ProtVar: mapping and contextualizing human missense variation.

Genomic variation can impact normal biological function in complex ways and so understanding variant effects requires a broad range of data to be coherently assimilated. Whilst the volume of human variant data and relevant annotations has increased, the corresponding increase in the breadth of participating fields, standards and versioning mean that moving between genomic, coding, protein and structure positions is increasingly complex. In turn this makes investigating variants in diverse formats and assimilating annotations from different resources challenging. ProtVar addresses these issues to facilitate the contextualization and interpretation of human missense variation with unparalleled flexibility and ease of accessibility for use by the broadest range of researchers. By precalculating all possible variants in the human proteome it offers near instantaneous mapping between all relevant data types. It also combines data and analyses from a plethora of resources to bring together genomic, protein sequence and function annotations as well as structural insights and predictions to better understand the likely effect of missense variation in humans. It is offered as an intuitive web server https://www.ebi.ac.uk/protvar where data can be explored and downloaded, and can be accessed programmatically via an API.

Nonclassical Crystallization Processes of Single-Crystalline Two-Dimensional Covalent Organic Frameworks.

Controlling the two-dimensional polymerization processes of two-dimensional covalent organic frameworks (2D COFs) is essential to fully realizing their distinct properties. Although most 2D COFs have been isolated as polycrystalline aggregates with only nanometer-scale crystalline domains, we have identified rapid, solvothermal conditions that provide micrometer-scale and larger single-crystal 2D polymers for a few 2D COFs. Yet it remains unclear why certain conditions produce far larger 2D polymers than others, which hinders generalizing these findings. The guiding principles for controlled two-dimensional polymerization in solution remain unclear. Here, we study the crystallization processes of both single-crystalline and polycrystalline 2D COFs using ultrasmall-angle X-ray scattering (USAXS) for the first time, through which we characterized COF formation conditions with scattering data collected every few seconds. In situ USAXS experiments revealed distinct growth mechanisms between single-crystalline and polycrystalline COFs and suggested a nonclassical particle fusion-based growth model for single-crystalline COFs that results in faceted, hexagonal particles. These findings were corroborated by in situ wide-angle X-ray scattering (WAXS) experiments and scanning electron microscopy (SEM). In contrast, polymerizations that provide polycrystalline COFs evolve as spherical aggregates that do not fuse in the same way. These insights into how micrometer-sized, crystalline 2D polymers are formed in solution point a way forward to establishing a robust connection between the 2D polymer structure and designed properties by controlling their polymerization processes.

Antigenic mapping of the hemagglutinin of the H9 subtype influenza A viruses using sera from Japanese quail (Coturnix c. japonica).

IMPORTANCE: Determining the relevant amino acids involved in antigenic drift on the surface protein hemagglutinin (HA) is critical to understand influenza virus evolution and efficient assessment of vaccine strains relative to current circulating strains. We used antigenic cartography to generate an antigenic map of the H9 hemagglutinin (HA) using sera produced in one of the most relevant minor poultry species, Japanese quail. Key antigenic positions were identified and tested to confirm their impact on the antigenic profile. This work provides a better understanding of the antigenic diversity of the H9 HA as it relates to reactivity to quail sera and will facilitate a rational approach for selecting more efficacious vaccines against poultry-origin H9 influenza viruses in minor poultry species.

Effects of the nematode Litylenchus crenatae subsp. mccannii and beech leaf disease on leaf fungal and bacterial communities on Fagus grandifolia (American beech).

