Oligodendrocyte myelin glycoprotein as a novel target for pathogenic autoimmunity in the CNS.

Autoimmune disorders of the central nervous system (CNS) comprise a broad spectrum of clinical entities. The stratification of patients based on the recognized autoantigen is of great importance for therapy optimization and for concepts of pathogenicity, but for most of these patients, the actual target of their autoimmune response is unknown. Here we investigated oligodendrocyte myelin glycoprotein (OMGP) as autoimmune target, because OMGP is expressed specifically in the CNS and there on oligodendrocytes and neurons. Using a stringent cell-based assay, we detected autoantibodies to OMGP in serum of 8/352 patients with multiple sclerosis, 1/28 children with acute disseminated encephalomyelitis and unexpectedly, also in one patient with psychosis, but in none of 114 healthy controls. Since OMGP is GPI-anchored, we validated its recognition also in GPI-anchored form. The autoantibodies to OMGP were largely IgG1 with a contribution of IgG4, indicating cognate T cell help. We found high levels of soluble OMGP in human spinal fluid, presumably due to shedding of the GPI-linked OMGP. Analyzing the pathogenic relevance of autoimmunity to OMGP in an animal model, we found that OMGP-specific T cells induce a novel type of experimental autoimmune encephalomyelitis dominated by meningitis above the cortical convexities. This unusual localization may be directed by intrathecal uptake and presentation of OMGP by meningeal phagocytes. Together, OMGP-directed autoimmunity provides a new element of heterogeneity, helping to improve the stratification of patients for diagnostic and therapeutic purposes.

A RAPGEF6 variant constitutes a major risk factor for laryngeal paralysis in dogs.

Laryngeal paralysis (LP) is the inability to abduct the arytenoid cartilages during inspiration, resulting in a partial to complete airway obstruction and consequent respiratory distress. Different forms of LP with varying age of onset exist in dogs. Hereditary early onset forms were reported in several dog breeds. In most breeds, hereditary LP is associated with other neurologic pathologies. Using a genome-wide association study and haplotype analyses, we mapped a major genetic risk factor for an early onset LP in Miniature Bull Terriers to a ~1.3 Mb interval on chromosome 11. Whole genome sequencing of an affected Miniature Bull Terrier and comparison to 598 control genomes revealed a 36 bp insertion into exon 15 of the RAPGEF6 gene (c.1793_1794ins36). The imperfect genotype-phenotype correlation suggested a complex mode of inheritance with a major genetic risk factor involving a recessive risk allele. Homozygosity for the insertion was associated with a 10- to 17-fold increased risk for LP. The insertion allele was only found in Miniature Bull Terriers and Bull Terriers. It was absent from >1000 control dogs of other dog breeds. The insertion sequence contains a splice acceptor motif leading to aberrant splicing in transcripts originating from the mutant allele (r.1732_1780del). This leads to a frameshift and a premature stop codon, p.(Ile587ProfsTer5), removing 64% of the open reading frame. Our results suggest an important role of RAPGEF6 in laryngeal nerve function and provide new clues to its physiological significance.

Pinpointed Stimulation of EphA2 Receptors via DNA-Templated Oligovalence.

DNA nanostructures enable the attachment of functional molecules to nearly any unique location on their underlying structure. Due to their single-base-pair structural resolution, several ligands can be spatially arranged and closely controlled according to the geometry of their desired target, resulting in optimized binding and/or signaling interactions. Here, the efficacy of SWL, an ephrin-mimicking peptide that binds specifically to EphrinA2 (EphA2) receptors, increased by presenting up to three of these peptides on small DNA nanostructures in an oligovalent manner. Ephrin signaling pathways play crucial roles in tumor development and progression. Moreover, Eph receptors are potential targets in cancer diagnosis and treatment. Here, the quantitative impact of SWL valency on binding, phosphorylation (key player for activation) and phenotype regulation in EphA2-expressing prostate cancer cells was demonstrated. EphA2 phosphorylation was significantly increased by DNA trimers carrying three SWL peptides compared to monovalent SWL. In comparison to one of EphA2's natural ligands ephrin-A1, which is known to bind promiscuously to multiple receptors, pinpointed targeting of EphA2 by oligovalent DNA-SWL constructs showed enhanced cell retraction. Overall, we show that DNA scaffolds can increase the potency of weak signaling peptides through oligovalent presentation and serve as potential tools for examination of complex signaling pathways.

Synthetic Transient Crosslinks Program the Mechanics of Soft, Biopolymer-Based Materials.

Actin networks are adaptive materials enabling dynamic and static functions of living cells. A central element for tuning their underlying structural and mechanical properties is the ability to reversibly connect, i.e., transiently crosslink, filaments within the networks. Natural crosslinkers, however, vary across many parameters. Therefore, systematically studying the impact of their fundamental properties like size and binding strength is unfeasible since their structural parameters cannot be independently tuned. Herein, this problem is circumvented by employing a modular strategy to construct purely synthetic actin crosslinkers from DNA and peptides. These crosslinkers mimic both intuitive and noncanonical mechanical properties of their natural counterparts. By isolating binding affinity as the primary control parameter, effects on structural and dynamic behaviors of actin networks are characterized. A concentration-dependent triphasic behavior arises from both strong and weak crosslinkers due to emergent structural polymorphism. Beyond a certain threshold, strong binding leads to a nonmonotonic elastic pulse, which is a consequence of self-destruction of the mechanical structure of the underlying network. The modular design also facilitates an orthogonal regulatory mechanism based on enzymatic cleaving. This approach can be used to guide the rational design of further biomimetic components for programmable modulation of the properties of biomaterials and cells.

