Monitoring off-resonance signals with SHARPER NMR - the MR-SHARPER experiment.

We demonstrate an extension to the SHARPER (Sensitive Homogenous and Refocussed Peaks in Real Time) NMR experiment which allows more than one signal to be monitored simultaneously, while still giving ultra-sharp, homo- and hetero-decoupled NMR signals. This is especially valuable in situations where magnetic field inhomogeneity would normally make NMR a problematic tool, for example when gas evolution is occurring during reaction monitoring. The originally reported SHARPER experiment only works for a single, on-resonance NMR signal, but here we demonstrate the Multiple Resonance SHARPER approach can be developed, which in principle can acquire multiple on-/off-resonance signals simultaneously while retaining the desirable properties of the parent sequence. In practice, the case of two resonances, e.g. those of a reactant and a product, will most of the time be considered for MR-SHARPER, as illustrated here.

Outcomes of assistance dog placement in the home for individuals with autism spectrum disorder and their families: A pilot study.

INTRODUCTION: Evidence indicates that assistance dogs placed in the home are effective in supporting individuals with Autism Spectrum Disorder (ASD) by increasing social and community participation and promoting quality of life. This study aimed to examine the outcomes of assistance dog placement on quality of life, independence, and participation of families including individuals with ASD placed with an assistance dog compared to families on the waiting list for an autism assistance dog and to evaluate the feasibility of the design for future studies. METHODS: A cross-sectional comparative study was conducted. The Adaptive Behaviour Analysis System, Social Responsiveness Scale, Autism Treatment Evaluation Checklist, Canadian Occupational Performance Measure, and Autism Family Experience Questionnaire were used to evaluate adaptive skills, behaviour, social difficulties, daily functioning, and family quality of life. RESULTS: Six families who had an autism assistance dog placed with them, and 12 families who were on the waiting list were recruited using purposeful sampling. The pilot data found no significant differences between the two groups. However, trends were observed which suggested that assistance dogs can increase desired social behaviours, decrease ASD severity, and improve family wellbeing. For families with an assistance dog, more positive outcomes were observed for families who were partnered with an assistance dog for longer. Parents (and sometimes individuals with ASD) were able to complete and return the outcome measures via mail to collect the outcome data for the study. CONCLUSION: These study findings add to the developing evidence about the use of assistance dogs with this population. A larger sample size may have allowed for significant associations to be detected. The methods used were feasible to be applied in a larger study. These results may assist health professionals advocate for funding for assistance dog placement to individuals with ASD and their families.

Understanding the participation outcomes for persons with disability when partnered with assistance dogs: A scoping review.

INTRODUCTION: Assistance dogs are trained to support persons living with disability and mitigate limitations that hinder their participation in everyday activities. Despite participation being a frequent challenge for people with disabilities, evidence linking assistance dog provision to improved participation outcomes is underdeveloped. This scoping review aimed to improve understanding by mapping the participation outcomes claimed in research on assistance dogs using the International Classification of Functioning (ICF), Disability and Health framework. METHODS: Using the Arksey and O'Malley's six-step framework, this scoping review searched six databases. Data were collected, mapped and summarised in accordance with the domains outlined in the ICF. RESULTS: In total, 38 studies across 41 papers met the inclusion criteria. Included studies investigated assistance dogs who were partnered with people living with physical disabilities, mental illness, autism and chronic conditions that require alerting (e.g., epilepsy and diabetes). Mapping of participation outcomes suggested that assistance dogs can have a positive impact on participation in many areas of daily life. CONCLUSION: Findings can assist practitioners, funders and policymakers to recognise the value of assistance dogs as a support for people with disability. However, further research is needed to address limitations regarding study designs, for example, the outcome measures used.

Rapid HIV-1 Capsid Interaction Screening Using Fluorescence Fluctuation Spectroscopy.

