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Natural prokaryotic gene repression systems often exploit DNA looping to increase the local concentration of gene repressor proteins at a regulated promoter via contributions from repressor proteins bound at distant sites. Using principles from the Escherichia coli lac operon we design analogous repression systems based on target sequence-programmable Transcription Activator-Like Effector dimer (TALED) proteins. Such engineered switches may be valuable for synthetic biology and therapeutic applications. Previous TALEDs with inducible non-covalent dimerization showed detectable, but limited, DNA loop-based repression due to the repressor protein dimerization equilibrium. Here, we show robust DNA loop-dependent bacterial promoter repression by covalent TALEDs and verify that DNA looping dramatically enhances promoter repression in E. coli. We characterize repression using a thermodynamic model that quantitates this favorable contribution of DNA looping. This analysis unequivocally and quantitatively demonstrates that optimized TALED proteins can drive loop-dependent promoter repression in E. coli comparable to the natural LacI repressor system. This work elucidates key design principles that set the stage for wide application of TALED-dependent DNA loop-based repression of target genes., (© The Author(s) 2024. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Reduced skeletal muscle mass and oxidative capacity coexist in patients with pulmonary emphysema and are independently associated with higher mortality. If reduced cellular respiration contributes to muscle atrophy in that setting remains unknown. Using a mouse with genetically induced pulmonary emphysema that recapitulates muscle dysfunction, we found that reduced activity of succinate dehydrogenase (SDH) is a hallmark of its myopathic changes. We generated an inducible, muscle-specific SDH knockout mouse that demonstrates lower mitochondrial oxygen consumption, myofiber contractility, and exercise endurance. Respirometry analyses show that in vitro complex I respiration is unaffected by loss of SDH subunit C in muscle mitochondria, which is consistent with the pulmonary emphysema animal data. SDH knockout initially causes succinate accumulation associated with a down-regulated transcriptome but modest proteome effects. Muscle mass, myofiber type composition, and overall body mass constituents remain unaltered in the transgenic mice. Thus, while SDH regulates myofiber respiration in experimental pulmonary emphysema, it does not control muscle mass or other body constituents.

In humans, loss of heterozygosity for defective alleles of any of the four subunits of mitochondrial tricarboxylic acid cycle enzyme succinate dehydrogenase (SDH, also Complex II of the electron transport chain) can lead to paraganglioma tumors in neuroendocrine cells. With the goal of developing mouse models of this rare disorder, we have developed various SDH conditional loss strategies. Based on recent lineage tracing studies, we hypothesized that conditional SDHC loss in early embryogenesis during migration of primordial neural crest cells that form the susceptible chromaffin cells of the adrenal medulla might induce paraganglioma. We triggered low levels of detectable SDHC loss in Sox10 + cells at E11.5 of mouse development. We report that, rather than developing adrenal medulla paraganglioma (pheochromocytoma), offspring survived with evidence of neural crest cell dysfunction. Phenotypes included mild lower extremity gait anomalies suggestive of neural tube closure defects and patches of unpigmented fur consistent with neural crest-derived melanocyte dysfunction. These defects were not observed in mice lacking Sdhc knockout. Our results add to existing data suggesting that, unlike humans, even early embryonic (Sox10-driven) SDHx loss is inadequate to trigger paraganglioma in mice of the genetic backgrounds that have been investigated. Instead, low levels of tricarboxylic acid cycle-deficient neural crest cells cause mild developmental defects in hind limb and melanocyte function. This new model may be of interest for studies of metabolism during early neural crest cell development., Competing Interests: The authors report no conflicts of interest., (© 2024 The Authors FASEB BioAdvances published by The Federation of American Societies for Experimental Biology.)

Despite recent advances in the understanding of brain tumor pathophysiology, challenges associated with tumor location and characteristics have prevented significant improvement in neuro-oncology therapies. Aptamers are short, single-stranded DNA or RNA oligonucleotides that fold into sequence-specific, 3-dimensional shapes that, like protein antibodies, interact with targeted ligands with high affinity and specificity. Aptamer technology has recently been applied to neuro-oncology as a potential approach to innovative therapy. Preclinical research has demonstrated the ability of aptamers to overcome some obstacles that have traditionally rendered neuro-oncology therapies ineffective. Potential aptamer advantages include their small size, ability in some cases to penetrate the blood-brain barrier, inherent lack of immunogenicity, and applicability for discovering novel biomarkers. Herein, we review recent reports of aptamer applications in neuro-oncology including aptamers found by cell- and in vivo- Systematic Evolution of Ligands by Exponential Enrichment approaches, aptamer-targeted therapeutic delivery modalities, and aptamers in diagnostics and imaging. We further identify crucial future directions for the field that will be important to advance aptamer-based drugs or tools to clinical application in neuro-oncology., (© The Author(s) 2023. Published by Oxford University Press on behalf of the Society for Neuro-Oncology.)

In this issue of Cell Chemical Biology, Bush et al. 1 report an in vitro selection method for optimizing CRISPR-Cas9 single-guide RNAs. This approach may be useful in targeting previously intractable genomic sequences. The results also provide insights into which positions in single-guide RNAs are most amenable to modification., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2023 Elsevier Ltd. All rights reserved.)

Whether TMPRSS2-ERG fusion and TP53 gene alteration coordinately promote prostate cancer (PCa) remains unclear. Here we demonstrate that TMPRSS2-ERG fusion and TP53 mutation / deletion co-occur in PCa patient specimens and this co-occurrence accelerates prostatic oncogenesis. p53 gain-of-function (GOF) mutants are now shown to bind to a unique DNA sequence in the CTNNB1 gene promoter and transactivate its expression. ERG and β-Catenin co-occupy sites at pyrimidine synthesis gene (PSG) loci and promote PSG expression, pyrimidine synthesis and PCa growth. β-Catenin inhibition by small molecule inhibitors or oligonucleotide-based PROTAC suppresses TMPRSS2-ERG- and p53 mutant-positive PCa cell growth in vitro and in mice. Our study identifies a gene transactivation function of GOF mutant p53 and reveals β-Catenin as a transcriptional target gene of p53 GOF mutants and a driver and therapeutic target of TMPRSS2-ERG- and p53 GOF mutant-positive PCa., (© 2023. Springer Nature Limited.)

