The Response Phase - The First Six Hours After Acute Airway Injury by SO2 Inhalation: An in vivo and in vitro Study

We have identified an airway epithelial response following acute injury that cannot be termed \u27repair\u27 or regeneration. It precedes these well characterized events and it is termed the \u27response phase\u27. We tested the hypothesis that for the first 6 h following acute injury to the tracheal mucosa, the initial cellular events of the response phase will continue as in vivo even if the tissue is maintained in vitro in an Ussing chamber. The tracheal mucosa of anesthetized, intubated mongrel dogs was injured by the inhalation of SO2 500 ppm for 1 h (7 dogs); controls (3 dogs) breathed filtered, compressed air for 1 h. 4 dogs were killed, in pairs, at 1 and 6 h after 500ppm of SO2; their tracheas were removed and fixed for microscopic examination. 3 dogs were killed immediately after the SO2 exposure, their tracheas were removed and epithelium isolated from the posterior-membranous sheath was mounted in Ussing chambers in oxygenated, Krebs-Henseleit buffer (8 per dog with aperature area of 1.5 cm2). These tissues (and those from control dogs prepared identically) were fixed after 1 and 6 h incubation for microscopic examination. Epithelial damage was not observed in any controls but was in all tissues exposed to SO2. A wide spectrum of mucosal cell injury during the response phase was observed. The patterns of exfoliation noted were: individual cells, rows (several cells wide) of mucosal cells and entire regions (several hundred μm2). At 1 h after exposure, in some lesions, the injury is difficult to assess because the tracheal surface was either blanketed in exfoliated cells or appeared in total disarray. By 6 h, the lesions were well defined and large flattened cells (130 μm2 in surface area) covered the basement membrane in areas where mucosal cells had exfoliated. Some ciliated cells still remained attached at their base in these areas. These were the findings whether the tissues were taken fresh from the animal or have been maintained in Ussing chambers for up to 6 h. These results show that cellular repair of the tracheal epithelium can be studied in vitro during the first 6 h after injury, even if the injury has occurred in situ

Stomatal Opening Involves Polar, Not Radial, Stiffening Of Guard Cells

It has long been accepted that differential radial thickening of guard cells plays an important role in the turgor-driven shape changes required for stomatal pore opening to occur [1-4]. This textbook description derives from an original interpretation of structure rather than measurement of mechanical properties. Here we show, using atomic force microscopy, that although mature guard cells display a radial gradient of stiffness, this is not present in immature guard cells, yet young stomata show a normal opening response. Finite element modeling supports the experimental observation that radial stiffening plays a very limited role in stomatal opening. In addition, our analysis reveals an unexpected stiffening of the polar regions of the stomata complexes, both in Arabidopsis and other plants, suggesting a widespread occurrence. Combined experimental data (analysis of guard cell wall epitopes and treatment of tissue with cell wall digesting enzymes, coupled with bioassay of guard cell function) plus modeling lead us to propose that polar stiffening reflects a mechanical, pectin-based pinning down of the guard cell ends, which restricts increase of stomatal complex length during opening. This is predicted to lead to an improved response sensitivity of stomatal aperture movement with respect to change of turgor pressure. Our results provide new insight into the mechanics of stomatal function, both negating an established view of the importance of radial thickening and providing evidence for a significant role for polar stiffening. Improved stomatal performance via altered cell-wall-mediated mechanics is likely to be of evolutionary and agronomic significance

'It's coming at things from a very different standpoint': evaluating the 'Supporting Self-Care in General Practice Programme' in NHS East of England

