Identification of the Human Erythrocyte Glucose Transporter (GLUT1) ATP Binding Domain: A Dissertation

The human erythrocyte glucose transport protein (GLUT1) interacts with, and is regulated by, cytosolic ATP. This study asks the following questions concerning ATP modulation of GLUT1 mediated sugar transport. 1) Which region(s) of GLUT1 form the adenine nucleotide-binding domain? 2) What factors influence ATP modulation of sugar transport? 3) Is ATP interaction with GLUT1 sufficient for sugar transport regulation? The first question was addressed through peptide mapping, n-terminal sequencing, and alanine scanning mutagenesis of GLUT1 using [32P]-azidoATP, a photoactivatable ATP analog. We then used a combination of transport measurements and photolabeling strategies to examine how glycolytic intermediates, pH, and transporter oligomeric structure affect ATP regulation of sugar transport. Finally, GLUT1 was reconstituted into proteoliposomes to determine whether ATP is sufficient for the modulation of GLUT1 function in-vitro. This thesis presents data supporting the hypothesis that residues 332-335 contribute to the efficiency of adenine nucleotide binding to GLUT1. In addition, we show that AMP, acidification, and conversion of the transporter to its dimeric form antagonize ATP regulation of sugar transport. Finally, we present results that support the proposal that ATP interaction with GLUT1 is sufficient for transport modulation

Process development approaches for expansion of adherent stem cells in microcarrier-based bioreactor culture

Industry trends in regenerative medicine show an increased need for scalable and closed manufacturing of cell therapies. Single-use bioreactor systems have proven suitable as a platform to meet the industry’s needs. Key process parameters for cell culture performance in these systems include pH, dissolved oxygen (DO) and agitation rates. Especially important is the understanding and application of appropriate solid-liquid mixing, which is essential for microcarrier-based cultures used for adherent stem cells. Agitation rates that are too high in microcarrier-based cultures can be correlated with smaller eddy lengths that impact cellular shear. Conversely, agitation rates that are low do not support the consistent microcarrier turnover required for cell access to nutrients, DO, and maintenance of pH. Moreover, suboptimal agitation rates may impact cell-to-microcarrier attachment and transfer. Here, we summarize a stepwise approach to optimizing pH, DO and agitation set-points in the Mobius® 3L single-use bioreactor for mesenchymal stem/stromal cells (MSCs). The theoretical agitation operating range best suited for microcarrier cultures was calculated based on the Zwietering equation for suspension of solids in stirred tanks, and verified experimentally with human bone marrow-derived MSCs. Upper agitation limits were defined by Kolmogorov\u27s theory of turbulent eddy lengths, and were substantially higher than the agitation rates required to keep microcarriers suspended. Identifying optimal pH, DO and agitation rates for microcarrier-based bioreactor expansion of adherent cells is paramount to developing a robust platform for use in a controlled manufacturing environment

Structural Basis of GLUT1 Inhibition by Cytoplasmic ATP

Cytoplasmic ATP inhibits human erythrocyte glucose transport protein (GLUT1)–mediated glucose transport in human red blood cells by reducing net glucose transport but not exchange glucose transport (Cloherty, E.K., D.L. Diamond, K.S. Heard, and A. Carruthers. 1996. Biochemistry. 35:13231–13239). We investigated the mechanism of ATP regulation of GLUT1 by identifying GLUT1 domains that undergo significant conformational change upon GLUT1–ATP interaction. ATP (but not GTP) protects GLUT1 against tryptic digestion. Immunoblot analysis indicates that ATP protection extends across multiple GLUT1 domains. Peptide-directed antibody binding to full-length GLUT1 is reduced by ATP at two specific locations: exofacial loop 7–8 and the cytoplasmic C terminus. C-terminal antibody binding to wild-type GLUT1 expressed in HEK cells is inhibited by ATP but binding of the same antibody to a GLUT1–GLUT4 chimera in which loop 6–7 of GLUT1 is substituted with loop 6–7 of GLUT4 is unaffected. ATP reduces GLUT1 lysine covalent modification by sulfo-NHS-LC-biotin by 40%. AMP is without effect on lysine accessibility but antagonizes ATP inhibition of lysine modification. Tandem electrospray ionization mass spectrometry analysis indicates that ATP reduces covalent modification of lysine residues 245, 255, 256, and 477, whereas labeling at lysine residues 225, 229, and 230 is unchanged. Exogenous, intracellular GLUT1 C-terminal peptide mimics ATP modulation of transport whereas C-terminal peptide-directed IgGs inhibit ATP modulation of glucose transport. These findings suggest that transport regulation involves ATP-dependent conformational changes in (or interactions between) the GLUT1 C terminus and the C-terminal half of GLUT1 cytoplasmic loop 6–7

A noncanonical autophagy pathway restricts Toxoplasma gondii growth in a strain-specific manner in IFN-γ-activated human cells