The efficient and specific delivery of functional cargos such as small-molecule drugs, proteins, or nucleic acids across lipid membranes and into subcellular compartments is a significant unmet need in nanomedicine and molecular biology. Systematic Evolution of Ligands by EXponential enrichment (SELEX) exploits vast combinatorial nucleic acid libraries to identify short, nonimmunogenic single-stranded DNA molecules (aptamers) capable of recognizing specific targets based on their 3D structures and molecular interactions. While SELEX has previously been applied to identify aptamers that bind specific cell types or gain cellular uptake, selection of aptamers capable of carrying cargos to specific subcellular compartments is challenging. Here, we describe peroxidase proximity selection (PPS), a generalizable subcellular SELEX approach. We implement local expression of engineered ascorbate peroxidase APEX2 to biotinylate naked DNA aptamers capable of gaining access to the cytoplasm of living cells without assistance. We discovered DNA aptamers that are preferentially taken up into endosomes by macropinocytosis, with a fraction apparently accessing APEX2 in the cytoplasm. One of these selected aptamers is capable of endosomal delivery of an IgG antibody., (© The Author(s) 2023. Published by Oxford University Press on behalf of National Academy of Sciences.)

Advances in peroxidase and biotin ligase-mediated signal amplification have enabled high-resolution subcellular mapping of endogenous RNA localization and protein-protein interactions. Application of these technologies has been limited to RNA and proteins because of the reactive groups required for biotinylation in each context. Here we report several novel methods for proximity biotinylation of exogenous oligodeoxyribonucleotides by application of well-established and convenient enzymatic tools. We describe approaches using simple and efficient conjugation chemistries to modify deoxyribonucleotides with "antennae" that react with phenoxy radicals or biotinoyl-5'-adenylate. In addition, we report chemical details of a previously undescribed adduct between tryptophan and a phenoxy radical group. These developments have potential application in the selection of exogenous nucleic acids capable of unaided entry into living cells.

The histone chaperone FACT (facilitates chromatin transcription) enhances transcription in eukaryotic cells, targeting DNA-protein interactions. FACT, a heterodimer in humans, comprises SPT16 and SSRP1 subunits. We measure nucleosome stability and dynamics in the presence of FACT and critical component domains. Optical tweezers quantify FACT/subdomain binding to nucleosomes, displacing the outer wrap of DNA, disrupting direct DNA-histone (core site) interactions, altering the energy landscape of unwrapping, and increasing the kinetics of DNA-histone disruption. Atomic force microscopy reveals nucleosome remodeling, while single-molecule fluorescence quantifies kinetics of histone loss for disrupted nucleosomes, a process accelerated by FACT. Furthermore, two isolated domains exhibit contradictory functions; while the SSRP1 HMGB domain displaces DNA, SPT16 MD/CTD stabilizes DNA-H2A/H2B dimer interactions. However, only intact FACT tethers disrupted DNA to the histones and supports rapid nucleosome reformation over several cycles of force disruption/release. These results demonstrate that key FACT domains combine to catalyze both nucleosome disassembly and reassembly., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2022 The Author(s). Published by Elsevier Inc. All rights reserved.)

The quantitative polymerase chain reaction (qPCR) with detection of duplex DNA yield by intercalator fluorescence is a common and essential technique in nucleic acid analysis. We encountered unexpected results when applying standard qPCR methods to the quantitation of random DNA libraries flanked by regions of fixed sequence, a configuration essential for in vitro selection experiments. Here we describe the results of experiments revealing why conventional qPCR methods can fail to allow automated analysis in such cases, and simple solutions to this problem. In particular we show that renaturation of PCR products containing random regions is incomplete in late PCR cycles when extension fails due to reagent depletion. Intercalator fluorescence can then be lost at standard interrogation temperatures. We show that qPCR analysis of random DNA libraries can be achieved simply by adjusting the step at which intercalator fluorescence is monitored so that the yield of annealed constant regions is detected rather than the yield of full duplex DNA products., (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)

Recent advances in peroxidase-mediated biotin tyramide (BT) signal amplification technology have resulted in high-resolution and subcellular compartment-specific mapping of protein and RNA localization. Horseradish peroxidase (HRP) in the presence of H 2 O 2 is known to activate phenolic compounds for phenoxy radical reaction with nucleic acids, where biotinylation by BT is a practical example. BT reactivity with RNA and DNA is not understood in detail. We report that BT phenoxy radicals react in a sequence-independent manner with guanosine bases in RNA. In contrast, DNA reactivity with BT cannot be detected by our methods under the same conditions. Remarkably, we show that fluorescein conjugates DNA rapidly and selectively reacts with BT phenoxy radicals, allowing convenient and practical biotinylation of DNA on fluorescein with retention of fluorescence., (© 2020 The Authors. ChemBioChem published by Wiley-VCH GmbH.)

Mutations in any of the genes encoding the four subunits of succinate dehydrogenase (SDH), a mitochondrial membrane-bound enzyme complex that is involved in both the tricarboxylic acid cycle and the electron transport chain, can lead to a variety of disorders. Recognized conditions with such mutations include Leigh syndrome and hereditary tumors such as pheochromocytoma and paraganglioma (PPGL), renal cell carcinoma, and gastrointestinal stromal tumor. Tumors appear in SDH mutation carriers with dominant inheritance due to loss of heterozygosity in susceptible cells. Here, we describe a mouse model intended to reproduce hereditary PPGL through Cre-mediated loss of SDHC in cells that express tyrosine hydroxylase (TH), a compartment where PPGL is known to originate. We report that while there is modest expansion of TH + glomus cells in the carotid body upon SDHC loss, PPGL is not observed in such mice, even in the presence of a conditional dominant negative p53 protein and chronic hypoxia. Instead, we report an unexpected phenotype of nondiabetic obesity beginning at about 20 weeks of age. We hypothesize that this obesity is caused by TH + cell loss or altered phenotype in key compartments of the central nervous system responsible for regulating feeding behavior, coupled with metabolic changes due to loss of peripheral catecholamine production., (© 2020 Federation of American Societies for Experimental Biology.)

