Identification of potential RNA substrates for the 3’-5’ polymerase BtTLP with RNA-Seq

Rustbelt RNA Meeting 2014 Poster Presenter AwardeeReverse (3'-5') polymerases are a relatively new discovery and are found in all three domains of life. The in vivo function of many of these proteins remains ambiguous. BtTLP, a reverse polymerase from the soil bacterium, Bacillus thuringiensis, has recently come under investigation through structural, genetic, and biochemical analysis. The overall goal of this study is to develop a new form of RNA-Seq to identify potential substrates for BtTLP in an engineered system (a previously characterized strain of Saccharomyces cerevisiae (baker's yeast) expressing BtTLP), with the ultimate goal of elucidating a more complete understanding of these unusual 3'-5' polymerases in biology. This project is being accomplished both from the wet and computational aspects. To start, a library of all small RNAs (approximately less than 200 bp) from the engineered system has been generated and sequenced. A computational pipeline has been developed to process the large amount of data that comes from deep-sequencing experiments. It is expected that multiple new substrates will be identified based on previous in vitro biochemical studies and an observed growth defect in the engineered system when compared to an isogenic control. This approach has significant advantages over the laborious alternative of testing each RNA in the cell individually. Traditionally, RNA-Seq has been used to identify functional elements in the genome, but here it is being used to detect post-transcriptional 5' nucleotide addition.SROP at OSUThe College of EngineeringNo embargoAcademic Major: Biomedical Engineerin

Metamorphic, Autonomous Symmetries

Unified signed models have led to many intuitive advances, including courseware and lambda calculus. In fact, few developers would disagree with the construction of the UNIVAC computer, which embodies the unfortunate principles of cryptoanalysis. We examine how DHCP can be applied to the evaluation of vacuum tubes

Equal mixing time enables scale-down and optimization of a CHO cell culture process using a shaken microbioreactor system

The advancement of microbioreactor technology in recent years has transformed early- and mid-stage process development. The monitoring and control capabilities of microbioreactors not only promote the quick accumulation of process knowledge but has also led to an increased scalability when compared to traditionally used systems such as shake flasks and microtitre plates. This study seeks to establish a framework for the micro-Matrix microbioreactor (Applikon-Biotechnology BV) as process development tool. Using the Dual Indicator System for Mixing Time, the system was initially characterized for mixing properties at varying operating conditions, which was found to yield mixing times between 0.9 and 41.8 s. A matched mixing time was proposed as scale-down criterion for an IgG4 producing GS-CHO fed-batch process between a 5 L stirred tank reactor (STR) and the micro-Matrix microbioreactor. Growth trends, maximum viable cell concentrations, final titre, and glycoprofiles were nearly identical at both scales. The scale-down model was then employed to optimize a bolus feeding regime using response surface methodology, which led to a 25.4% increase of the space-time yield and a 25% increase of the final titre. The optimized feeding strategy was validated at the small-scale and successfully scaled up to the 5 L STR. This work for the first time provides a framework of how the micro-Matrix microbioreactor can be implemented in a bioprocess development workflow and demonstrates scalability of growth and production kinetics as well as IgG4 glycosylation between the micro-Matrix and a benchtop-scale STR system. Graphical Abstract and Lay Summary: (Figure presented.) Microbioreactor technology has become an essential part of early- and mid-stage bioprocess development. This study provides a framework of how the shaken micro-Matrix system can be used for cell culture process development of a mammalian CHO cell line producing a monoclonal antibody. Following scale-down from a 5 L stirred tank bioreactor, the micro-Matrix was employed for the optimisation of a feeding strategy and the optimised protocol was successfully scaled up

