Exploring the relevance of NUP93 variants in steroid-resistant nephrotic syndrome using next generation sequencing and a fly kidney model.

BACKGROUND: Variants in genes encoding nuclear pore complex (NPC) proteins are a newly identified cause of paediatric steroid-resistant nephrotic syndrome (SRNS). Recent reports describing NUP93 variants suggest these could be a significant cause of paediatric onset SRNS. We report NUP93 cases in the UK and demonstrate in vivo functional effects of Nup93 depletion in a fly (Drosophila melanogaster) nephrocyte model. METHODS: Three hundred thirty-seven paediatric SRNS patients from the National cohort of patients with Nephrotic Syndrome (NephroS) were whole exome and/or whole genome sequenced. Patients were screened for over 70 genes known to be associated with Nephrotic Syndrome (NS). D. melanogaster Nup93 knockdown was achieved by RNA interference using nephrocyte-restricted drivers. RESULTS: Six novel homozygous and compound heterozygous NUP93 variants were detected in 3 sporadic and 2 familial paediatric onset SRNS characterised histologically by focal segmental glomerulosclerosis (FSGS) and progressing to kidney failure by 12 months from clinical diagnosis. Silencing of the two orthologs of human NUP93 expressed in D. melanogaster, Nup93-1, and Nup93-2 resulted in significant signal reduction of up to 82% in adult pericardial nephrocytes with concomitant disruption of NPC protein expression. Additionally, nephrocyte morphology was highly abnormal in Nup93-1 and Nup93-2 silenced flies surviving to adulthood. CONCLUSION: We expand the spectrum of NUP93 variants detected in paediatric onset SRNS and demonstrate its incidence within a national cohort. Silencing of either D. melanogaster Nup93 ortholog caused a severe nephrocyte phenotype, signaling an important role for the nucleoporin complex in podocyte biology. A higher resolution version of the Graphical abstract is available as Supplementary information.

Long-read nanopore sequencing resolves a TMEM231 gene conversion event causing Meckel-Gruber syndrome.

The diagnostic deployment of massively parallel short-read next-generation sequencing (NGS) has greatly improved genetic test availability, speed, and diagnostic yield, particularly for rare inherited disorders. Nonetheless, diagnostic approaches based on short-read sequencing have a poor ability to accurately detect gene conversion events. We report on the genetic analysis of a family in which 3 fetuses had clinical features consistent with the autosomal recessive disorder Meckel-Gruber syndrome (MKS). Targeted NGS of 29 known MKS-associated genes revealed a heterozygous TMEM231 splice donor variant c.929+1A>G. Comparative read-depth analysis, performed to identify a second pathogenic allele, revealed an apparent heterozygous deletion of TMEM231 exon 4. To verify this result we performed single-molecule long-read sequencing of a long-range polymerase chain reaction product spanning this locus. We identified four missense variants that were absent from the short-read dataset due to the preferential mapping of variant-containing reads to a downstream TMEM231 pseudogene. Consistent with the parental segregation analysis, we demonstrate that the single-molecule long reads could be used to show that the variants are arranged in trans. Our experience shows that robust validation of apparent dosage variants remains essential to avoid the pitfalls of short-read sequencing and that new third-generation long-read sequencing technologies can already aid routine clinical care.

Affective responses to coherence in high and low risk scenarios.

Presenting information in a coherent fashion has been shown to increase processing fluency, which in turn influences affective responses. The pattern of responses have been explained by two apparently competing accounts: hedonic marking (response to fluency is positive) and fluency amplification (response to fluency can be positive or negative, depending on stimuli valence). This paper proposes that these accounts are not competing explanations, but separate mechanisms, serving different purposes. Therefore, their individual contributions to overall affective responses should be observable. In three experiments, participants were presented with businesses scenarios, with riskiness (valence) and coherence (fluency) manipulated, and affective responses recorded. Results suggested that increasing the fluency of stimuli increases positive affect. If the stimulus is negative, then increasing fluency simultaneously increases negative affect. These affective responses appeared to cancel each other out (Experiment 1) when measured using self-report bipolar scales. However, separate measurement of positive and negative affect, either using unipolar scales (Experiment 2) or using facial electromyography (Experiment 3), provided evidence for co-occurring positive and negative affective responses, and therefore the co-existence of hedonic marking and fluency amplification mechanisms.