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.

Gene regulation by control of transcription initiation is a fundamental property of living cells. Much of our understanding of gene repression originated from studies of the Escherichia coli lac operon switch, in which DNA looping plays an essential role. To validate and generalize principles from lac for practical applications, we previously described artificial DNA looping driven by designed transcription activator-like effector dimer (TALED) proteins. Because TALE monomers bind the idealized symmetrical lac operator sequence in two orientations, our prior studies detected repression due to multiple DNA loops. We now quantitatively characterize gene repression in living E. coli by a collection of individual TALED loops with systematic loop length variation. Fitting of a thermodynamic model allows unequivocal demonstration of looping and comparison of the engineered TALED repression system with the natural lac repressor system., (Copyright © 2020 Biophysical Society. Published by Elsevier Inc. All rights reserved.)

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 1 H-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., Competing Interests: The authors declare no competing financial interest.

RNA-binding proteins (RBPs) regulate RNA metabolism at multiple levels by affecting splicing of nascent transcripts, RNA folding, base modification, transport, localization, translation, and stability. Despite their central role in RNA function, the RNA-binding specificities of most RBPs remain unknown or incompletely defined. To address this, we have assembled a genome-scale collection of RBPs and their RNA-binding domains (RBDs) and assessed their specificities using high-throughput RNA-SELEX (HTR-SELEX). Approximately 70% of RBPs for which we obtained a motif bound to short linear sequences, whereas ∼30% preferred structured motifs folding into stem-loops. We also found that many RBPs can bind to multiple distinctly different motifs. Analysis of the matches of the motifs in human genomic sequences suggested novel roles for many RBPs. We found that three cytoplasmic proteins-ZC3H12A, ZC3H12B, and ZC3H12C-bound to motifs resembling the splice donor sequence, suggesting that these proteins are involved in degradation of cytoplasmic viral and/or unspliced transcripts. Structural analysis revealed that the RNA motif was not bound by the conventional C3H1 RNA-binding domain of ZC3H12B. Instead, the RNA motif was bound by the ZC3H12B's PilT N terminus (PIN) RNase domain, revealing a potential mechanism by which unconventional RBDs containing active sites or molecule-binding pockets could interact with short, structured RNA molecules. Our collection containing 145 high-resolution binding specificity models for 86 RBPs is the largest systematic resource for the analysis of human RBPs and will greatly facilitate future analysis of the various biological roles of this important class of proteins., (© 2020 Jolma et al.; Published by Cold Spring Harbor Laboratory Press.)

Mitochondrial disorders arise from defects in nuclear genes encoding enzymes of oxidative metabolism. Mutations of metabolic enzymes in somatic tissues can cause cancers due to oncometabolite accumulation. Paraganglioma and pheochromocytoma are examples, whose etiology and therapy are complicated by the absence of representative cell lines or animal models. These tumors can be driven by loss of the tricarboxylic acid cycle enzyme succinate dehydrogenase. We exploit the relationship between succinate accumulation, hypoxic signaling, egg-laying behavior, and morphology in C. elegans to create genetic and pharmacological models of succinate dehydrogenase loss disorders. With optimization, these models may enable future high-throughput screening efforts., Competing Interests: The authors have declared that no competing interests exist.

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.

Repression of a promoter by entrapment within a tightly bent DNA loop is a common mechanism of gene regulation in bacteria. Besides the mechanical properties of the looped DNA and affinity of the protein that anchors the loop, cellular energetics and DNA negative supercoiling are likely factors determining the stability of the repression loop. E. coli cells undergo numerous highly regulated and dynamic transitions as resources are depleted during bacterial growth. We hypothesized that the probability of DNA looping depends on the growth status of the E. coli culture. We utilized a well-characterized repression loop model assembled from elements of the lac operon to measure loop length-dependent repression at three different culture densities. Remarkably, even with changes in supercoiling, there exists a dynamic compensation in which the contribution of DNA looping to gene repression remains essentially constant., Competing Interests: None.

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 activation 1 . 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 function 2 . 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 (T H 1) cells by biochemically uncoupling these two processes. We find that the tricarboxylic acid cycle is required for the terminal effector function of T H 1 cells through succinate dehydrogenase (complex II), but that the activity of succinate dehydrogenase suppresses T H 1 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.

Background: Succinate dehydrogenase (SDH) loss and mastermind-like 3 (MAML3) translocation are two clinically important genetic alterations that correlate with increased rates of metastasis in subtypes of human paraganglioma and pheochromocytoma (PPGL) neuroendocrine tumors. Although hypotheses propose that succinate accumulation after SDH loss poisons dioxygenases and activates pseudohypoxia and epigenomic hypermethylation, it remains unclear whether these mechanisms account for oncogenic transcriptional patterns. Additionally, MAML3 translocation has recently been identified as a genetic alteration in PPGL, but is poorly understood. We hypothesize that a key to understanding tumorigenesis driven by these genetic alterations is identification of the transcription factors responsible for the observed oncogenic transcriptional changes., Methods: We leverage publicly-available human tumor gene expression profiling experiments (N = 179) to reconstruct a PPGL tumor-specific transcriptional network. We subsequently use the inferred transcriptional network to perform master regulator analyses nominating transcription factors predicted to control oncogenic transcription in specific PPGL molecular subtypes. Results are validated by analysis of an independent collection of PPGL tumor specimens (N = 188). We then perform a similar master regulator analysis in SDH-loss mouse embryonic fibroblasts (MEFs) to infer aspects of SDH loss master regulator response conserved across species and tissue types., Results: A small number of master regulator transcription factors are predicted to drive the observed subtype-specific gene expression patterns in SDH loss and MAML3 translocation-positive PPGL. Interestingly, although EPAS1 perturbation is detectible in SDH-loss and VHL-loss tumors, it is by no means the most potent factor driving observed patterns of transcriptional dysregulation. Analysis of conserved SDH-loss master regulators in human tumors and MEFs implicated ZNF423, a known modulator of retinoic acid response in neuroblastoma. Subsequent functional analysis revealed a blunted cell death response to retinoic acid in SDH-loss MEFs and blunted differentiation response in SDH-inhibited SH-SY5Y neuroblastoma cells., Conclusions: The unbiased analyses presented here nominate specific transcription factors that are likely drivers of oncogenic transcription in PPGL tumors. This information has the potential to be exploited for targeted therapy. Additionally, the observation that SDH loss or inhibition results in blunted retinoic acid response suggests a potential developmental etiology for this tumor subtype.