Beech leaf disease (BLD) is a newly emerging disease in North America that affects American beech (Fagus grandifolia). It is increasingly recognized that BLD is caused by a subspecies of the anguinid nematode Litylenchus crenatae subsp. mccannii (hereafter L. crenatae), which is likely native to East Asia. How nematode infestation of leaves affects the leaf microbiome and whether changes in the microbiome could contribute to BLD symptoms remain uncertain. In this study, we examined bacterial and fungal communities associated with the leaves of F. grandifolia across nine sites in Ohio and Pennsylvania that were either symptomatic or asymptomatic for BLD and used qPCR to measure relative nematode infestation levels. We found significantly higher levels of infestation at sites visibly symptomatic for BLD. Low levels of nematode infestation were also observed at asymptomatic sites, which suggests that nematodes can be present without visible symptoms evident. Bacterial and fungal communities were significantly affected by sampling site and symptomology, but only fungal communities were affected by nematode presence alone. We found many significant indicators of both bacteria and fungi related to symptoms of BLD, with taxa generally occurring in both asymptomatic and symptomatic leaves, suggesting that microbes are not responsible for BLD but could act as opportunistic pathogens. Of particular interest was the fungal genus Erysiphe, which is common in the Fagaceae and is reported to overwinter in buds-a strategy consistent with L. crenatae. The specific role microbes play in opportunistic infection of leaves affected by L. crenatae will require additional study. IMPORTANCE: Beech leaf disease (BLD) is an emerging threat to American beech (Fagus grandifolia) and has spread quickly throughout the northeastern United States and into southern Canada. This disease leads to disfigurement of leaves and is marked by characteristic dark, interveinal banding, followed by leaf curling and drop in more advanced stages. BLD tends to especially affect understory leaves, which can lead to substantial thinning of the forest understory where F. grandifolia is a dominant tree species. Understanding the cause of BLD is necessary to employ management strategies that protect F. grandifolia and the forests where it is a foundation tree species. Current research has confirmed that the foliar nematode Litylenchus crenatae subsp. mccannii is required for BLD, but whether other organisms are involved is currently unknown. Here, we present a study that investigated leaf-associated fungi and bacteria of F. grandifolia to understand more about how microorganisms may contribute to BLD.

Tuning Crystallinity and Stacking of Two-Dimensional Covalent Organic Frameworks through Side-Chain Interactions.

Two-dimensional covalent organic frameworks (2D COFs) form as layered 2D polymers whose sheets stack through high-surface-area, noncovalent interactions that can give rise to different interlayer arrangements. Manipulating the stacking of 2D COFs is crucial since it dictates the effective size and shape of the pores as well as the specific interactions between functional aromatic systems in adjacent layers, both of which will strongly influence the emergent properties of 2D COFs. However, principles for tuning layer stacking are not yet well understood, and many 2D COFs are disordered in the stacking direction. Here, we investigate effects of pendant chain length through a series of 2D imine-linked COFs functionalized with n-alkyloxy chains varying in length from one carbon (C1 COF) to 11 carbons (C11 COF). This series reveals previously unrecognized and unanticipated trends in both the stacking geometry and crystallinity. C1 COF adopts an averaged eclipsed geometry with no apparent offset between layers. In contrast, all subsequent chain lengths lead to some degree of unidirectional slip stacking. As pendant chain length is increased, trends show average layer offset increasing to a maximum of 2.07 Å in C5 COF and then decreasing as chain length is extended through C11 COF. Counterintuitively, shorter chains (C2-C4) give rise to lower yields of weakly crystalline materials, while longer chains (C6-C9) produce greater yields of highly crystalline materials, as confirmed by powder X-ray diffraction and scanning electron microscopy. Molecular dynamics simulations corroborate these observations, suggesting that long alkyl chains can interact favorably to promote the self-assembly of sheets. In situ proton NMR spectroscopy provides insights into the reaction equilibrium as well as the relationship between low COF yields and low crystallinity. These results provide fundamental insights into principles of supramolecular assembly in 2D COFs, demonstrating an opportunity for harnessing favorable side-chain interactions to produce highly crystalline materials.

Synthesis, Hole Doping, and Electrical Properties of a Semiconducting Azatriangulene-Based Covalent Organic Framework.