Energy stability analysis for a hybrid fluid-kinetic plasma model

In plasma physics, a hybrid fluid-kinetic model is composed of a magnetohydrodynamics (MHD) part that describes a bulk fluid component and a Vlasov kinetic theory part that describes an energetic plasma component. While most hybrid models in the plasma literature are non-Hamiltonian, this paper investigates a recent Hamiltonian variant in its two-dimensional configuration. The corresponding Hamiltonian structure is described along with its Casimir invariants. Then, the energy-Casimir method is used to derive explicit sufficient stability conditions, which imply a stable spectrum and suggest nonlinear stability

L1cam as an e-selectin ligand in colon cancer

Metastasis is the main cause of death among colorectal cancer (CRC) patients. E-selectin and its carbohydrate ligands, including sialyl Lewis X (sLeX) antigen, are key players in the binding of circulating tumor cells to the endothelium, which is one of the major events leading to organ invasion. Nevertheless, the identity of the glycoprotein scaffolds presenting these glycans in CRC remains unclear. In this study, we firstly have characterized the glycoengineered cell line SW620 transfected with the fucosyltransferase 6 (FUT6) coding for the \u3b11,3-fucosyltransferase 6 (FUT6), which is the main enzyme responsible for the synthesis of sLeX in CRC. The SW620FUT6 cell line expressed high levels of sLeX antigen and E-selectin ligands. Moreover, it displayed increased migration ability. E-selectin ligand glycoproteins were isolated from the SW620FUT6 cell line, identified by mass spectrometry, and validated by flow cytometry and Western blot (WB). The most prominent E-selectin ligand we identified was the neural cell adhesion molecule L1 (L1CAM). Previous studies have shown association of L1CAM with metastasis in cancer, thus the novel role as E-selectin counter-receptor contributes to understand the molecular mechanism involving L1CAM in metastasis formation

Analysis of three epoetin alpha products by LC and LC-MS indicates differences in glycosylation critical quality attributes, including sialic acid content

Erythropoietin (EPO) is one of the main therapeutics used to treat anaemic patients, greatly improving their quality of life. In this study, biosimilars Binocrit and a development product, called here CIGB-EPO, were compared to the originator product, Eprex. All three are epoetin alpha products, reputed to have similar glycosylation profiles. The quality, safety and efficacy of this biotherapeutic depend on the following glycosylation critical quality attributes (GCQAs): sialylation, N-glycolyl-neuraminic acid (Neu5Gc) content, branching, N-acetyl-lactosamine (LacNAc) extensions and O-acetylation pattern. Reverse-phase ultra high pressure liquid chromatography (RP-UHPLC) analysis of acid-released, 1,2-diamino-4,5-methylenedioxybenzene (DMB) labelled sialic acid derivatives and hydrophilic interaction liquid chromatography (HILIC) in combination with mass spectrometry (HILIC-UHPLC-MS) of procainamide (PROC) labelled N-glycans were the analytical tools used. An automated method for enzymatic release and PROC labelling was applied for the first time to the erythropoiesis stimulating agent (ESA) products, which facilitated novel, in-depth characterisation, and allowed identification of precise structural features including the location of O-acetyl groups on sialic acid (SA) moie-ties. Samples were digested by a sialate-O-acetylesterase (NanS) to confirm the presence of O-acetyl groups. It was found that Eprex contained the greatest relative abundance of O-acetylated derivatives, Binocrit expressed the least Neu5Gc, and CIGB-EPO showed the greatest variety of high-mannose-phosphate structures. The sialylation and LacNAc extension patterns of the three ESAs were similar, with a maximum of four N-acetyl-neuraminic acid (Neu5Ac) moieties detected per glycan. Such differences in SA derivatisation, particularly O-acetylation, could have consequences for the quality and safety of a biotherapeutic, as well as its efficacy

