The rhizoferrin biosynthetic gene in the fungal pathogen Rhizopus delemar is a novel member of the NIS gene family

This work was supported by the Natural Sciences and Engineering Research Council of Canada award to MM (grant number 611181). C. Carroll thanks Simon Fraser University for a travel and research award.Iron is essential for growth and in low iron environments such as serum many bacteria and fungi secrete ferric iron-chelating molecules called siderophores. All fungi produce hydroxamate siderophores with the exception of Mucorales fungi, which secrete rhizoferrin, a polycarboxylate siderophore. Here we investigated the biosynthesis of rhizoferrin by the opportunistic human pathogen, Rhizopus delemar. We searched the genome of R. delemar 99–880 for a homologue of the bacterial NRPS-independent siderophore (NIS) protein, SfnaD that is involved in biosynthesis of staphyloferrin A in Staphylococcus aureus. A protein was identified in R. delemar with 22% identity and 37% similarity with SfnaD, containing an N-terminal IucA/IucC family domain, and a C-terminal conserved ferric iron reductase FhuF-like transporter domain. Expression of the putative fungal rhizoferrin synthetase (rfs) gene was repressed by iron. The rfs gene was cloned and expressed in E.coli and siderophore biosynthesis from citrate and diaminobutane was confirmed using high resolution LC–MS. Substrate specificity was investigated showing that Rfs produced AMP when oxaloacetic acid, tricarballylic acid, ornithine, hydroxylamine, diaminopentane and diaminopropane were employed as substrates. Based on the production of AMP and the presence of a mono-substituted rhizoferrin, we suggest that Rfs is a member of the superfamily of adenylating enzymes. We used site-directed mutagenesis to mutate selected conserved residues predicted to be in the Rfs active site. These studies revealed that H484 is essential for Rfs activity and L544 may play a role in amine recognition by the enzyme. This study on Rfs is the first characterization of a fungal NIS enzyme. Future work will determine if rhizoferrin biosynthesis is required for virulence in Mucorales fungi.PostprintPeer reviewe

Microbial Diversity in a Hypersaline Sulfate Lake: A Terrestrial Analog of Ancient Mars

Life can persist under severe osmotic stress and low water activity in hypersaline environments. On Mars, evidence for the past presence of saline bodies of water is prevalent and resulted in the widespread deposition of sulfate and chloride salts. Here we investigate Spotted Lake (British Columbia, Canada), a hypersaline lake with extreme (>3 M) levels of sulfate salts as an exemplar of the conditions thought to be associated with ancient Mars. We provide the first characterization of microbial structure in Spotted Lake sediments through metagenomic sequencing, and report a bacteria-dominated community with abundant Proteobacteria, Firmicutes, and Bacteroidetes, as well as diverse extremophiles. Microbial abundance and functional comparisons reveal similarities to Ace Lake, a meromictic Antarctic lake with anoxic and sulfidic bottom waters. Our analysis suggests that hypersaline-associated species occupy niches characterized foremost by differential abundance of Archaea, uncharacterized Bacteria, and Cyanobacteria. Potential biosignatures in this environment are discussed, specifically the likelihood of a strong sulfur isotopic fractionation record within the sediments due to the presence of sulfate reducing bacteria. With its high sulfate levels and seasonal freeze-thaw cycles, Spotted Lake is an analog for ancient paleolakes on Mars in which sulfate salt deposits may have offered periodically habitable environments, and could have concentrated and preserved organic materials or their biomarkers over geologic time

Nucleic Acid-based Detection of Bacterial Pathogens Using Integrated Microfluidic Platform Systems

The advent of nucleic acid-based pathogen detection methods offers increased sensitivity and specificity over traditional microbiological techniques, driving the development of portable, integrated biosensors. The miniaturization and automation of integrated detection systems presents a significant advantage for rapid, portable field-based testing. In this review, we highlight current developments and directions in nucleic acid-based micro total analysis systems for the detection of bacterial pathogens. Recent progress in the miniaturization of microfluidic processing steps for cell capture, DNA extraction and purification, polymerase chain reaction, and product detection are detailed. Discussions include strategies and challenges for implementation of an integrated portable platform