The HIV capsid is a multifunctional protein capsule that mediates the delivery of the viral genetic material into the nucleus of the target cell. Host cell proteins bind to a number of repeating binding sites on the capsid to regulate steps in the replication cycle. Here, we develop a fluorescence fluctuation spectroscopy method using self-assembled capsid particles as the bait to screen for fluorescence-labeled capsid-binding analytes ("prey" molecules) in solution. The assay capitalizes on the property of the HIV capsid as a multivalent interaction platform, facilitating high sensitivity detection of multiple prey molecules that have accumulated onto capsids as spikes in fluorescence intensity traces. By using a scanning stage, we reduced the measurement time to 10 s without compromising on sensitivity, providing a rapid binding assay for screening libraries of potential capsid interactors. The assay can also identify interfaces for host molecule binding by using capsids with defects in known interaction interfaces. Two-color coincidence detection using the fluorescent capsid as the bait further allows the quantification of binding levels and determination of binding affinities. Overall, the assay provides new tools for the discovery and characterization of molecules used by the HIV capsid to orchestrate infection. The measurement principle can be extended for the development of sensitive interaction assays, utilizing natural or synthetic multivalent scaffolds as analyte-binding platforms.

Rapid Estimation of T1 for Quantitative NMR.

Quantitative NMR spectroscopy (qNMR) is an essential tool in organic chemistry, with applications including reaction monitoring, mechanistic analysis, and purity determination. Establishing the correct acquisition rate for consecutive qNMR scans requires knowledge of the longitudinal relaxation time constants (T1) for all of the nuclei being monitored. We report a simple method that is about 10-fold faster than the conventional inversion recovery technique for the estimation of T1.

Locally produced personal protective equipment can offer hospital staff protection against Covid-19 if combined with surgical masks and rigorous personal protective equipment cleaning routine.

Locally made, washable and reusable personal protective equipment (PPE), used in combination with N95 masks that were reused safely, has proven to be a viable alternative to disposable gowns and caps for hospital staff in low- and middle-income countries. Muhimbili University Hospital's children's cancer ward in Dar es Salaam, Tanzania, developed locally made PPE and created rigorous cleaning and disinfecting protocols, when the daily use of imported, disposable materials were not an option. These items continue to protect staff, children and parents. The novel PPE approach was able to prevent staff from becoming infected during the pandemic despite the fact that several parents, and subsequently their children, became infected with Covid-19 during cancer treatment at the facility.

Sequential ubiquitination and deubiquitination enzymes synchronize the dual sensor and effector functions of TRIM21.

Tripartite motif (TRIM) 21 is a cytosolic antibody receptor that neutralizes antibody-coated viruses that penetrate the cell and simultaneously activates innate immunity. Here we show that the conjugation of TRIM21 with K63-linked ubiquitin (Ub-(63)Ub) catalyzed by the sequential activity of nonredundant E2 Ub enzymes is required for its dual antiviral functions. TRIM21 is first labeled with monoubiquitin (monoUb) by the E2 Ube2W. The monoUb is a substrate for the heterodimeric E2 Ube2N/Ube2V2, resulting in TRIM21-anchored Ub-(63)Ub. Depletion of either E2 abolishes Ub-(63)Ub and Ub-(48)Ub conjugation of TRIM21, NF-κB signaling, and virus neutralization. The formation of TRIM21-Ub-(63)Ub precedes proteasome recruitment, and we identify an essential role for the 19S-resident and degradation-coupled deubiquitinase Poh1 in TRIM21 neutralization, signaling, and cytokine induction. This study elucidates a complex mechanism of step-wise ubiquitination and deubiquitination activities that allows contemporaneous innate immune signaling and neutralization by TRIM21.

The Staphylococcus aureus Protein IsdH Inhibits Host Hemoglobin Scavenging to Promote Heme Acquisition by the Pathogen.

Hemolysis is a complication in septic infections with Staphylococcus aureus, which utilizes the released Hb as an iron source. S. aureus can acquire heme in vitro from hemoglobin (Hb) by a heme-sequestering mechanism that involves proteins from the S. aureus iron-regulated surface determinant (Isd) system. However, the host has its own mechanism to recapture the free Hb via haptoglobin (Hp) binding and uptake of Hb-Hp by the CD163 receptor in macrophages. It has so far remained unclear how the Isd system competes with this host iron recycling system in situ to obtain the important nutrient. By binding and uptake studies, we now show that the IsdH protein, which serves as an Hb receptor in the Isd system, directly interferes with the CD163-mediated clearance by binding the Hb-Hp complex and inhibiting CD163 recognition. Analysis of truncated IsdH variants including one or more of three near iron transporter domains, IsdHN1, IsdHN2, and IsdHN3, revealed that Hb binding of IsdHN1 and IsdHN2 accounted for the high affinity for Hb-Hp complexes. The third near iron transporter domain, IsdHN3, exhibited redox-dependent heme extraction, when Hb in the Hb-Hp complex was in the oxidized met form but not in the reduced oxy form. IsdB, the other S. aureus Hb receptor, failed to extract heme from Hb-Hp, and it was a poor competitor for Hb-Hp binding to CD163. This indicates that Hb recognition by IsdH, but not by IsdB, sterically inhibits the receptor recognition of Hb-Hp. This function of IsdH may have an overall stimulatory effect on S. aureus heme acquisition and growth.

