Backbone NMR resonance assignments for the C terminal domain of the Streptococcus mutans adhesin P1.

Adhesin P1 (aka AgI/II) plays a pivotal role in mediating Streptococcus mutans attachment in the oral cavity, as well as in regulating biofilm development and maturation. P1's naturally occurring truncation product, Antigen II (AgII), adopts both soluble, monomeric and insoluble, amyloidogenic forms within the bacterial life cycle. Monomers are involved in important quaternary interactions that promote cell adhesion and the functional amyloid form promotes detachment of mature biofilms. The heterologous, 51-kD C123 construct comprises most of AgII and was previously characterized by X-ray crystallography. C123 contains three structurally homologous domains, C1, C2, and C3. NMR samples made using the original C123 construct, or its C3 domain, yielded moderately resolved NMR spectra. Using Alphafold, we re-analyzed the P1 sequence to better identify domain boundaries for C123, and in particular the C3 domain. We then generated a more tractable construct for NMR studies of the monomeric form, including quaternary interactions with other proteins. The addition of seven amino acids at the C-terminus greatly improved the spectral dispersion for C3 relative to the prior construct. Here we report the backbone NMR resonance assignments for the new construct and characterize some of its quaternary interactions. These data are in good agreement with the structure predicted by Alphafold, which contains additional ß-sheet secondary structure compared to the C3 domain in the C123 crystal structure for a construct lacking the seven C-terminal amino acids. Its quaternary interactions with known protein partners are in good agreement with prior competitive binding assays. This construct can be used for further NMR studies, including protein-protein interaction studies and assessing the impact of environmental conditions on C3 structure and dynamics within C123 as it transitions from monomer to amyloid form.

Analysis of Anxiety, Depression and Fear of Progression at 12 Months Post-Cytoreductive Surgery in the SOCQER-2 (Surgery in Ovarian Cancer-Quality of Life Evaluation Research) Prospective, International, Multicentre Study.

Patients with ovarian cancer (OC) often experience anxiety, depression and fear of progression (FOP); however, it is unclear whether surgical complexity has a role to play. We investigated the prevalence of anxiety, depression and FOP at 12 months post-cytoreductive surgery and investigated associations with surgical complexity, patient (age, ethnicity, performance status, BMI) and tumour (stage, disease load) factors. One hundred and forty-one patients with FIGO Stage III-IV OC, who did not have disease progression at 12 months post-surgery, completed the Hospital Anxiety and Depression Scale and FOP short-form questionnaire. Patients underwent surgery with low (40.4%), intermediate (31.2%) and high (28.4%) surgical complexity scores. At 12 months post-surgery, 99 of 141 (70%) patients with advanced OC undergoing surgery experienced clinically significant anxiety, 21 of 141 (14.9%) patients experienced moderate to severe depression and 37 of 140 (26.4%) experienced dysfunctional FOP. No associations were identified between the three different surgical complexity groups with regards to anxiety, depression or FOP scores. Unsurprisingly, given the natural history of the disease, most patients with OC suffer from anxiety, depression and fear of progression after completion of first-line cancer treatment. Surgical complexity at the time of surgery is not associated with a deleterious impact on anxiety, depression or FOP for patients with OC. Patients with OC experience a profound mental health impact and should be offered mental health support throughout their cancer journey.

Investigating the Impact of Ultra-Radical Surgery on Survival in Advanced Ovarian Cancer Using Population-Based Data in a Multicentre UK Study.