ABSTRACT A core set of autophagy proteins is required for gamma interferon (IFN-γ)-mediated clearance of Toxoplasma gondii in the mouse because of their control of several downstream effectors, including immunity-related GTPases (IRGs) and guanylate-binding proteins (GBPs). However, these effectors are absent (i.e., IRGs) from or nonessential (i.e., GBPs) in IFN-γ-activated human cells, raising the question of how these cells control parasite replication. Here, we define a novel role for ubiquitination and recruitment of autophagy adaptors in the strain-specific control of T. gondii replication in IFN-γ-activated human cells. Vacuoles containing susceptible strains of T. gondii became ubiquitinated, recruited the adaptors p62 and NDP52, and were decorated with LC3. Parasites within LC3-positive vacuoles became enclosed in multiple layers of host membranes, resulting in stunting of parasite replication. However, LC3-positive T. gondii-containing vacuoles did not fuse with endosomes and lysosomes, indicating that this process is fundamentally different from xenophagy, a form of autophagy involved in the control of intracellular bacterial pathogens. Genetic knockout of ATG16L or ATG7 reverted the membrane encapsulation and restored parasite replication, indicating that core autophagy proteins involved in LC3 conjugation are important in the control of parasite growth. Despite a role for the core autophagy machinery in this process, upstream activation through Beclin 1 was not sufficient to enhance the ubiquitination of T. gondii-containing vacuoles, suggesting a lack of reliance on canonical autophagy. These findings demonstrate a new mechanism for IFN-γ-dependent control of T. gondii in human cells that depends on ubiquitination and core autophagy proteins that mediate membrane engulfment and restricted growth

Closing the sea surface mixed layer temperature budget from in situ observations alone: Operation Advection during BoBBLE

Sea surface temperature (SST) is a fundamental driver of tropical weather systems such as monsoon rainfall and tropical cyclones. However, understanding of the factors that control SST variability is lacking, especially during the monsoons when in situ observations are sparse. Here we use a ground-breaking observational approach to determine the controls on the SST variability in the southern Bay of Bengal. We achieve this through the first full closure of the ocean mixed layer energy budget derived entirely from in situ observations during the Bay of Bengal Boundary Layer Experiment (BoBBLE). Locally measured horizontal advection and entrainment contribute more significantly than expected to SST evolution and thus oceanic variability during the observation period. These processes are poorly resolved by state-of-the-art climate models, which may contribute to poor representation of monsoon rainfall variability. The novel techniques presented here provide a blueprint for future observational experiments to quantify the mixed layer heat budget on longer time scales and to evaluate these processes in models

The role of multisystemic resilience in fostering critical agency : UK adolescents during the COVID-19 pandemic

DATA AVAILABILITY STATEMENT : TThe datasets generated during and/or analysed during the current study are available in the University of Leicester Figshare repository: https://doi.org/10.25392/leicester.data.c.6079419.v2.Critical agency (CA) refers to an individual’s feeling of power in relation to social inequalities. Research has demonstrated that high CA is associated with positive adolescent outcomes, however, less is known about what supports are important for its development. Moreover, a large majority of the literature is based on studies from the US and various countries in Africa; although the UK is saturated with inequalities there is little research within a UK context. In this paper we examine (a) the validity of using an existing measure of CA with a sample of UK adolescents and (b) the extent to which resilience supports account for variance in CA. Our analysis identified two distinct factors of CA: justiceoriented and community-oriented. High CA in both factors was explained by resilience supports associated with peer relationships (p < 0.01). Our findings push us towards new relational, ecological ways of understanding adolescent CA. We close by instantiating a translational framework for those devising policies in support of youth resilience and CA.FUNDING : Arts and Humanities Research Council (AH/V015060/1).http://link.springer.com/journal/12144am2024Educational PsychologySDG-03:Good heatlh and well-bein

The role of multisystemic resilience in fostering critical agency: UK adolescents during the COVID-19 Pandemic

Critical agency (CA) refers to an individual’s feeling of power in relation to social inequalities. Research has demonstrated that high CA is associated with positive adolescent outcomes, however, less is known about what supports are important for its development. Moreover, a large majority of the literature is based on studies from the US and various countries in Africa; although the UK is saturated with inequalities there is little research within a UK context. In this paper we examine (a) the validity of using an existing measure of CA with a sample of UK adolescents and (b) the extent to which resilience supports account for variance in CA. Our analysis identified two distinct factors of CA: justice-oriented and community-oriented. High CA in both factors was explained by resilience supports associated with peer relationships (p < 0.01). Our findings push us towards new relational, ecological ways of understanding adolescent CA. We close by instantiating a translational framework for those devising policies in support of youth resilience and CA

Flow-based membrane technology to engineer T-cells

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Moyo Vol. VIII N 1

Durica, Paul Editor\u27s Letter . 4. Thackeray, Alex. Postcard from the sXe . 5. Ward, Luc. Gods & Monsters (Hook-Up at Church) . 6. Anshuman, Karan. The India Nobody Knows (Mysticism and Misconceptions Revealed) . 8. Clements, Nina and Betsy Falconer. God as One of Us: Diverse Faiths Thrive at Denison . 10. Million, Chris. Splendor in the Fall (First Year Love Bittersweet) . 16. Grindstaff, Michelle. Beer by Night, Bed by Morning . 17. Hart, Madeline and Meredith Newman. Smoke Alarm: Reading This may Cause Lung Cancer, Heart Disease, Emphysema, and Complicate Pregnancy . 21. Almirall, Sara and Kirsten Werne. 20 Best Spots to Smoke on Campus . 22. Werne, Kirsten. An Interview with Painted Thin . 23. Burt, Kara. All in All, We\u27re Just Paper o the Wall (Dorm Art Clue to Denison Identity) . 25. Levine, Robert. Less Talk, Moore Rock (Thurston\u27 Sound Uplifts Soul) . 30. Almirall, R.R. Turtles . 31. Almirall, RR. The Warthog Feels He Has Much in Common With Paul Newman, Others Don\u27t . 20