Delineation of Duchenne muscular dystrophy gene therapy using genetically engineered mice

Duchenne muscular dystrophy (DMD) is a genetically inherited debilitating muscle disorder affecting young boys due to the loss of dystrophin protein in muscle and the heart. Affected individuals lose their mobility and become confined to a wheel chair in early teens. They develop heart disease towards the end stage of the disease. Heart failure or breathing complications leads to death. Currently there is no cure for DMD. Gene therapy has shown great promise to restore the lost dystrophin protein in DMD. During my PhD, I have addressed several important aspects of dystrophin gene therapy. The findings from these studies will benefit development of an effective gene therapy for DMD and will open the door for some important future studies. Here I briefly describe the findings in my research. In the first study I addressed whether muscle-only rescue affects the heart. I used mice genetically engineered (alias transgenic) to carry dystrophin in skeletal muscle but not in the heart. To more closely mimic the human heart condition, I aged these mice to 23-months, an aged dystrophin-null mice show heart failure similar to that of DMD patients. Evaluation of heart pathology, ECG and pump function revealed that the heart in transgenic mice does not notably differ from the dystrophin deficient mice. In other words, selective muscle only treatment did not improve or heighten heart disease in DMD. In the next study I evaluated whether continuous therapeutic dystrophin expression is essential for muscle and heart health. To evaluate this in a mouse model, I created two mouse models carrying therapeutic dystrophin in the heart or muscle. These mice were engineered in a way that permits intentional removal of the therapeutic gene from the heart or muscle using viral mediated enzyme delivery. I delivered the viral mediated enzyme to muscle or the heart of the respective mouse strain in adult mice. After 12-15 months of therapeutic dystrophin removal, I evaluated muscle and the heart from individual strain. The removal of muscle therapeutic dystrophin resulted muscle deterioration, reduced muscle weight, size and force. The heart pump function was noticeably weakened after removal of cardiac therapeutic dystrophin. These findings indicated that uninterrupted therapeutic dystrophin expression is essential to maintain muscle and heart health. In the third study, I proposed, based on patient data, that dystrophin may contain a heart protection region between repeats 16-19 (R16-19) of dystrophin. I studied this by comparing the heart pathology, ECG and pump function in two transgenic mouse models that express dystrophin in the heart with or without R16-19. In support of my hypothesis, addition of R16-19 completely rescued ECG and corrected an important heart function parameter that were not rescued in mice that lack R16-19. Lastly, I looked at whether very low levels of dystrophin can benefit DMD heart. To test this, I used a mouse model referred to as mdx3cv that was genetically modified using chemical induced mutations. These mice expressed about 3.3% of dystrophin compared to normal mice. The heart of mdx3cv mice showed similar level of damage as dystrophin deficient mice. Surprisingly, ECG and hemodynamic function were improved in mdx3cv mice. These results suggest that marginal level dystrophin expression can still help the heart but it is far from sufficient for full recovery

Evolutionary Dynamics of Genomes and Endosymbionts in Plant-Parasitic Nematodes

Plant-parasitic nematodes cause more than US $100 billion in annual agriculture loss worldwide. Thorough knowledge of their genetic diversity, and interactions with endosymbionts and environment have the potential to provide valuable insights into the basic biology of these animals, and assist future efforts aimed at management of these plant parasites. In this dissertation, genetics and genomic strategies were utilized to advance our understanding of plant-parasitic nematode evolution and symbiosis. In chapter 2, a molecular-genetic strategy that targeted the 16S rRNA gene was used to assess the prevalence and distribution of two endosymbiotic bacteria, Wolbachia and Cardinium, in plant-parasitic Pratylenchus penetrans field populations collected from varying plant hosts. The study also explored the potential effects of Wolbachia on reproduction in this nematode species. Variable abundances and distributions for both endosymbionts were observed in the P. penetrans populations examined. The complete absence of Wolbachia in some populations and low prevalence in others suggest that this endosymbiont is not an obligate mutualist of P. penetrans, making this system distinct from the obligately mutualist Wolbachia-filarial nematode symbioses. The observed sex ratio bias in Wolbachia-infected nematode populations suggests that Wolbachia acts as a reproductive manipulator, also unlike the well-studied Wolbachia-filarial nematode symbiosis. In chapter 3, a genome skimming strategy was used to investigate Wolbachia and Cardinium occurrence in economically important plant-parasitic nematode populations representing twelve different species, including samples from North and South America. The study further investigated the population level genetic variability among the detected endosymbionts. Limited occurrence of Wolbachia (in P. penetrans) and Cardinium (in P. penetrans and Heterodera glycines) across plant-parasitic nematode species was observed suggesting non-obligatory function of both endosymbionts in these nematodes. Wolbachia genomes were highly similar across populations infecting P. penetrans. Cardinium displayed dissimilarities in their genomes from different nematode species, P. penetrans and H. glycines, suggesting potential to act in a species-specific manner in plant-parasitic nematodes. In chapter 4, population genetic analyses were performed to identify genome-wide patterns of genetic variation of the plant-parasitic nematode Globodera pallida across Idaho field populations to evaluate the origin, and potential evolutionary responses since the time of infestation. Most of the nematode genetic variation was shared among the Idaho fields, indicating that the infestation likely resulted from a single introduction. The significantly differentiated allele frequencies at certain loci among G. pallida populations across time suggested adaptive potential of this parasite invading new environments, and identified candidate genetic loci under positive selection. The dissertation provided important new insights into plant-parasitic nematode biology and evolution through providing information on (i) microbial endosymbionts that have the potential for future development in biological control applications, and (ii) evolutionary adaptive potential of plant-parasitic nematode populations invading new environments