Clinical genetic testing using a custom-designed steroid-resistant nephrotic syndrome gene panel: analysis and recommendations.

BACKGROUND: There are many single-gene causes of steroid-resistant nephrotic syndrome (SRNS) and the list continues to grow rapidly. Prompt comprehensive diagnostic testing is key to realising the clinical benefits of a genetic diagnosis. This report describes a bespoke-designed, targeted next-generation sequencing (NGS) diagnostic gene panel assay to detect variants in 37 genes including the ability to identify copy number variants (CNVs). METHODS: This study reports results of 302 patients referred for SRNS diagnostic gene panel analysis. Phenotype and clinical impact data were collected using a standard proforma. Candidate variants detected by NGS were confirmed by Sanger sequencing/Multiplex Ligation-dependent Probe Amplification with subsequent family segregation analysis where possible. RESULTS: Clinical presentation was nephrotic syndrome in 267 patients and suspected Alport syndrome (AS) in 35. NGS panel testing determined a likely genetic cause of disease in 44/220 (20.0%) paediatric and 10/47 (21.3%) adult nephrotic cases, and 17/35 (48.6%) of haematuria/AS patients. Of 71 patients with genetic disease, 32 had novel pathogenic variants without a previous disease association including two with deletions of one or more exons of NPHS1 or NPHS2. CONCLUSION: Gene panel testing provides a genetic diagnosis in a significant number of patients presenting with SRNS or suspected AS. It should be undertaken at an early stage of the care pathway and include the ability to detect CNVs as an emerging mechanism for genes associated with this condition. Use of clinical genetic testing after diagnosis of SRNS has the potential to stratify patients and assist decision-making regarding management.

Therapy influences goal attainment following botulinum neurotoxin injection for focal spasticity in adults with neurological conditions.

OBJECTIVE: To determine whether therapy influenced goal attainment following botulinum toxin (BoNT-A) injection for focal spasticity in adults with neurological conditions. METHODS: A prospective observational cohort study conducted in a large metropolitan spasticity clinic on adults with focal spasticity of any origin. Participants were provided with a therapy programme, designed to maximise therapeutic outcome. The primary outcome measure was Goal Attainment Scaling. To measure adherence, participants completed a therapy-recording tool each day. Goal attainment, and the rate of adherence to the therapy programme, was evaluated after 10 weeks. RESULTS: Active indications for BoNT-A treatment made up the majority of the goals (80.30%). Goals were achieved in 43/76 cases (56.60%; 95% CI = 42.40 to 69.80%). Therapy adherence was associated with significantly greater goal attainment (OR = 1.02, p = 0.03, 95% CI = 1.00 to 1.04). Greater adherence to therapy increased the odds of goal achievement for active indications but not for passive indications, suggesting a possible statistical interaction between the indication for injection and adherence to therapy (p < 0.01). CONCLUSION: Therapy adherence was associated with greater goal attainment. Active indications for BoNT-A were more reliant on adherence to prescribed therapy programmes than passive indications, although further investigation is required.

MAGI2 Mutations Cause Congenital Nephrotic Syndrome.