Accurate measurement of long range proton-carbon scalar coupling constants.

The accuracy and practicality of measuring heteronuclear scalar coupling constants, nJCH, from modern NMR experimental methods is examined, based on F1 or F2 evolution of nJCH in HSQMBC (including EXSIDE) and HMBC experiments. The results from these methods are compared to both robust experimental data (derived from coupled 13C spectra), computed (Density Functional Theory) and literature values where available. We report on the accuracy, ease of use and time efficiency of these multi-dimensional methods and highlight their extent and limitations.

Structure of the hemoglobin-IsdH complex reveals the molecular basis of iron capture by Staphylococcus aureus.

Staphylococcus aureus causes life-threatening disease in humans. The S. aureus surface protein iron-regulated surface determinant H (IsdH) binds to mammalian hemoglobin (Hb) and extracts heme as a source of iron, which is an essential nutrient for the bacteria. However, the process of heme transfer from Hb is poorly understood. We have determined the structure of IsdH bound to human Hb by x-ray crystallography at 4.2 Å resolution, revealing the structural basis for heme transfer. One IsdH molecule is bound to each α and ß Hb subunit, suggesting that the receptor acquires iron from both chains by a similar mechanism. Remarkably, two near iron transporter (NEAT) domains in IsdH perform very different functions. An N-terminal NEAT domain binds α/ß globin through a site distant from the globin heme pocket and, via an intervening structural domain, positions the C-terminal heme-binding NEAT domain perfectly for heme transfer. These data, together with a 2.3 Å resolution crystal structure of the isolated N-terminal domain bound to Hb and small-angle x-ray scattering of free IsdH, reveal how multiple domains of IsdH cooperate to strip heme from Hb. Many bacterial pathogens obtain iron from human hemoglobin using proteins that contain multiple NEAT domains and other domains whose functions are poorly understood. Our results suggest that, rather than acting as isolated units, NEAT domains may be integrated into higher order architectures that employ multiple interaction interfaces to efficiently extract heme from host proteins.

The structure of haemoglobin bound to the haemoglobin receptor IsdH from Staphylococcus aureus shows disruption of the native α-globin haem pocket.

Staphylococcus aureus is a common and serious cause of infection in humans. The bacterium expresses a cell-surface receptor that binds to, and strips haem from, human haemoglobin (Hb). The binding interface has previously been identified; however, the structural changes that promote haem release from haemoglobin were unknown. Here, the structure of the receptor-Hb complex is reported at 2.6 Å resolution, which reveals a conformational change in the α-globin F helix that disrupts the haem-pocket structure and alters the Hb quaternary interactions. These features suggest potential mechanisms by which the S. aureus Hb receptor induces haem release from Hb.

IsdB-dependent hemoglobin binding is required for acquisition of heme by Staphylococcus aureus.

Staphylococcus aureus is a Gram-positive pathogen responsible for tremendous morbidity and mortality. As with most bacteria, S. aureus requires iron to cause disease, and it can acquire iron from host hemoglobin. The current model for staphylococcal hemoglobin-iron acquisition proposes that S. aureus binds hemoglobin through the surface-exposed hemoglobin receptor IsdB. IsdB removes heme from bound hemoglobin and transfers this cofactor to other proteins of the Isd system, which import and degrade heme to release iron in the cytoplasm. Here we demonstrate that the individual components of the Isd system are required for growth on low nanomolar concentrations of hemoglobin as a sole source of iron. An in-depth study of hemoglobin binding by IsdB revealed key residues that are required for hemoglobin binding. Further, we show that these residues are necessary for heme extraction from hemoglobin and growth on hemoglobin as a sole iron source. These processes are found to contribute to the pathogenicity of S. aureus in a murine model of infection. Together these results build on the model for Isd-mediated hemoglobin binding and heme-iron acquisition during the pathogenesis of S. aureus infection.