Two-dimensional covalent organic frameworks (2D COFs) containing heterotriangulenes have been theoretically identified as semiconductors with tunable, Dirac-cone-like band structures, which are expected to afford high charge-carrier mobilities ideal for next-generation flexible electronics. However, few bulk syntheses of these materials have been reported, and existing synthetic methods provide limited control of network purity and morphology. Here, we report transimination reactions between benzophenone-imine-protected azatriangulenes (OTPA) and benzodithiophene dialdehydes (BDT), which afforded a new semiconducting COF network, OTPA-BDT. The COFs were prepared as both polycrystalline powders and thin films with controlled crystallite orientation. The azatriangulene nodes are readily oxidized to stable radical cations upon exposure to an appropriate p-type dopant, tris(4-bromophenyl)ammoniumyl hexachloroantimonate, after which the network's crystallinity and orientation are maintained. Oriented, hole-doped OTPA-BDT COF films exhibit electrical conductivities of up to 1.2 × 10-1 S cm-1, which are among the highest reported for imine-linked 2D COFs to date.

Development of Primers Specific for Detection of Litylenchus crenatae, the Causal Agent of Beech Leaf Disease, in Plant Tissue.

Beech leaf disease (BLD), an emerging threat to American beech (Fagus grandifolia) in the northern United States and Canada, was recently confirmed to be caused by the nematode Litylenchus crenatae subsp. mccannii (hereafter L. crenatae). Consequently, there is a need for a rapid, sensitive, and accurate method for detecting L. crenatae for both diagnostic as well as control purposes. This research developed a new set of DNA primers that specifically amplify L. crenatae and allow for accurate detection of the nematode in plant tissue. These primers have also been used in quantitative PCR (qPCR) to determine relative differences in gene copy number between samples. This primer set provides an improved, effective tool for monitoring and detecting L. crenatae in temperate tree leaf tissue which is necessary to understand the spread of this emerging forest pest and to develop management strategies.

Single-Crystalline Imine-Linked Two-Dimensional Covalent Organic Frameworks Separate Benzene and Cyclohexane Efficiently.

Two-dimensional (2D) covalent organic frameworks (COFs) are composed of structurally precise, permanently porous, layered macromolecular sheets, which are traditionally synthesized as polycrystalline solids with crystalline domain lengths smaller than 100 nm. Here, we polymerize imine-linked 2D COFs as suspensions of faceted single crystals in as little as 5 min at moderate temperature and ambient pressure. Single crystals of two imine-linked 2D COFs were prepared, consisting of a rhombic 2D COF (TAPPy-PDA) and a hexagonal 2D COF (TAPB-DMPDA). The sizes of TAPPy-PDA and TAPB-DMPDA crystals were tuned from 720 nm to 4 µm and 450 nm to 20 µm in width, respectively. High-resolution transmission electron microscopy revealed that the COF crystals consist of layered, 2D polymers comprising single-crystalline domains. Continuous rotation electron diffraction resolved the unit cell and crystal structure of both COFs, which are single-crystalline in the a-b plane but disordered in the stacking c dimension. Single crystals of both COFs were incorporated into gas chromatography separation columns and exhibited unusual selective retention of cyclohexane over benzene, with single-crystalline TAPPy-PDA significantly outperforming single-crystalline TAPB-DMPDA. Polycrystalline TAPPy-PDA exhibited no separation, while polycrystalline TAPB-DMPDA exhibited poor separation and the opposite order of elution, retaining benzene more than cyclohexane, indicating the importance of improved material quality for COFs to exhibit properties that derive from their precise, crystalline structures. This work represents the first example of synthesizing imine-linked 2D COF single crystals at ambient pressure and short reaction times and demonstrates the promise of high-quality COFs for molecular separations.

Thermally conductive ultra-low-k dielectric layers based on two-dimensional covalent organic frameworks.

