Method for RNA extraction and transcriptomic analysis of single fungal spores

Transcriptomic analysis of single cells has been increasingly in demand in recent years, thanks to technological and methodological advances as well as growing recognition of the importance of individuals in biological systems. However, the majority of these studies have been performed in mammalian cells, due to their ease of lysis and high RNA content. No single cell transcriptomic analysis has yet been applied to microbial spores, even though it is known that heterogeneity at the phenotype level exists among individual spores. Transcriptomic analysis of single spores is challenging, in part due to the physically robust nature of the spore wall. This precludes the use of methods commonly used for mammalian cells. Here, we describe a simple method for extraction and amplification of transcripts from single fungal conidia (asexual spores), and its application in single-cell transcriptomics studies. The method can also be used for studies of small numbers of fungal conidia, which may be necessary in the case of limited sample availability, low-abundance transcripts or interest in small subpopulations of conidia.• The method allows detection of transcripts from single conidia of Aspergillus niger• The method allows detection of genomic DNA from single conidia of Aspergillus nige

Food insecurity and severe mental illness: understanding the hidden problem and how to ask about food access during routine healthcare

SUMMARY Food insecurity occurs when an individual lacks the financial resources to ensure reliable access to sufficient food to meet their dietary, nutritional and social needs. Adults living with mental ill health, particularly severe mental illness, are more likely to experience food insecurity than the general adult population. Despite this, most interventions and policy reforms in recent years have been aimed at children and families, with little regard for other vulnerable groups. Initiating a conversation about access to food can be tricky and assessing for food insecurity does not happen in mental health settings. This article provides an overview of food insecurity and how it relates to mental ill health. With reference to research evidence, the reader will gain an understanding of food insecurity, how it can be assessed and how food-insecure individuals with severe mental illness can be supported. Finally, we make policy recommendations to truly address this driver of health inequality.</jats:p

Mentalizing in first-episode psychosis: Correlates with symptomatology and traits of borderline personality disorder

Aim: To explore the associations between mentalizing, positive and negative symptoms of psychosis, and traits of borderline personality disorder, in a sample of patients with first-episode psychosis, and in a non-clinical sample. Methods: A quantitative cross-sectional design was employed. Thirty-two adults with first-episode psychosis and 148 non-clinical participants were assessed using the reflective functioning questionnaire. The questionnaire measures two dimensions of mentalizing, certainty and uncertainty about mental states. Traits of borderline personality disorder and symptoms of psychosis were measured using the self-report version of the Zanarini rating scale, the Community Assessment of Psychotic Experiences, and the Green et al., paranoid thought scale. Results: Patients with first-episode psychosis reported increased mentalizing impairments, characterized as hypomentalizing tendencies, compared to the non-clinical group. Regression analysis showed significant associations between higher scores on the uncertainty about mental states scale and negative symptoms of psychosis in both groups. No associations were found between mentalizing impairments and traits of borderline personality disorder in the clinical sample, although associations were found in the non-clinical sample. Conclusions: The present findings suggests that impairments in mentalizing may be associated with negative symptoms of psychosis across both clinical and non-clinical samples. Mentalizing impairments was found to be associated with traits of borderline personality disorder, but this finding was only confirmed in the non-clinical sample. Mentalizing should therefore be considered in the early assessment and treatment of patients experiencing difficulties with negative symptoms of psychosis

Augmented reality meeting table: a novel multi-user interface for architectural design

Immersive virtual environments have received widespread attention as providing possible replacements for the media and systems that designers traditionally use, as well as, more generally, in providing support for collaborative work. Relatively little attention has been given to date however to the problem of how to merge immersive virtual environments into real world work settings, and so to add to the media at the disposal of the designer and the design team, rather than to replace it. In this paper we report on a research project in which optical see-through augmented reality displays have been developed together with prototype decision support software for architectural and urban design. We suggest that a critical characteristic of multi user augmented reality is its ability to generate visualisations from a first person perspective in which the scale of rendition of the design model follows many of the conventions that designers are used to. Different scales of model appear to allow designers to focus on different aspects of the design under consideration. Augmenting the scene with simulations of pedestrian movement appears to assist both in scale recognition, and in moving from a first person to a third person understanding of the design. This research project is funded by the European Commission IST program (IST-2000-28559)

Weak Acid Resistance A (WarA), a Novel Transcription Factor Required for Regulation of Weak-Acid Resistance and Spore-Spore Heterogeneity in Aspergillus niger

