Insights into Ligand-Mediated Activation of an Oligomeric Ring-Shaped Gene-Regulatory Protein from Solution- and Solid-State NMR.

The 91 kDa oligomeric ring-shaped ligand binding protein TRAP (trp RNA binding attenuation protein) regulates the expression of a series of genes involved in tryptophan (Trp) biosynthesis in bacilli. When cellular Trp levels rise, the free amino acid binds to sites buried in the interfaces between each of the 11 (or 12, depending on the species) protomers in the ring. Crystal structures of Trp-bound TRAP show the Trp ligands are sequestered from solvent by a pair of loops from adjacent protomers that bury the bound ligand via polar contacts to several threonine residues. Binding of the Trp ligands occurs cooperatively, such that successive binding events occur with higher apparent affinity but the structural basis for this cooperativity is poorly understood. We used solution methyl-TROSY NMR relaxation experiments focused on threonine and isoleucine sidechains, as well as magic angle spinning solid-state NMR 13C-13C and 15N-13C chemical shift correlation spectra on uniformly labeled samples recorded at 800 and 1200 MHz, to characterize the structure and dynamics of the protein. Methyl 13C relaxation dispersion experiments on ligand-free apo TRAP revealed concerted exchange dynamics on the µs-ms time scale, consistent with transient sampling of conformations that could allow ligand binding. Cross-correlated relaxation experiments revealed widespread disorder on fast timescales. Chemical shifts for methyl-bearing side chains in apo- and Trp-bound TRAP revealed subtle changes in the distribution of sampled sidechain rotameric states. These observations reveal a pathway and mechanism for induced conformational changes to generate homotropic Trp-Trp binding cooperativity.

Hydrogen bonding in duplex DNA probed by DNP enhanced solid-state NMR N-H bond length measurements.

Numerous biological processes and mechanisms depend on details of base pairing and hydrogen bonding in DNA. Hydrogen bonds are challenging to quantify by X-ray crystallography and cryo-EM due to difficulty of visualizing hydrogen atom locations but can be probed with site specificity by NMR spectroscopy in solution and the solid state with the latter particularly suited to large, slowly tumbling DNA complexes. Recently, we showed that low-temperature dynamic nuclear polarization (DNP) enhanced solid-state NMR is a valuable tool for distinguishing Hoogsteen base pairs (bps) from canonical Watson-Crick bps in various DNA systems under native-like conditions. Here, using a model 12-mer DNA duplex containing two central adenine-thymine (A-T) bps in either Watson-Crick or Hoogsteen confirmation, we demonstrate DNP solid-state NMR measurements of thymine N3-H3 bond lengths, which are sensitive to details of N-H···N hydrogen bonding and permit hydrogen bonds for the two bp conformers to be systematically compared within the same DNA sequence context. For this DNA duplex, effectively identical TN3-H3 bond lengths of 1.055 ± 0.011 Å and 1.060 ± 0.011 Å were found for Watson-Crick A-T and Hoogsteen A (syn)-T base pairs, respectively, relative to a reference amide bond length of 1.015 ± 0.010 Å determined for N-acetyl-valine under comparable experimental conditions. Considering that prior quantum chemical calculations which account for zero-point motions predict a somewhat longer effective peptide N-H bond length of 1.041 Å, in agreement with solution and solid-state NMR studies of peptides and proteins at ambient temperature, to facilitate direct comparisons with these earlier studies TN3-H3 bond lengths for the DNA samples can be readily scaled appropriately to yield 1.083 Å and 1.087 Å for Watson-Crick A-T and Hoogsteen A (syn)-T bps, respectively, relative to the 1.041 Å reference peptide N-H bond length. Remarkably, in the context of the model DNA duplex, these results indicate that there are no significant differences in N-H···N A-T hydrogen bonds between Watson-Crick and Hoogsteen bp conformers. More generally, high precision measurements of N-H bond lengths by low-temperature DNP solid-state NMR based methods are expected to facilitate detailed comparative analysis of hydrogen bonding for a range of DNA complexes and base pairing environments.

A Service Reconfiguration Bundle for Expanding Access to Peritoneal Dialysis Including for Older Frailer Patients.