As the features of microprocessors are miniaturized, low-dielectric-constant (low-k) materials are necessary to limit electronic crosstalk, charge build-up, and signal propagation delay. However, all known low-k dielectrics exhibit low thermal conductivities, which complicate heat dissipation in high-power-density chips. Two-dimensional (2D) covalent organic frameworks (COFs) combine immense permanent porosities, which lead to low dielectric permittivities, and periodic layered structures, which grant relatively high thermal conductivities. However, conventional synthetic routes produce 2D COFs that are unsuitable for the evaluation of these properties and integration into devices. Here, we report the fabrication of high-quality COF thin films, which enable thermoreflectance and impedance spectroscopy measurements. These measurements reveal that 2D COFs have high thermal conductivities (1 W m-1 K-1) with ultra-low dielectric permittivities (k = 1.6). These results show that oriented, layered 2D polymers are promising next-generation dielectric layers and that these molecularly precise materials offer tunable combinations of useful properties.

Measuring impact of a quality improvement initiative on glaucoma clinic flow using an automated real-time locating system.

BACKGROUND: Lean methodology helps maximize value by reducing waste, first by defining what value and waste are in a system. In ophthalmology clinics, value is determined by the number of patients flowing through the clinic for a given time. We aimed to increase value using a lean-methodology guided policy change, then assessed its impact on clinic flow using an automated radiofrequency identification (RFID) based real-time locating system (RTLS). METHODS: A total of 6813 clinical visits occurred at a single academic institution's outpatient glaucoma clinic between January 5, 2018 to July 3, 2018. Over that period, 1589 patients comprising 1972 (29%) of visits were enrolled, with 1031 clinical visits occurring before and 941 visits after a policy change. The original policy was to refract all patients that improved with pinhole testing. The policy change was not to refract patients with a visual acuity ≥20/30 unless a specific request was made by the patient. Pre-post analysis of an automated time-motion study was conducted for the data collected 3 months before and 3 months after the policy change occurred on March 30, 2018. Changes to process and wait times were summarized using descriptive statistics and fitted to linear mixed regression models adjusting for appointment type, clinic volume, and daily clinic trends. RESULTS: One thousand nine hundred twenty-three visits with 1588 patients were included in the analysis. Mean [SD] age was 65.9 [14.7] years and 892 [56.2%] were women. After the policy change, technician process time decreased by 2.9 min (p < 0.0001) while daily clinical patient volume increased from 51.9 ± 16.8 patients to 58.4 ± 17.4 patients (p < 0.038). No significant difference was found in total wait time (p = 0.18) or total visit time (p = 0.83). CONCLUSIONS: Real-time locating systems are effective at capturing clinical flow data and assessing clinical practice change initiatives. The refraction policy change was associated with reduced technician process time and overall the clinic was able to care for 7 more patients per day without significantly increasing patient wait time.

Polycrystalline Covalent Organic Framework Films Act as Adsorbents, Not Membranes.

Covalent organic framework (COF) membranes are of great promise for energy-efficient separations. Thick, polycrystalline COF films have been reported to separate dyes, salts, bacteria, and nanoparticles on the basis of size-selective transport through ordered pores. Here, we show that these materials function as adsorbents, not as size-sieving membranes. Binding isotherms of several dyes typical of the COF membrane literature to three COF powder samples illustrate that COFs are high-capacity adsorbents with affinities that span a range of 3 orders of magnitude, trends which map onto previously reported separation behavior. Computational results suggest that observed differences in adsorption can be correlated to variable entropic gains driving the adsorption process. Polycrystalline COF pellets show volume-dependent and flow-rate dependent "rejection" of dyes, consistent with an adsorption-based removal mechanism. Previous reports of thick, polycrystalline COF membranes used low flow rates and small dye volumes to probe rejection capabilities, where membrane and adsorbent behavior is not distinguishable. A mixed dye separation experiment in flow shows affinity-dependent performance. These results necessitate a careful reexamination of the COF membrane literature, as separations based on differential transport through 2D COF pores remain an important yet unrealized frontier.

Electrophysiological investigation of motor axonal excitability in a mouse model of nerve constriction injury.