We investigated URS and impact on survival in whole patient cohorts with AOC treated within gynaecological cancer centres that participated in the previously presented SOCQER 2 study. National cancer registry datasets were used to identify FIGO Stage 3,4 and unknown stage patients from 11 cancer centres that had previously participated in the SOCQER2 study. Patient outcomes' association with surgical ethos were evaluated using logistic regression and Cox proportional hazards. Centres were classified into three groups based on their surgical complexity scores (SCS); those practicing mainly low complexity, (5/11 centres with >70% low SCS procedures, 759 patients), mainly intermediate (3/11, 35−50% low SCS, 356 patients), or mainly high complexity surgery (3/11, >35% high SCS, 356 patients). Surgery rates were 43.2% vs. 58.4% vs. 60.9%. across mainly low, intermediate and high SCS centres, respectively, p < 0.001. Combined surgery and chemotherapy rates were 39.2% vs. 51.8% vs. 38.3% p < 0.000 across mainly low, intermediate and high complexity groups, respectively. Median survival was 23.1 (95% CI 19.0 to 27.2) vs. 22.0 (95% CI 17.6 to 26.3) vs. 17.9 months (95% CI 15.7 to 20.1), p = 0.043 in mainly high SCS, intermediate, and low SCS centres, respectively. In an age and deprivation adjusted model, compared to patients in the high SCS centres, patients in the low SCS group had an HR of 1.21 (95% CI 1.03 to 1.40) for death. Mainly high/intermediate SCS centres have significantly higher surgery rates and better survival at a population level. Centres that practice mainly low complexity surgery should change practice. This study provides support for the utilization of URS for patients with advanced OC.

Amyloid Aggregates Are Localized to the Nonadherent Detached Fraction of Aging Streptococcus mutans Biofilms.

The number of bacterial species recognized to utilize purposeful amyloid aggregation within biofilms continues to grow. The oral pathogen Streptococcus mutans produces several amyloidogenic proteins, including adhesins P1 (also known as AgI/II, PAc) and WapA, whose truncation products, namely, AgII and AgA, respectively, represent the amyloidogenic moieties. Amyloids demonstrate common biophysical properties, including recognition by Thioflavin T (ThT) and Congo red (CR) dyes that bind to the cross ß-sheet quaternary structure of amyloid aggregates. Previously, we observed amyloid formation to occur only after 60 h or more of S. mutans biofilm growth. Here, we extend those findings to investigate where amyloid is detected within 1- and 5-day-old biofilms, including within tightly adherent compared with those in nonadherent fractions. CR birefringence and ThT uptake demonstrated amyloid within nonadherent material removed from 5-day-old cultures but not within 1-day-old or adherent samples. These experiments were done in conjunction with confocal microscopy and immunofluorescence staining with AgII- and AgA-reactive antibodies, including monoclonal reagents shown to discriminate between monomeric protein and amyloid aggregates. These results also localized amyloid primarily to the nonadherent fraction of biofilms. Lastly, we show that the C-terminal region of P1 loses adhesive function following amyloidogenesis and is no longer able to competitively inhibit binding of S. mutans to its physiologic substrate, salivary agglutinin. Taken together, our results provide new evidence that amyloid aggregation negatively impacts the functional activity of a widely studied S. mutans adhesin and are consistent with a model in which amyloidogenesis of adhesive proteins facilitates the detachment of aging biofilms. IMPORTANCE Streptococcus mutans is a keystone pathogen and causative agent of human dental caries, commonly known as tooth decay, the most prevalent infectious disease in the world. Like many pathogens, S. mutans causes disease in biofilms, which for dental decay begins with bacterial attachment to the salivary pellicle coating the tooth surface. Some strains of S. mutans are also associated with bacterial endocarditis. Amyloid aggregation was initially thought to represent only a consequence of protein mal-folding, but now, many microorganisms are known to produce functional amyloids with biofilm environments. In this study, we learned that amyloid formation diminishes the activity of a known S. mutans adhesin and that amyloid is found within the nonadherent fraction of older biofilms. This finding suggests that the transition from adhesin monomer to amyloid facilitates biofilm detachment. Knowing where and when S. mutans produces amyloid will help in developing therapeutic strategies to control tooth decay and other biofilm-related diseases.

The interaction and orientation of Peptide KL4 in model membranes.

We report on the orientation and location of synthetic pulmonary surfactant peptide KL4, (KLLLL)4K, in model lipid membranes. The partitioning depths of selectively deuterated leucine residues within KL4 were determined in DPPC:POPG (4:1) and POPC:POPG (4:1) bilayers by oriented neutron diffraction. These measurements were combined with an NMR-generated model of the peptide structure to determine the orientation and partitioning of the peptide at the lipid-water interface. The results demonstrate KL4 adopting an orientation that interacts with a single membrane leaflet. These observations are consistent with past 2H NMR and EPR studies (Antharam et al., 2009; Turner et al., 2014).