Interpretable surface-based detection of focal cortical dysplasias:a Multi-centre Epilepsy Lesion Detection study

One outstanding challenge for machine learning in diagnostic biomedical imaging is algorithm interpretability. A key application is the identification of subtle epileptogenic focal cortical dysplasias (FCDs) from structural MRI. FCDs are difficult to visualize on structural MRI but are often amenable to surgical resection. We aimed to develop an open-source, interpretable, surface-based machine-learning algorithm to automatically identify FCDs on heterogeneous structural MRI data from epilepsy surgery centres worldwide. The Multi-centre Epilepsy Lesion Detection (MELD) Project collated and harmonized a retrospective MRI cohort of 1015 participants, 618 patients with focal FCD-related epilepsy and 397 controls, from 22 epilepsy centres worldwide. We created a neural network for FCD detection based on 33 surface-based features. The network was trained and cross-validated on 50% of the total cohort and tested on the remaining 50% as well as on 2 independent test sites. Multidimensional feature analysis and integrated gradient saliencies were used to interrogate network performance. Our pipeline outputs individual patient reports, which identify the location of predicted lesions, alongside their imaging features and relative saliency to the classifier. On a restricted 'gold-standard' subcohort of seizure-free patients with FCD type IIB who had T1 and fluid-attenuated inversion recovery MRI data, the MELD FCD surface-based algorithm had a sensitivity of 85%. Across the entire withheld test cohort the sensitivity was 59% and specificity was 54%. After including a border zone around lesions, to account for uncertainty around the borders of manually delineated lesion masks, the sensitivity was 67%. This multicentre, multinational study with open access protocols and code has developed a robust and interpretable machine-learning algorithm for automated detection of focal cortical dysplasias, giving physicians greater confidence in the identification of subtle MRI lesions in individuals with epilepsy

Event-based modelling in temporal lobe epilepsy demonstrates progressive atrophy from cross-sectional data

OBJECTIVE: Recent work has shown that people with common epilepsies have characteristic patterns of cortical thinning, and that these changes may be progressive over time. Leveraging a large multi-centre cross-sectional cohort, we investigated whether regional morphometric changes occur in a sequential manner, and whether these changes in people with mesial temporal lobe epilepsy and hippocampal sclerosis (MTLE-HS) correlate with clinical features. METHODS: We extracted regional measures of cortical thickness, surface area and subcortical brain volumes from T1-weighted (T1W) MRI scans collected by the ENIGMA-Epilepsy consortium, comprising 804 people with MTLE-HS and 1,625 healthy controls from 25 centres. Features with a moderate case-control effect size (Cohen's d≥0.5) were used to train an Event-Based Model (EBM), which estimates a sequence of disease-specific biomarker changes from cross-sectional data and assigns a biomarker-based fine-grained disease stage to individual patients. We tested for associations between EBM disease stage and duration of epilepsy, age of onset and anti-seizure medicine (ASM) resistance. RESULTS: In MTLE-HS, decrease in ipsilateral hippocampal volume along with increased asymmetry in hippocampal volume was followed by reduced thickness in neocortical regions, reduction in ipsilateral thalamus volume and, finally, increase in ipsilateral lateral ventricle volume. EBM stage was correlated to duration of illness (Spearman's ρ=0.293, p=7.03x10-16 ), age of onset (ρ=-0.18, p=9.82x10-7 ) and ASM resistance (AUC=0.59, p=0.043, Mann-Whitney U test). However, associations were driven by cases assigned to EBM stage zero, which represents MTLE-HS with mild or non-detectable abnormality on T1W MRI. SIGNIFICANCE: From cross-sectional MRI, we reconstructed a disease progression model that highlights a sequence of MRI changes that aligns with previous longitudinal studies. This model could be used to stage MTLE-HS subjects in other cohorts and help establish connections between imaging-based progression staging and clinical features

White matter abnormalities across different epilepsy syndromes in adults: an ENIGMA-Epilepsy study