We previously reported the in vitro selection and characterization of a DNA aptamer capable of stimulating remyelination in a mouse model of multiple sclerosis. This aptamer was selected for its ability to bind to suspensions of crude murine myelin in vitro . Our initial studies in vitro and in vivo involved a 40-nucleotide derivative (LJM-3064) of the original 100-nucleotide aptamer. LJM-3064 retained robust myelin-binding properties. Structural characterization of LJM-3064 revealed that the guanosine-rich 5' half of the sequence forms different G-quadruplex-type structures that are variably stable in the presence of physiologically relevant ions. We hypothesized that this structured domain is sufficient for myelin binding. In this study, we confirm that a 20-nucleotide DNA, corresponding to the 5' half of LJM-3064, retains myelin-binding properties. We then optimize this minimal myelin-binding aptamer via systematic evolution of ligands by exponential enrichment after sparse rerandomization. We report a sequence variant (LJM-5708) of the 20-nucleotide myelin-binding aptamer with enhanced myelin-binding properties and the ability to bind cultured human oligodendroglioma cells in vitro , providing the first evidence of cross-species reactivity of this myelin-binding aptamer. As our formulation of DNA aptamers for in vivo remyelination therapy involves conjugation to streptavidin, we verified that the myelin-binding properties of LJM-5708 were retained in conjugates to avidin, streptavidin, and neutravidin. DNA aptamer LJM-5708 is a lead for further preclinical development of remyelinating aptamer technologies.

Introduction : Multiple sclerosis is a serious demyelinating disease of the central nervous system (CNS) with treatments generally restricted to immunosuppression to reduce attack rate and for symptom management. Glial cells may be useful targets for future CNS regenerative therapies to reverse disease. Areas covered : In this review, the authors cover currently available multiple sclerosis treatments and examine potential upcoming therapies targeting glial cells. The potential for new therapeutic approaches in the treatment of progressive multiple sclerosis is examined. Expert opinion : Microglia, astrocytes, and oligodendrocytes are each promising targets for the disease-altering treatment of multiple sclerosis. Though challenging, the opportunities presented have great potential for CNS regeneration and further investigation of glial cells in therapy is warranted. Patient-specific combinatorial therapy targeting the three glial cell types is expected to be the future of MS treatment.

Nucleosome disruption plays a key role in many nuclear processes including transcription, DNA repair and recombination. Here we combine atomic force microscopy (AFM) and optical tweezers (OT) experiments to show that high mobility group B (HMGB) proteins strongly disrupt nucleosomes, revealing a new mechanism for regulation of chromatin accessibility. We find that both the double box yeast Hmo1 and the single box yeast Nhp6A display strong binding preferences for nucleosomes over linker DNA, and both HMGB proteins destabilize and unwind DNA from the H2A-H2B dimers. However, unlike Nhp6A, Hmo1 also releases half of the DNA held by the (H3-H4)2 tetramer. This difference in nucleosome destabilization may explain why Nhp6A and Hmo1 function at different genomic sites. Hmo1 is enriched at highly transcribed ribosomal genes, known to be depleted of histones. In contrast, Nhp6A is found across euchromatin, pointing to a significant difference in cellular function.

rHIgM22 is a recombinant human monoclonal IgM designed to promote remyelination, and it is currently in Phase I clinical trials in patients with multiple sclerosis (MS). In animal models of demyelination, a single low dose of rHIgM22 stimulates oligodendrocyte maturation, induces remyelination, preserves axons, and slows the decline of locomotor deficits. Natural autoantibodies like rHIgM22 typically bind to multiple antigens with weak affinity. rHIgM22 binds to oligodendrocytes and myelin. Because the antigens for rHIgM22 is prevalent within and exclusive to central nervous system (CNS) myelin, we used CNS myelin particles in combination with surface plasmon resonance to determine the kinetic and affinity constants for the interaction of rHIgM22 to myelin. We found that both the serum and recombinant forms of the antibody bind to myelin with very small dissociation constants in the 100 pM range, which is highly unusual for natural autoantibodies. The extraordinary affinity between rHIgM22 and myelin may explain why such a low effective dose can stimulate CNS repair in animal models of demyelination and underlie the accumulation of rHIgM22 in the CSF in treated MS patients by targeting myelin.

Leveraging structural biology tools, we report the results of experiments seeking to determine if the different mechanical properties of DNA polymers with base analog substitutions can be attributed, at least in part, to induced changes from classical B-form DNA. The underlying hypothesis is that different inherent bending and twisting flexibilities may characterize non-canonical B-DNA, so that it is inappropriate to interpret mechanical changes caused by base analog substitution as resulting simply from 'electrostatic' or 'base stacking' influences without considering the larger context of altered helical geometry. Circular dichroism spectra of inosine-substituted oligonucleotides and longer base-substituted DNAs in solution indicated non-canonical helical conformations, with the degree of deviation from a standard B-form geometry depending on the number of I⋅C pairs. X-ray diffraction of a highly inosine-substituted DNA decamer crystal (eight I⋅C and two A⋅T pairs) revealed an A-tract-like conformation with a uniformly narrow minor groove, reduced helical rise, and the majority of sugars adopting a C1'-exo (southeastern) conformation. This contrasts with the standard B-DNA geometry with C2'-endo sugar puckers (south conformation). In contrast, the crystal structure of a decamer with only four I⋅C pairs has a geometry similar to that of the reference duplex with eight G⋅C and two A⋅T pairs. The unique crystal geometry of the inosine-rich duplex is noteworthy given its unusual CD signature in solution and the altered mechanical properties of some inosine-containing DNAs.