Steroid-resistant nephrotic syndrome (SRNS), a heterogeneous disorder of the renal glomerular filtration barrier, results in impairment of glomerular permselectivity. Inheritance of genetic SRNS may be autosomal dominant or recessive, with a subset of autosomal recessive SRNS presenting as congenital nephrotic syndrome (CNS). Mutations in 53 genes are associated with human SRNS, but these mutations explain ≤30% of patients with hereditary cases and only 20% of patients with sporadic cases. The proteins encoded by these genes are expressed in podocytes, and malfunction of these proteins leads to a universal end point of podocyte injury, glomerular filtration barrier disruption, and SRNS. Here, we identified novel disease-causing mutations in membrane-associated guanylate kinase, WW, and PDZ domain-containing 2 (MAGI2) through whole-exome sequencing of a deeply phenotyped cohort of patients with congenital, childhood-onset SRNS. Although MAGI2 has been shown to interact with nephrin and regulate podocyte cytoskeleton and slit diaphragm dynamics, MAGI2 mutations have not been described in human SRNS. We detected two unique frameshift mutations and one duplication in three patients (two families); two siblings shared the same homozygous frameshift mutation, whereas one individual with sporadic SRNS exhibited compound heterozygosity. Two mutations were predicted to introduce premature stop codons, and one was predicted to result in read through of the normal translational termination codon. Immunohistochemistry in kidney sections from these patients revealed that mutations resulted in lack of or diminished podocyte MAGI2 expression. Our data support the finding that mutations in the MAGI2 gene are causal for congenital SRNS.

Alcohol use and substance use disorders in Gulf War, Afghanistan, and Iraq War veterans compared with nondeployed military personnel.

Although recent veterans have been found to be at increased risk of psychiatric disorders, limited research has focused on alcohol or substance use disorders. This systematic review and meta-analysis examined whether alcohol or substance use disorders were more common in Gulf War, Afghanistan, and Iraq War veterans compared with military comparison groups nondeployed to the corresponding conflict, including never deployed personnel. Literature was searched (1990-2014) in multiple electronic databases. Studies were assessed for eligibility and quality, including risk of bias. Eighteen studies (1997-2014) met inclusion criteria. Pooled analysis based on a random-effects model yielded a summary odds ratio of 1.33 (95% confidence interval (CI): 1.22, 1.46) for alcohol (7 studies) and 2.13 (95% CI: 0.96, 4.72) for substance use (3 studies) disorders among Gulf War veterans, as well as 1.36 (95% CI: 1.11, 1.66) for alcohol (7 studies) and 1.14 (95% CI: 1.04, 1.25) for substance use (4 studies) disorders among Iraq/Afghanistan veterans; meta-regressions found no statistically significant association between theater of war and alcohol use or substance use disorders. Our findings indicate that Gulf and Iraq/Afghanistan war veterans are at higher alcohol use disorder risk than nondeployed veterans, but further studies with increased power are needed to assess substance use disorder risk in Gulf War veteran populations.

Long-term structural changes after mTBI and their relation to post-concussion symptoms.

PRIMARY OBJECTIVE: To investigate sustained structural changes in the long-term (>1 year) after mild traumatic brain injury (mTBI) and their relationship to ongoing post-concussion syndrome (PCS). RESEARCH DESIGN: Morphological and structural connectivity magnetic resonance imaging (MRI) data were acquired from 16 participants with mTBI and nine participants without previous head injury. MAIN OUTCOMES AND RESULTS: Participants with mTBI had less prefrontal grey matter and lower fractional anisotropy (FA) in the anterior corona radiata and internal capsule. Furthermore, PCS severity was associated with less parietal lobe grey matter and lower FA in the corpus callosum. CONCLUSIONS: There is evidence for both white and grey matter damage in participants with mTBI over 1 year after injury. Furthermore, these structural changes are greater in those that report more PCS symptoms, suggesting a neurophysiological basis for these persistent symptoms.

Comparing methods for determining motor-hand lateralization based on fTCD signals.

The lateralization index (LI) as determined from functional transcranial Doppler sonography (fTCD) can be used to determine the hemispheric organization of neural activation during a behavioral task. Previous studies have proposed different methods to determine this index, but to our knowledge no studies have compared the performance of these methods. In this study, we compare two established methods with a simpler method proposed here. The aim was to see whether similar results could be achieved with a simpler method and to give an indication of the analysis steps required to determine the LI. A simple unimanual motor task was performed while fTCD was acquired, and the LI determined by each of these methods was compared. In addition, LI determined by each method was related to behavioural output in the form of degree of handedness. The results suggest that although the methods differed in complexity, they yielded similar results when determining the lateralization of motor functions, and its correlation with behavior. Further investigation is needed to expand the conclusions of this preliminary study, however the new method proposed in the paper has great potential as it is much simpler than the more established methods yet yields similar results.