INTRODUCTION: Rates of peritoneal dialysis (PD) have been traditionally low in Northern Ireland. With rising numbers of patients reaching end-stage kidney disease, PD is a more cost-effective treatment than haemodialysis and aligns with international goals to increase home-based dialysis options. The aim of our study was to highlight how a service reconfiguration bundle expanded access to PD in Northern Ireland. METHODS: The service reconfiguration bundle consisted of the appointment of a surgical lead, a dedicated interventional radiologist for fluoroscopically guided PD catheter insertion, and a nephrology-led ultrasound-guided PD catheter insertion service in an area of particular need. All patients in Northern Ireland who had a PD catheter inserted in the year following service reconfigurations were included and prospectively followed up for one-year. Patient demographics, PD catheter insertion technique, setting of procedure, and outcome data were summarised. RESULTS: The number of patients receiving PD catheter insertion doubled to 66 in the year following service reconfigurations. The range of approaches to PD catheter insertion (laparoscopic n = 41, percutaneous n = 24 and open n = 1) allowed a wide range of patients to benefit from PD. Six patients had emergency PD catheter insertion, with four receiving urgent or early start PD. Nearly half (48%, 29/60) of the PD catheters inserted electively were in smaller elective hubs rather than the regional unit. A total of 97% of patients successfully started PD. Patients who experienced percutaneous PD catheter insertion were older [median age 76 (range 37-88) vs. 56 (range 18-84), p < 0.0001] and had less previous abdominal surgery than patients who experienced laparoscopic PD catheter insertion (25%, 6/24 vs. 54%, 22/41, p = 0.05). DISCUSSION: Through a service reconfiguration bundle, we were able to double our annual incident PD population. This study highlights how flexible models of service delivery introduced as a bundle can quickly deliver expanded access to PD and home therapy.

Probing Watson-Crick and Hoogsteen base pairing in duplex DNA using dynamic nuclear polarization solid-state NMR spectroscopy.

The majority of base pairs in double-stranded DNA exist in the canonical Watson-Crick geometry. However, they can also adopt alternate Hoogsteen conformations in various complexes of DNA with proteins and small molecules, which are key for biological function and mechanism. While detection of Hoogsteen base pairs in large DNA complexes and assemblies poses considerable challenges for traditional structural biology techniques, we show here that multidimensional dynamic nuclear polarization-enhanced solid-state NMR can serve as a unique spectroscopic tool for observing and distinguishing Watson-Crick and Hoogsteen base pairs in a broad range of DNA systems based on characteristic NMR chemical shifts and internuclear dipolar couplings. We illustrate this approach using a model 12-mer DNA duplex, free and in complex with the antibiotic echinomycin, which features two central adenine-thymine base pairs with Watson-Crick and Hoogsteen geometry, respectively, and subsequently extend it to the ∼200 kDa Widom 601 DNA nucleosome core particle.

Accessing Photoredox Transformations with an Iron(III) Photosensitizer and Green Light.

Efficient excited-state electron transfer between an iron(III) photosensitizer and organic electron donors was realized with green light irradiation. This advance was enabled by the use of the previously reported iron photosensitizer, [Fe(phtmeimb)2]+ (phtmeimb = {phenyl[tris(3-methyl-imidazolin-2-ylidene)]borate}, that exhibited long-lived and luminescent ligand-to-metal charge-transfer (LMCT) excited states. A benchmark dehalogenation reaction was investigated with yields that exceed 90% and an enhanced stability relative to the prototypical photosensitizer [Ru(bpy)3]2+. The initial catalytic step is electron transfer from an amine to the photoexcited iron sensitizer, which is shown to occur with a large cage-escape yield. For LMCT excited states, this reductive electron transfer is vectorial and may be a general advantage of Fe(III) photosensitizers. In-depth time-resolved spectroscopic methods, including transient absorption characterization from the ultraviolet to the infrared regions, provided a quantitative description of the catalytic mechanism with associated rate constants and yields.

Kinetic Evidence That the Solvent Barrier for Electron Transfer Is Absent in the Electric Double Layer.

Classical capacitance studies have revealed that the first layer of water present at an aqueous metal-electrolyte interface has a dielectric constant less than 1/10th of that of bulk water. Modern theory indicates that the barrier for electron transfer will decrease substantially in this layer; yet, this important prediction has not been tested experimentally. Here, we report the interfacial electron transfer kinetics for molecules positioned at variable distances within the electric double layer of a transparent conductive oxide as a function of the Gibbs free energy change. The data indicate that the solvent reorganization is indeed near zero and increases to bulk values only when the molecules are positioned greater than 15 Å from the conductive electrode. Consistent with this conclusion, lateral intermolecular electron transfer, parallel to a semiconducting oxide electrode, was shown to be more rapid when the molecules were within the electric double layer. The results provide much needed feedback for theoretical studies and also indicate a huge kinetic advantage for aqueous electron transfer and redox catalysis that takes place proximate to a solid interface.

Targeted production of reactive oxygen species in mitochondria to overcome cancer drug resistance.

Multidrug resistance is a major challenge to cancer chemotherapy. The multidrug resistance phenotype is associated with the overexpression of the adenosine triphosphate (ATP)-driven transmembrane efflux pumps in cancer cells. Here, we report a lipid membrane-coated silica-carbon (LSC) hybrid nanoparticle that targets mitochondria through pyruvate, to specifically produce reactive oxygen species (ROS) in mitochondria under near-infrared (NIR) laser irradiation. The ROS can oxidize the NADH into NAD+ to reduce the amount of ATP available for the efflux pumps. The treatment with LSC nanoparticles and NIR laser irradiation also reduces the expression and increases the intracellular distribution of the efflux pumps. Consequently, multidrug-resistant cancer cells lose their multidrug resistance capability for at least 5 days, creating a therapeutic window for chemotherapy. Our in vivo data show that the drug-laden LSC nanoparticles in combination with NIR laser treatment can effectively inhibit the growth of multidrug-resistant tumors with no evident systemic toxicity.