Peripheral nerve injuries caused by focal constriction are characterised by local nerve ischaemia, axonal degeneration, demyelination, and neuroinflammation. The aim of this study was to understand temporal changes in the excitability properties of injured motor axons in a mouse model of nerve constriction injury (NCI). The excitability of motor axons following unilateral sciatic NCI was studied in male C57BL/6J mice distal to the site of injury at the acute (6 hours-1 week) and chronic (up to 20 weeks) phases of injury, using threshold tracking. Multiple measures of nerve excitability, including strength-duration properties, threshold electrotonus, current-threshold relationship, and recovery cycle were examined using the automated nerve excitability protocol (TRONDNF). Acutely, injured motor axons developed a pattern of excitability characteristic of ischemic depolarisation. In most cases, the sciatic nerve became transiently inexcitable. When a liminal compound muscle action potential could again be recorded, it had an increase in threshold and latency, compared to both pre-injury baseline and sham-injured groups. These axons showed a greater threshold change in response to hyperpolarising threshold electrotonus and a significant upward shift in the recovery cycle. Mathematical modelling suggested that the changes seen in chronically injured axons involve shortened internodes, reduced myelination, and exposed juxtaparanodal fast K+ conductances. The findings of this study demonstrate long-term changes in motor excitability following NCI (involving alterations in axonal properties and ion channel activity) and are important for understanding the mechanisms of neurapraxic injuries and traumatic mononeuropathies.

Plasticity of Amino Acid Residue 145 Near the Receptor Binding Site of H3 Swine Influenza A Viruses and Its Impact on Receptor Binding and Antibody Recognition.

The hemagglutinin (HA), a glycoprotein on the surface of influenza A virus (IAV), initiates the virus life cycle by binding to terminal sialic acid (SA) residues on host cells. The HA gradually accumulates amino acid substitutions that allow IAV to escape immunity through a mechanism known as antigenic drift. We recently confirmed that a small set of amino acid residues are largely responsible for driving antigenic drift in swine-origin H3 IAV. All identified residues are located adjacent to the HA receptor binding site (RBS), suggesting that substitutions associated with antigenic drift may also influence receptor binding. Among those substitutions, residue 145 was shown to be a major determinant of antigenic evolution. To determine whether there are functional constraints to substitutions near the RBS and their impact on receptor binding and antigenic properties, we carried out site-directed mutagenesis experiments at the single-amino-acid level. We generated a panel of viruses carrying substitutions at residue 145 representing all 20 amino acids. Despite limited amino acid usage in nature, most substitutions at residue 145 were well tolerated without having a major impact on virus replication in vitro All substitution mutants retained receptor binding specificity, but the substitutions frequently led to decreased receptor binding. Glycan microarray analysis showed that substitutions at residue 145 modulate binding to a broad range of glycans. Furthermore, antigenic characterization identified specific substitutions at residue 145 that altered antibody recognition. This work provides a better understanding of the functional effects of amino acid substitutions near the RBS and the interplay between receptor binding and antigenic drift.IMPORTANCE The complex and continuous antigenic evolution of IAVs remains a major hurdle for vaccine selection and effective vaccination. On the hemagglutinin (HA) of the H3N2 IAVs, the amino acid substitution N 145 K causes significant antigenic changes. We show that amino acid 145 displays remarkable amino acid plasticity in vitro, tolerating multiple amino acid substitutions, many of which have not yet been observed in nature. Mutant viruses carrying substitutions at residue 145 showed no major impairment in virus replication in the presence of lower receptor binding avidity. However, their antigenic characterization confirmed the impact of the 145 K substitution in antibody immunodominance. We provide a better understanding of the functional effects of amino acid substitutions implicated in antigenic drift and its consequences for receptor binding and antigenicity. The mutation analyses presented in this report represent a significant data set to aid and test the ability of computational approaches to predict binding of glycans and in antigenic cartography analyses.

Methylphenidate improves executive functions in patients with traumatic brain injuries: a feasibility trial via the idiographic approach.

