Energy coupling and stoichiometry of Zn2+/H+ antiport by the prokaryotic cation diffusion facilitator YiiP.

YiiP from Shewanella oneidensis is a prokaryotic Zn2+/H+ antiporter that serves as a model for the Cation Diffusion Facilitator (CDF) superfamily, members of which are generally responsible for homeostasis of transition metal ions. Previous studies of YiiP as well as related CDF transporters have established a homodimeric architecture and the presence of three distinct Zn2+ binding sites named A, B, and C. In this study, we use cryo-EM, microscale thermophoresis and molecular dynamics simulations to address the structural and functional roles of individual sites as well as the interplay between Zn2+ binding and protonation. Structural studies indicate that site C in the cytoplasmic domain is primarily responsible for stabilizing the dimer and that site B at the cytoplasmic membrane surface controls the structural transition from an inward facing conformation to an occluded conformation. Binding data show that intramembrane site A, which is directly responsible for transport, has a dramatic pH dependence consistent with coupling to the proton motive force. A comprehensive thermodynamic model encompassing Zn2+ binding and protonation states of individual residues indicates a transport stoichiometry of 1 Zn2+ to 2-3 H+ depending on the external pH. This stoichiometry would be favorable in a physiological context, allowing the cell to use the proton gradient as well as the membrane potential to drive the export of Zn2+.

Thermodynamically consistent determination of free energies and rates in kinetic cycle models.

Kinetic and thermodynamic models of biological systems are commonly used to connect microscopic features to system function in a bottom-up multiscale approach. The parameters of such models-free energy differences for equilibrium properties and in general rates for equilibrium and out-of-equilibrium observables-have to be measured by different experiments or calculated from multiple computer simulations. All such parameters necessarily come with uncertainties so that when they are naively combined in a full model of the process of interest, they will generally violate fundamental statistical mechanical equalities, namely detailed balance and an equality of forward/backward rate products in cycles due to Hill. If left uncorrected, such models can produce arbitrary outputs that are physically inconsistent. Here, we develop a maximum likelihood approach (named multibind) based on the so-called potential graph to combine kinetic or thermodynamic measurements to yield state-resolved models that are thermodynamically consistent while being most consistent with the provided data and their uncertainties. We demonstrate the approach with two theoretical models, a generic two-proton binding site and a simplified model of a sodium/proton antiporter. We also describe an algorithm to use the multibind approach to solve the inverse problem of determining microscopic quantities from macroscopic measurements and, as an example, we predict the microscopic pKa values and protonation states of a small organic molecule from 1D NMR data. The multibind approach is applicable to any thermodynamic or kinetic model that describes a system as transitions between well-defined states with associated free energy differences or rates between these states. A Python package multibind, which implements the approach described here, is made publicly available under the MIT Open Source license.

Thermodynamically consistent determination of free energies and rates in kinetic cycle models.