Imaging active site chemistry and protonation states: NMR crystallography of the tryptophan synthase α-aminoacrylate intermediate.

NMR-assisted crystallography-the integrated application of solid-state NMR, X-ray crystallography, and first-principles computational chemistry-holds significant promise for mechanistic enzymology: by providing atomic-resolution characterization of stable intermediates in enzyme active sites, including hydrogen atom locations and tautomeric equilibria, NMR crystallography offers insight into both structure and chemical dynamics. Here, this integrated approach is used to characterize the tryptophan synthase α-aminoacrylate intermediate, a defining species for pyridoxal-5'-phosphate-dependent enzymes that catalyze ß-elimination and replacement reactions. For this intermediate, NMR-assisted crystallography is able to identify the protonation states of the ionizable sites on the cofactor, substrate, and catalytic side chains as well as the location and orientation of crystallographic waters within the active site. Most notable is the water molecule immediately adjacent to the substrate ß-carbon, which serves as a hydrogen bond donor to the ε-amino group of the acid-base catalytic residue ßLys87. From this analysis, a detailed three-dimensional picture of structure and reactivity emerges, highlighting the fate of the L-serine hydroxyl leaving group and the reaction pathway back to the preceding transition state. Reaction of the α-aminoacrylate intermediate with benzimidazole, an isostere of the natural substrate indole, shows benzimidazole bound in the active site and poised for, but unable to initiate, the subsequent bond formation step. When modeled into the benzimidazole position, indole is positioned with C3 in contact with the α-aminoacrylate Cß and aligned for nucleophilic attack. Here, the chemically detailed, three-dimensional structure from NMR-assisted crystallography is key to understanding why benzimidazole does not react, while indole does.

Quality of life from cytoreductive surgery in advanced ovarian cancer: Investigating the association between disease burden and surgical complexity in the international, prospective, SOCQER-2 cohort study.

OBJECTIVE: To investigate quality of life (QoL) and association with surgical complexity and disease burden after surgical resection for advanced ovarian cancer in centres with variation in surgical approach. DESIGN: Prospective multicentre observational study. SETTING: Gynaecological cancer surgery centres in the UK, Kolkata, India, and Melbourne, Australia. SAMPLE: Patients undergoing surgical resection (with low, intermediate or high surgical complexity score, SCS) for late-stage ovarian cancer. MAIN OUTCOME MEASURES: Primary: change in global score on the European Organisation for Research and Treatment of Cancer (EORTC) core quality-of-life questionnaire (QLQ-C30). Secondary: EORTC ovarian cancer module (OV28), progression-free survival. RESULTS: Patients' preoperative disease burden and SCS varied between centres, confirming differences in surgical ethos. QoL response rates were 90% up to 18 months. Mean change from the pre-surgical baseline in the EORTC QLQ-C30 was 3.4 (SD 1.8, n = 88) in the low, 4.0 (SD 2.1, n = 55) in the intermediate and 4.3 (SD 2.1, n = 52) in the high-SCS group after 6 weeks (p = 0.048), and 4.3 (SD 2.1, n = 51), 5.1 (SD 2.2, n = 41) and 5.1 (SD 2.2, n = 35), respectively, after 12 months (p = 0.133). In a repeated-measures model, there were no clinically or statistically meaningful differences in EORTC QLQ-C30 global scores between the three SCS groups (p = 0.840), but there was a small statistically significant improvement in all groups over time (p < 0.001). The high-SCS group experienced small to moderate decreases in physical (p = 0.004), role (p = 0.016) and emotional (p = 0.001) function at 6 weeks post-surgery, which resolved by 6-12 months. CONCLUSIONS: The global QoL of patients undergoing low-, intermediate- and high-SCS surgery improved at 12 months after surgery and was no worse in patients undergoing extensive surgery. TWEETABLE ABSTRACT: Compared with surgery of lower complexity, extensive surgery does not result in poorer quality of life in patients with advanced ovarian cancer.

Dynamic Nuclear Polarization of Biomembrane Assemblies.

