433 research outputs found

    Engineering of Second-Generation Acoustic Reporter Genes

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    A major outstanding challenge in the fields of biological research, synthetic biology, and cell-based medicine is visualizing the functions of natural and engineered cells noninvasively inside opaque organisms. Ultrasound imaging has the potential to address this challenge as a widely available technique with a tissue penetration of several centimeters and spatial resolution below 100 Āµm. Recently, the first genetically encoded acoustic reporters were developed based on bacterial gas vesicles (GVs) to link ultrasound signals to molecular and cellular function. However, the properties of these first-generation acoustic reporter genes (ARGs) resulted in limited sensitivity and specificity for imaging gene expression in vivo. The goal of my thesis work has been to engineer second-generation ARGs with improved acoustic and expression phenotypes compared to the existing first-generation constructs. I took two complementary engineering approaches to developing these constructs: homolog screening and directed evolution, sometimes referred to as the ā€œnature and nurtureā€ of protein engineering. The resulting constructs offer major qualitative and quantitative improvements, including much stronger ultrasound contrast, the ability to produce nonlinear signals distinguishable from background tissue in vivo, stable long-term expression, and compatibility with in vitro multiplexed imaging. In collaboration with others in the lab, we demonstrate the capabilities of these next-generation ARGs by imaging in situ gene expression in mouse models of breast cancer and tumor-homing therapeutic bacteria, noninvasively revealing the unique spatial distributions of tumor growth and colonization by therapeutic cells in living subjects and providing real-time guidance for interventions such as needle biopsies. This thesis is organized as follows: in the first two chapters, I introduce the key background needed to understand both the importance and properties of ARGS, and how they have been and could be engineered. In the next two chapters, I detail specific efforts to engineer themā€”one involving the construction of a high-throughput, semi-automated setup for acoustic phenotyping of cells and its application to ARG directed evolution, and another involving the screening of several GV cluster homologs to identify ones suitable for use as improved ARGs. Finally, I conclude with insights gleaned from these two ARG engineering projects and suggestions for future ones. The approaches, results, and ideas presented in this thesis represent the current state-of-the-art in ARG engineering and application. While recent technology development in this field has unlocked exciting new use cases for ARGs in noninvasive biological imaging, most of their potential for basic science and disease diagnosis and treatment has yet to be realized.</p

    The Host-Microbiota Axis in Chronic Wound Healing

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    Chronic, non-healing skin wounds represent a substantial area of unmet clinical need, leading to debilitating morbidity and mortality in affected individuals. Due to their high prevalence and recurrence, chronic wounds pose a significant economic burden. Wound infection is a major component of healing pathology, with up to 70% of wound-associated lower limb amputations preceded by infection. Despite this, the wound microbiome remains poorly understood. Studies outlined in this thesis aimed to characterise the wound microbiome and explore the complex interactions that occur in the wound environment. Wound samples were analysed using a novel long-read nanopore sequencing-based approach that delivers quantitative species-level taxonomic identification. Clinical wound specimens were collected at both the point of lower-extremity amputation and via a pilot clinical trial evaluating extracorporeal shockwave therapy (ESWT) for wound healing. Combining microbial community composition, host tissue transcriptional (RNAseq) profiling, with clinical parameters has provided new insight into healing pathology. Specific commensal and pathogenic organisms appear mechanistically linked to healing, eliciting unique host response signatures. Patient- and site-specific shifts in microbial abundance and communitycomposition were observed in individuals with chronic wounds versus healthy skin. Transcriptional profiling (RNAseq) of the wound tissue revealed important insight into functional elements of the host-microbe interaction. Finally, ESWT was shown to confer beneficial effects on both cellular and microbial aspects of healing. High-resolution long-read sequencing offers clinically important genomic insights, including rapid wide-spectrum pathogen identification and antimicrobial resistance profiling, which are not possible using current culture-based diagnostic approaches. Thus, data presented in this thesis provides important new insight into complex host-microbe interactions within the wound microbiome, providing new and exciting future avenues for diagnostic and therapeutic approaches to wound management

