Large-scale studies and biophysical analysis of systems involved in plant immunity.

The field of plant immunity has progressed significantly in the last decade, driven primarily by both forward and reverse genetics and to a lesser extent by molecular biology techniques. However, many unknowns still remain before a more complete picture of this system can be achieved, which hinders our capacity to develop biotechnological solutions to ensure food safety for our growing population. Some of the problems that still need to be tackled relate to the multi-system involvement of some proteins, the interrelation of the different hormones, such as in trade-off systems, and the challenges of translating existing molecular knowledge into crop protection strategies. The goal of this thesis was to develop new methods and to adapt existing ones to address the challenges and push the boundaries of our knowledge of plant immunity as a system. We have adapted ClueGO analyses to visualize functionally grouped Gene Ontology (GO) terms specific to Arabidopsis. We developed a transcription factor- coregulator identification strategy based on double-transcriptome analyses. Finally, we have adapted a biophysical method, differential scanning fluorimetry (DSF). We tested the usefulness of these methods by interrogating different immune proteins/genes of the model plant Arabidopsis thaliana. Here is a summary of the major results obtained. In the realm of basal immunity, we discovered that clade I TGA transcription factors positively regulate this system by repressing WRKY transcription factors, which are negative regulators of the process. Furthermore, we have demonstrated that clade I TGA integrates into the growth- immunity trade-off system regulated by brassinosteroids by antagonizing the brassinosteroids-dependent suppression of basal immunity. In the realm of systemic acquired resistance (SAR), we have demonstrated that clade I TGA recruits a specific novel glutaredoxin as a corepressor to dampen the expression of a set of SAR-regulated genes controlled by salicylic acid (SA) and the SAR-orchestrator, NPR1. Finally, we demonstrated that NPR1 binds SA and that this interaction leads to the destabilization of NPR1. More importantly, the method used to show the latter is scalable and can be used to develop novel chemistries capable of deploying plant immunity in the field

Biosensors for Diagnosis and Monitoring

Biosensor technologies have received a great amount of interest in recent decades, and this has especially been the case in recent years due to the health alert caused by the COVID-19 pandemic. The sensor platform market has grown in recent decades, and the COVID-19 outbreak has led to an increase in the demand for home diagnostics and point-of-care systems. With the evolution of biosensor technology towards portable platforms with a lower cost on-site analysis and a rapid selective and sensitive response, a larger market has opened up for this technology. The evolution of biosensor systems has the opportunity to change classic analysis towards real-time and in situ detection systems, with platforms such as point-of-care and wearables as well as implantable sensors to decentralize chemical and biological analysis, thus reducing industrial and medical costs. This book is dedicated to all the research related to biosensor technologies. Reviews, perspective articles, and research articles in different biosensing areas such as wearable sensors, point-of-care platforms, and pathogen detection for biomedical applications as well as environmental monitoring will introduce the reader to these relevant topics. This book is aimed at scientists and professionals working in the field of biosensors and also provides essential knowledge for students who want to enter the field

DNA Sequencing

This book illustrates methods of DNA sequencing and its application in plant, animal and medical sciences. It has two distinct sections. The one includes 2 chapters devoted to the DNA sequencing methods and the second includes 6 chapters focusing on various applications of this technology. The content of the articles presented in the book is guided by the knowledge and experience of the contributing authors. This book is intended to serve as an important resource and review to the researchers in the field of DNA sequencing

Single Cell Analysis

Cells are the most fundamental building block of all living organisms. The investigation of any type of disease mechanism and its progression still remains challenging due to cellular heterogeneity characteristics and physiological state of cells in a given population. The bulk measurement of millions of cells together can provide some general information on cells, but it cannot evolve the cellular heterogeneity and molecular dynamics in a certain cell population. Compared to this bulk or the average measurement of a large number of cells together, single-cell analysis can provide detailed information on each cell, which could assist in developing an understanding of the specific biological context of cells, such as tumor progression or issues around stem cells. Single-cell omics can provide valuable information about functional mutation and a copy number of variations of cells. Information from single-cell investigations can help to produce a better understanding of intracellular interactions and environmental responses of cellular organelles, which can be beneficial for therapeutics development and diagnostics purposes. This Special Issue is inviting articles related to single-cell analysis and its advantages, limitations, and future prospects regarding health benefits

Studies in the development and evaluation of in-house molecular tools to identify and characterise the Mycobacterium tuberculosis in resource limited settings

Munish Puri developed and validated in-house molecular based tools to identify and characterise the Mycobacterium tuberculosis (Mtb) from rural PNG. He found that TaqMan based assays could be used to identify the Mtb and that high resolution melt may play a role in screening for drug resistance mutations. These outcomes could be further developed to provide cost effective approaches to lab-based diagnosis of TB in resource poor settings

Are molecular tools solving the challenges posed by detection of plant pathogenic bacteria and viruses?

