Steroidal glycoalkaloids contribute to anthracnose resistance in Solanum lycopersicum.

Anthracnose is a widespread plant disease caused by various species of the fungal pathogen Colletotrichum. In solanaceous plants such as tomato (Solanum lycopersicum), Colletotrichum infections exhibit a quiescent, asymptomatic state in developing fruit, followed by a transition to necrotrophic infections in ripe fruit. Through analysis of fruit tissue extracts of 95L368, a tomato breeding line that yields fruit with enhanced anthracnose resistance, we identified a role for steroidal glycoalkaloids (SGAs) in anthracnose resistance. The SGA α-tomatine and several of its derivatives accumulated at higher levels, in comparison with fruit of the susceptible tomato cultivar US28, and 95L368 fruit extracts displayed fungistatic activity against Colletotrichum. Correspondingly, ripe and unripe 95L368 fruit displayed enhanced expression of glycoalkaloid metabolic enzyme (GAME) genes, which encode key enzymes in SGA biosynthesis. Metabolomics analysis incorporating recombinant inbred lines generated from 95L368 and US28 yielded strong positive correlations between anthracnose resistance and accumulation of α-tomatine and several derivatives. Lastly, transient silencing of expression of the GAME genes GAME31 and GAME5 in anthracnose-susceptible tomato fruit yielded enhancements to anthracnose resistance. Together, our data support a role for SGAs in anthracnose defense in tomato, with a distinct SGA metabolomic profile conferring resistance to virulent Colletotrichum infections in ripe fruit.

Detached Leaf Assays to Simplify Gene Expression Studies in Potato During Infestation by Chewing Insect Manduca sexta.

The multitrophic nature of gene expression studies of insect herbivory demands large numbers of biological replicates, creating the need for simpler, more streamlined herbivory protocols. Perturbations of chewing insects are usually studied in whole plant systems. While this whole organism strategy is popular, it is not necessary if similar observations can be replicated in a single detached leaf. The assumption is that basic elements required for signal transduction are present within the leaf itself. In the case of early events in signal transduction, cells need only to receive the signal from the perturbation and transmit that signal to neighboring cells which are assayed for gene expression. The proposed method simply changes the timing of the detachment. In whole plant experiments, larvae are confined to a single leaf which is eventually detached from the plant and assayed for gene expression. If the order of excision is reversed, from last in whole plant studies, to first in the detached study, the feeding experiment is simplified. Solanum tuberosum var. Kennebec is propagated by nodal transfer in a simple tissue culture medium and transferred to soil for further growth if desired. Leaves are excised from the parent plant and relocated to Petri dishes where the feeding assay is conducted with the larval stages of M. sexta. Damaged leaf tissue is assayed for the expression of relatively early events in signal transduction. Gene expression analysis identified infestation-specific Cys2-His2 (C2H2) transcription factors, confirming the success of using detached leaves in early response studies. The method is easier to perform than whole plant infestations and uses less space.

In vivo coupling of tau pathology and cortical thinning in Alzheimer's disease.

INTRODUCTION: The deposition of neurofibrillary tangles in neurodegenerative disorders is associated with neuronal loss on autopsy; however, their in vivo associations with atrophy across the continuum of Alzheimer's disease (AD) remain unclear. METHODS: We estimated cortical thickness, tau ([18F]-AV-1451), and amyloid ß (Aß) status ([11C]-PiB) in 47 subjects who were stratified into Aß- (14 healthy controls and six mild cognitive impairment-Aß-) and Aß+ (14 mild cognitive impairment-Aß+ and 13 AD) groups. RESULTS: Compared with the Aß- group, tau was increased in widespread regions whereas cortical thinning was restricted to the temporal cortices. Increased tau binding was associated with cortical thinning in each Aß group. Locally, regional tau was associated with temporoparietal atrophy. DISCUSSION: These findings position tau as a promising therapeutic target. Further studies are needed to elucidate the casual relationships between tau pathology and trajectories of atrophy in AD.

[18F]AV-1451 binding is increased in frontotemporal dementia due to C9orf72 expansion.

The PET ligand [18F]AV-1451 was developed to bind tau pathology in Alzheimer's disease, but increased binding has been shown in both genetic tauopathies and in semantic dementia, a disease strongly associated with TDP-43 pathology. Here we assessed [18F]AV-1451 binding in behavioral variant frontotemporal dementia due to a hexanucleotide repeat expansion in C9orf72, characterized by TDP-43 pathology. We show that the C9orf72 mutation increases binding in frontotemporal cortex, with a distinctive distribution of binding compared with healthy controls.