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.

Succinylation is a post-translational protein acylation modification that converts the cationic lysine side chain to an anion with large potential impacts on protein structure and function. Here we characterize the epigenome-wide distribution of succinyllysine marks in chromatin using chromatin immuno-precipitation sequencing (ChIP-seq). We estimate that more than one-third of all nucleosomes contain lysine succinylation marks and demonstrate a potential role of chromatin succinylation in modulating gene expression. We further demonstrate that defective tricarboxylic acid (TCA) cycle metabolism perturbs the succinyllysine distribution in chromatin, correlating with transcriptional responses. This is consistent with previous observations linking nucleosome succinylation with enhanced in vitro transcription. We additionally demonstrate that defective TCA cycle metabolism results in a DNA repair defect and sensitivity to genotoxic agents, consistent with previously reported chromatin hypersuccinylation effects observed in the context of SIRT7 depletion. Chromatin succinylation may thus represent a mechanism by which metabolism modulates both genome-wide transcription and DNA repair activities., Competing Interests: DECLARATION OF INTERESTS The authors declare no competing interests.

Multiple sclerosis (MS) is a debilitating disease for which regenerative therapies are sought. We have previously described human antibodies and DNA aptamer-streptavidin conjugates that promote remyelination after systemic injection into mice infected by Theiler's murine encephalomyelitis virus. Here, we report an in vitro assay of myelin binding with results that correlate with remyelination outcome in vivo , as shown for data from a set of DNA aptamer complexes of different size and formulation. This in vitro assay will be valuable for future screening of MS regenerative therapies targeting remyelination.

Genetic switches must alternate between states whose probabilities are dependent on regulatory signals. Classical examples of transcriptional control in bacteria depend on repressive DNA loops anchored by proteins whose structures are sensitive to small molecule inducers or co-repressors. We are interested in exploiting these natural principles to engineer artificial switches for transcriptional control of bacterial genes. Here, we implement designed homodimeric DNA looping proteins ('Transcription Activator-Like Effector Dimers'; TALEDs) for this purpose in living bacteria. Using well-studied FKBP dimerization domains, we build switches that mimic regulatory characteristics of classical Escherichia coli lactose, galactose and tryptophan operon promoters, including induction or co-repression by small molecules. Engineered DNA looping using TALEDs is thus a new approach to tuning gene expression in bacteria. Similar principles may also be applicable for gene control in eukaryotes.

Atomic force microscopy (AFM) is widely used to image and study biological molecules. As an example, we have utilized AFM to investigate how the mechanical properties of DNA polymers depend on electrostatics and the strength of DNA base stacking by studying double-stranded DNA molecules incorporating several different neutral and charged base modifications. Here, we describe ten complementary approaches for determining DNA persistence length by AFM imaging. The combination of different approaches provides increased confidence and statistical reliability over existing methods utilizing only a single approach.

The occurrence of DNA looping is ubiquitous. This process plays a well-documented role in the regulation of prokaryotic gene expression, such as the Escherichia coli lactose (lac) operon. Here, we present two complementary methods for high-resolution in vivo detection of DNA/protein binding within the bacterial nucleoid by using either chromatin immunoprecipitation combined with phage λ exonuclease digestion (ChIP-exo) or chromatin endogenous cleavage (ChEC), coupled with ligation-mediated polymerase chain reaction (LM-PCR) and Southern blot analysis. As an example we apply these in vivo protein-mapping methods to E. coli to show direct binding of architectural proteins in the Lac repressor-mediated DNA repression loop.

Succinate dehydrogenase (SDH)-loss pheochromocytoma and paraganglioma (PPGL) are tumors driven by metabolic derangement. SDH loss leads to accumulation of intracellular succinate, which competitively inhibits dioxygenase enzymes, causing activation of pseudohypoxic signaling and hypermethylation of histones and DNA. The mechanisms by which these alterations lead to tumorigenesis are unclear, however. In an effort to fundamentally understand how SDH loss reprograms cell biology, we developed an immortalized mouse embryonic fibroblast cell line with conditional disruption of Sdhc and characterize the kinetics of Sdhc gene rearrangement, SDHC protein loss, succinate accumulation, and the resultant hypoproliferative phenotype. We further perform global transcriptomic, epigenomic, and proteomic characterization of changes resulting from SDHC loss, identifying specific perturbations at each biological level. We compare the observed patterns of epigenomic derangement to another previously-described immortalized mouse chromaffin cell model of SDHB loss, and compare both models to human SDH-loss tumors. Finally, we perform analysis of SDHC synthetic lethality with lactate dehydrogenase A (LDHA) and pyruvate carboxylase (PCX), which are important for regeneration of NAD+ and aspartate biosynthesis, respectively. Our data show that SDH-loss cells are selectively vulnerable to LDH genetic knock-down or chemical inhibition, suggesting that LDH inhibition may be an effective therapeutic strategy for SDH-loss PPGL., Competing Interests: CONFLICTS OF INTEREST None declared.