Kinetic and thermodynamic models of biological systems are commonly used to connect microscopic features to system function in a bottom-up multiscale approach. The parameters of such models-free energy differences for equilibrium properties and in general rates for equilibrium and out-of-equilibrium observables-have to be measured by different experiments or calculated from multiple computer simulations. All such parameters necessarily come with uncertainties so that when they are naively combined in a full model of the process of interest, they will generally violate fundamental statistical mechanical equalities, namely detailed balance and an equality of forward/backward rate products in cycles due to T. Hill. If left uncorrected, such models can produce arbitrary outputs that are physically inconsistent. Here we develop a maximum likelihood approach (named multibind ) based on the so-called potential graph to combine kinetic or thermodynamic measurements to yield state resolved models that are thermodynamically consistent while being most consistent with the provided data and their uncertainties. We demonstrate the approach with two theoretical models, a generic two-proton binding site and a simplified model of a sodium/proton antiporter. We also describe an algorithm to use the multibind approach to solve the inverse problem of determining microscopic quantities from macroscopic measurements and as an example we predict the microscopic p K a s and protonation states of a small organic molecule from 1D NMR data. The multibind approach is applicable to any thermodynamic or kinetic model that describes a system as transitions between well-defined states with associated free energy differences or rates between these states. A Python package multibind , which implements the approach described here, is made publicly available under the MIT Open Source license. WHY IT MATTERS: The increase in computational efficiency and rapid advances in methodology for quantitative free energy and rate calculations has allowed for the construction of increasingly complex thermodynamic or kinetic "bottom-up" models of chemical and biological processes. These multi-scale models serve as a framework for analyzing aspects of cellular function in terms of microscopic, molecular properties and provide an opportunity to connect molecular mechanisms to cellular function. The underlying model parameters-free energy differences or rates-are constrained by thermodynamic identities over cycles of states but these identities are not necessarily obeyed during model construction, thus potentially leading to inconsistent models. We address these inconsistencies through the use of a maximum likelihood approach for free energies and rates to adjust the model parameters in such a way that they are maximally consistent with the input parameters and exactly fulfill the thermodynamic cycle constraints. This approach enables formulation of thermodynamically consistent multi-scale models from simulated or experimental measurements.

Energy Coupling and Stoichiometry of Zn2+/H+ Antiport by the Cation Diffusion Facilitator YiiP.

YiiP is a prokaryotic Zn2+/H+ antiporter that serves as a model for the Cation Diffusion Facilitator (CDF) superfamily, members of which are generally responsible for homeostasis of transition metal ions. Previous studies of YiiP as well as related CDF transporters have established a homodimeric architecture and the presence of three distinct Zn2+ binding sites named A, B, and C. In this study, we use cryo-EM, microscale thermophoresis and molecular dynamics simulations to address the structural and functional roles of individual sites as well as the interplay between Zn2+ binding and protonation. Structural studies indicate that site C in the cytoplasmic domain is primarily responsible for stabilizing the dimer and that site B at the cytoplasmic membrane surface controls the structural transition from an inward facing conformation to an occluded conformation. Binding data show that intramembrane site A, which is directly responsible for transport, has a dramatic pH dependence consistent with coupling to the proton motive force. A comprehensive thermodynamic model encompassing Zn2+ binding and protonation states of individual residues indicates a transport stoichiometry of 1 Zn2+ to 2-3 H+ depending on the external pH. This stoichiometry would be favorable in a physiological context, allowing the cell to use the proton gradient as well as the membrane potential to drive the export of Zn2+.

Crystal structure of the Na+/H+ antiporter NhaA at active pH reveals the mechanistic basis for pH sensing.

The strict exchange of protons for sodium ions across cell membranes by Na+/H+ exchangers is a fundamental mechanism for cell homeostasis. At active pH, Na+/H+ exchange can be modelled as competition between H+ and Na+ to an ion-binding site, harbouring either one or two aspartic-acid residues. Nevertheless, extensive analysis on the model Na+/H+ antiporter NhaA from Escherichia coli, has shown that residues on the cytoplasmic surface, termed the pH sensor, shifts the pH at which NhaA becomes active. It was unclear how to incorporate the pH senor model into an alternating-access mechanism based on the NhaA structure at inactive pH 4. Here, we report the crystal structure of NhaA at active pH 6.5, and to an improved resolution of 2.2 Å. We show that at pH 6.5, residues in the pH sensor rearrange to form new salt-bridge interactions involving key histidine residues that widen the inward-facing cavity. What we now refer to as a pH gate, triggers a conformational change that enables water and Na+ to access the ion-binding site, as supported by molecular dynamics (MD) simulations. Our work highlights a unique, channel-like switch prior to substrate translocation in a secondary-active transporter.

SAMPL6: calculation of macroscopic pKa values from ab initio quantum mechanical free energies.