    A High-Throughput and Genomics-Based Approach to Combat Antimicrobial Resistance

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    Antimicrobial resistance (AMR) is becoming an increasingly large threat to global health and economics. In 2019, there were approximately 1.27 million deaths directly attributable to bacterial AMR and 4.95 million deaths associated with bacterial AMR. These numbers are expected to increase to 10 million by the year 2050. The use of Adjuvant therapeutics has been proposed as a strategy to mitigate antimicrobial resistance. Adjuvants can help resensitize resistant bacteria to clinically-relevant antibiotics, while also prolonging resistance from occurring. Here I present two high-throughput screens: one that identifies robust adjuvant compounds that target resistant bacteria, and one that repurposes drug-like compounds for antimicrobial use against Gram-negative bacteria. From these screens, one lead adjuvant candidate and four repurposed drug-like antimicrobials were taken forward for a mix of analog generation studies, mechanistic studies, resistance evolution studies and genomic analysis. This work will help play a role in bringing novel therapies to the clinic and prolong the evolution of resistance from occurring

    Investigation of de novo mutations in human genomes using whole genome sequencing data

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    De novo mutations (DNMs) are novel mutations which occur for the first time in an offspring and are not inherited from the parents. High-Throughput Sequencing (HTS) technologies such as whole genome sequencing (WGS) and whole exome sequencing (WES) of trios have allowed the investigation of DNMs and their role in diseases. Increased contribution of DNMs in both rare monogenic and common complex disorders is now known. Identification of DNMs from WGS is challenging since the error rates in the HTS data are much higher than the expected DNM rate. To facilitate the evaluation of existing DNM callers and development of new callers, I developed TrioSim, the first automated tool to generate simulated WGS datasets for trios with a feature to spike-in DNMs in the offspring WGS data. Several computational methods have been developed to call DNMs from HTS data. I performed the first systematic evaluation of current DNM callers for WGS trio data using real dataset and simulated trio datasets and found that DNM callers have high sensitivity and can detect the majority of true DNMs. However, they suffer from very low specificity with thousands of false positive calls made by each caller. To address this, I developed MetaDeNovo, a consensus-based ensemble computational method to call DNMs using cloud-based technologies. MetaDeNovo is a fully automated methodology that utilises existing DNM callers and integrates their results. It demonstrates much higher specificity than all other callers while maintaining high sensitivity. Congenital Heart Disease (CHD) is the most common birth disorder worldwide. DNMs have been found to contribute to CHD causation. Most CHD cases are sporadic, suggesting role of DNMs in large proportion of them. I applied MetaDeNovo to detect DNMs in a WGS dataset of CHD trios to aid with genetic variant prioritisation. MetaDeNovo can dramatically reduce the number of false positive DNMs as compared to individual DNM callers. This has improved the current practices of identifying the genetic causes of disease in such cohorts. MetaDeNovo is applicable to other trio WGS datasets of other genetic diseases. This thesis has contributed new knowledge by in depth exploration of existing DNM callers, development of a novel tool (TrioSim) to simulate trio WGS data and an ensemble improved automated tool (MetaDeNovo) to identify DNMs with high specificity. MetaDeNovo demonstrates its use to identify disease-causing mutations in a trio analysis using WGS

    The Host-Microbiota Axis in Chronic Wound Healing

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    Chronic, non-healing skin wounds represent a substantial area of unmet clinical need, leading to debilitating morbidity and mortality in affected individuals. Due to their high prevalence and recurrence, chronic wounds pose a significant economic burden. Wound infection is a major component of healing pathology, with up to 70% of wound-associated lower limb amputations preceded by infection. Despite this, the wound microbiome remains poorly understood. Studies outlined in this thesis aimed to characterise the wound microbiome and explore the complex interactions that occur in the wound environment. Wound samples were analysed using a novel long-read nanopore sequencing-based approach that delivers quantitative species-level taxonomic identification. Clinical wound specimens were collected at both the point of lower-extremity amputation and via a pilot clinical trial evaluating extracorporeal shockwave therapy (ESWT) for wound healing. Combining microbial community composition, host tissue transcriptional (RNAseq) profiling, with clinical parameters has provided new insight into healing pathology. Specific commensal and pathogenic organisms appear mechanistically linked to healing, eliciting unique host response signatures. Patient- and site-specific shifts in microbial abundance and community composition were observed in individuals with chronic wounds versus healthy skin. Transcriptional profiling (RNAseq) of the wound tissue revealed important insight into functional elements of the host-microbe interaction. Finally, ESWT was shown to confer beneficial effects on both cellular and microbial aspects of healing. High-resolution long-read sequencing offers clinically important genomic insights, including rapid wide-spectrum pathogen identification and antimicrobial resistance profiling, which are not possible using current culture-based diagnostic approaches. Thus, data presented in this thesis provides important new insight into complex host-microbe interactions within the wound microbiome, providing new and exciting future avenues for diagnostic and therapeutic approaches to wound management