The epilepsies are commonly accompanied by widespread abnormalities in cerebral white matter. ENIGMA-Epilepsy is a large quantitative brain imaging consortium, aggregating data to investigate patterns of neuroimaging abnormalities in common epilepsy syndromes, including temporal lobe epilepsy, extratemporal epilepsy, and genetic generalized epilepsy. Our goal was to rank the most robust white matter microstructural differences across and within syndromes in a multicentre sample of adult epilepsy patients. Diffusion-weighted MRI data were analysed from 1069 healthy controls and 1249 patients: temporal lobe epilepsy with hippocampal sclerosis (n = 599), temporal lobe epilepsy with normal MRI (n = 275), genetic generalized epilepsy (n = 182) and non-lesional extratemporal epilepsy (n = 193). A harmonized protocol using tract-based spatial statistics was used to derive skeletonized maps of fractional anisotropy and mean diffusivity for each participant, and fibre tracts were segmented using a diffusion MRI atlas. Data were harmonized to correct for scanner-specific variations in diffusion measures using a batch-effect correction tool (ComBat). Analyses of covariance, adjusting for age and sex, examined differences between each epilepsy syndrome and controls for each white matter tract (Bonferroni corrected at P < 0.001). Across ‘all epilepsies’ lower fractional anisotropy was observed in most fibre tracts with small to medium effect sizes, especially in the corpus callosum, cingulum and external capsule. There were also less robust increases in mean diffusivity. Syndrome-specific fractional anisotropy and mean diffusivity differences were most pronounced in patients with hippocampal sclerosis in the ipsilateral parahippocampal cingulum and external capsule, with smaller effects across most other tracts. Individuals with temporal lobe epilepsy and normal MRI showed a similar pattern of greater ipsilateral than contralateral abnormalities, but less marked than those in patients with hippocampal sclerosis. Patients with generalized and extratemporal epilepsies had pronounced reductions in fractional anisotropy in the corpus callosum, corona radiata and external capsule, and increased mean diffusivity of the anterior corona radiata. Earlier age of seizure onset and longer disease duration were associated with a greater extent of diffusion abnormalities in patients with hippocampal sclerosis. We demonstrate microstructural abnormalities across major association, commissural, and projection fibres in a large multicentre study of epilepsy. Overall, patients with epilepsy showed white matter abnormalities in the corpus callosum, cingulum and external capsule, with differing severity across epilepsy syndromes. These data further define the spectrum of white matter abnormalities in common epilepsy syndromes, yielding more detailed insights into pathological substrates that may explain cognitive and psychiatric co-morbidities and be used to guide biomarker studies of treatment outcomes and/or genetic research

Topographic divergence of atypical cortical asymmetry and atrophy patterns in temporal lobe epilepsy

Temporal lobe epilepsy, a common drug-resistant epilepsy in adults, is primarily a limbic network disorder associated with predominant unilateral hippocampal pathology. Structural MRI has provided an in vivo window into whole-brain grey matter structural alterations in temporal lobe epilepsy relative to controls, by either mapping (i) atypical inter-hemispheric asymmetry; or (ii) regional atrophy. However, similarities and differences of both atypical asymmetry and regional atrophy measures have not been systematically investigated. Here, we addressed this gap using the multisite ENIGMA-Epilepsy dataset comprising MRI brain morphological measures in 732 temporal lobe epilepsy patients and 1418 healthy controls. We compared spatial distributions of grey matter asymmetry and atrophy in temporal lobe epilepsy, contextualized their topographies relative to spatial gradients in cortical microstructure and functional connectivity calculated using 207 healthy controls obtained from Human Connectome Project and an independent dataset containing 23 temporal lobe epilepsy patients and 53 healthy controls and examined clinical associations using machine learning. We identified a marked divergence in the spatial distribution of atypical inter-hemispheric asymmetry and regional atrophy mapping. The former revealed a temporo-limbic disease signature while the latter showed diffuse and bilateral patterns. Our findings were robust across individual sites and patients. Cortical atrophy was significantly correlated with disease duration and age at seizure onset, while degrees of asymmetry did not show a significant relationship to these clinical variables. Our findings highlight that the mapping of atypical inter-hemispheric asymmetry and regional atrophy tap into two complementary aspects of temporal lobe epilepsy-related pathology, with the former revealing primary substrates in ipsilateral limbic circuits and the latter capturing bilateral disease effects. These findings refine our notion of the neuropathology of temporal lobe epilepsy and may inform future discovery and validation of complementary MRI biomarkers in temporal lobe epilepsy.11Nsciescopu