Background & Aims: Depletion of interstitial cells of Cajal (ICCs) is common in diabetic gastroparesis. However, in approximately 20% of patients with diabetes, gastric emptying (GE) is accelerated. GE also occurs faster in obese individuals, and is associated with increased blood levels of glucose in patients with type 2 diabetes. To understand the fate of ICCs in hyperinsulinemic, hyperglycemic states characterized by rapid GE, we studied mice with mutation of the leptin receptor (Lepr db/db ), which in our colony had accelerated GE. We also investigated hyperglycemia-induced signaling in the ICC lineage and ICC dependence on glucose oxidative metabolism in mice with disruption of the succinate dehydrogenase complex, subunit C gene (Sdhc)., Methods: Mice were given breath tests to analyze GE of solids. ICCs were studied by flow cytometry, intracellular electrophysiology, isometric contractility measurement, reverse-transcription polymerase chain reaction, immunoblot, immunohistochemistry, enzyme-linked immunosorbent assays, and metabolite assays; cells and tissues were manipulated pharmacologically and by RNA interference. Viable cell counts, proliferation, and apoptosis were determined by methyltetrazolium, Ki-67, proliferating cell nuclear antigen, bromodeoxyuridine, and caspase-Glo 3/7 assays. Sdhc was disrupted in 2 different strains of mice via cre recombinase., Results: In obese, hyperglycemic, hyperinsulinemic female Lepr db/db mice, GE was accelerated and gastric ICC and phasic cholinergic responses were increased. Female Kit K641E/+ mice, which have genetically induced hyperplasia of ICCs, also had accelerated GE. In isolated cells of the ICC lineage and gastric organotypic cultures, hyperglycemia stimulated proliferation by mitogen-activated protein kinase 1 (MAPK1)- and MAPK3-dependent stabilization of ets variant 1-a master transcription factor for ICCs-and consequent up-regulation of v-kit Hardy-Zuckerman 4 feline sarcoma viral oncogene homolog (KIT) receptor tyrosine kinase. Opposite changes occurred in mice with disruption of Sdhc., Conclusions: Hyperglycemia increases ICCs via oxidative metabolism-dependent, MAPK1- and MAPK3-mediated stabilization of ets variant 1 and increased expression of KIT, causing rapid GE. Increases in ICCs might contribute to the acceleration in GE observed in some patients with diabetes., (Copyright © 2017 AGA Institute. Published by Elsevier Inc. All rights reserved.)

Protein-DNA interactions can be characterized and quantified using single molecule methods such as optical tweezers, magnetic tweezers, atomic force microscopy, and fluorescence imaging. In this review, we discuss studies that characterize the binding of high-mobility group B (HMGB) architectural proteins to single DNA molecules. We show how these studies are able to extract quantitative information regarding equilibrium binding as well as non-equilibrium binding kinetics. HMGB proteins play critical but poorly understood roles in cellular function. These roles vary from the maintenance of chromatin structure and facilitation of ribosomal RNA transcription (yeast high-mobility group 1 protein) to regulatory and packaging roles (human mitochondrial transcription factor A). We describe how these HMGB proteins bind, bend, bridge, loop and compact DNA to perform these functions. We also describe how single molecule experiments observe multiple rates for dissociation of HMGB proteins from DNA, while only one rate is observed in bulk experiments. The measured single-molecule kinetics reveals a local, microscopic mechanism by which HMGB proteins alter DNA flexibility, along with a second, much slower macroscopic rate that describes the complete dissociation of the protein from DNA.

DNA-protein loops can be essential for gene regulation. The Escherichia coli lactose (lac) operon is controlled by DNA-protein loops that have been studied for decades. Here we adapt this model to test the hypothesis that negative superhelical strain facilitates the formation of short-range (6-8 DNA turns) repression loops in E. coli. The natural negative superhelicity of E. coli DNA is regulated by the interplay of gyrase and topoisomerase enzymes, adding or removing negative supercoils, respectively. Here, we measured quantitatively DNA looping in three different E. coli strains characterized by different levels of global supercoiling: wild type, gyrase mutant (gyrB226), and topoisomerase mutant (ΔtopA10). DNA looping in each strain was measured by assaying repression of the endogenous lac operon, and repression of ten reporter constructs with DNA loop sizes between 70-85 base pairs. Our data are most simply interpreted as supporting the hypothesis that negative supercoiling facilitates gene repression by small DNA-protein loops in living bacteria., Competing Interests: The authors have declared that no competing interests exist.

Cyclin A2 activates the cyclin-dependent kinases Cdk1 and Cdk2 and is expressed at elevated levels from S phase until early mitosis. We found that mutant mice that cannot elevate cyclin A2 are chromosomally unstable and tumor-prone. Underlying the chromosomal instability is a failure to up-regulate the meiotic recombination 11 (Mre11) nuclease in S phase, which leads to impaired resolution of stalled replication forks, insufficient repair of double-stranded DNA breaks, and improper segregation of sister chromosomes. Unexpectedly, cyclin A2 controlled Mre11 abundance through a C-terminal RNA binding domain that selectively and directly binds Mre11 transcripts to mediate polysome loading and translation. These data reveal cyclin A2 as a mechanistically diverse regulator of DNA replication combining multifaceted kinase-dependent functions with a kinase-independent, RNA binding-dependent role that ensures adequate repair of common replication errors., (Copyright © 2016, American Association for the Advancement of Science.)

Transcription factors (TFs) are DNA-binding proteins that play critical roles in regulating gene expression. These proteins control all major cellular processes, including growth, development, and homeostasis. Because of their pivotal role, cells depend on proper TF function. It is, therefore, not surprising that TF deregulation is linked to disease. The therapeutic drug targeting of TFs has been proposed as a frontier in medicine. RNA aptamers make interesting candidates for TF modulation because of their unique characteristics. The products of in vitro selection, aptamers are short nucleic acids (DNA or RNA) that bind their targets with high affinity and specificity. Aptamers can be expressed on demand from transgenes and are intrinsically amenable to recognition by nucleic acid-binding proteins such as TFs. In this study, we review several natural prokaryotic and eukaryotic examples of RNAs that modulate the activity of TFs. These examples include 5S RNA, 6S RNA, 7SK, hepatitis delta virus-RNA (HDV-RNA), neuron restrictive silencer element (NRSE)-RNA, growth arrest-specific 5 (Gas5), steroid receptor RNA activator (SRA), trophoblast STAT utron (TSU), the 3' untranslated region of caudal mRNA, and heat shock RNA-1 (HSR1). We then review examples of unnatural RNA aptamers selected to inhibit TFs nuclear factor-kappaB (NF-κB), TATA-binding protein (TBP), heat shock factor 1 (HSF1), and runt-related transcription factor 1 (RUNX1). The field of RNA aptamers for DNA-binding proteins continues to show promise.

