Symptoms and impacts of ambulatory nonsense mutation Duchenne muscular dystrophy: a qualitative study and the development of a patient-centred conceptual model.

Background: Duchenne muscular dystrophy is a rare genetic neuromuscular disorder, which can result in early death due to disease progression. Ataluren is indicated for the treatment of nonsense mutation Duchenne muscular dystrophy, in ambulatory individuals aged two years and older. This study explored the symptoms and impacts of nonsense mutation Duchenne muscular dystrophy and experience with ataluren.

A qualitative study on the impact of caring for an ambulatory individual with nonsense mutation Duchenne muscular dystrophy.

Background: Duchenne muscular dystrophy is a rare genetic neuromuscular disorder, which can result in early death due to disease progression. Ataluren is indicated for the treatment of nonsense mutation Duchenne muscular dystrophy, in ambulatory individuals aged two years and older. This study explored the impact of caring for an ambulatory individual with nonsense mutation Duchenne muscular dystrophy, as well as the impact of treatment with ataluren on the caregiver experience, using retrospective recall.

Algebraic expressions for Carr-Purcell-Meiboom-Gill relaxation dispersion for N-site chemical exchange.

The Carr-Purcell-Meiboom-Gill (CPMG) NMR relaxation dispersion experiment measures the effective relaxation rate constant during a train of spin-echo pulse sequence elements as a function of the echo time. The CPMG experiment is a powerful method for characterizing chemical and conformational dynamic processes, termed chemical and conformational exchange, on μs-ms time scales, comparable to the experimentally accessible echo times. Approximate theoretical expressions for the effective relaxation rate constant for N-site chemical exchange have been reported (H.

Compact expressions for R relaxation for N-site chemical exchange using Schur decomposition.

The rotating-frame spin relaxation rate constant, R, is a powerful probe of macromolecular chemical and conformational dynamics in relaxation dispersion, CEST, and DEST NMR experiments. The R relaxation rate constant is given by the absolute value of the largest (least negative) eigenvalue of the Bloch-McConnell evolution matrix; however, estimation of this eigenvalue require inversion of 3 N × 3 N dimensional matrices, in which N is the number of interconverting sites or states for a given nuclear spin in a molecule. The Schur complement is used to reduce the problem of calculating the characteristic polynomial of a 3 N × 3 N matrix to that of calculating the characteristic polynomial of a 3 × 3 matrix.

Effect of monovalent ion binding on molecular dynamics of the S100-family calcium-binding protein calbindin D.

Calbindin D is a member of the S100 subfamily of EF-hand calcium binding proteins, and has served as an important model system for biophysical studies. The fast timescale dynamics of the calcium-free (apo) state is characterized using molecular dynamics simulations. Order parameters for the backbone NH bond vectors are determined from the simulations and compared with experimentally derived values, with a focus on the dynamics of calcium-binding site I.

Defining a Canonical Ligand-Binding Pocket in the Orphan Nuclear Receptor Nurr1.

Nuclear receptor-related 1 protein (Nurr1/NR4A2) is an orphan nuclear receptor (NR) that is considered to function without a canonical ligand-binding pocket (LBP). A crystal structure of the Nurr1 ligand-binding domain (LBD) revealed no physical space in the conserved region where other NRs with solvent accessible apo-protein LBPs bind synthetic and natural ligands. Using solution nuclear magnetic resonance spectroscopy, hydrogen/deuterium exchange mass spectrometry, and molecular dynamics simulations, we show that the putative canonical Nurr1 LBP is dynamic with high solvent accessibility, exchanges between two or more conformations on the microsecond-to-millisecond timescale, and can expand from the collapsed crystallized conformation to allow binding of unsaturated fatty acids.

General Expressions for Carr-Purcell-Meiboom-Gill Relaxation Dispersion for N-Site Chemical Exchange.

The Carr-Purcell-Meiboom-Gill (CPMG) nuclear magnetic resonance experiment is widely used to characterize chemical exchange phenomena in biological macromolecules. Theoretical expressions for the nuclear spin relaxation rate constant for two-site chemical exchange during CPMG pulse trains valid for all time scales are well-known as are descriptions of N-site exchange in the fast limit. We have obtained theoretical expressions for N-site exchange outside of the fast limit by using approximations to an average Liouvillian describing the decay of magnetization during a CPMG pulse train.