Staphylococcus aureus uses a novel multidomain receptor to break apart human hemoglobin and steal its heme.

Staphylococcus aureus is a leading cause of life-threatening infections in the United States. It requires iron to grow, which must be actively procured from its host to successfully mount an infection. Heme-iron within hemoglobin (Hb) is the most abundant source of iron in the human body and is captured by S. aureus using two closely related receptors, IsdH and IsdB. Here we demonstrate that each receptor captures heme using two conserved near iron transporter (NEAT) domains that function synergistically. NMR studies of the 39-kDa conserved unit from IsdH (IsdH(N2N3), Ala(326)-Asp(660)) reveals that it adopts an elongated dumbbell-shaped structure in which its NEAT domains are properly positioned by a helical linker domain, whose three-dimensional structure is determined here in detail. Electrospray ionization mass spectrometry and heme transfer measurements indicate that IsdH(N2N3) extracts heme from Hb via an ordered process in which the receptor promotes heme release by inducing steric strain that dissociates the Hb tetramer. Other clinically significant Gram-positive pathogens capture Hb using receptors that contain multiple NEAT domains, suggesting that they use a conserved mechanism.

α-Hemoglobin-stabilizing protein (AHSP) perturbs the proximal heme pocket of oxy-α-hemoglobin and weakens the iron-oxygen bond.

α-Hemoglobin (αHb)-stabilizing protein (AHSP) is a molecular chaperone that assists hemoglobin assembly. AHSP induces changes in αHb heme coordination, but how these changes are facilitated by interactions at the αHb·AHSP interface is not well understood. To address this question we have used NMR, x-ray absorption spectroscopy, and ligand binding measurements to probe αHb conformational changes induced by AHSP binding. NMR chemical shift analyses of free CO-αHb and CO-αHb·AHSP indicated that the seven helical elements of the native αHb structure are retained and that the heme Fe(II) remains coordinated to the proximal His-87 side chain. However, chemical shift differences revealed alterations of the F, G, and H helices and the heme pocket of CO-αHb bound to AHSP. Comparisons of iron-ligand geometry using extended x-ray absorption fine structure spectroscopy showed that AHSP binding induces a small 0.03 Å lengthening of the Fe-O2 bond, explaining previous reports that AHSP decreases αHb O2 affinity roughly 4-fold and promotes autooxidation due primarily to a 3-4-fold increase in the rate of O2 dissociation. Pro-30 mutations diminished NMR chemical shift changes in the proximal heme pocket, restored normal O2 dissociation rate and equilibrium constants, and reduced O2-αHb autooxidation rates. Thus, the contacts mediated by Pro-30 in wild-type AHSP promote αHb autooxidation by introducing strain into the proximal heme pocket. As a chaperone, AHSP facilitates rapid assembly of αHb into Hb when ßHb is abundant but diverts αHb to a redox resistant holding state when ßHb is limiting.

Enhancing 19F Benchtop NMR Spectroscopy by Combining para-Hydrogen Hyperpolarization and Multiplet Refocusing.

Benchtop NMR spectrometers provide a promising alternative to high-field NMR for applications that are limited by instrument size and/or cost. 19F benchtop NMR is attractive due to the larger chemical shift range of 19F relative to 1H and the lack of background signal in most applications. However, practical applications of benchtop 19F NMR are limited by its low sensitivity due to the relatively weak field strengths of benchtop NMR spectrometers. Here we present a sensitivity-enhancement strategy that combines SABRE (Signal Amplification By Reversible Exchange) hyperpolarization with the multiplet refocusing method SHARPER (Sensitive, Homogeneous, And Resolved PEaks in Real time). When applied to a range of fluoropyridines, SABRE-SHARPER achieves overall signal enhancements of up to 5700-fold through the combined effects of hyperpolarization and line-narrowing. This approach can be generalized to the analysis of mixtures through the use of a selective variant of the SHARPER sequence, selSHARPER. The ability of SABRE-selSHARPER to simultaneously boost sensitivity and discriminate between two components of a mixture is demonstrated, where selectivity is achieved through a combination of selective excitation and the choice of polarization transfer field during the SABRE step.

SHARPER-DOSY: Sensitivity enhanced diffusion-ordered NMR spectroscopy.