Background: Putative G-quadruplex-forming sequences (PQS) have long been implicated in regulation of transcription, though the actual mechanisms are not well understood. One proposed mechanism involves the activity of PQS-specific helicases belonging to the RecQ helicase family. However, patterns of PQS that correlate with transcriptional sensitivity to RecQ helicases are not well studied, and no adequate transcriptional model exists to account for PQS effects., Methods: To better understand PQS transcriptional effects, we analyze PQS motifs in genes differentially-transcribed in Bloom Syndrome (BS) and Werner Syndrome (WS), two disorders resulting in loss of PQS-interacting RecQ helicases.  We also correlate PQS genome-wide with transcription in multiple human cells lines while controlling for epigenetic status.  Finally, we perform neural network clustering of PQS motifs to assess whether certain motifs are over-represented in genes sensitive to RecQ helicase loss., Results: By analyzing PQS motifs in promoters of genes differentially-transcribed in BS and WS, we demonstrate that abundance of promoter PQS is generally higher in down-regulated genes and lower in up-regulated genes, and show that these effects are position-dependent. To interpret these correlations we determined genome-wide PQS correlations with transcription while controlling for epigenetic status. Our results identify multiple discrete transcription start site-proximal positions where PQS are correlated with either increased or decreased transcription. Finally, we report neural network clustering analysis of PQS motifs demonstrating that genes differentially-expressed in BS and WS are significantly biased in PQS motif composition., Conclusions: Our findings unveil unappreciated detail in the relationship between PQS, RecQ helicases, and transcription. We show that promoter PQS are generally correlated with reduced gene expression, and that this effect is relieved by RecQ helicases. We also show that PQS at certain positions on the downstream sense strand are correlated with increased transcription. We therefore propose a new transcriptional model in which promoter PQS have at least two distinct types of transcriptional regulatory effects.

Hypoxia inducible factors (HIFs) play vital roles in cellular maintenance of oxygen homeostasis. These transcription factors are responsible for the expression of genes involved in angiogenesis, metabolism, and cell proliferation. Here, we generate a detailed mathematical model for the enzyme kinetics of α-ketoglutarate-dependent HIF prolyl 4-hydroxylase domain (PHD) dioxygenases to simulate our in vitro data showing synergistic PHD inhibition by succinate and hypoxia in experimental models of succinate dehydrogenase loss, which phenocopy familial paraganglioma. Our mathematical model confirms the inhibitory synergy of succinate and hypoxia under physiologically-relevant conditions. In agreement with our experimental data, the model predicts that HIF1α is not stabilized under atmospheric oxygen concentrations, as observed. Further, the model confirms that addition of α-ketoglutarate can reverse PHD inhibition by succinate and hypoxia in SDH-deficient cells., (© 2015. Published by The Company of Biologists Ltd.)

Fluoroquinolones (FQ) are powerful broad-spectrum antibiotics whose side effects include renal damage and, strangely, tendinopathies. The pathological mechanisms underlying these toxicities are poorly understood. Here, we show that the FQ drugs norfloxacin, ciprofloxacin, and enrofloxacin are powerful iron chelators comparable with deferoxamine, a clinically useful iron-chelating agent. We show that iron chelation by FQ leads to epigenetic effects through inhibition of α-ketoglutarate-dependent dioxygenases that require iron as a co-factor. Three dioxygenases were examined in HEK293 cells treated with FQ. At sub-millimolar concentrations, these antibiotics inhibited jumonji domain histone demethylases, TET DNA demethylases, and collagen prolyl 4-hydroxylases, leading to accumulation of methylated histones and DNA and inhibition of proline hydroxylation in collagen, respectively. These effects may explain FQ-induced nephrotoxicity and tendinopathy. By the same reasoning, dioxygenase inhibition by FQ was predicted to stabilize transcription factor HIF-1α by inhibition of the oxygen-dependent hypoxia-inducible transcription factor prolyl hydroxylation. In dramatic contrast to this prediction, HIF-1α protein was eliminated by FQ treatment. We explored possible mechanisms for this unexpected effect and show that FQ inhibit HIF-1α mRNA translation. Thus, FQ antibiotics induce global epigenetic changes, inhibit collagen maturation, and block HIF-1α accumulation. We suggest that these mechanisms explain the classic renal toxicities and peculiar tendinopathies associated with FQ antibiotics., (© 2015 by The American Society for Biochemistry and Molecular Biology, Inc.)

Restriction endonucleases (REases) recognize and cleave short palindromic DNA sequences, protecting bacterial cells against bacteriophage infection by attacking foreign DNA. We are interested in the potential of folded RNA to mimic DNA, a concept that might be applied to inhibition of DNA-binding proteins. As a model system, we sought RNA aptamers against the REases BamHI, PacI and KpnI using systematic evolution of ligands by exponential enrichment (SELEX). After 20 rounds of selection under different stringent conditions, we identified the 10 most enriched RNA aptamers for each REase. Aptamers were screened for binding and specificity, and assayed for REase inhibition. We obtained eight high-affinity (Kd ∼12-30 nM) selective competitive inhibitors (IC50 ∼20-150 nM) for KpnI. Predicted RNA secondary structures were confirmed by in-line attack assay and a 38-nt derivative of the best anti-KpnI aptamer was sufficient for inhibition. These competitive inhibitors presumably act as KpnI binding site analogs, but lack the primary consensus KpnI cleavage sequence and are not cleaved by KpnI, making their potential mode of DNA mimicry fascinating. Anti-REase RNA aptamers could have value in studies of REase mechanism and may give clues to a code for designing RNAs that competitively inhibit DNA binding proteins including transcription factors., (© The Author(s) 2015. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Double-stranded DNA is a locally inflexible polymer that resists bending and twisting over hundreds of base pairs. Despite this, tight DNA bending is biologically important for DNA packaging in eukaryotic chromatin and tight DNA looping is important for gene repression in prokaryotes. We and others have previously shown that sequence nonspecific DNA kinking proteins, such as Escherichia coli heat unstable and Saccharomyces cerevisiae non-histone chromosomal protein 6A (Nhp6A), facilitate lac repressor (LacI) repression loops in E. coli. It has been unknown if this facilitation involves direct protein binding to the tightly bent DNA loop or an indirect effect promoting global negative supercoiling of DNA. Here we adapt two high-resolution in vivo protein-mapping techniques to demonstrate direct binding of the heterologous Nhp6A protein at a LacI repression loop in living E. coli cells.