Post-concussion syndrome: prevalence after mild traumatic brain injury in comparison with a sample without head injury.

PRIMARY OBJECTIVE: To compare the prevalence of persistent post-concussion syndrome (PCS; >1 year post-injury) in participants with mild traumatic brain injury (mTBI) and those without head injury. RESEARCH DESIGN: A cross-sectional sample of 119 participants with mTBI and 246 without previous head injury. METHODS: Online questionnaires collected data about post-concussion symptoms, cognitive failures, anxiety, depression, sleep behaviour and post-traumatic stress disorder. Variability within the sample was addressed by splitting by PCS diagnosis to create four groups: mTBI + PCS, mTBI-PCS, Control + PCS and Control-PCS. PCS was diagnosed using ICD-10 criteria in all groups, with controls not requiring previous head injury. MAIN OUTCOMES AND RESULTS: PCS was present to a similar extent in participants with no head injury (34%) compared to those with mTBI (31%). Only report of headaches, which could be caused by expectation bias, distinguished between mTBI + PCS and Control + PCS groups. In addition, significantly higher cognitive problems were observed in participants with mTBI compared with the control group. CONCLUSIONS: Persistent PCS, as currently defined, is not specific to mTBI. These data suggest that somatic and cognitive symptoms are most likely to be able to distinguish PCS after mTBI from that present in the general population. Further research is necessary into these factors in order to create more specific PCS diagnostic criteria.

In search for the most optimal EEG method: A practical evaluation of a water-based electrode EEG system.

The study assessed a mobile electroencephalography system with water-based electrodes for its applicability in cognitive and behavioural neuroscience. It was compared to a standard gel-based wired system. Electroencephalography was recorded on two occasions (first with gel-based, then water-based system) as participants completed the flanker task. Technical and practical considerations for the application of the water-based system are reported based on participant and experimenter experiences. Empirical comparisons focused on electroencephalography data noise levels, frequency power across four bands (theta, alpha, low beta and high beta) and event-related components (P300 and ERN). The water-based system registered more noise compared to the gel-based system which resulted in increased loss of data during artefact rejection. Signal-to-noise ratio was significantly lower for the water-based system in the parietal channels which affected the observed parietal beta power. It also led to a shift in topography of the maximal P300 activity from parietal to frontal regions. The water-based system may be prone to slow drift noise which may affect the reliability and consistency of low-frequency band analyses. Practical considerations for the use of water-based electrode electroencephalography systems are provided.

A novel ATM-dependent X-ray-inducible gene is essential for both plant meiosis and gametogenesis.

DNA damage in Arabidopsis thaliana seedlings results in upregulation of hundreds of genes. One of the earliest and highest levels of induction is displayed by a previously uncharacterized gene that we have termed X-ray induced 1 (XRI1). Analysis of plants carrying a null xri1 allele revealed two distinct requirements for this gene in plant fertility. XRI1 was important for the post-meiotic stages of pollen development, leading to inviability of xri(-) pollen and abnormal segregation of the mutant allele in heterozygous xri1(+/-) plants. In addition, XRI1 was essential for male and female meiosis, as indicated by the complete sterility of homozygous xri1 mutants due to extensive chromosome fragmentation visible in meiocytes. Abolition of programmed DNA double-strand breaks in a spo11-1 mutant background failed to rescue the DNA fragmentation of xri1 mutants, suggesting that XRI1 functions at an earlier stage than SPO11-1 does. Yeast two-hybrid studies identified an interaction between XRI1 and a novel component of the Arabidopsis MND1/AHP2 complex, indicating possible requirements for XRI1 in meiotic DNA repair.

Live Intravital Imaging of Cellular Trafficking in the Cardiac Microvasculature-Beating the Odds.