Integrated Biosensor Systems: Automated Microfluidic Pathogen Detection Platforms And Microcantilever-Based Monitoring Of Biological Activity

Recent advances in micro- and nanofabrication technology have led to the development of a wide range of integrated biosensor platforms. Though multiple sensors and assays have been developed for the rapid and sensitive detection of cells, proteins, and other small molecules, few systems have successfully integrated sample preparation, sample handing, and detection components onto a single platform. This dissertation has aimed to tackle some of the challenges associated with developing innovative and portable biosensor platforms. In this work, microfluidic modules for fluid handling using electrohydraulic pumps, magnetic bead-based sample preparation protocols, DNA extraction, purification, and real-time amplification detection were individually investigated and ultimately integrated into a single microfluidic chip platform. A LabView®-based instrument was designed to automate the platform, allowing the user to easily interface with the instrument and modify parameters via laptop-controlled software. Although technical challenges still remain for increasing cell capture efficiencies, detection sensitivities, and assay optimization, the platform presented here provides a solution for the portable field-deployable detection of foodborne pathogens with raw-sampleto-result capabilities. In addition to an integrated microfluidic pathogen detection platform, we also developed a cantilever-based biosensing system for the monitoring of changes in biological activity of single cells. We fabricated an array of tipless gold-coated silicon nitride cantilevers with an exposed nitride pad at the tip for chemical functionalization to capture single cells. As part of these investigations, experiments were performed which successfully demonstrated the monitoring changes in the increased activity of bacterial flagella and detection of capture and subsequent immunogenic events in RBL mast cells. Our efforts in developing integrated biosensor systems not only demonstrates improvements in microfluidic devices and cantilever-based sensing technologies, but also provides exciting new avenues toward the development of innovative integrated biosensor systems

The Role of Parent-Adolescent Relationship Quality in the Functioning of Anxious Adolescents