While atomic scale structural and dynamic information are hallmarks of nuclear magnetic resonance (NMR) methodologies, sensitivity is a fundamental limitation in NMR studies. Fully exploiting NMR capabilities to study membrane proteins is further hampered by their dilution within biological membranes. Recent developments in dynamic nuclear polarization (DNP), which can transfer the relatively high polarization of unpaired electrons to nuclear spins, show promise for overcoming the sensitivity bottleneck and enabling NMR characterization of membrane proteins under native-like conditions. Here we discuss fundamental aspects of DNP-enhanced solid-state NMR spectroscopy, experimental details relevant to the study of lipid assemblies and incorporated proteins, and sensitivity gains which can be realized in biomembrane-based samples. We also present unique insights which can be gained from DNP measurements and prospects for further development of the technique for elucidating structures and orientations of membrane proteins in native lipid environments.

Adeno-Associated Virus (AAV) Capsid Stability and Liposome Remodeling During Endo/Lysosomal pH Trafficking.

Adeno-associated viruses (AAVs) are small, non-pathogenic ssDNA viruses being used as therapeutic gene delivery vectors for the treatment of a variety of monogenic diseases. An obstacle to successful gene delivery is inefficient capsid trafficking through the endo/lysosomal pathway. This study aimed to characterize the AAV capsid stability and dynamics associated with this process for a select number of AAV serotypes, AAV1, AAV2, AAV5, and AAV8, at pHs representative of the early and late endosome, and the lysosome (6.0, 5.5, and 4.0, respectively). All AAV serotypes displayed thermal melt temperatures that varied with pH. The stability of AAV1, AAV2, and AAV8 increased in response to acidic conditions and then decreased at pH 4.0. In contrast, AAV5 demonstrated a consistent decrease in thermostability in response to acidification. Negative-stain EM visualization of liposomes in the presence of capsids at pH 5.5 or when heat shocked showed induced remodeling consistent with the externalization of the PLA2 domain of VP1u. These observations provide clues to the AAV capsid dynamics that facilitate successful infection. Finally, transduction assays revealed a pH and temperature dependence with low acidity and temperatures > 4 °C as detrimental factors.

Frugal filtering optical lenses for point-of-care diagnostics.

Infectious diseases are the leading cause of morbidity and mortality in low and middle income countries (LMICs). Rapid diagnosis of infections in LMICs presents many challenges, especially in rural areas where access to health care, including diagnostics, is poor. Microscopy is one of the most commonly used platforms to diagnose bacterial infections on clinical samples. Fluorescence microscopy has high sensitivity and specificity but to date is mostly performed within a laboratory setting due to the high-cost, low portability and highly specialist nature of equipment. Point-of-care diagnostics could offer a solution to the challenge of infection diagnosis in LMICs. In this paper we present frugal, easy to manufacture, doped polydimethylsiloxane filtering optical lenses that can be integrated into smartphone microscopes for immediate detection of fluorescently labelled bacteria. This provides a breakthrough technology platform for point-of-care diagnostics.

Enhanced purification coupled with biophysical analyses shows cross-ß structure as a core building block for Streptococcus mutans functional amyloids.

Streptococcus mutans is an etiologic agent of human dental caries that forms dental plaque biofilms containing functional amyloids. Three amyloidogenic proteins, P1, WapA, and Smu_63c were previously identified. C123 and AgA are naturally occurring amyloid-forming fragments of P1 and WapA, respectively. We determined that four amyloidophilic dyes, ThT, CDy11, BD-oligo, and MK-H4, differentiate C123, AgA, and Smu_63c amyloid from monomers, but non-specific binding to bacterial cells in the absence of amyloid precludes their utility for identifying amyloid in biofilms. Congo red-induced birefringence is a more specific indicator of amyloid formation and differentiates biofilms formed by wild-type S. mutans from a triple ΔP1/WapA/Smu_63c mutant with reduced biofilm forming capabilities. Amyloid accumulation is a late event, appearing in older S. mutans biofilms after 60 hours of growth. Amyloid derived from pure preparations of all three proteins is visualized by electron microscopy as mat-like structures. Typical amyloid fibers become evident following protease digestion to eliminate non-specific aggregates and monomers. Amyloid mats, similar in appearance to those reported in S. mutans biofilm extracellular matrices, are reconstituted by co-incubation of monomers and amyloid fibers. X-ray fiber diffraction of amyloid mats and fibers from all three proteins demonstrate patterns reflective of a cross-ß amyloid structure.

