Indeed, VISA emerges from hVISA when VISA acquires a reduced autolysis caused by a down-regulation of autolysin genes, atl/lytM, and a reduction of the net negative cell-envelope charge via dltA over-expression. Vancomycin and daptomycin, acting in a similar manner in hVISA and VISA, can influence their cross-resistance mechanisms promoting VISA behavior in hVISA and enhancing the cell-wall pathways responsible for the intermediate vancomycin resistance in VISA. Daptomycin can also induce a charge repulsion mechanism both in hVISA and VISA increasing the activity of the mprF

Engineering Translation in Mammalian Cell Factories to Increase Protein Yield: The Unexpected Use of Long Non-Coding SINEUP RNAs

Mammalian cells are an indispensable tool for the production of recombinant proteins in contexts where function depends on post-translational modifications. Among them, Chinese Hamster Ovary (CHO) cells are the primary factories for the production of therapeutic proteins, including monoclonal antibodies (MAbs). To improve expression and stability, several methodologies have been adopted, including methods based on media formulation, selective pressure and cell- or vector engineering. This review presents current approaches aimed at improving mammalian cell factories that are based on the enhancement of translation. Among well-established techniques (codon optimization and improvement of mRNA secondary structure), we describe SINEUPs, a family of antisense long non-coding RNAs that are able to increase translation of partially overlapping protein-coding mRNAs. By exploiting their modular structure, SINEUP molecules can be designed to target virtually any mRNA of interest, and thus to increase the production of secreted proteins. Thus, synthetic SINEUPs represent a new versatile tool to improve the production of secreted proteins in biomanufacturing processes. \ua9 2016 The Author

An assessment of refrigeration system performance with a particular emphasis on the effects of frosting and defrosting

SIGLEAvailable from British Library Document Supply Centre- DSC:DX87039 / BLDSC - British Library Document Supply CentreGBUnited Kingdo

Isolation and Characterization of the Promoter and Partial Enhancer Region of the Porcine Inter-α-Trypsin Inhibitor Heavy Chain 4 Gene

A porcine genomic library was screened for clones containing the promoter of the major acute-phase protein in pigs, inter-α-trypsin heavy chain 4 (ITIH4). Following isolation of the promoter, a functional analysis was performed with Hep3B cells. The promoter was induced by interleukin-6 (IL-6) but not by IL-1β. However, IL-1β was shown to inhibit the IL-6-induced activation of the porcine ITIH4 promoter

Oral history interview with Karen Harraghy (OH-009)

In this interview, Karen Harraghy, a member of Congressman John Joseph Moakley’s district staff from 1983 to 2001, recalls her time working for the congressman. The interview covers Ms. Harraghy’s responsibilities as a district staff member; Congressman Moakley’s involvement in immigration and human rights issues in El Salvador; his relationships with the Massachusetts congressional delegation and other members of Congress; and his reputation as a bread-and-butter politician. She concludes by reflecting on Congressman Moakley’s legacy of public service and political leadership.https://dc.suffolk.edu/moh/1010/thumbnail.jp