Since its discovery in mid-20th century, the sensitivity of Nuclear Magnetic Resonance (NMR) has increased steadily, in part due to the design of new, sophisticated NMR experiments. Here we report on a liquid-state NMR methodology that significantly increases the sensitivity of diffusion coefficient measurements of pure compounds, allowing to estimate their sizes using a much reduced amount of material. In this method, the diffusion coefficients are being measured by analysing narrow and intense singlets, which are invariant to magnetic field inhomogeneities. The singlets are obtained through signal acquisition embedded in short (<0.5 ms) spin-echo intervals separated by non-selective 180° or 90° pulses, suppressing the chemical shift evolution of resonances and their splitting due to J couplings. The achieved 10-100 sensitivity enhancement results in a 100-10000-fold time saving. Using high field cryoprobe NMR spectrometers, this makes it possible to measure a diffusion coefficient of a medium-size organic molecule in a matter of minutes with as little as a few hundred nanograms of material.

Trim-Away ubiquitinates and degrades lysine-less and N-terminally acetylated substrates.

TRIM proteins are the largest family of E3 ligases in mammals. They include the intracellular antibody receptor TRIM21, which is responsible for mediating targeted protein degradation during Trim-Away. Despite their importance, the ubiquitination mechanism of TRIM ligases has remained elusive. Here we show that while Trim-Away activation results in ubiquitination of both ligase and substrate, ligase ubiquitination is not required for substrate degradation. N-terminal TRIM21 RING ubiquitination by the E2 Ube2W can be inhibited by N-terminal acetylation, but this doesn't prevent substrate ubiquitination nor degradation. Instead, uncoupling ligase and substrate degradation prevents ligase recycling and extends functional persistence in cells. Further, Trim-Away degrades substrates irrespective of whether they contain lysines or are N-terminally acetylated, which may explain the ability of TRIM21 to counteract fast-evolving pathogens and degrade diverse substrates.

Structural basis for hemoglobin capture by Staphylococcus aureus cell-surface protein, IsdH.

Pathogens must steal iron from their hosts to establish infection. In mammals, hemoglobin (Hb) represents the largest reservoir of iron, and pathogens express Hb-binding proteins to access this source. Here, we show how one of the commonest and most significant human pathogens, Staphylococcus aureus, captures Hb as the first step of an iron-scavenging pathway. The x-ray crystal structure of Hb bound to a domain from the Isd (iron-regulated surface determinant) protein, IsdH, is the first structure of a Hb capture complex to be determined. Surface mutations in Hb that reduce binding to the Hb-receptor limit the capacity of S. aureus to utilize Hb as an iron source, suggesting that Hb sequence is a factor in host susceptibility to infection. The demonstration that pathogens make highly specific recognition complexes with Hb raises the possibility of developing inhibitors of Hb binding as antibacterial agents.

SHARPER-enhanced benchtop NMR: improving SNR by removing couplings and approaching natural linewidths.

We present a signal enhancement strategy for benchtop NMR that produces SNR increases on the order of 10 to 30 fold by collapsing the target resonance into an extremely narrow singlet. Importantly, the resultant signal is amenable to quantitative interpretation and therefore can be applied to analytical applications such as reaction monitoring.

Evasion of cGAS and TRIM5 defines pandemic HIV.

Of the 13 known independent zoonoses of simian immunodeficiency viruses to humans, only one, leading to human immunodeficiency virus (HIV) type 1(M) has become pandemic, causing over 80 million human infections. To understand the specific features associated with pandemic human-to-human HIV spread, we compared replication of HIV-1(M) with non-pandemic HIV-(O) and HIV-2 strains in myeloid cell models. We found that non-pandemic HIV lineages replicate less well than HIV-1(M) owing to activation of cGAS and TRIM5-mediated antiviral responses. We applied phylogenetic and X-ray crystallography structural analyses to identify differences between pandemic and non-pandemic HIV capsids. We found that genetic reversal of two specific amino acid adaptations in HIV-1(M) enables activation of TRIM5, cGAS and innate immune responses. We propose a model in which the parental lineage of pandemic HIV-1(M) evolved a capsid that prevents cGAS and TRIM5 triggering, thereby allowing silent replication in myeloid cells. We hypothesize that this capsid adaptation promotes human-to-human spread through avoidance of innate immune response activation.