Comparison of Multivendor Single-Voxel MR Spectroscopy Data Acquired in Healthy Brain at 26 Sites.

Background The hardware and software differences between MR vendors and individual sites influence the quantification of MR spectroscopy data. An analysis of a large data set may help to better understand sources of the total variance in quantified metabolite levels. Purpose To compare multisite quantitative brain MR spectroscopy data acquired in healthy participants at 26 sites by using the vendor-supplied single-voxel point-resolved spectroscopy (PRESS) sequence. Materials and Methods An MR spectroscopy protocol to acquire short-echo-time PRESS data from the midparietal region of the brain was disseminated to 26 research sites operating 3.0-T MR scanners from three different vendors. In this prospective study, healthy participants were scanned between July 2016 and December 2017. Data were analyzed by using software with simulated basis sets customized for each vendor implementation. The proportion of total variance attributed to vendor-, site-, and participant-related effects was estimated by using a linear mixed-effects model. P values were derived through parametric bootstrapping of the linear mixed-effects models (denoted Pboot). Results In total, 296 participants (mean age, 26 years ± 4.6; 155 women and 141 men) were scanned. Good-quality data were recorded from all sites, as evidenced by a consistent linewidth of N-acetylaspartate (range, 4.4-5.0 Hz), signal-to-noise ratio (range, 174-289), and low Cramér-Rao lower bounds (≤5%) for all of the major metabolites. Among the major metabolites, no vendor effects were found for levels of myo-inositol (Pboot > .90), N-acetylaspartate and N-acetylaspartylglutamate (Pboot = .13), or glutamate and glutamine (Pboot = .11). Among the smaller resonances, no vendor effects were found for ascorbate (Pboot = .08), aspartate (Pboot > .90), glutathione (Pboot > .90), or lactate (Pboot = .28). Conclusion Multisite multivendor single-voxel MR spectroscopy studies performed at 3.0 T can yield results that are coherent across vendors, provided that vendor differences in pulse sequence implementation are accounted for in data analysis. However, the site-related effects on variability were more profound and suggest the need for further standardization of spectroscopic protocols. © RSNA, 2020 Online supplemental material is available for this article.

Characterization of an intermolecular quaternary interaction between discrete segments of the Streptococcus mutans adhesin P1 by NMR spectroscopy.

Cell surface-localized P1 adhesin (aka Antigen I/II or PAc) of the cariogenic bacterium Streptococcus mutans mediates sucrose-independent adhesion to tooth surfaces. Previous studies showed that P1's C-terminal segment (C123, AgII) is also liberated as a separate polypeptide, contributes to cellular adhesion, interacts specifically with intact P1 on the cell surface, and forms amyloid fibrils. Identifying how C123 specifically interacts with P1 at the atomic level is essential for understanding related virulence properties of S. mutans. However, with sizes of ~ 51 and ~ 185 kDa, respectively, C123 and full-length P1 are too large to achieve high-resolution data for full structural analysis by NMR. Here, we report on biologically relevant interactions of the individual C3 domain with A3VP1, a polypeptide that represents the apical head of P1 as it is projected on the cell surface. Also evaluated are C3's interaction with C12 and the adhesion-inhibiting monoclonal antibody (MAb) 6-8C. NMR titration experiments with 15 N-enriched C3 demonstrate its specific binding to A3VP1. Based on resolved C3 assignments, two binding sites, proximal and distal, are identified. Complementary NMR titration of A3VP1 with a C3/C12 complex suggests that binding of A3VP1 occurs on the distal C3 binding site, while the proximal site is occupied by C12. The MAb 6-8C binding interface to C3 overlaps with that of A3VP1 at the distal site. Together, these results identify a specific C3-A3VP1 interaction that serves as a foundation for understanding the interaction of C123 with P1 on the bacterial surface and the related biological processes that stem from this interaction. DATABASE: BMRB submission code: 27935.

Quality of life outcomes following surgery for advanced ovarian cancer: a systematic review and meta-analysis.