Aim: To undertake a service evaluation of the NHS East of England Supporting Self-Care in General Practice programme. Background: The number of people purported to live with long-term conditions continues to rise generating increasing policy emphasis on the importance of self-care. Previous work has highlighted barriers to implementing self-care interventions in general practice, including a lack of organisational approaches to providing self-care and limited engagement and training of healthcare professionals. In response to these barriers and policy drivers, NHS East of England Strategic Health Authority developed and commenced the Supporting Self-care in General Practice (SSCiGP) programme, which seeks to transform the relationships between people with long-term conditions and primary care practitioners. Methods: This was a mixed methods study, carried out over two phases, which included interviews, survey work and practice-based case studies. Results: This paper focuses on findings related to clinician and practice level change. Clinicians reported changes in their perceptions and in consultation practices following attendance on the SSCiGP programme. These changes were linked to empathy and patient-centredness that mirrored what patients valued in interactions with clinicians. There were qualitative and descriptive differences, but no statistically significant differences between clinicians who had and had not attended the SSCiGP programme. Time was recognised as a significant barrier to implementing, and sustaining skills learnt from the SSCiGP programme. Greater impact at practice level could be achieved when there was whole practice commitment to values that underpinned the SSCiGP programme. There was evidence that such approaches are being incorporated to change practice systems and structures to better facilitate self-care, particularly in practices who were early programme adopters. Conclusion: This evaluation demonstrates that training around clinician change can be effective in shifting service delivery when sat within a cultural framework that genuinely situates patients at the centre of consultations and practice activity

Design and Administration of Patient-Centred Outcome Measures: The Perspectives of Children and Young People with Life-Limiting or Life-Threatening Conditions and Their Family Members

BACKGROUND: Self-reported health data from children with life-limiting conditions is rarely collected. To improve acceptability and feasibility of child and family-centred outcome measures for children, they need to be designed in a way that reflects preferences, priorities and abilities. OBJECTIVES: The aim was to identify preferences for patient-reported outcome measure design (recall period, response format, length, administration mode) to improve the feasibility, acceptability, comprehensibility and relevance of a child and family-centred outcome measure, among children with life-limiting conditions and their family members. METHOD: A semi-structured qualitative interview study seeking the perspectives of children with life-limiting conditions, their siblings and parents on measure design was conducted. Participants were purposively sampled and recruited from nine UK sites. Verbatim transcripts were analysed using framework analysis. RESULTS: A total of 79 participants were recruited: 39 children aged 5–17 years (26 living with a life-limiting condition; 13 healthy siblings) and 40 parents (of children aged 0–17 years). Children found a short recall period and a visually appealing measure with ten questions or fewer most acceptable. Children with life-limiting conditions were more familiar with using rating scales such as numeric and Likert than their healthy siblings. Children emphasised the importance of completing the measure alongside interactions with a healthcare professional to enable them to talk about their responses. While parents assumed that electronic completion methods would be most feasible and acceptable, a small number of children preferred paper. CONCLUSIONS: This study demonstrates that children with life-limiting conditions can engage in communicating preferences regarding the design of a patient-centred outcome measure. Where possible, children should be given the opportunity to participate in the measure development process to enhance acceptability and uptake in clinical practice. Results of this study should be considered in future research on outcome measure development in children

Examining the effect of evaluation sample size on the sensitivity and specificity of COVID-19 diagnostic tests in practice: a simulation study

Background In response to the global COVID-19 pandemic, many in vitro diagnostic (IVD) tests for SARS-CoV-2 have been developed. Given the urgent clinical demand, researchers must balance the desire for precise estimates of sensitivity and specificity against the need for rapid implementation. To complement estimates of precision used for sample size calculations, we aimed to estimate the probability that an IVD will fail to perform to expected standards after implementation, following clinical studies with varying sample sizes. Methods We assumed that clinical validation study estimates met the ‘desirable’ performance (sensitivity 97%, specificity 99%) in the target product profile (TPP) published by the Medicines and Healthcare products Regulatory Agency (MHRA). To estimate the real-world impact of imprecision imposed by sample size we used Bayesian posterior calculations along with Monte Carlo simulations with 10,000 independent iterations of 5,000 participants. We varied the prevalence between 1 and 15% and the sample size between 30 and 2,000. For each sample size, we estimated the probability that diagnostic accuracy would fail to meet the TPP criteria after implementation. Results For a validation study that demonstrates ‘desirable’ sensitivity within a sample of 30 participants who test positive for COVID-19 using the reference standard, the probability that real-world performance will fail to meet the ‘desirable’ criteria is 10.7–13.5%, depending on prevalence. Theoretically, demonstrating the 'desirable' performance in 90 positive participants would reduce that probability to below 5%. A marked reduction in the probability of failure to hit ‘desirable’ specificity occurred between samples of 100 (19.1–21.5%) and 160 (4.3–4.8%) negative participants. There was little further improvement above sample sizes of 160 negative participants. Conclusion Based on imprecision alone, small evaluation studies can lead to the acceptance of diagnostic tests which are likely to fail to meet performance targets when deployed. There is diminished return on uncertainty surrounding an accuracy estimate above a total sample size of 250 (90 positive and 160 negative)