Macroscopic pKa values were calculated for all compounds in the SAMPL6 blind prediction challenge, based on quantum chemical calculations with a continuum solvation model and a linear correction derived from a small training set. Microscopic pKa values were derived from the gas-phase free energy difference between protonated and deprotonated forms together with the Conductor-like Polarizable Continuum Solvation Model and the experimental solvation free energy of the proton. pH-dependent microstate free energies were obtained from the microscopic pKas with a maximum likelihood estimator and appropriately summed to yield macroscopic pKa values or microstate populations as function of pH. We assessed the accuracy of three approaches to calculate the microscopic pKas: direct use of the quantum mechanical free energy differences and correction of the direct values for short-comings in the QM solvation model with two different linear models that we independently derived from a small training set of 38 compounds with known pKa. The predictions that were corrected with the linear models had much better accuracy [root-mean-square error (RMSE) 2.04 and 1.95 pKa units] than the direct calculation (RMSE 3.74). Statistical measures indicate that some systematic errors remain, likely due to differences in the SAMPL6 data set and the small training set with respect to their interactions with water. Overall, the current approach provides a viable physics-based route to estimate macroscopic pKa values for novel compounds with reasonable accuracy.

Prediction of cyclohexane-water distribution coefficients for the SAMPL5 data set using molecular dynamics simulations with the OPLS-AA force field.

All-atom molecular dynamics simulations were used to predict water-cyclohexane distribution coefficients [Formula: see text] of a range of small molecules as part of the SAMPL5 blind prediction challenge. Molecules were parameterized with the transferable all-atom OPLS-AA force field, which required the derivation of new parameters for sulfamides and heterocycles and validation of cyclohexane parameters as a solvent. The distribution coefficient was calculated from the solvation free energies of the compound in water and cyclohexane. Absolute solvation free energies were computed by an established protocol using windowed alchemical free energy perturbation with thermodynamic integration. This protocol resulted in an overall root mean square error in [Formula: see text] of almost 4 log units and an overall signed error of -3 compared to experimental data. There was no substantial overall difference in accuracy between simulating in NVT and NPT ensembles. The signed error suggests a systematic error but the experimental [Formula: see text] data on their own are insufficient to uncover the source of this error. Preliminary work suggests that the major source of error lies in the hydration free energy calculations.

Paediatric urinary tract infections: a retrospective application of the National Institute of Clinical Excellence guidelines to a large general practitioner referred historical cohort.

BACKGROUND: The National Institute for Clinical Excellence (NICE) is a United Kingdom nondepartmental public body accountable to the Department of Health. Before the introduction of the NICE guidelines in the United Kingdom most children younger than 1 year of age had a urinary tract ultrasound, cyclic micturating cystourethrogram and dimercaptosuccinic acid scintigraphy, the latter delayed 6 months post infection. Children older than 1 year had a urinary tract ultrasound only, and further imaging if necessary. OBJECTIVE: Identify who would have been investigated had the NICE imaging strategy been used and who would not. Compare the diagnostic yield and patient outcome with the previous imaging protocol using our prospectively collected historical data. MATERIALS AND METHODS: We applied the new imaging strategy to a historic cohort of 934 patients with a urinary tract infection (UTI) referred by general practitioners to a specialist children's hospital between 1996 and 2002. RESULTS: Of the 934 patients referred, 218 would have been investigated according to the NICE guidelines. In total, there were 105 patients with abnormal imaging findings, and 44 of these (42%) would have been investigated under the NICE guidelines. CONCLUSION: Applying the NICE guidelines to children presenting with UTI will reduce the number imaged by 77% and will lead to missed identification of 58% of imaging abnormalities in the group. The majority of these abnormalities may be important. While supporting conservative investigation protocols, we are concerned that many abnormalities might go undetected.

Dysbaric osteonecrosis in recreational divers: a study using magnetic resonance imaging.

