Author Correction: Distinct modes of mitochondrial metabolism uncouple T cell differentiation and function.

An Amendment to this paper has been published and can be accessed via a link at the top of the paper.

Distinct modes of mitochondrial metabolism uncouple T cell differentiation and function.

Activated CD4 T cells proliferate rapidly and remodel epigenetically before exiting the cell cycle and engaging acquired effector functions. Metabolic reprogramming from the naive state is required throughout these phases of activation1. In CD4 T cells, T-cell-receptor ligation-along with co-stimulatory and cytokine signals-induces a glycolytic anabolic program that is required for biomass generation, rapid proliferation and effector function2. CD4 T cell differentiation (proliferation and epigenetic remodelling) and function are orchestrated coordinately by signal transduction and transcriptional remodelling. However, it remains unclear whether these processes are regulated independently of one another by cellular biochemical composition. Here we demonstrate that distinct modes of mitochondrial metabolism support differentiation and effector functions of mouse T helper 1 (TH1) cells by biochemically uncoupling these two processes. We find that the tricarboxylic acid cycle is required for the terminal effector function of TH1 cells through succinate dehydrogenase (complex II), but that the activity of succinate dehydrogenase suppresses TH1 cell proliferation and histone acetylation. By contrast, we show that complex I of the electron transport chain, the malate-aspartate shuttle and mitochondrial citrate export are required to maintain synthesis of aspartate, which is necessary for the proliferation of T helper cells. Furthermore, we find that mitochondrial citrate export and the malate-aspartate shuttle promote histone acetylation, and specifically regulate the expression of genes involved in T cell activation. Combining genetic, pharmacological and metabolomics approaches, we demonstrate that the differentiation and terminal effector functions of T helper cells are biochemically uncoupled. These findings support a model in which the malate-aspartate shuttle, mitochondrial citrate export and complex I supply the substrates needed for proliferation and epigenetic remodelling early during T cell activation, whereas complex II consumes the substrates of these pathways, which antagonizes differentiation and enforces terminal effector function. Our data suggest that transcriptional programming acts together with a parallel biochemical network to enforce cell state.

An in Vitro Selection Strategy Identifying Naked DNA That Localizes to Cell Nuclei.

Combinatorial chemistry drives the biological generation of protein structural diversity in antibodies and T-cell receptors. When applied to nucleic acids, vast engineered random libraries of DNA and RNA strands allow selection of affinity reagents ("aptamers") against molecular targets. Selection involves cycles rewarding target binding affinity with amplification. Despite the success of this approach, delivery of selected aptamers across cell membranes and to specific subcellular compartments is an unmet need in chemical biology. Here, we address this challenge, demonstrating in vitro selection of DNA aptamers capable of homing to nuclei of cultured cells without transfection agents or viral transduction. Selection of such folded karyophilic DNA aptamers (∼100 nucleotides) is achieved by a biosensor strategy that rewards exposure to nuclear DNA ligase. Identified DNA molecules are preferentially delivered to cell nuclei within minutes. Related strategies can be envisioned to select aptamers that home to other subcellular compartments.

Motivating Self-Efficacy in Diverse Biomedical Science Post-baccalaureate and Graduate Students Through Scientific Conference Implementation.

Tactics to increase the number of underrepresented (UR) students in biomedical research PhD training programs have not yet translated to UR faculty numbers that reflect the diversity of the United States. Continued interventions are required to build skills beyond those that result in placement into a PhD program. We hypothesize that successful interventions must build skills that give UR students foundations for confident self-efficacy in leadership. We seek interventions that allow UR students to envision themselves as successful faculty. We posit that development of such skills is difficult in the classroom or laboratory alone. Therefore, novel interventions are required. As part of the NIH-funded Post-baccalaureate Research Education Program (PREP) and Initiative for Maximizing Student Development (IMSD) at the Mayo Clinic Graduate School of Biomedical Sciences, we designed and implemented a unique intervention to support development of student leadership skills: a biannual student-organized and student-led national research conference titled "Scientific Innovation Through Diverse Perspectives" (SITDP). This initiative is based on the concept that students who actively live out realistic roles as scientific leaders will be encouraged to persist to scientific leadership as faculty. Here we describe the motivation for, design of, and outcomes from, the first three pilot conferences of this series. We further discuss approaches needed to rigorously evaluate the effectiveness of such interventions in the future.

Sensing and Liquid-Liquid Extraction of Dicarboxylates Using Dicopper Cryptates.

We report the investigation of dicopper(II) bistren cryptate, containing naphthyl spacers between the tren subunits, as a receptor for polycarboxylates in neutral aqueous solution. An indicator displacement assay for dicarboxylates was also developed by mixing the azacryptate with the fluorescent indicator 5-carboxyfluorescein in a 50:1 molar ratio. Fluorimetric studies showed a significant restoration of fluorophore emission upon addition of fumarate anions followed by succinate and isophthalate. The introduction of hexyl chains on the naphthalene groups created a novel hydrophobic cage; the corresponding dicopper complex was investigated as an extractant for dicarboxylates from neutral water into dichloromethane. The liquid-liquid extraction of succinate-as a model anion-was successfully achieved by exploiting the high affinity of this anionic guest for the azacryptate cavity. Extraction was monitored through the changes in the UV-visible spectrum of the dicopper complex in dichloromethane and by measuring the residual concentration of succinate in the aqueous phase by HPLC-UV. The successful extraction was also confirmed by 1H-NMR spectroscopy. Considering the relevance of polycarboxylates in biochemistry and in the environmental field, e.g., as waste products of industrial processes, our results open new perspectives for research in all contexts where recognition, sensing, or extraction of polycarboxylates is required.