    Detection of the select agent Coniothyrium glycines, causal pathogen of red leaf blotch of soybeans using high-throughput sequencing data

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    The select agent Coniothyrium glycines, the causal pathogen of the disease red leaf blotch of soybeans, has not been identified in the U.S. Although C. glycines is listed as a select agent by the Federal Government, little information about its biology, evolution, and genomics is available, which poses a challenge to developing diagnostic tools. This research aimed to expand the general molecular and genomic knowledge of C. glycines and apply the generated information for detecting the pathogen using high-throughput sequencing (HTS) data. During this research, fourteen C. glycines isolates obtained from Zambia and Zimbabwe were used. A multilocus phylogenetic analysis was performed using two non-coding and two coding genes, revealing that isolates from matching locations form monophyletic clades. The results also suggested the movement of the fungus across borders since some isolates from different countries had the same common ancestor. Based on the topology of the phylogenetic tree, five representative isolates were selected, and their whole genome was assembled using Oxford Nanopore Technologies and Illumina sequencing data. Finally, the generated assemblies and the MiFiĀ® web application were used to develop and validate three e-probe sets for the detection and differentiation of C. glycines isolates. The limit of detection (LOD), sensitivity, and specificity was estimated using in silico, in vitro, and in vivo approaches. LOD was influenced by the number of e-probes, sequencing read length, and sequencing platform. Once the LOD was exceeded, the sensitivity and specificity were 100%, allowing a reliable detection and discrimination of C. glycines isolates. The obtained results contribute to the molecular and genomic knowledge of C. glycines, facilitating future research on this organism. Additionally, these findings provide valuable guidelines for using HTS-based e-probe detection and discrimination of C. glycines to improve current biosecurity measures

    Effective Utilization of Molecular Genetic Screening of Patients with Sickle Cell Disease and Beta Thalassemia Major in Saudi Arabia

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    Hereditary blood diseases are prevalent in the Kingdom of Saudi Arabia. The majority of these blood disorders are sickle cell disease and Ī²-thalassemia with variants located on the beta globin gene (HBB). Aim: To determine the profile of novel or previously reported causative mutations in more than 150 transfusion dependent individuals using TaqMan genotyping and next-generation DNA sequencing. In addition, I explored the genomic variation in a family with transfusion dependency but without a definitive genetic diagnosis related to HBB. I also attempted to detect unknown genetic variations in functionally related genes and applied in-silico analysis of the detected variants to propose candidate genes that may contribute to the severe etiology of thalassemia within a family. Methods: To identify HBB variants, I conducted Taqman genotyping tests using SCD, c.92+5G>C, c.92+1G>A, c.93-21G>A, c.27dupG, and c.118C>T as the most frequently identified HBB variants within the Saudi population. After that, targeted next generation sequencing was performed on samples with either negative or only heterozygous results for these variants. The use of different molecular techniques including MLPA alpha thalassemia, whole exome sequencing, cytoscan HD array, and whole genome sequencing was undertaken on samples that needed further investigation. Implementation of different data filtering approaches and several in-silico techniques were utilized to investigate the detected variants. Results: After Taqman genotyping of the 154 DNA samples, 100 samples were either homozygous or compound heterozygous for the most frequently known HBB variants. The rest of these samples were sequenced using targeted NGS and 20 different common and rare HBB variants were identified. Three out of the 154 samples did not have any apparent HBB mutation and further investigation was applied using additional molecular techniques. This led to the identification of two gene candidates, SMC5 and TALDO1, with possible novel associations in increasing the severity of clinical manifestation in transfusion-dependent patients with heterozygous pathogenic variant of beta thalassemia. Conclusion: Beta thalassemia is a heterogenous disease with a wide range of clinical severity and the steps towards identification of the underlying genetic cause of the phenotype is different from case to case and may require a combination of several molecular techniques. Therefore, the interaction of illness-causing variations with the rest of an individual's genome is crucial to gaining a complete understanding of the condition. Excellent detection rates in less time may be achieved with a specialized filtering technique and strategy, making this an option for primary laboratory workflow