Although mortality rates from cardiovascular disease in the developed world are falling, the prevalence of cardiovascular disease (CVD) is not. Each year, the number of people either being diagnosed as suffering with CVD or undergoing a surgical procedure related to it, such as percutaneous coronary intervention, continues to increase. In order to ensure that we can effectively manage these diseases in the future, it is critical that we fully understand their basic physiology and their underlying causative factors. Over recent years, the important role of the cardiac microcirculation in both acute and chronic disorders of the heart has become clear. The recruitment of inflammatory cells into the cardiac microcirculation and their subsequent activation may contribute significantly to tissue damage, adverse remodeling, and poor outcomes during recovery. However, our basic understanding of the cardiac microcirculation is hampered by an historic inability to image the microvessels of the beating heart-something we have been able to achieve in other organs for over 100 years. This stems from a couple of clear and obvious difficulties related to imaging the heart-firstly, it has significant inherent contractile motion and is affected considerably by the movement of lungs. Secondly, it is located in an anatomically challenging position for microscopy. However, recent microscopic and technological developments have allowed us to overcome some of these challenges and to begin to answer some of the basic outstanding questions in cardiac microvascular physiology, particularly in relation to inflammatory cell recruitment. In this review, we will discuss some of the historic work that took place in the latter part of last century toward cardiac intravital, before moving onto the advanced work that has been performed since. This work, which has utilized technology such as spinning-disk confocal and multiphoton microscopy, has-along with some significant advancements in algorithms and software-unlocked our ability to image the "business end" of the cardiac vascular tree. This review will provide an overview of these techniques, as well as some practical pointers toward software and other tools that may be useful for other researchers who are considering utilizing this technique themselves.

Tify: A quality-based frame selection tool for improving the output of unstable biomedical imaging.

The ability to image biological tissues is critical to our understanding of a range of systems and processes. In the case of in situ living tissue, such imaging is hampered by the innate mechanical properties of the tissue. In many cases, this provides challenges in how to process large amounts of image data which may contain aberrations from movement. Generally, current tools require the provision of reference images and are unable to maintain temporal correlations within an image set. Here, we describe a tool-Tify-which can accurately predict a numerical quality score versus human scoring and can analyse image sets in a manner that allows the maintenance of temporal relationships. The tool uses regression-based techniques to link image statistics to image quality based on user provided scores from a sample of images. Scores calculated by the software correlate strongly with the scores provided by human users. We identified that, in most cases, the software requires users to score between 20-30 frames in order to be able to accurately calculate the remaining images. Importantly, our results suggest that the software can use coefficients generated from consolidated image sets to process images without the need for additional manual scoring. Finally, the tool is able to use a frame windowing technique to identify the highest quality frame from a moving window, thus retaining macro-chronological connections between frames. In summary, Tify is able to successfully predict the quality of images in an image set based on a small number of sample scores provided by end-users. This software has the potential to improve the effectiveness of biological imaging techniques where motion artefacts, even in the presence of stabilisation, pose a significant problem.

Neural correlates of movement preparation in healthy ageing.

Motor disorders increase dramatically with age; however, little is known about non-clinical ageing of motor control mechanisms and their respective neural correlates. With the present experiment we aimed to study age effects on advance movement preparation, a key characteristic of motor behaviour that is known to involve premotor and primary motor circuits. The respective brain regions are subject to age-related brain atrophy of grey and white matter, and we therefore hypothesized that motor preparation mechanisms may be altered in older persons. Using a motor priming paradigm, performance data and event-related potentials were recorded in older (68-83 years) and younger (21-25 years) participants. The effect pattern observed for the younger group fully replicated previous findings, showing significant reaction time benefits and greater foreperiod activity for valid trials, as well as lateralized activation over motor regions. In older participants, the validity effect was insignificant, which corresponded to markedly reduced foreperiod amplitudes and the absence of lateralized activity. At the same time, the event-related potential showed a frontocentrally distributed positive component peaking in the P300 latency range after presentation of the prime. The amplitude of this potential was enhanced in elderly compared with young participants. The data suggest that the information processing related to the anticipation and preparation of an upcoming response changes substantially with age. In contrast to younger participants, older participants show no indication of effector-specific activation and recruit frontal areas in anticipation of a response signal. It is therefore not only movement execution that changes with age but also motor cognition.