Remyelination-Promoting DNA Aptamer Conjugate Myaptavin-3064 Binds to Adult Oligodendrocytes In Vitro.

We previously applied Systematic Evolution of Ligands by EXponential enrichment (SELEX) technology to identify myelin-specific DNA aptamers, using crude mouse central nervous system myelin as bait. This selection identified a 40-nucleotide aptamer (LJM-3064). Multiple biotinylated LJM-3064 molecules were conjugated to a streptavidin core to mimic a multimeric immunoglobulin M (IgM) antibody, generating 3064-BS-streptavidin (Myaptavin-3064). We previously showed that Myaptavin-3064 induces remyelination in the Theiler's murine encephalomyelitis virus (TMEV) model of chronic spinal cord demyelination. While details of target binding and the mechanism of action remain unclear, we hypothesized that Myaptavin-3064 induces remyelination by binding to oligodendrocytes (OLs). We now report the results of binding assays using the human oligodendroglioma (HOG) cell line, applying both flow cytometry and immunocytochemistry (IC) to assay aptamer conjugate binding to cells. IC assays were applied to compare aptamer conjugate binding to primary embryonic mouse mixed cortical cultures and primary adult rat mixed glial cultures. We show that Myaptavin-3064 binds to HOG cells, with increased binding upon differentiation. In contrast, a negative control aptamer conjugate, 3060-BS, which did not promote central nervous system (CNS) remyelination, does not bind to HOG cells. Myaptavin-3064 did not bind to lung (L2) or kidney (BHK) cell lines. Total internal reflection fluorescence (TIRF) imaging indicates that Myaptavin-3064 binds at the cell membrane of live cells. In addition to HOG cells, Myaptavin-3064 binds to adult rat OLs, but not to embryonic mouse mixed cortical cultures. These data support the hypothesis that Myaptavin-3064 binds to a surface molecule on both rodent and human OLs in a manner that triggers a remyelination signal pathway.

DNA on a tube: electrostatic contribution to stiffness.

Two simple models are used to estimate the electrostatic contributions to the stiffness of short DNA fragments. The first model views DNA as two strands that are appropriately parametrized and are wrapped helically around a straight cylinder radius equal to the radius of the DNA molecule. The potential energy of the DNA due to phosphate-phosphate electrostatic interactions is evaluated assuming that the charges interact through Debye-Hückel potentials. This potential energy is compared with the potential energy as computed using our second model in which DNA is viewed as two helical strands wrapping around a curved tube whose cross-section is a disk of radius equal to the radius of the DNA. We find that the electrostatic persistence length for B-DNA molecules in the range of 105-130 bp is 125.64 angstroms (37 bp) and 76.05 angstroms (23 bp) at 5 and 10 mM monovalent salt concentration, respectively. If the condensed fraction theta is taken to be 0.715 at 10 mM, then the electrostatic persistence length is 108.28 angstroms (32 bp), while that based on taking into account end effects is 72.87 angstroms (21 bp). At 5 mM monovalent salt, the total persistence length for DNA fragments in this length range is approximately 575.64 angstroms (171 bp), using the best estimate for nonelectrostatic contribution to persistence length. Electrostatic effects thus contribute 21.8% to DNA stiffness at 5 mM for fragments between 105- to 130-bp. In contrast, electrostatics are calculated to make a negligible contribution to the DNA persistence length at physiological monovalent cation concentration. The results are compared with counterion condensation models and experimental data.

A comparison of human natural monoclonal antibodies and aptamer conjugates for promotion of CNS remyelination: where are we now and what comes next?

INTRODUCTION: Multiple sclerosis (MS) is a chronic and progressive inflammatory demyelinating disease of the human central nervous system (CNS) and is the most common disabling neurological condition in young adults, resulting in severe neurological defects. No curative or long-term progression-inhibiting therapy has yet been developed. However, recent investigation has revealed potential strategies that do not merely modulate potentially pathogenic autoimmune responses, but stimulate remyelination within CNS lesions. AREAS COVERED: We discuss the history and development of natural human IgM-isotype immunoglobulins (HIgMs) and recently-identified aptamer-conjugates that have been shown to enhance endogenous myelin repair in animal models of demyelination by acting on myelin-producing oligodendrocytes (OLs) or oligodendrocyte progenitor cells (OPCs) within CNS lesions. We also discuss future development aims and applications for these important novel technologies. EXPERT OPINION: Aptamer conjugate Myaptavin-3064 and recombinant human IgM-isotype antibody rHIgM22 regenerate CNS myelin, thereby reducing axonal degeneration and offering the potential of recovery from MS relapses, reversal of disability and prevention of disease progression. Advancement of these technologies into the clinic for MS treatment is therefore a top priority. It remains unclear to what extent the therapeutic modalities of remyelinating antibodies and aptamers may synergize with other currently-approved therapies to yield enhanced therapeutic effects.