    Investigation into calcium regulation and mitochondrial metabolism in chronic myeloid leukaemia

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    Chronic myeloid leukaemia (CML) is a myeloproliferative disease characterised by accumulation of myeloid cells in the bone marrow (BM) and blood circulation. On a molecular level, CML is caused by a chromosomal translocation between chromosome 9 and chromosome 22, resulting in a formation of deformed short chromosome called Philadelphia chromosome. This translocation results in a replacement of a negative regulator domain in Abelson (ABL) tyrosine kinase with breakpoint cluster region (BCR), leading to constitutive ABL kinase protein activity. Since the BCR-ABL kinase inhibitor imatinib was discovered in 2000, the survival rates of CML patients have dramatically improved. Nevertheless, not only mutations within BCR-ABL limit BCR-ABL inhibitor potency but also the fact that CML is a haematopoietic stem cell (HSC) driven disease (BCR-ABL positive HSCs are referred to as leukaemic stem cells; LSCs). LSCs adopt both intrinsic and BM extrinsic factors to resist current therapy and underpin the disease persistence experienced in the clinic. It has been previously reported that LSCs rely more on mitochondrial OXPHOS for their survival compared to their normal HSCs counterparts. Targeting such metabolic vulnerability sensitised those LSCs to eradication by BCR-ABL inhibitors. Since current OXPHOS inhibitors have drawbacks in the clinic, such as short half-life or residual side effects, screening for novel and safe OXPHOS inhibitors is critical. To deliver this approach, the Helgason lab conducted a drug repurposing screen to identify novel OXPHOS inhibitors which can be safely used in clinics. The drug screening analysis identified the Ca2+ channel blocker lomerizine as one of the most promising OXPHOS inhibitor. Ca2+ is one of the main ions in BM niche and has been shown to catalyse mitochondrial OXPHOS in HSCs. Our transcriptome analysis of LSCs revealed that Ca2+ channels such as TRPC6 and CACNA1D are upregulated in CD34+CD38- LSCs when compared with their normal counterparts. Also, stem cell enriched CML CD34+ cells have higher possession of ER mass and subsequent mitochondrial Ca2+ level than normal counterparts. Both in vitro and ex vivo studies using CML cell lines and CD34+ patient samples revealed that imatinib therapy does not affect the CACNA1D and TRPC6 mediated Ca2+ influx, which is targeted by lomerizine single treatment or when combined with imatinib. Also, by deleting genes using clustered regularly interspaced short palindromic repeats (CRISPR) Cas9, we confirmed the reliance of CML on TRPC6 and CACNA1D to provide ER and mitochondria with Ca2+ ions as a cofactor for tricyclic acid (TCA) cycle dehydrogenases. We also performed in vitro and ex vivo metabolomics, cell growth, and stem cell functional assays on CML cell lines and stem cell enriched patient samples that supported the impact of lomerizine on CML through inhibition of mitochondrial Ca2+ and impacting metabolism. Finally, we applied our findings to preclinical models of CML. We confirmed the effective combination between lomerizine and imatinib using well-established CML murine models where combination treatment enhanced survival of mice xenografted with CML KCL22 cells. Also, combination of lomerizine with imatinib significantly decreased level of LSCs in BM of mice transplanted with primary CD34+ CML cells. Overall, we showed for first time that Ca2+ influx via CACNA1D and TRPC6 is important for OXPHOS in therapy resistant LSCs, rendering them sensitive to the combination of lomerizine, as a Ca2+ channel blocker, and imatinib
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