OBJECTIVE: We set out to identify whether magnetic resonance imaging (MRI) would identify evidence of dysbaric osteonecrosis (DON) in a group of experienced recreational scuba divers. DESIGN: Local British Sub Aqua Club divers of at least Trainee Dive Leader grade were offered MRI scans (T1 and TIRM sequences) of hips, femora and shoulders. Anonymous images were interpreted separately by two radiologists, and cases not considered unequivocally normal were discussed for consensus opinion. RESULTS: Of 26 divers imaged, five merited discussion. Four of these were considered to show unrelated normal variants or incidental findings. Only one case (abnormalities in the right humerus and left femur) could have possibly represented osteonecrotic lesions. After obtaining plain radiographs and more detailed clinical and dive history, these lesions were considered "indeterminate" but probably not DON by both reviewers and after taking further specialist musculoskeletal MRI opinion. CONCLUSION: This study found no evidence that DON is a significant risk in recreational scuba diving and as such concurs with prevailing current opinion.

A rare case of 'blow-up' fracture of the orbit in a child.

We present a case of blow-out fracture of the superomedial orbital wall in a 6-year-old boy. The initial plain radiograph showed an intact orbital margin and opacification of the ethmoid sinus. A fine-cut CT scan of the facial bones revealed a complex fracture of the medial orbital wall extending into the orbital roof, with migration of fracture fragments into the anterior cranial fossa. Suspicion for unusual orbital fractures is crucial when assessing a child for orbital trauma, especially when plain radiographs do not display the typical signs.

The Grellier twins, Norman (1886-1949) and Bernard (1886-1957), radiologists of East Sussex.

Identical twins Bernard and Norman Grellier (born Epsom, 1886) attended Epsom College before entering Dental School at the Royal Dental Hospital of London in 1904, graduating in 1910. Then they trained in medicine at Charing Cross Hospital. Bernard graduated in 1913 and Norman in 1915. In 1915 they joined the Royal Army Medical Corps (RAMC), serving to the end of World War I (WWI), each being awarded the Military Cross for gallantry. After WWI, they trained as radiologists and moved to St Leonards-on-Sea in West Sussex, taking up Consultant posts at the Royal East Sussex Hospital and the Municipal Hospital in Hastings, and the Eversfield Chest Hospital in St Leonards. In 1940 they rejoined the RAMC as radiologists, serving throughout World War II. They remained unmarried, devoted to each other, to their practice and to their loves of model engineering and flying, the latter nearly causing their deaths in an air crash in 1936.

Is sonographically demonstrated mild distal ureteric dilatation predictive of vesicoureteric reflux as seen on micturating cystourethrography?

BACKGROUND: In most paediatric units, the micturating cystourethrogram (MCU) is the gold standard in the diagnosis of vesicoureteric reflux (VUR). Because of the well-known difficulties associated with MCUs, there is interest in any imaging finding which may be predictive of the absence of VUR with enough confidence to avoid the necessity for an MCU. OBJECTIVE: To evaluate whether incorporation of measurement of the internal diameter of the retrovesical ureter into a routine urinary tract US protocol can provide a useful predictor of VUR. MATERIALS AND METHODS: The radiology information system at the Royal Alexandra Children's Hospital in Brighton was searched to identify children who had urinary tract US and an MCU within 3 months of each other. This identified 285 renal units in 144 patients. The presence and grade of VUR on the MCU was then compared with the presence or absence of mild-to-moderate distal ureteric dilation, using 3.5 mm as the upppr limit of normal for the retrovesical ureter on US. RESULTS: A distal ureteric diameter of more than 3.5 mm on US is predictive of VUR with a sensitivity of 0.63 and specificity of 0.78. Figures for dilating VUR (grades 3-5) were 0.78 and 0.77, respectively. The negative predictive value of a ureteric calibre less than 3.5 mm in excluding dilating reflux was 0.96. Interestingly, all three solitary renal units had ureteric diameters of more than 3.5 mm but no VUR. CONCLUSIONS: Absence of distal ureteric dilation on US, on its own, cannot reliably exclude VUR. It does, however, make dilating reflux unlikely. We believe US measurement of the distal ureteric diameter is a useful additional tool in everyday assessment of children who might have reflux.