Stomatal Function Requires Pectin De-methyl-esterification of the Guard Cell Wall

Stomatal opening and closure depends on changes in turgor pressure acting within guard cells to alter cell shape. The extent of these shape changes is limited by the mechanical properties of the cells, which will be largely dependent on the structure of the cell walls. Although it has long been observed that guard cells are anisotropic due to differential thickening and the orientation of cellulose microfibrils, our understanding of the composition of the cell wall that allows them to undergo repeated swelling and deflation remains surprisingly poor. Here, we show that the walls of guard cells are rich in unesterified pectins. We identify a pectin methylesterase gene, PME6, which is highly expressed in guard cells and required for stomatal function. pme6-1 mutant guard cells have walls enriched in methyl-esterified pectin and show a decreased dynamic range in response to triggers of stomatal opening/closure, including elevated osmoticum, suggesting that abrogation of stomatal function reflects a mechanical change in the guard cell wall. Altered stomatal function leads to increased conductance and evaporative cooling, as well as decreased plant growth. The growth defect of the pme6-1 mutant is rescued by maintaining the plants in elevated CO2, substantiating gas exchange analyses, indicating that the mutant stomata can bestow an improved assimilation rate. Restoration of PME6 rescues guard cell wall pectin methyl-esterification status, stomatal function, and plant growth. Our results establish a link between gene expression in guard cells and their cell wall properties, with a corresponding effect on stomatal function and plant physiology

A Feedback Quenched Oscillator Produces Turing Patterning with One Diffuser

Efforts to engineer synthetic gene networks that spontaneously produce patterning in multicellular ensembles have focused on Turing's original model and the “activator-inhibitor” models of Meinhardt and Gierer. Systems based on this model are notoriously difficult to engineer. We present the first demonstration that Turing pattern formation can arise in a new family of oscillator-driven gene network topologies, specifically when a second feedback loop is introduced which quenches oscillations and incorporates a diffusible molecule. We provide an analysis of the system that predicts the range of kinetic parameters over which patterning should emerge and demonstrate the system's viability using stochastic simulations of a field of cells using realistic parameters. The primary goal of this paper is to provide a circuit architecture which can be implemented with relative ease by practitioners and which could serve as a model system for pattern generation in synthetic multicellular systems. Given the wide range of oscillatory circuits in natural systems, our system supports the tantalizing possibility that Turing pattern formation in natural multicellular systems can arise from oscillator-driven mechanisms

X-Linked Genes and Risk of Orofacial Clefts: Evidence from Two Population-Based Studies in Scandinavia

Background: Orofacial clefts are common birth defects of complex etiology, with an excess of males among babies with cleft lip and palate, and an excess of females among those with cleft palate only. Although genes on the X chromosome have been implicated in clefting, there has been no association analysis of X-linked markers. Methodology/Principal Findings: We added new functionalities in the HAPLIN statistical software to enable association analysis of X-linked markers and an exploration of various causal scenarios relevant to orofacial clefts. Genotypes for 48 SNPs in 18 candidate genes on the X chromosome were analyzed in two population-based samples from Scandinavia (562 Norwegian and 235 Danish case-parent triads). For haplotype analysis, we used a sliding-window approach and assessed isolated cleft lip with or without cleft palate (iCL/P) separately from isolated cleft palate only (iCPO). We tested three statistical models in HAPLIN, allowing for: i) the same relative risk in males and females, ii) sex-specific relative risks, and iii) X-inactivation in females. We found weak but consistent associations with the oral-facial-digital syndrome 1 (OFD1) gene (formerly known as CXORF5) in the Danish iCL/P samples across all models, but not in the Norwegian iCL/P samples. In sex-specific analyses, the association with OFD1 was in male cases only. No analyses showed associations with iCPO in either the Norwegian or the Danish sample. Conclusions: The association of OFD1 with iCL/P is plausible given the biological relevance of this gene. However, the lack of replication in the Norwegian samples highlights the need to verify these preliminary findings in other large datasets. More generally, the novel analytic methods presented here are widely applicable to investigations of the role of X-linked genes in complex traits