Water-enhanced antegrade MR pyelography in pregnancy: a novel radiation-free approach.

Our aim was to determine if water-enhanced antegrade magnetic resonance (MR) pyelography can be an alternative to conventional antegrade pyelography in pregnant patients who require percutaneous nephrostomy placement for urosepsis and/or obstructive uropathy. The pregnant patient was placed supine in a 1.5-T MRI scanner seven days after percutaneous nephrostomy placement using ultrasound. Serial axial and coronal T2-weighted echo-planar fast spin-echo sequences were performed before and after injection of the catheter. The right nephrostomy catheter hub was sterilised using chlorhexidine. Sixty millilitres of sterile water were slowly injected. No Gd-based contrast agent was utilised due to safety concerns for the foetus. MR antegrade pyelography demonstrated the level of ureteric obstruction and the absence of renal calculi using sterile water as a contrast medium injected through a percutaneous nephrostomy followed by T2-weighted imaging. Air bubbles in the injected solution were differentiated from calculi due to their mobility on serial scans and their anti-dependent position. Water-enhanced antegrade MR pyelography was a safe and effective method of imaging the pregnant patient. It served as an alternative to conventional antegrade pyelography and minimised potential risks to the foetus.

Incidence of primary cancers and intracranial tumour recurrences in GH-treated and untreated adult hypopituitary patients: analyses from the Hypopituitary Control and Complications Study.

OBJECTIVE: Speculation remains that GH treatment is associated with increased neoplasia risk. Studies in GH-treated childhood cancer survivors suggested higher rates of second neoplasms, while cancer risk data for GH-treated and untreated hypopituitary adults have been variable. We present primary cancer risk data from the Hypopituitary Control and Complications Study (HypoCCS) with a focus on specific cancers, and assessment of recurrence rates for pituitary adenomas (PA) and craniopharyngiomas (CP). DESIGN: Incident neoplasms during HypoCCS were evaluated in 8418 GH-treated vs 1268 untreated patients for primary malignancies, 3668 GH-treated vs 720 untreated patients with PA history, and 956 GH-treated vs 102 untreated patients with CP history. METHODS: Using population cancer rates, standardised incidence ratios (SIRs) were calculated for all primary cancers, breast, prostate, and colorectal cancers. Neoplasm rates in GH-treated vs untreated patients were analysed after propensity score adjustment of baseline treatment group imbalances. RESULTS: During mean follow-up of 4.8 years, 225 primary cancers were identified in GH-treated patients, with SIR of 0.82 (95% CI 0.71-0.93). SIRs (95% CI) for GH-treated patients were 0.59 (0.36-0.90) for breast, 0.80 (0.57-1.10) for prostate, and 0.62 (0.38-0.96) for colorectal cancers. Cancer risk was not statistically different between GH-treated and untreated patients (relative risk (RR)=1.00 (95% CI 0.70-1.41), P=0.98). Adjusted RR for recurrence was 0.91 (0.68-1.22), P=0.53 for PA and 1.32 (0.53-3.31), P=0.55 for CP. CONCLUSIONS: There was no increased risk for all-site cancers: breast, prostate or colorectal primary cancers in GH-treated patients during HypoCCS. GH treatment did not increase the risk of PA and CP recurrences.

Use of the Hardman index in predicting mortality in endovascular repair of ruptured abdominal aortic aneurysms.

PURPOSE: The Hardman index is a predictor of 30-day mortality after open ruptured abdominal aneurysm repair through the use of preoperative patient factors. The aim of this study was to assess the Hardman index in patients undergoing endovascular repair of ruptured aortic aneurysms. MATERIALS AND METHODS: A retrospective analysis of 95 patients undergoing emergency endovascular repairs of computed tomography-confirmed ruptured aneurysms from 1994 to 2008 in a university hospital was performed. All relevant patient variables, calculations of the Hardman index, and the incidence of 30-day mortality were collected in these patients. Correlation of the relationship between each variable and the overall score with the incidence of 30-day mortality was undertaken. RESULTS: The 24-hour mortality was 16% and 30-day mortality 36%. Increasing scores on the Hardman index showed an increasing mortality rate. Thirty-day mortality in patients with a score of 0 to 2 was 30.5%, and in those with a score of ≥3 was 69.2% (P = .01, risk ratio = 2.26, 95% confidence interval = 0.98 to 5.17). This is lower than predicted in both patient groups based on Hardman index score. Loss of consciousness was the only statistically significant independent predictor of 30-day mortality with a risk ratio of 3.16 (95% confidence interval = 2.00-4.97, P < .001). CONCLUSION: These data suggest that the Hardman index can predict an increased risk of 30-day mortality from endovascular repairs of ruptured aortic aneurysms. However, mortality from endovascular repair is much lower than would be predicted in open repair and it therefore cannot be used clinically as a tool for exclusion from intervention.