L1cam as an e-selectin ligand in colon cancer

POCI-01-0145-FEDER-007728 ref. 140_596817822Metastasis is the main cause of death among colorectal cancer (CRC) patients. E-selectin and its carbohydrate ligands, including sialyl Lewis X (sLeX) antigen, are key players in the binding of circulating tumor cells to the endothelium, which is one of the major events leading to organ invasion. Nevertheless, the identity of the glycoprotein scaffolds presenting these glycans in CRC remains unclear. In this study, we firstly have characterized the glycoengineered cell line SW620 transfected with the fucosyltransferase 6 (FUT6) coding for the α1,3-fucosyltransferase 6 (FUT6), which is the main enzyme responsible for the synthesis of sLeX in CRC. The SW620FUT6 cell line expressed high levels of sLeX antigen and E-selectin ligands. Moreover, it displayed increased migration ability. E-selectin ligand glycoproteins were isolated from the SW620FUT6 cell line, identified by mass spectrometry, and validated by flow cytometry and Western blot (WB). The most prominent E-selectin ligand we identified was the neural cell adhesion molecule L1 (L1CAM). Previous studies have shown association of L1CAM with metastasis in cancer, thus the novel role as E-selectin counter-receptor contributes to understand the molecular mechanism involving L1CAM in metastasis formation.publishersversionpublishe

Sialylation on O-linked glycans protects von Willebrand factor from macrophage galactose lectin mediated clearance

Terminal sialylation determines the plasma half-life of von Willebrand factor (VWF). A role for macrophage galactose lectin (MGL) in regulating hyposialylated VWF clearance has recently been proposed. In this study, we showed that MGL influences physiological plasma VWF clearance. MGL inhibition was associated with a significantly extended mean residence time and 3-fold increase in endogenous plasma VWF antigen levels (P<0.05). Using a series of VWF truncations, we further demonstrated that the A1 domain of VWF is predominantly responsible for enabling the MGL interaction. Binding of both full-length and VWF-A1-A2-A3 to MGL was significantly enhanced in the presence of ristocetin (P<0.05), suggesting that the MGL-binding site in A1 is not fully accessible in globular VWF. Additional studies using different VWF glycoforms demonstrated that VWF O-linked glycans, clustered at either end of the A1 domain, play a key role in protecting VWF against MGLmediated clearance. Reduced sialylation has been associated with pathological, increased clearance of VWF in patients with von Willebrand disease. Herein, we demonstrate that specific loss of α2-3 linked sialylation from O-glycans results in markedly increased MGL-binding in vitro, and markedly enhanced MGL-mediated clearance of VWF in vivo. Our data further show that the asialoglycoprotein receptor (ASGPR) does not have a significant role in mediating the increased clearance of VWF following loss of O-sialylation. Conversely however, we observed that loss of N-linked sialylation from VWF drives enhanced circulatory clearance predominantly via the ASGPR. Collectively, our data support the hypothesis that in addition to regulating physiological VWF clearance, the MGL receptor works in tandem with ASGPR to modulate enhanced clearance of aberrantly sialylated VWF in the pathogenesis of von Willebrand disease

"The daily grunt": middle class bias and vested interests in the 'Getting in Early' and 'Why Can't They Read?' reports.

It is a long-standing and commonly held belief in the UK and elsewhere that the use of elite forms of language reflects superior intellect and education. Expert opinion from sociolinguistics, however, contends that such a view is the result of middle-class bias and cannot be scientifically justified. In the 1960s and 1970s,such luminaries as Labov (1969) and Trudgill (1975) were at pains to point out to educationalists, with some success, that this 'deficit 'view of working-class children's communicative competence is not a helpful one. However, a close reading of recent think-tank reports and policy papers on language and literacy teaching in schools reveals that the linguistic deficit hypothesis has resurfaced and is likely to influence present-day educational policy and practice. In this paper I examine in detail the findings, claims and recommendations of the reports and I argue that they are biased, poorly researched and reflect the vested interests of certain specialist groups, such as speech and language therapists and companies who sell literacy materials to schools. I further argue that we need to, once again, inject the debate with the social dimensions of educational failure, and we need to move away from the pathologisation of working-class children's language patterns