BACKGROUND: Road traffic accidents are known to be the main cause of traumatic brain injury (TBI). TBI is also a leading cause of death and disability. This study, by means of the idiographic approach (single-case experimental designs using multiple-baseline designs), has examined whether methylphenidate (MPH - trade name Ritalin) had a differential effect on cognitive measures among patients with TBI with the sequel of acute and chronic post-concussion syndromes. The effect on gender was also explored. METHODS: In comparison with healthy controls, patients with TBI (acute and chronic) and accompanying mild cognitive impairment (MCI) were screened for their integrity of executive functioning. Twenty-four patients exhibiting executive dysfunction (ED) were then instituted with the pharmacological intervention methylphenidate (MPH). The methylphenidate was administered using an uncontrolled, open label design. RESULTS: The administration of methylphenidate impacted ED in the TBI group but had no effect on mood. Attenuation of ED was more apparent in the chronic phases of TBI. The effect on gender was not statistically significant with regard to the observed changes. CONCLUSIONS: To our knowledge, this is the first feasibility trial from the Arabian Gulf to report the performance of a TBI population with mild cognitive impairment according to the IQCODE Arabic version. This investigation confirms anecdotal observations of methylphenidate having the potential to attenuate cognitive impairment; particularly those functions that are critically involved in the integrity of executive functioning. The present feasibility trial should be followed by nomothetic studies such as those that adhere to the protocol of the randomized controlled trial. This evidence-based research is the foundation for intervention and future resource allocation by policy- or public health decision-makers.

Designing and validating a low-cost real time locating system to continuously assess patient wait times.

OBJECTIVE: Outpatient clinics lack infrastructure to easily measure and understand patient wait times. Our objective was to design a low-cost, portable passive real time locating system within an outpatient clinic setting to measure patient wait times and patient-provider interactions. MATERIALS AND METHODS: Direct observation was used to determine workflow in an outpatient glaucoma clinic at the University of Michigan. We used off-the shelf, antenna-integrated ultra-high frequency (UHF) RFID readers (ThingMagic, Astra-Ex, Woburn, MA) and UHF re-useable passive RFID tags (Zebra Impinj Monza 4QT, Seattle, WA). We designed a custom RFID management application in the Java programming language that was equipped with 'live' device administration to collect time and location data from patients and providers. These hardware choices enabled low cost system installation. Hidden Markov Modeling (HMM) was used to smooth patient and provider location data. Location data were validated against direct observations and EHR evaluation. RESULTS: The HMM smoothed RFID system data accurately predicted patient location 80.6% of the time and provider location 79.1% of the time, compared to direct observation locations, an improvement over the raw RFID location data (65.0% and 77.9% accurate, respectively). Patient process time was on average 42.8 min (SD = 27.5) and wait time was 47.9 min (SD = 33.1). The installation and recurring capital costs of the system are approximately 10% of available commercially-supplied patient/provider tracking systems. DISCUSSION: Passive RFID time study systems can enable real-time localization of people in clinic, facilitating continuous capture of patient wait times and patient-provider interactions. The system must be tailored to the clinic to accurately reflect patient and provider movement. CONCLUSIONS: Capturing wait time data continuously and passively can empower continuous clinical quality improvement initiatives to enhance the patient experience.

Medication Adherence and Liquid Level Tracking System for Healthcare Provider Feedback.

A common problem for healthcare providers is accurately tracking patients' adherence to medication and providing real-time feedback on the management of their medication regimen. This is a particular problem for eye drop medications, as the current commercially available monitors focus on measuring adherence to pills, and not to eye drops. This work presents an intelligent bottle sleeve that slides onto a prescription eye drop medication bottle. The intelligent sleeve is capable of detecting eye drop use, measuring fluid level, and sending use information to a healthcare team to facilitate intervention. The electronics embedded into the sleeve measure fluid level, dropper orientation, the state of the dropper top (on/off), and rates of angular motion during an application. The sleeve was tested with ten patients (age ≥65) and successfully identified and timestamped 94% of use events. On-board processing enabled event detection and the measurement of fluid levels at a 0.4 mL resolution. These data were communicated to the healthcare team using Bluetooth and Wi-Fi in real-time, enabling rapid feedback to the subject. The healthcare team can therefore monitor a log of medication use behavior to make informed decisions on treatment or support for the patient.