Copyright © 2020 Geoghegan et al. Propionic, sorbic, and benzoic acids are organic weak acids that are widely used as food preservatives, where they play a critical role in preventing microbial growth. In this study, we uncovered new mechanisms of weak-acid resistance in molds. By screening a library of 401 transcription factor deletion strains in Aspergillus fumigatus for sorbic acid hypersensitivity, a previously uncharacterized transcription factor was identified and named weak acid resistance A (WarA). The orthologous gene in the spoilage mold Aspergillus niger was identified and deleted. WarA was required for resistance to a range of weak acids, including sorbic, propionic, and benzoic acids. A transcriptomic analysis was performed to characterize genes regulated by WarA during sorbic acid treatment in A. niger Several genes were significantly upregulated in the wild type compared with a ΔwarA mutant, including genes encoding putative weak-acid detoxification enzymes and transporter proteins. Among these was An14g03570, a putative ABC-type transporter which we found to be required for weak-acid resistance in A. niger We also show that An14g03570 is a functional homologue of the Saccharomyces cerevisiae protein Pdr12p and we therefore name it PdrA. Last, resistance to sorbic acid was found to be highly heterogeneous within genetically uniform populations of ungerminated A. niger conidia, and we demonstrate that pdrA is a determinant of this heteroresistance. This study has identified novel mechanisms of weak-acid resistance in A. niger which could help inform and improve future food spoilage prevention strategies.IMPORTANCE Weak acids are widely used as food preservatives, as they are very effective at preventing the growth of most species of bacteria and fungi. However, some species of molds can survive and grow in the concentrations of weak acid employed in food and drink products, thereby causing spoilage with resultant risks for food security and health. Current knowledge of weak-acid resistance mechanisms in these fungi is limited, especially in comparison to that in yeasts. We characterized gene functions in the spoilage mold species Aspergillus niger which are important for survival and growth in the presence of weak-acid preservatives. Such identification of weak-acid resistance mechanisms in spoilage molds will help in the design of new strategies to reduce food spoilage in the future

Circadian dynamics in measures of cortical excitation and inhibition balance

Several neuropsychiatric and neurological disorders have recently been characterized as dysfunctions arising from a ‘final common pathway’ of imbalanced excitation to inhibition within cortical networks. How the regulation of a cortical E/I ratio is affected by sleep and the circadian rhythm however, remains to be established. Here we addressed this issue through the analyses of TMS-evoked responses recorded over a 29h sleep deprivation protocol conducted in young and healthy volunteers. Spectral analyses of TMS-evoked responses in frontal cortex revealed non-linear changes in gamma band evoked oscillations, compatible with an influence of circadian timing on inhibitory interneuron activity. In silico inferences of cell-to-cell excitatory and inhibitory connectivity and GABA/Glutamate receptor time constant based on neural mass modeling within the Dynamic causal modeling framework, further suggested excitation/inhibition balance was under a strong circadian influence. These results indicate that circadian changes in EEG spectral properties, in measure of excitatory/inhibitory connectivity and in GABA/glutamate receptor function could support the maintenance of cognitive performance during a normal waking day, but also during overnight wakefulness. More generally, these findings demonstrate a slow daily regulation of cortical excitation/inhibition balance, which depends on circadian-timing and prior sleep-wake history

How are normal sleeping controls selected? A systematic review of cross-sectional insomnia studies, and a standardised method to select healthy controls for sleep research

There appears to be some inconsistency in how normal sleepers (controls) are selected and screened for participation in research studies for comparison with insomnia patients. The purpose of the current study is to assess and compare methods of identifying normal sleepers in insomnia studies, with reference to published standards. We systematically reviewed the literature on insomnia patients which included control subjects. The resulting 37 articles were systematically reviewed with reference to the five criteria for normal sleep specified by Edinger et al. (2004). In summary, these criteria are: evidence of sleep disruption; sleep scheduling; general health; substance/medication use; and other sleep disorders. We found sleep diaries, PSG, and clinical screening examinations to be widely used with both control subjects and insomnia participants. However, there are differences between research groups in the precise definitions applied to the components of normal sleep. We found that none of reviewed studies applied all of the Edinger et al. criteria, and 16% met four criteria. In general, screening is applied most rigorously at the level of a clinical disorder, whether physical, psychiatric, or sleep. While the Edinger et al. criteria seem to be applied in some form by most researchers, there is scope to improve standards and definitions in this area. Ideally, different methods such as sleep diaries and questionnaires would be used concurrently with objective measures to ensure normal sleepers are identified, and descriptive information for control subjects would be reported. Here, we have devised working criteria and methods to be used for assessment of normal sleepers. This would help clarify the nature of the control group, in contrast to insomnia subjects and other patient groups