UMOD: A Device for Monitoring Postoperative Urination.

A Urine Monitoring Device (UMOD) has been designed and implemented for monitoring postoperative urination. This device has been created primarily to assist nurses and doctors monitor patients during their postoperative and recovery period. Furthermore, to reduce the burden of the nursing staff required to regularly monitor and empty the urine bags saving them precious time. The device consists of a stand and a load cell where the urine bag is attached. The stand is light and can easily move shall the patient require to move. An ESP Wi-Fi microprocessor module is used to calculate the rate of flow of urine in real time, identify and ignore any false readings due to accidental movements of the urine bag using an accelerometer and transmit the readings to a server / cloud through the local Wi-Fi.

A Small Cellulose-Binding-Domain Protein (CBD1) in Phytophthora is Highly Variable in the Non-binding Amino Terminus.

The small cellulose-binding-domain protein CBD1 is tightly bound to the cellulosic cell wall of the plant pathogenic stramenopile Phytophthora infestans. Transgene expression of the protein in potato plants also demonstrated binding to plant cell walls. A study was undertaken using 47 isolates of P. infestans from a worldwide collection, along with 17 other Phytophthora species and a related pathogen Plasmopara halstedii, to determine if the critical cell wall protein is subject to amino acid variability. Within the amino acid sequence of the secreted portion of CBD 1, encoded by the P. infestans isolates, 30 were identical with each other, and with P. mirabilis. Four isolates had one amino acid difference, each in a different location, while one isolate had two amino acid substitutions. The remaining 13 isolates had five amino acid changes that were each in identical locations (D17/G, D31/G, I32/S, T43/A, and G50/A), suggesting a single origin. Comparison of P. infestans CBD1 with other Phytophthora species identified extensive amino acid variation among the 60 amino acids at the amino terminus of the protein, and a high level of conservation from G61, where the critical cellulose-binding domain sequences begin, to the end of the protein (L110). While the region needed to bind to cellulose is conserved, the region that is available to interact with other cell wall components is subject to considerable variation, a feature that is evident even in the related genus Plasmopara. Specific changes can be used in determining intra- and inter-species relatedness. Application of this information allowed for the design of species-specific primers for PCR detection of P. infestans and P. sojae, by combining primers from the highly conserved and variable regions of the CBD1 gene.

The Artificial Pancreas: Reducing Safety Risk via Intra-Peritoneal Insulin Delivery.

The Artificial Pancreas is a medical device, currently in development, that aims to provide automatic regulation of blood glucose (BG) levels in ambulatory type 1 diabetes mellitus sufferers. The most popular approach utilises subcutaneous (SC) continuous glucose monitoring (CGM) combined with SC insulin injection controlled by a Model Predictive Control (MPC) algorithm. From a safety risk perspective problematic elements include the performance robustness of the CGM and the need for a pre-meal insulin bolus to provide satisfactory post prandial BG regulation. This contribution presents preliminary in silico studies which indicate that the use of an alternative approach to insulin delivery - intra-peritoneal (IP) infusion - could provide a similar level of BG regulation (as obtained with SC delivery), without the need for the pre-meal bolus. This simplified control architecture would remove the patient safety risk related to the administration (or not) of pre-meal bolus.

Telehealth and the Re-Design of Emergency Medical Services.

As patient numbers continue to rise Emergency Department's (ED's) are struggling to not only control patient wait times but also to maintain the quality of patient care. Improving patient flow through the ED has been a priority for many years with techniques such as Lean Six-Sigma being implemented specifically to help alleviate the problem. The Institute for Healthcare Improvement recently stated that the best opportunities to improving patient flow relate to the front-end of the ED, namely triage. This contribution examines the use of Telehealth initiatives at the front-end of the ED, specifically tele-consultation, to reduce patient loading, provide timelier healthcare (with improved patient outcomes) and reduce costs.

Totally Connected Healthcare with TV White Spaces.

Recent technological advances in electronics, wireless communications and low cost medical sensors generated a plethora of Wearable Medical Devices (WMDs), which are capable of generating considerably large amounts of new, unstructured real-time data. This contribution outlines how this data can be propagated to a healthcare system through the internet, using long distance Radio Access Networks (RANs) and proposes a novel communication system architecture employing White Space Devices (WSD) to provide seamless connectivity to its users. Initial findings indicate that the proposed communication system can facilitate broadband services over a large geographical area taking advantage of the freely available TV White Spaces (TVWS).