Plant pathogenic bacteria, phytoplasmas, viruses and viroids are difficult to control, and preventive measures are essential to minimize the losses they cause each year in different crops. In this context, rapid and accurate methods for detection and diagnosis of these plant pathogens are required to apply treatments, undertake agronomic measures or proceed with eradication practices, particularly for quarantine pathogens. In recent years, there has been an exponential increase in the number of protocols based on nucleic-acid tools being those based on PCR or RT-PCR now routinely applied worldwide. Nucleic acid extraction is still necessary in many cases and in practice inhibition problems are decreasing the theoretical sensitivity of molecular detection. For these reasons, integrated protocols that include the use of molecular techniques as screening methods, followed by confirmation by other techniques supported by different biological principles are advisable. Overall, molecular techniques based on different types of PCR amplification and very especially on real-time PCR are leading to high throughput, faster and more accurate detection methods for the most severe plant pathogens, with important benefits for agriculture. Other technologies, such as isothermal amplification, microarrays, etc. have great potential, but their practical development in plant pathology is still underway. Despite these advances, there are some unsolved problems concerning the detection of many plant pathogens due to their low titre in the plants, their uneven distribution, the existence of latent infections and the lack of validated sampling protocols. Research based on genomic advances and innovative detection methods as well as better knowledge of the pathogens' lifecycle, will facilitate their early and accurate detection, thus improving the sanitary status of cultivated plants in the near future

Application of rapid sequencing for the detection and epidemiology of respiratory pathogens

Lower respiratory tract infections (LRTI) are a leading cause of morbidity and mortality globally, and the rise of Antimicrobial Resistance (AMR) complicates their treatment. To achieve the best patient outcomes and avoid contributing to the rise of AMR, timely and appropriate antimicrobial treatment needs to be prescribed. However, the current gold standard for aetiological investigation of LRTIs (microbiological culture) is too slow to guide initial therapy. Clinical metagenomics (CMg) has emerged as a potential solution to this problem; however, existing methods are too laborious. In this study, we optimise our previously published CMg pipeline to achieve a sensitive workflow with a 3.5 hour turnaround time. Evaluating the workflow, we show efficient depletion (>99.8%) of host DNA with our new 15 minute host depletion method. Sensitivity and specificity are 90.5% and 62.5%, respectively, rising to 96.6% and 100% when qPCR is used to investigate discordance. We also show that 30 minutes of sequencing is sufficient to make an accurate pathogen call. For pathogen surveillance, targeted sequencing approaches are more appropriate. Sequencing of SARS-CoV-2 for genomic epidemiology became a valuable tool during the ongoing COVID-19 pandemic. However, early on, methods were low-throughput and inflexible. We responded to this by developing a high-throughput library preparation method, CoronaHiT, which can be used for sequencing SARS-CoV-2 on Illumina or Oxford Nanopore Technologies platforms. The method was shown to be cheap and accurate, while also being more robust for samples with lower viral loads. CoronaHiT has subsequently been used to sequence hundreds of thousands of SARS-CoV-2 genomes in the UK. In conclusion, we have developed and optimised two different approaches for investigating respiratory infections (CMg and targeted) for two different applications, demonstrating the potential of rapid sequencing. Methods like these will continue to reshape diagnostics and public health in the future

Evaluation of rapid low-cost colorimetric methods for diagnosis of multidrug-resistant tuberculosis in limited-resource settings

One third of the world\u27s population is currently infected with tuberculosis (TB), a consuming airborne disease whose main causative agent is Mycobacterium tuberculosis. The majority of these patients are found in the world\u27s poorest areas. Treatment of TB is a lengthy and demanding process utilizing a cocktail of powerful drugs; however, multidrug resistant TB (MDR-TB) strains, defined by resistance to both isoniazid (INH) and rifampicin (RIF), are now emerging worldwide and threatening disease control efforts. The major problem facing efforts to combat MDR-TB spread is its early detection. Conventional fairly affordable methods for drug resistance detection are based on solid culture and are highly time consuming (3-6 weeks in addition to initial pathogen culturing). On the other hand, the more rapid liquid culture-based automated systems are costly to set up and maintain while the very rapid molecular assays (hours to few days) are simply too complex and unaffordable and non-sustainable in limited resource settings. The objective of this work was to evaluate the performance of two liquid culture-based colorimetric assays for detection of drug resistance; nitrate reducate assay (NRA) and colorimetric redox indicator (CRI) methods for detection of MDR-TB. The assays were tested on mycobacterial isolates from Egyptian patients and their performance was compared with microscopic observation drug susceptibility assay (MODS) and the commercial automated culture system MGIT 960. Concordance was 96.7% for CRI and 93.3%, at almost one-tenth of the MGIT cost, and close to that of MODS without the need for an inverted microscope. The NRA format used in this study is more convenient and higher in throughput than the initially developed format. Additionally, DNA was extracted from the mycobacterial isolates and16S rDNA was amplified and sequenced to gain insight on the molecular diversity of Egyptian strains. Moreover, the molecular basis of strain resistance was investigated by DNA sequencing of the genes most commonly containing resistance conferring mutations. Analysis of the 16S rDNA sequencing results confirmed the identity of the samples as mycobacterium tuberculosis and suggested possible presence of two different strains. On the other hand, the analysis of the resistance related genes found common resistance conferring mutations in the MDR samples