Lysine Side-Chain Dynamics in the Binding Site of Homeodomain/DNA Complexes As Observed by NMR Relaxation Experiments and Molecular Dynamics Simulations.

An important but poorly characterized contribution to the thermodynamics of protein-DNA interactions is the loss of entropy that occurs from restricting the conformational freedom of amino acid side chains. The effect of restricting the flexibility of several side chains at a protein-DNA interface may be comparable in many cases to the other factors that determine the binding thermodynamics and may, therefore, play a key role in dictating the binding affinity and/or specificity. Because the entropic contributions, including the presence and influence of side-chain dynamics, are especially difficult to estimate based on structural information, it is important to pursue experimental and theoretical studies that can provide direct information regarding these issues.

Enhanced spectral density mapping through combined multiple-field deuterium CHD methyl spin relaxation NMR spectroscopy.

Quadrupolar relaxation of H (D) nuclear spins is a powerful probe of conformational dynamics in biological macromolecules. Deuterium relaxation rate constants are determined by the spectral density function for reorientation of the C-D bond vector at zero, single-quantum, and double-quantum H frequencies. In the present work, H relaxation rate constants were measured for an E.

General expressions for R relaxation for N-site chemical exchange and the special case of linear chains.

Exploration of dynamic processes in proteins and nucleic acids by spin-locking NMR experiments has been facilitated by the development of theoretical expressions for the R relaxation rate constant covering a variety of kinetic situations. Herein, we present a generalized approximation to the chemical exchange, R, component of R for arbitrary kinetic schemes, assuming the presence of a dominant major site population, derived from the negative reciprocal trace of the inverse Bloch-McConnell evolution matrix. This approximation is equivalent to first-order truncation of the characteristic polynomial derived from the Bloch-McConnell evolution matrix.

Cost-effectiveness of linaclotide compared to antidepressants in the treatment of irritable bowel syndrome with constipation in Scotland.

Presently, linaclotide is the only EMA-approved therapy indicated for the treatment of irritable bowel syndrome with constipation (IBS-C). This study sought to determine the cost-effectiveness of linaclotide compared to antidepressants for the treatment of adults with moderate to severe IBS-C who have previously received antispasmodics and/or laxatives. A Markov model was created to estimate costs and QALYs over a 5-year time horizon from the perspective of NHS Scotland.

Burden of irritable bowel syndrome in an increasingly cost-aware National Health Service.

Background: The National Health Service (NHS) is faced with increasing cost pressures that make the efficient use of resources paramount. Irritable bowel syndrome (IBS) places a large burden on the NHS as it has been estimated that at least 12% of the UK population is affected. However, poor clinical coding makes accurate assessment of this burden challenging.

Targeting substrate-site in Jak2 kinase prevents emergence of genetic resistance.

Emergence of genetic resistance against kinase inhibitors poses a great challenge for durable therapeutic response. Here, we report a novel mechanism of JAK2 kinase inhibition by fedratinib (TG101348) that prevents emergence of genetic resistance. Using in vitro drug screening, we identified 211 amino-acid substitutions conferring resistance to ruxolitinib (INCB018424) and cross-resistance to the JAK2 inhibitors AZD1480, CYT-387 and lestaurtinib.

Structural mechanism for signal transduction in RXR nuclear receptor heterodimers.

A subset of nuclear receptors (NRs) function as obligate heterodimers with retinoid X receptor (RXR), allowing integration of ligand-dependent signals across the dimer interface via an unknown structural mechanism. Using nuclear magnetic resonance (NMR) spectroscopy, x-ray crystallography and hydrogen/deuterium exchange (HDX) mass spectrometry, here we show an allosteric mechanism through which RXR co-operates with a permissive dimer partner, peroxisome proliferator-activated receptor (PPAR)-γ, while rendered generally unresponsive by a non-permissive dimer partner, thyroid hormone (TR) receptor. Amino acid residues that mediate this allosteric mechanism comprise an evolutionarily conserved network discovered by statistical coupling analysis (SCA).