Cybersecurity and the Medical Device Product Development Lifecycle.

Protecting connected medical devices from evolving cyber related threats, requires a continuous lifecycle approach whereby cybersecurity is integrated within the product development lifecycle and both complements and re-enforces the safety risk management processes therein. This contribution reviews the guidance relating to medical device cybersecurity within the product development lifecycle.

Implications and Modelling of Data Quality on Confidence of Clinical Decision Support: A Conceptual Framework.

Integrated care paradigms depend on multiple sources of data. The quality of data used in decision-making will ultimately affect the delivered care to the patient. Quality includes several dimensions, which may affect the result. This paper presents how data quality dimensions may affect the delivered service, and propose a conceptual framework for the classification of confidence in data used in clinical decision-making for integrated care.

A framework for synthesis of safety justification for digitally enabled healthcare services.

BACKGROUND: Digitally enabled healthcare services combine socio-technical resources to deliver the required outcomes to patients. Unintended operation of these services may result in adverse effects to the patient. Eliminating avoidable harm requires a systematic way of analysing the causal conditions, identifying opportunities for intervention. Operators of such services may be required to justify, and communicate, their safety. For example, the UK Standardisation Committee for Care Information (SCCI) standards 0129 and 0160 require a safety justification for health IT (superseded versions were known as the Information Standards Board (ISB) 0129 & 0160. Initial as well as current standards are maintained by the NHS Digital. METHOD: A framework was designed, and applied as proof of concept, to an IT-supported clinical emergencies (A&E) service. Evaluation was done qualitatively based on the authors' experience, identifying potential benefits of the approach. RESULTS: The applied framework encapsulates analysis, and structures the generated information, into a skeleton of an evidence-based case for safety. The framework improved management of the safety activities, assigning ownership to stakeholders (e.g. IT developer), also creating a clear and compelling safety justification. CONCLUSIONS: Application of the framework significantly contributed to systematising an exploratory approach for analysing the service, in addition to existing methods such as reporting. Its application made the causal chain to harm more diaphanous. Constructing a safety case contributed to: (a) identifying potential assurance gaps, (b) planning production of information and evidence, and (c) communication of the justification by graphical unambiguous means.

The Challenges of Balancing Safety and Security in Implantable Medical Devices.

Modern Implantable Medical Devices (IMDs), implement capabilities that have contributed significantly to patient outcomes, as well as quality of life. The ever increasing connectivity of IMD's does raise security concerns though there are instances where implemented security measures might impact on patient safety. The paper discusses challenges of addressing both of these attributes in parallel.

Using Event Trees to Inform Quantitative Analysis of Healthcare Services.

The paper illustrates how event tree diagrams, used in safety engineering, can be applied to test the design of a healthcare service. Event tree diagrams can be employed to inform quantitative approaches to quality, by providing justification with respect to safety, of operational aspects to be monitored and measured.

Root Cause Analysis and Health Informatics.

Root Cause Analysis (RCA) is the most widely used system analysis tool for investigating safety related incidents in healthcare. This contribution reviews RCA techniques, using a Health Informatics example, and discusses barriers to their successful uptake by healthcare organisations. It is concluded that a critical assessment to examine the uptake and evaluate the success of RCA, and other safety related techniques, within healthcare is long overdue.

A cellulose binding domain protein restores female fertility when expressed in transgenic Bintje potato.

BACKGROUND: Expression of a gene encoding the family 1 cellulose binding domain protein CBD1, identified in the cellulosic cell wall of the potato late blight pathogen Phytophthora infestans, was tested in transgenic potato to determine if it had an influence on plant cell walls and resistance to late blight. RESULTS: Multiple regenerants of potato (cv. Bintje) were developed and selected for high expression of CBD 1 transcripts. Tests with detached leaflets showed no evidence of increased or decreased resistance to P. infestans, in comparison with the blight susceptible Bintje controls, however, changes in plant morphology were evident in CBD 1 transgenics. Plant height increases were evident, and most importantly, the ability to produce seed berries from a previously sterile cultivar. Immunolocalization of CBD 1 in seed berries revealed the presence throughout the tissue. While Bintje control plants are male and female sterile, CBD 1 transgenics were female fertile. Crosses made using pollen from the late blight resistant Sarpo Mira and transgenic CBD1 Bintje as the female parent demonstrated the ability to introgress P. infestans targeted resistance genes, as well as genes responsible for color and tuber shape, into Bintje germplasm. CONCLUSIONS: A family 1 cellulose-binding domain (CBD 1) encoding gene from the potato late blight pathogen P. infestans was used to develop transgenic Bintje potato plants. Transgenic plants became female fertile, allowing for a previously sterile cultivar to be used in breeding improvement. Selection for the absence of the CBD transgene in progeny should allow for immediate use of a genetically enhanced material. Potential for use in other Solanaceous crops is proposed.