DNA Nanotubes as a Versatile Tool to Study Semiflexible Polymers.

Mechanical properties of complex, polymer-based soft matter, such as cells or biopolymer networks, can be understood in neither the classical frame of flexible polymers nor of rigid rods. Underlying filaments remain outstretched due to their non-vanishing backbone stiffness, which is quantified via the persistence length (lp), but they are also subject to strong thermal fluctuations. Their finite bending stiffness leads to unique, non-trivial collective mechanics of bulk networks, enabling the formation of stable scaffolds at low volume fractions while providing large mesh sizes. This underlying principle is prevalent in nature (e.g., in cells or tissues), minimizing the high molecular content and thereby facilitating diffusive or active transport. Due to their biological implications and potential technological applications in biocompatible hydrogels, semiflexible polymers have been subject to considerable study. However, comprehensible investigations remained challenging since they relied on natural polymers, such as actin filaments, which are not freely tunable. Despite these limitations and due to the lack of synthetic, mechanically tunable, and semiflexible polymers, actin filaments were established as the common model system. A major limitation is that the central quantity lp cannot be freely tuned to study its impact on macroscopic bulk structures. This limitation was resolved by employing structurally programmable DNA nanotubes, enabling controlled alteration of the filament stiffness. They are formed through tile-based designs, where a discrete set of partially complementary strands hybridize in a ring structure with a discrete circumference. These rings feature sticky ends, enabling the effective polymerization into filaments several microns in length, and display similar polymerization kinetics as natural biopolymers. Due to their programmable mechanics, these tubes are versatile, novel tools to study the impact of lp on the single-molecule as well as the bulk scale. In contrast to actin filaments, they remain stable over weeks, without notable degeneration, and their handling is comparably straightforward.

Tuning Synthetic Semiflexible Networks by Bending Stiffness.

The mechanics of complex soft matter often cannot be understood in the classical physical frame of flexible polymers or rigid rods. The underlying constituents are semiflexible polymers, whose finite bending stiffness (κ) leads to nontrivial mechanical responses. A natural model for such polymers is the protein actin. Experimental studies of actin networks, however, are limited since the persistence length (l_{p}∝κ) cannot be tuned. Here, we experimentally characterize this parameter for the first time in entangled networks formed by synthetically produced, structurally tunable DNA nanotubes. This material enabled the validation of characteristics inherent to semiflexible polymers and networks thereof, i.e., persistence length, inextensibility, reptation, and mesh size scaling. While the scaling of the elastic plateau modulus with concentration G_{0}∝c^{7/5} is consistent with previous measurements and established theories, the emerging persistence length scaling G_{0}∝l_{p} opposes predominant theoretical predictions.

Probing selected structural regions in the secreted phospholipase A2 from Arabidopsis thaliana for their impact on stability and activity.

In contrast to the well characterized secreted phospholipases A2 (sPLA2) from animals, their homologues from plants have been less explored. Their production in purified form is more difficult, and no data on their stability are known. In the present paper, different variants of the sPLA2 isoform α from Arabidopsis thaliana (AtPLA2α) were designed using a new homology model with the aim to probe the impact of regions that are assumed to be important for stability and catalysis. Moreover tryptophan residues were introduced in critical regions to enable stability studies by fluorescence spectroscopy. The variants were expressed in Escherichia coli and the purified enzymes were analyzed to get first insights into the peculiarities of structure stability and structure activity relationships in plant sPLA2s in comparison with the well-characterized homologous enzymes from bee venom and porcine pancreas. Stability data of the AtPLA2 variants obtained by fluorescence or CD measurements of the reversible unfolding by guanidine hydrochloride and urea showed that all enzyme variants are less stable than the enzymes from animal sources although a similar tertiary core structure can be assumed based on molecular modeling. More extended loop structures at the N-terminus in AtPLA2α are suggested to be the main reasons for the much lower thermodynamic stabilities and cooperativities of the transition curves. Modifications in the N-terminal region (insertion, deletion, substitution by a Trp residue) exhibited a strong positive effect on activity whereas amino acid exchanges in other regions of the protein such as the Ca(2+)-binding loop and the loop connecting the two central helices were deleterious with respect to activity.

Anesthesia and Duchenne or Becker muscular dystrophy: review of 117 anesthetic exposures.

BACKGROUND: Duchenne muscular dystrophy (DMD) and Becker muscular dystrophy (BMD) are associated with life-threatening perioperative complications, including rhabdomyolysis, hyperkalemia, and hyperthermia. Current recommendations contraindicate use of succinylcholine and volatile anesthetics; however, the latter recommendation remains controversial. OBJECTIVE: To review the perioperative outcomes of patients with DMD and BMD. METHODS: We reviewed records of patients with DMD or BMD who underwent anesthetic management at our institution from January 1990 through December 2011. RESULTS: We identified 47 patients (DMD, 37; BMD, 10) who underwent 117 anesthetic exposures (DMD, 101; BMD, 16). Volatile anesthetic agents were used 66 times (DMD, 59; BMD, 7). One patient with undiagnosed BMD received succinylcholine and developed acute rhabdomyolysis and hyperkalemic cardiac arrest. All other major complications were attributed to the procedure (i.e., large bleeding), to preexisting comorbidities (i.e., respiratory failure, cardiac disease), or to both. CONCLUSIONS: Use of succinylcholine in children with dystrophinopathy is contraindicated. These patients have significant comorbidities and are frequently undergoing extensive operations; complications related to these factors can develop, as evidenced by our series. These complications may occur with use of volatile and nonvolatile anesthetics. However, because most of our patients were older than 8 years at the time of surgery, our observation cannot be generalized to younger dystrophin-deficient children.