Rotation operator propagators for time-varying radiofrequency pulses in NMR spectroscopy: applications to shaped pulses and pulse trains.

The propagator for trains of radiofrequency pulses can be directly integrated numerically or approximated by average Hamiltonian approaches. The former provides high accuracy and the latter, in favorable cases, convenient analytical formula. The Euler-angle rotation operator factorization of the propagator provides insights into performance that are not as easily discerned from either of these conventional techniques.

Effects of interleukin-10 on cutaneous wounds and scars in humans of African continental ancestral origin.

Scars in humans of African continental ancestry heal with an exaggerated inflammatory response and a generally wider scar. Interleukin-10 is an anti-inflammatory and antifibrotic cytokine. A randomized controlled trial in Caucasians found that exogenous interleukin-10 resulted in improved macroscopic scar appearance and reduced scar redness.

The N-terminal basolateral targeting signal unlikely acts alone in the differential trafficking of membrane transporters in MDCK cells.

We have shown previously, using confocal imaging and transport assays, that the N-terminus of sodium-dependent vitamin C transporter 2 (SVCT2) can redirect apical SVCT1 to the basolateral membrane. Here, the SVCT model was used to further characterize the basolateral targeting peptide signal. Both the length (31 amino acids) and sequence accuracy of the N-terminus of SVCT2 were found to be important in basolateral targeting activity, suggesting a structural requirement.

Ligand and receptor dynamics contribute to the mechanism of graded PPARγ agonism.

Ligand binding to proteins is not a static process, but rather involves a number of complex dynamic transitions. A flexible ligand can change conformation upon binding its target. The conformation and dynamics of a protein can change to facilitate ligand binding.

Structural and biophysical insights into the ligand-free Pitx2 homeodomain and a ring dermoid of the cornea inducing homeodomain mutant.

The homeodomain-containing transcription factor Pitx2 (pituitary homeobox protein 2) is present in many developing embryonic tissues, including the heart. Its homeodomain is responsible for the recognition and binding to target DNA sequences and thus constitutes a major functional unit in the Pitx2 protein. Nuclear magnetic resonance techniques were employed to determine the solution structure of the native Pitx2 homeodomain and a R24H mutant that causes autosomal dominantly inherited ring dermoid of the cornea syndrome.

¹H, ¹³C and ¹⁵N chemical shift assignments for the human Pitx2 homeodomain in complex with a 22-base hairpin DNA.

As one of the most important eukaryotic DNA-binding motifs, the homeodomain has been identified in over one thousand proteins. Transcription factors containing homeodomains play critical roles in diverse biological processes and are often implicated in human genetic disorders. The human Pitx2 protein is a central downstream transcription factor in both the TGF-β and Wnt/β-catenin signaling pathways, and involved in the regulation of cell differentiation, cell pattern formation and tissue maintenance.

Observing selected domains in multi-domain proteins via sortase-mediated ligation and NMR spectroscopy.

NMR spectroscopy has distinct advantages for providing insight into protein structures, but faces significant resolution challenges as protein size increases. To alleviate such resonance overlap issues, the ability to produce segmentally labeled proteins is beneficial. Here we show that the S.

1H, 13C and 15N chemical shift assignments for the human Pitx2 homeodomain and a R24H homeodomain mutant.

The homeodomain is one of the most important eukaryotic DNA-binding motifs and has been identified in over one thousand proteins. Homeodomain proteins play critical roles in diverse biological processes, including cell differentiation and cell pattern formation. The human Pitx2 homeodomain binds several different DNA sequences and is a pivotal component of both the TGF-β and Wnt/β-catenin signaling pathways.

Structural and motional contributions of the Bacillus subtilis ClpC N-domain to adaptor protein interactions.

The AAA(+) (ATPases associated with a variety of cellular activities) superfamily protein ClpC is a key regulator of cell development in Bacillus subtilis. As part of a large oligomeric complex, ClpC controls an array of cellular processes by recognizing, unfolding, and providing misfolded and aggregated proteins as substrates for the ClpP peptidase. ClpC is unique compared to other HSP100/Clp proteins, as it requires an adaptor protein for all fundamental activities.