BACKGROUND: Quality of life after ovarian cancer treatment is an important goal for patients. Complex debulking surgeries and platinum based chemotherapy are often required but quality of life after surgery is rarely reported. OBJECTIVES: To describe quality of life outcomes after surgery for advanced ovarian cancer in a systematic review and meta-analysis. SEARCH STRATEGY: MEDLINE, EMBASE, and CENTRAL through March 2019 with no language restrictions. SELECTION CRITERIA: Included studies reported quality of life in women diagnosed with primary advanced ovarian cancer, fallopian tube carcinoma or primary peritoneal cancer undergoing cytoreduction surgery. DATA COLLECTION AND ANALYSIS: Data on extent and timing of surgery, quality of life outcomes, and surgical complications were extracted and study quality assessed. RESULTS: Three randomized controlled trials comparing primary surgery to neoadjuvant chemotherapy had heterogeneous quality of life outcomes with no difference between arms, although there was a clinical improvement in global quality of life scores in both arms at 6 months compared with baseline. Data from two observational studies showed no meaningful difference in quality of life scores between patients undergoing standard or extensive surgery at 6 months. CONCLUSIONS: There was no clinically important difference in the quality of life of patients undergoing either primary debulking surgery or neoadjuvant chemotherapy. There is insufficient evidence on quality of life outcomes of patients undergoing extensive or ultra-radical surgery compared with those undergoing less extensive surgery. Quality of life outcomes matter to patients, but there is little evidence to inform patient choice regarding the extent of surgery.

Direct dynamic nuclear polarization of 15N and 13C spins at 14.1 T using a trityl radical and magic angle spinning.

We investigate solid-state dynamic nuclear polarization of 13C and 15N nuclei using monoradical trityl OX063 as a polarizing agent in a magnetic field of 14.1 T with magic angle spinning at ∼100 K. We monitored the field dependence of direct 13C and 15N polarization for frozen [13C, 15N] urea and achieved maximum absolute enhancement factors of 240 and 470, respectively. The field profiles are consistent with polarization of 15N spins via either the solid effect or the cross effect, and polarization of 13C spins via a combination of cross effect and solid effect. For microcrystalline, 15N-enriched tryptophan synthase sample containing trityl radical, a 1500-fold increase in 15N signal was observed under microwave irradiation. These results show the promise of trityl radicals and their derivatives for direct polarization of low gamma, spin-½ nuclei at high magnetic fields and suggest a novel approach for selectively polarizing specific moieties or for polarizing systems which have low levels of protonation.

Big GABA II: Water-referenced edited MR spectroscopy at 25 research sites.

Accurate and reliable quantification of brain metabolites measured in vivo using 1H magnetic resonance spectroscopy (MRS) is a topic of continued interest. Aside from differences in the basic approach to quantification, the quantification of metabolite data acquired at different sites and on different platforms poses an additional methodological challenge. In this study, spectrally edited γ-aminobutyric acid (GABA) MRS data were analyzed and GABA levels were quantified relative to an internal tissue water reference. Data from 284 volunteers scanned across 25 research sites were collected using GABA+ (GABA + co-edited macromolecules (MM)) and MM-suppressed GABA editing. The unsuppressed water signal from the volume of interest was acquired for concentration referencing. Whole-brain T1-weighted structural images were acquired and segmented to determine gray matter, white matter and cerebrospinal fluid voxel tissue fractions. Water-referenced GABA measurements were fully corrected for tissue-dependent signal relaxation and water visibility effects. The cohort-wide coefficient of variation was 17% for the GABA + data and 29% for the MM-suppressed GABA data. The mean within-site coefficient of variation was 10% for the GABA + data and 19% for the MM-suppressed GABA data. Vendor differences contributed 53% to the total variance in the GABA + data, while the remaining variance was attributed to site- (11%) and participant-level (36%) effects. For the MM-suppressed data, 54% of the variance was attributed to site differences, while the remaining 46% was attributed to participant differences. Results from an exploratory analysis suggested that the vendor differences were related to the unsuppressed water signal acquisition. Discounting the observed vendor-specific effects, water-referenced GABA measurements exhibit similar levels of variance to creatine-referenced GABA measurements. It is concluded that quantification using internal tissue water referencing is a viable and reliable method for the quantification of in vivo GABA levels.