Recombination rate and selection strength in HIV intra-patient evolution

The evolutionary dynamics of HIV during the chronic phase of infection is driven by the host immune response and by selective pressures exerted through drug treatment. To understand and model the evolution of HIV quantitatively, the parameters governing genetic diversification and the strength of selection need to be known. While mutation rates can be measured in single replication cycles, the relevant effective recombination rate depends on the probability of coinfection of a cell with more than one virus and can only be inferred from population data. However, most population genetic estimators for recombination rates assume absence of selection and are hence of limited applicability to HIV, since positive and purifying selection are important in HIV evolution. Here, we estimate the rate of recombination and the distribution of selection coefficients from time-resolved sequence data tracking the evolution of HIV within single patients. By examining temporal changes in the genetic composition of the population, we estimate the effective recombination to be r=1.4e-5 recombinations per site and generation. Furthermore, we provide evidence that selection coefficients of at least 15% of the observed non-synonymous polymorphisms exceed 0.8% per generation. These results provide a basis for a more detailed understanding of the evolution of HIV. A particularly interesting case is evolution in response to drug treatment, where recombination can facilitate the rapid acquisition of multiple resistance mutations. With the methods developed here, more precise and more detailed studies will be possible, as soon as data with higher time resolution and greater sample sizes is available.Comment: to appear in PLoS Computational Biolog

The effect of luminal content and rate of occlusion on the interpretation of colonic manometry

This is the accepted version of the following article: [Arkwright, J. W., Dickson, A., Maunder, S. A., Blenman, N. G., Lim, J., O’Grady, G., Archer, R., Costa, M., Spencer, N. J., Brookes, S., Pullan, A. and Dinning, P. G. (2013), The effect of luminal content and rate of occlusion on the interpretation of colonic manometry. Neurogastroenterology & Motility, 25: e52–e59.], which has been published in final form at [http://dx.doi.org/10.1111/nmo.12051]. In addition, authors may also transmit, print and share copies with colleagues, provided that there is no systematic distribution of the submitted version, e.g. posting on a listserve, network or automated delivery.Background Manometry is commonly used for diagnosis of esophageal and anorectal motility disorders. In the colon, manometry is a useful tool, but clinical application remains uncertain. This uncertainty is partly based on the belief that manometry cannot reliably detect non-occluding colonic contractions and, therefore, cannot identify reliable markers of dysmotility. This study tests the ability of manometry to record pressure signals in response to non-lumen-occluding changes in diameter, at different rates of wall movement and with content of different viscosities. Methods A numerical model was built to investigate pressure changes caused by localized, non-lumen-occluding reductions in diameter, similar to those caused by contraction of the gut wall. A mechanical model, consisting of a sealed pressure vessel which could produce localized reductions in luminal diameter, was used to validate the model using luminal segments formed from; (i) natural latex; and (ii) sections of rabbit proximal colon. Fluids with viscosities ranging from 1 to 6800 mPa s-1 and luminal contraction rates over the range 5-20 mmHg s-1 were studied. Key Results Manometry recorded non-occluding reductions in diameter, provided that they occurred with sufficiently viscous content. The measured signal was linearly dependent on the rate of reduction in luminal diameter and also increased with increasing viscosity of content (R2 = 0.62 and 0.96 for 880 and 1760 mPa s-1, respectively). Conclusions & Inferences Manometry reliably registers non-occluding contractions in the presence of viscous content, and is therefore a viable tool for measuring colonic motility. Interpretation of colonic manometric data, and definitions based on manometric results, must consider the viscosity of luminal content.Australian National Health & Medical Research Counci

Analysis of high-depth sequence data for studying viral diversity: a comparison of next generation sequencing platforms using Segminator II

Background: Next generation sequencing provides detailed insight into the variation present within viral populations, introducing the possibility of treatment strategies that are both reactive and predictive. Current software tools, however, need to be scaled up to accommodate for high-depth viral data sets, which are often temporally or spatially linked. In addition, due to the development of novel sequencing platforms and chemistries, each with implicit strengths and weaknesses, it will be helpful for researchers to be able to routinely compare and combine data sets from different platforms/chemistries. In particular, error associated with a specific sequencing process must be quantified so that true biological variation may be identified. Results: Segminator II was developed to allow for the efficient comparison of data sets derived from different sources. We demonstrate its usage by comparing large data sets from 12 influenza H1N1 samples sequenced on both the 454 Life Sciences and Illumina platforms, permitting quantification of platform error. For mismatches median error rates at 0.10 and 0.12%, respectively, suggested that both platforms performed similarly. For insertions and deletions median error rates within the 454 data (at 0.3 and 0.2%, respectively) were significantly higher than those within the Illumina data (0.004 and 0.006%, respectively). In agreement with previous observations these higher rates were strongly associated with homopolymeric stretches on the 454 platform. Outside of such regions both platforms had similar indel error profiles. Additionally, we apply our software to the identification of low frequency variants. Conclusion: We have demonstrated, using Segminator II, that it is possible to distinguish platform specific error from biological variation using data derived from two different platforms. We have used this approach to quantify the amount of error present within the 454 and Illumina platforms in relation to genomic location as well as location on the read. Given that next generation data is increasingly important in the analysis of drug-resistance and vaccine trials, this software will be useful to the pathogen research community. A zip file containing the source code and jar file is freely available for download from http://www.bioinf.manchester.ac.uk/segminator/