There is growing recognition that the reciprocal relationships between adolescents and\ua0their parents play an important role in the development and maintenance of mental health\ua0problems for young people. However, the unique contributions of the different aspects of parent-adolescent\ua0relationship quality to adolescent functioning remain unclear. This thesis aimed to test\ua0a theoretical model of how parent-adolescent relationship quality (i.e., connectedness and\ua0hostility) mediated the relationship between adolescent anxiety and adolescent functioning (i.e.,\ua0positive development and oppositional defiant behaviour), and to evaluate the effectiveness of a\ua0brief parenting intervention in improving adolescents’ relationships with parents and adolescent\ua0outcomes.Three studies were conducted with parents and their adolescents aged 11 to 17 years\ua0living in Australia. Study I was a single time-point community survey completed by 723 parents\ua0and 72 adolescents designed to investigate the links between demographics, adolescent anxiety,\ua0parental wellbeing, parenting practices, parent-adolescent relationship quality and adolescent\ua0functioning. Multivariate correlational analyses demonstrated that parent-adolescent\ua0connectedness and hostility were significant correlates of adolescent functioning. Other\ua0significant predictors of adolescent functioning included sociodemographic variables (parent age,\ua0parent cultural and educational backgrounds, adolescent gender and age and household financial\ua0stress), level of adolescent anxiety, as well as parental psychological distress, parental\ua0psychological flexibility (cognitive defusion, committed action and acceptance) and parenting\ua0practices (positive parenting, inconsistent discipline and poor supervision). Moreover,\ua0hierarchical multiple regression analyses showed that parent-adolescent connectedness and\ua0hostility were unique predictors of adolescent functioning after accounting for demographics,\ua0adolescent anxiety and parental factors. Parent-adolescent connectedness was not identified as a\ua0potential mediator on the link between adolescent anxiety and adolescent functioning. However,\ua0mediation analyses indicated that parent-adolescent hostility had an impact on reducing the\ua0influence of adolescent anxiety on positive development as well as on oppositional defiant\ua0behaviour. This provided some support for the role of parent-adolescent relationship quality in\ua0increasing positive outcomes and reducing the risk of developing disruptive and oppositional\ua0behaviour for adolescents with higher levels of anxiety.Study II was an observational study conducted with 15 parent-adolescent dyads that\ua0investigated the utility and psychometric properties of a parent-adolescent observation task and\ua0coding scheme in measuring the quality of parent-adolescent interactions. The observational\ua0measure was found to have good internal consistency and discriminant validity. However, results\ua0for construct validity were mixed. Significant correlations were revealed between two of the\ua0eleven items evaluated on the Connectedness/Hostility subscale of the observational measure\ua0(i.e., adolescent’s response and adolescent’s focus) and corresponding subscales measuring the\ua0constructs of connectedness and hostility on an existing self-report measure of parent-adolescent\ua0relationships, the Parent-Adolescent Relationship Scale (PARS). Participants were assigned to an\ua0Anxiety Disorder group or a No Disorder group based on DSM-5 criteria for anxiety disorders.\ua0Statistically significant differences were found between groups on the PARS Hostility subscale,\ua0as well as three items on the observational measure’s Connectedness/Hostility subscale (i.e.,\ua0General Mood, Adolescent’s Response and Adolescent’s Focus).\ua0Study III was a pre-post evaluation of the effectiveness a 2-hour Triple P Discussion\ua0Group on promoting parent-adolescent relationships and adolescent functioning with 12 parentadolescent\ua0dyads. No significant differences were revealed when comparing scores on the\ua0questionnaires measuring adolescent anxiety, parental psychological distress, parental\ua0psychological flexibility, parenting practices, parent-adolescent relationship quality and\ua0adolescent functioning across the two time points, with the exception of a significant decrease in\ua0PARS Connectedness reported by parents. Three of the eleven items evaluated on the\ua0observational measure’s Connectedness/Hostility subscale (i.e., general mood, adolescent’s\ua0response and adolescent’s focus) showed statistically significant differences from pre- to postintervention,\ua0but did not meet criteria for reliable change, which suggested the changes observed\ua0were not clinically significant.Overall, these findings supported the notion that parent-adolescent relationship quality is\ua0a critical factor impacting adolescent functioning and should be addressed when determining\ua0ways to improve behavioural outcomes for anxious adolescents. The present study also\ua0highlighted the value of including both parent and adolescent perspectives and multiple\ua0assessment modalities in adolescent developmental research. Further modification and\ua0development of the observational tool as a valid measure of parent-adolescent relationship\ua0quality is recommended. Although results from the intervention study were not found to be\ua0clinically significant, it was unclear if this was due to the small sample size and relatively\ua0homogenous profile of participants recruited to Studies II and III or the effectiveness of the\ua0intervention. Suggestions are made for investigating a modified version of the observational\ua0measure that could be sensitive to intervention effects with a larger and more diverse sample

The role of mother-adolescent relationship quality in moderating the effect of adolescent anxiety on psychosocial functioning

ABSTRACTObjective The effects of anxiety on adolescents’ psychosocial outcomes are well established, but little consideration has been given to the potential influence of the parent-adolescent relationship in moderating these effects. This study examined the moderating role of parent-adolescent connectedness and hostility in the association between anxiety and adolescent psychosocial functioning (measured by positive development [PD] and oppositional defiant behaviour [ODB]) within a community sample of mothers of adolescents.Method Participants were 723 Australian mothers (M age = 44.05 years, SD = 5.97) of adolescents aged 11 to 17 years (M = 14.32 years, SD = 5.97; 49% male). Participants completed an online survey comprising measures of parent-adolescent relationships, parenting practices, parental psychological distress, and adolescent anxiety and psychosocial functioning.Results Consistent with the first hypothesis, results from hierarchical regression analyses revealed that adolescent anxiety, connectedness, and hostility were independent predictors of PD and ODB. Inconsistent with predictions, parent-reported anxiety had a stronger, negative association with PD when mothers viewed the relationship with their adolescents as more connected and less hostile. Neither parent-adolescent connectedness nor hostility moderated the association between maternal reported adolescent anxiety and ODB.Conclusions Further longitudinal research is needed to understand how the parent-adolescent relationship context might affect outcomes and inform family-based prevention and intervention efforts for at-risk youth with anxiety symptomatology