Tetra-phenyl-phospho-nium hydrogen oxalate.

In the title compound, C(24)H(20)P(+)·C(2)HO(4) (-), two symmetry-independent ion pairs are present. The cations aggregate into puckered sheets via zigzag infinite chains of sixfold phenyl embraces and parallel fourfold phenyl embraces, while the anions form hydrogen-bonded chains between the sheets of cations. In the two independent oxalate anions, the angles between the normals to the two least-squares carboxyl-ate COO planes are unusually large, viz. 72.5 (1) and 82.1 (1)°.

Potential for use of creatine supplementation following mild traumatic brain injury.

There is significant overlap between the neuropathology of mild traumatic brain injury (mTBI) and the cellular role of creatine, as well as evidence of neural creatine alterations after mTBI. Creatine supplementation has not been researched in mTBI, but shows some potential as a neuroprotective when administered prior to or after TBI. Consistent with creatine's cellular role, supplementation reduced neuronal damage, protected against the effects of cellular energy crisis and improved cognitive and somatic symptoms. A variety of factors influencing the efficacy of creatine supplementation are highlighted, as well as avenues for future research into the potential of supplementation as an intervention for mTBI. In particular, the slow neural uptake of creatine may mean that greater effects are achieved by pre-emptive supplementation in at-risk groups.

A Metal-Insulator Transition of the Buried MnO2 Monolayer in Complex Oxide Heterostructure.

A novel artificially created MnO2 monolayer system is demonstrated in atomically controlled epitaxial perovskite heterostructures. With careful design of different electrostatic boundary conditions, a magnetic transition as well as a metal-insulator transition of the MnO2 monolayer is unveiled, providing a fundamental understanding of dimensionality-confined strongly correlated electron systems and a direction to design new electronic devices.

Efficient conformational sampling of local side-chain flexibility.

Side-chain flexibility of ligand-binding sites needs to be considered in the rational design of novel inhibitors. We have developed a method to generate conformational ensembles that efficiently sample local side-chain flexibility from a single crystal structure. The rotamer-based approach is tested here for the S1' pocket of human collagenase-1 (MMP-1), which is known to undergo conformational changes in multiple side-chains upon binding of certain inhibitors. First, a raw ensemble consisting of a large number of conformers of the S1' pocket was generated using an exhaustive search of rotamer combinations on a template crystal structure. A combination of principal component analysis and fuzzy clustering was then employed to successfully identify a core ensemble consisting of a low number of representatives from the raw ensemble. The core ensemble contained geometrically diverse conformers of stable nature, as indicated in several cases by a relative energy lower than that of the minimised template crystal structure. Through comparisons with X-ray crystallography and NMR structural data we show that the core ensemble occupied a conformational space similar to that observed under experimental conditions. The synthetic inhibitor RS-104966 is known to induce a conformational change in the side-chains of the S1' pocket of MMP-1 and could not be docked in the template crystal structure. However, the experimental binding mode was reproduced successfully using members of the core ensemble as the docking target, establishing the usefulness of the method in drug design.

Receptor flexibility in de novo ligand design and docking.

One of the major problems in computational drug design is incorporation of the intrinsic flexibility of protein binding sites. This is particularly crucial in ligand binding events, when induced fit can lead to protein structure rearrangements. As a consequence of the huge conformational space available to protein structures, receptor flexibility is rarely considered in ligand design procedures. In this work, we present an algorithm for integrating protein binding-site flexibility into de novo ligand design and docking processes. The approach allows dynamic rearrangement of amino acid side chains during the docking and design simulations. The impact of protein conformational flexibility is investigated in the docking of highly active inhibitors in the binding sites of acetylcholinesterase and human collagenase (matrix metalloproteinase-1) and in the design of ligands in the S1' pocket of MMP-1. The results of corresponding simulations for both rigid and flexible binding sites are compared in order to gauge the influence of receptor flexibility in drug discovery protocols.