Acid Exfoliation of Imine-linked Covalent Organic Frameworks Enables Solution Processing into Crystalline Thin Films.

Covalent organic frameworks (COFs) are highly modular porous crystalline polymers that are of interest for applications such as charge-storage devices, nanofiltration membranes, and optoelectronic devices. COFs are typically synthesized as microcrystalline powders, which limits their performance in these applications, and their limited solubility precludes large-scale processing into more useful morphologies and devices. We report a general, scalable method to exfoliate two-dimensional imine-linked COF powders by temporarily protonating their linkages. The resulting suspensions were cast into continuous crystalline COF films up to 10 cm in diameter, with thicknesses ranging from 50 nm to 20 µm depending on the suspension composition, concentration, and casting protocol. Furthermore, we demonstrate that the film fabrication process proceeds through a partial depolymerization/repolymerization mechanism, providing mechanically robust films that can be easily separated from their substrates.

Ih contributes to increased motoneuron excitability in restless legs syndrome.

KEY POINTS: Restless legs patients complain about sensory and motor symptoms leading to sleep disturbances. Symptoms include painful sensations, an urge to move and involuntary leg movements. The responsible mechanisms of restless legs syndrome are still not known, although current studies indicate an increased neuronal network excitability. Reflex studies indicate the involvement of spinal structures. Peripheral mechanisms have not been investigated so far. In the present study, we provide evidence of increased hyperpolarization-activated cyclic nucleotide-gated (HCN) channel-mediated inward rectification in motor axons. The excitability of sensory axons was not changed. We conclude that, in restless legs syndrome, an increased HCN current in motoneurons may play a pathophysiological role, such that these channels could represent a valuable target for pharmaceutical intervention. ABSTRACT: Restless legs syndrome is a sensorimotor network disorder. So far, the responsible pathophysiological mechanisms are poorly understood. In the present study, we provide evidence that the excitability of peripheral motoneurons contributes to the pathophysiology of restless legs syndrome. In vivo excitability studies on motor and sensory axons of the median nerve were performed on patients with idiopathic restless legs syndrome (iRLS) who were not currently on treatment. The iRLS patients had greater accommodation in motor but not sensory axons to long-lasting hyperpolarization compared to age-matched healthy subjects, indicating greater inward rectification in iRLS. The most reasonable explanation is that hyperpolarization-activated cyclic nucleotide-gated (HCN) channels open at less hyperpolarized membrane potentials, a view supported by mathematical modelling. The half-activation potential for HCN channels (Bq) was the single best parameter that accounted for the difference between normal controls and iRLS data. A 6 mV depolarization of Bq reduced the discrepancy between the normal control model and the iRLS data by 92.1%. Taken together, our results suggest an increase in the excitability of motor units in iRLS that could enhance the likelihood of leg movements. The abnormal axonal properties are consistent with other findings indicating that the peripheral system is part of the network involved in iRLS.

Epistatic interactions can moderate the antigenic effect of substitutions in haemagglutinin of influenza H3N2 virus.

We previously showed that single amino acid substitutions at seven positions in haemagglutinin determined major antigenic change of influenza H3N2 virus. Here, the impact of two such substitutions was tested in 11 representative H3 haemagglutinins to investigate context-dependence effects. The antigenic effect of substitutions introduced at haemagglutinin position 145 was fully independent of the amino acid context of the representative haemagglutinins. Antigenic change caused by substitutions introduced at haemagglutinin position 155 was variable and context-dependent. Our results suggest that epistatic interactions with contextual amino acids in the haemagglutinin can moderate the magnitude of antigenic change.