Mining biological information from 3D short time-series gene expression data: the OPTricluster algorithm

<p>Abstract</p> <p>Background</p> <p>Nowadays, it is possible to collect expression levels of a set of genes from a set of biological samples during a series of time points. Such data have three dimensions: gene-sample-time (GST). Thus they are called 3D microarray gene expression data. To take advantage of the 3D data collected, and to fully understand the biological knowledge hidden in the GST data, novel subspace clustering algorithms have to be developed to effectively address the biological problem in the corresponding space.</p> <p>Results</p> <p>We developed a subspace clustering algorithm called Order Preserving Triclustering (OPTricluster), for 3D short time-series data mining. OPTricluster is able to identify 3D clusters with coherent evolution from a given 3D dataset using a combinatorial approach on the sample dimension, and the order preserving (OP) concept on the time dimension. The fusion of the two methodologies allows one to study similarities and differences between samples in terms of their temporal expression profile. OPTricluster has been successfully applied to four case studies: immune response in mice infected by malaria (<it>Plasmodium chabaudi</it>), systemic acquired resistance in <it>Arabidopsis thaliana</it>, similarities and differences between inner and outer cotyledon in <it>Brassica napus </it>during seed development, and to <it>Brassica napus </it>whole seed development. These studies showed that OPTricluster is robust to noise and is able to detect the similarities and differences between biological samples.</p> <p>Conclusions</p> <p>Our analysis showed that OPTricluster generally outperforms other well known clustering algorithms such as the TRICLUSTER, gTRICLUSTER and K-means; it is robust to noise and can effectively mine the biological knowledge hidden in the 3D short time-series gene expression data.</p

Gene coexpression clusters and putative regulatory elements underlying seed storage reserve accumulation in Arabidopsis

Abstract Background In Arabidopsis, a large number of genes involved in the accumulation of seed storage reserves during seed development have been characterized, but the relationship of gene expression and regulation underlying this physiological process remains poorly understood. A more holistic view of this molecular interplay will help in the further study of the regulatory mechanisms controlling seed storage compound accumulation. Results We identified gene coexpression networks in the transcriptome of developing Arabidopsis (Arabidopsis thaliana) seeds from the globular to mature embryo stages by analyzing publicly accessible microarray datasets. Genes encoding the known enzymes in the fatty acid biosynthesis pathway were found in one coexpression subnetwork (or cluster), while genes encoding oleosins and seed storage proteins were identified in another subnetwork with a distinct expression profile. In the triacylglycerol assembly pathway, only the genes encoding diacylglycerol acyltransferase 1 (DGAT1) and a putative cytosolic "type 3" DGAT exhibited a similar expression pattern with genes encoding oleosins. We also detected a large number of putative cis-acting regulatory elements in the promoter regions of these genes, and promoter motifs for LEC1 (LEAFY COTYLEDON 1), DOF (DNA-binding-with-One-Finger), GATA, and MYB transcription factors (TF), as well as SORLIP5 (Sequences Over-Represented in Light-Induced Promoters 5), are overrepresented in the promoter regions of fatty acid biosynthetic genes. The conserved CCAAT motifs for B3-domain TFs and binding sites for bZIP (basic-leucine zipper) TFs are enriched in the promoters of genes encoding oleosins and seed storage proteins. Conclusions Genes involved in the accumulation of seed storage reserves are expressed in distinct patterns and regulated by different TFs. The gene coexpression clusters and putative regulatory elements presented here provide a useful resource for further experimental characterization of protein interactions and regulatory networks in this process.</p