Application of succulent plant leaves for Agrobacterium infiltration-mediated protein production.

When expressing plant cell wall degrading enzymes in the widely used tobacco (Nicotiana benthamiana) after Agrobacterium infiltration, difficulties arise due to the thin leaf structure. Thick leaved succulents, Kalanchoe blossfeldiana and Hylotelephium telephium, were tested as alternatives. A xyloglucanase, as well as a xyloglucanase inhibitor protein was successfully produced.

Herbivory responsive C2H2 zinc finger transcription factor protein StZFP2 from potato.

While C2H2 zinc finger transcription factors (TF) are often regulated by abiotic stress, their role during insect infestation has been overlooked. This study demonstrates that the transcripts of the zinc finger transcription factors StZFP1 and StZFP2 are induced in potato (Solanum tuberosum L.) upon infestation by either the generalist tobacco hornworm (THW, Manduca sexta L.) or the specialist Colorado potato beetle (CPB, Leptinotarsa decemlineata Say). StZFP1 has been previously characterized as conferring salt tolerance to transgenic tobacco and its transcript is induced by Phytophthora infestans and several abiotic stresses. StZFP2 has not been characterized previously, but contains the hallmarks of a C2H2 zinc finger TF, with two conserved zinc finger domains and DLN motif, which encodes a transcriptional repressor domain. Expression studies demonstrate that StZFP2 transcript is also induced by tobacco hornworm and Colorado potato beetle. These observations expand the role of the C2H2 transcription factor in potato to include the response to chewing insect pests.

Novel cellulose-binding-domain protein in Phytophthora is cell wall localized.

Cellulose binding domains (CBD) in the carbohydrate binding module family 1 (CBM1) are structurally conserved regions generally linked to catalytic regions of cellulolytic enzymes. While widespread amongst saprophytic fungi that subsist on plant cell wall polysaccharides, they are absent amongst most plant pathogenic fungal cellulases. A genome wide survey for CBM1 was performed on the highly destructive plant pathogen Phytophthora infestans, a fungal-like Stramenopile, to determine if it harbored cellulolytic enzymes with CBM1. Only five genes were found to encode CBM1, and none were associated with catalytic domains. Surveys of other genomes indicated that the CBM1-containing proteins, lacking other domains, represent a unique group of proteins largely confined to the Stramenopiles. Immunolocalization of one of these proteins, CBD1, indicated that it is embedded in the hyphal cell wall. Proteins with CBM1 domains can have plant host elicitor activity, but tests with Agrobacterium-mediated in planta expression and synthetic peptide infiltration failed to identify plant hypersensitive elicitation with CBD1. A structural basis for differential elicitor activity is proposed.

Genome sequence and analysis of the Irish potato famine pathogen Phytophthora infestans.

Phytophthora infestans is the most destructive pathogen of potato and a model organism for the oomycetes, a distinct lineage of fungus-like eukaryotes that are related to organisms such as brown algae and diatoms. As the agent of the Irish potato famine in the mid-nineteenth century, P. infestans has had a tremendous effect on human history, resulting in famine and population displacement. To this day, it affects world agriculture by causing the most destructive disease of potato, the fourth largest food crop and a critical alternative to the major cereal crops for feeding the world's population. Current annual worldwide potato crop losses due to late blight are conservatively estimated at $6.7 billion. Management of this devastating pathogen is challenged by its remarkable speed of adaptation to control strategies such as genetically resistant cultivars. Here we report the sequence of the P. infestans genome, which at approximately 240 megabases (Mb) is by far the largest and most complex genome sequenced so far in the chromalveolates. Its expansion results from a proliferation of repetitive DNA accounting for approximately 74% of the genome. Comparison with two other Phytophthora genomes showed rapid turnover and extensive expansion of specific families of secreted disease effector proteins, including many genes that are induced during infection or are predicted to have activities that alter host physiology. These fast-evolving effector genes are localized to highly dynamic and expanded regions of the P. infestans genome. This probably plays a crucial part in the rapid adaptability of the pathogen to host plants and underpins its evolutionary potential.