Familial paraganglioma (PGL) is a rare neuroendocrine cancer associated with defects in the genes encoding the subunits of succinate dehydrogenase (SDH), a tricarboxylic acid (TCA) cycle enzyme. For unknown reasons, a higher prevalence of PGL has been reported for humans living at higher altitude, with increased disease aggressiveness and morbidity. In this study, we evaluate the effects of oxygen on epigenetic changes due to succinate accumulation in three SDH loss cell culture models. We test the hypothesis that the mechanism of α-ketoglutarate (α-KG)-dependent dioxygenase enzymes explains the inhibitory synergy of hypoxia and succinate accumulation. We confirm that SDH loss leads to profound succinate accumulation. We further show that hypoxia and succinate accumulation synergistically inhibit α-KG-dependent dioxygenases leading to increased stabilization of transcription factor HIF1α, HIF2α, and hypermethylation of histones and DNA. Increasing oxygen suppresses succinate inhibition of α-KG-dependent dioxygenases. This result provides a possible explanation for the association between hypoxia and PGL, and suggests hyperoxia as a potential novel therapy.

Context: Germline mutations in genes coding succinate dehydrogenase (SDH) subunits A, B, C, and D have been identified in familial paragangliomas (PGLs)/pheochromocytomas (PHEOs) and other tumors. We described a GH-secreting pituitary adenoma (PA) caused by SDHD mutation in a patient with familial PGLs. Additional patients with PAs and SDHx defects have since been reported., Design: We studied 168 patients with unselected sporadic PA and with the association of PAs, PGLs, and/or pheochromocytomas, a condition we named the 3P association (3PAs) for SDHx germline mutations. We also studied the pituitary gland and hormonal profile of Sdhb(+/-) mice and their wild-type littermates at different ages., Results: No SDHx mutations were detected among sporadic PA, whereas three of four familial cases were positive for a mutation (75%). Most of the SDHx-deficient PAs were either prolactinomas or somatotropinomas. Pituitaries of Sdhb(+/-) mice older than 12 months had an increased number mainly of prolactin-secreting cells and several ultrastructural abnormalities such as intranuclear inclusions, altered chromatin nuclear pattern, and abnormal mitochondria. Igf-1 levels of mutant mice tended to be higher across age groups, whereas Prl and Gh levels varied according to age and sex., Conclusion: The present study confirms the existence of a new association that we termed 3PAs. It is due mostly to germline SDHx defects, although sporadic cases of 3PAs without SDHx defects also exist. Using Sdhb(+/-) mice, we provide evidence that pituitary hyperplasia in SDHx-deficient cells may be the initial abnormality in the cascade of events leading to PA formation.

DNA aptamer oligonucleotides and their protein conjugates show promise as therapeutics in animal models of diseases such as multiple sclerosis. These molecules are large and highly charged, raising questions about their biodistribution and pharmacokinetics in mammals. Here we exploit the power of quantitative polymerase chain reaction to accurately quantitate the tissue distribution of 40-nucleotide DNA aptamers and their streptavidin conjugates after intraperitoneal injection in mice. We show remarkably rapid distribution to peripheral tissues including the central nervous system. Modeling of tissue distribution data reveals the importance of DNA aptamer sequence, 3' modification, and protein conjugation in enhancing tissue exposure. These data help to interpret the previously observed effectiveness of aptamer conjugates, as opposed to free aptamers, in stimulating central nervous system remyelination in a mouse model of multiple sclerosis.

It is counterintuitive that metabolic defects reducing ATP production can cause, rather than protect from, cancer. Yet this is precisely the case for familial paraganglioma, a form of neuroendocrine malignancy caused by loss of succinate dehydrogenase in the tricarboxylic acid cycle. Here we review biochemical, genetic, and epigenetic considerations in succinate dehydrogenase loss and present leading models and mysteries associated with this fascinating and important tumor.

The regulation of chromatin structure in eukaryotic cells involves abundant architectural factors such as high mobility group B (HMGB) proteins. It is not understood how these factors control the interplay between genome accessibility and compaction. In vivo, HMO1 binds the promoter and coding regions of most ribosomal RNA genes, facilitating transcription and possibly stabilizing chromatin in the absence of histones. To understand how HMO1 performs these functions, we combine single molecule stretching and atomic force microscopy (AFM). By stretching HMO1-bound DNA, we demonstrate a hierarchical organization of interactions, in which HMO1 initially compacts DNA on a timescale of seconds, followed by bridge formation and stabilization of DNA loops on a timescale of minutes. AFM experiments demonstrate DNA bridging between strands as well as looping by HMO1. Our results support a model in which HMO1 maintains the stability of nucleosome-free chromatin regions by forming complex and dynamic DNA structures mediated by protein-protein interactions., (© The Author(s) 2014. Published by Oxford University Press on behalf of Nucleic Acids Research.)

This work probes the mystery of what balance of forces creates the extraordinary mechanical stiffness of DNA to bending and twisting. Here we explore the relationship between base stacking, functional group occupancy of the DNA minor and major grooves, and DNA mechanical properties. We study double-helical DNA molecules substituting either inosine for guanosine or 2,6-diaminopurine for adenine. These DNA variants, respectively, remove or add an amino group from the DNA minor groove, with corresponding changes in hydrogen-bonding and base stacking energy. Using the techniques of ligase-catalyzed cyclization kinetics, atomic force microscopy, and force spectroscopy with optical tweezers, we show that these DNA variants have bending persistence lengths within the range of values reported for sequence-dependent variation of the natural DNA bases. Comparison with seven additional DNA variants that modify the DNA major groove reveals that DNA bending stiffness is not correlated with base stacking energy or groove occupancy. Data from circular dichroism spectroscopy indicate that base analog substitution can alter DNA helical geometry, suggesting a complex relationship among base stacking, groove occupancy, helical structure, and DNA bend stiffness., (Copyright © 2014 Biophysical Society. Published by Elsevier Inc. All rights reserved.)