Characterization of Brain Metabolism by Nuclear Magnetic Resonance.

The noninvasive, quantitative ability of nuclear magnetic resonance (NMR) spectroscopy to characterize small molecule metabolites has long been recognized as a major strength of its application in biology. Numerous techniques exist for characterizing metabolism in living, excised, or extracted tissue, with a particular focus on 1 H-based methods due to the high sensitivity and natural abundance of protons. With the increasing use of high magnetic fields, the utility of in vivo 1 H magnetic resonance spectroscopy (MRS) has markedly improved for measuring specific metabolite concentrations in biological tissues. Higher fields, coupled with recent developments in hyperpolarization, also enable techniques for complimenting 1 H measurements with spectroscopy of other nuclei, such as 31 P and 13 C, and for combining measurements of metabolite pools with metabolic flux measurements. We compare ex vivo and in vivo methods for studying metabolism in the brain using NMR and highlight insights gained through using higher magnetic fields, the advent of dissolution dynamic nuclear polarization, and combining in vivo MRS and ex vivo NMR approaches.

Gadolinium based endohedral metallofullerene Gd2@C79N as a relaxation boosting agent for dissolution DNP at high fields.

We show increased dynamic nuclear polarization by adding a low dosage of a S = 15/2 Gd based endohedral metallofullerene (EMF) to DNP samples. By adding 60 µM Gd2@C79N, the nuclear polarization of 1H and 13C spins from 40 mM 4-oxo-TEMPO increases by approximately 40% and 50%, respectively, at 5 T and 1.2 K. Electron-electron double resonance (ELDOR) measurements show that the high spin EMF shortens the electron relaxation times and increases electron spectral diffusion leading to the increased DNP enhancement.

A quasi-optical and corrugated waveguide microwave transmission system for simultaneous dynamic nuclear polarization NMR on two separate 14.1 T spectrometers.

Nuclear magnetic resonance (NMR) is an intrinsically insensitive technique, with Boltzmann distributions of nuclear spin states on the order of parts per million in conventional magnetic fields. To overcome this limitation, dynamic nuclear polarization (DNP) can be used to gain up to three orders of magnitude in signal enhancement, which can decrease experimental time by up to six orders of magnitude. In DNP experiments, nuclear spin polarization is enhanced by transferring the relatively larger electron polarization to NMR active nuclei via microwave irradiation. Here, we describe the design and performance of a quasi-optical system enabling the use of a single 395 GHz gyrotron microwave source to simultaneously perform DNP experiments on two different 14.1 T (1H 600 MHz) NMR spectrometers: one configured for magic angle spinning (MAS) solid state NMR; the other configured for solution state NMR experiments. In particular, we describe how the high power microwave beam is split, transmitted, and manipulated between the two spectrometers. A 13C enhancement of 128 is achieved via the cross effect for alanine, using the nitroxide biradical AMUPol, under MAS-DNP conditions at 110 K, while a 31P enhancement of 160 is achieved via the Overhauser effect for triphenylphosphine using the monoradical BDPA under solution NMR conditions at room temperature. The latter result is the first demonstration of Overhauser DNP in the solution state at a field of 14.1 T (1H 600 MHz). Moreover these results have been produced with large sample volumes (∼100 µL, i.e. 3 mm diameter NMR tubes).

Mind over bladder: Women, aging, and bladder health.

The purpose of this study was to explore the efficacy and acceptability of an innovative, electronically delivered self-management intervention for urinary incontinence (UI) that included daily mindfulness practice, completion of sequential bladder diaries, and bladder health education to improve UI in older women living independently in a retirement community. A mixed methods pilot study was conducted over ten weeks using a custom website or CD. Ten women were recruited and 8 completed the study; 5 of those (71%) experienced fewer daily UI episodes post intervention (p = 0.055). The women also reported a statistically significant decrease in the impact UI had on their everyday life (p = 0.04). Seventy-one percent (N = 5) reported subjective improvement in UI, and high acceptability scores also were achieved. The intervention was both effective in helping older women self-manage UI and acceptable to the population group. Further research is needed with a larger and diverse population of older women.