Implementation of a perception system for autonomous vehicles using a detection-segmentation network in SoC FPGA

Perception and control systems for autonomous vehicles are an active area of scientific and industrial research. These solutions should be characterised by high efficiency in recognising obstacles and other environmental elements in different road conditions, real-time capability, and energy efficiency. Achieving such functionality requires an appropriate algorithm and a suitable computing platform. In this paper, we have used the MultiTaskV3 detection-segmentation network as the basis for a perception system that can perform both functionalities within a single architecture. It was appropriately trained, quantised, and implemented on the AMD Xilinx Kria KV260 Vision AI embedded platform. By using this device, it was possible to parallelise and accelerate the computations. Furthermore, the whole system consumes relatively little power compared to a CPU-based implementation (an average of 5 watts, compared to the minimum of 55 watts for weaker CPUs, and the small size (119mm x 140mm x 36mm) of the platform allows it to be used in devices where the amount of space available is limited. It also achieves an accuracy higher than 97% of the mAP (mean average precision) for object detection and above 90% of the mIoU (mean intersection over union) for image segmentation. The article also details the design of the Mecanum wheel vehicle, which was used to test the proposed solution in a mock-up city.Comment: The paper was accepted for the 19th International Symposium on Applied Reconfigurable Computing - ARC 2023, Cottbus - German

Towards an Open Source Localisation Orchestration Framework

This paper describes the design and implementation of a novel environment for the inter-connectivity of distributed localisation components - both open source and proprietary - in response to the scarcity of relevant research in this area, as we view interoperability as being the key to the seamless integration of different entities. The proposed solution promotes interoperability through the adoption of a Service Oriented Architecture (SOA) framework based on established localisation standards. We describe a generic use scenario and the architecture of the environment that allows us to study interoperability issues in localisation processes. This environment was successfully demonstrated at the CNGL Public Showcase in Microsoft, Ireland, November 2010.Aquest article descriu el disseny i la implementació d'un nou entorn per a la interconnectivitat de components de localització distribuïts, tant de codi obert com a propietari, donant així resposta a l'escassetat d'estudis d'investigació rellevants en aquesta àrea, ja que considerem que la interoperabilitat és clau per aconseguir la integració completa de diferents entitats. La solució que proposem permet la interoperabilitat mitjançant un esquema d'arquitectura orientada a serveis (SOA) que es basa en els estàndards de localització habituals. Descriurem un escenari d'ús genèric i l'arquitectura de l'entorn que ens permetrà estudiar qüestions d'interoperabilitat en els processos de localització. Aquest entorn va ser presentat amb èxit en el CNGL Public Showcase que es va celebrar a la seu de Microsoft, Irlanda, el novembre de 2010.Este artículo describe el diseño y la implementación de un nuevo entorno para la interconectividad de componentes de localización distribuidos, tanto de código abierto como propietario, con el propósito de paliar la escasez de estudios de investigación relevantes en esta área. A nuestro entender, la interoperabilidad es clave para conseguir la integración completa de diferentes entidades. La solución que proponemos permite la interoperabilidad mediante un esquema de arquitectura orientada a servicios (SOA) que se basa en los estándares de localización habituales. Describiremos un escenario de uso genérico y la arquitectura del entorno que nos permitirá estudiar cuestiones de interoperabilidad en los procesos de localización. Este entorno fue presentado con éxito en el CNGL Public Showcase que se celebró en la sede de Microsoft en Irlanda en noviembre de 2010

Delaying the Ripening of Banana (Musa sp.) cv Kandula through the Application of 1-Methylcyclopropane (1-MCP)

 Climacteric fruits are treated with 1-Methylcyclopropene (1-MCP) to delay the ripening process, thereby to reduce postharvest losses. This study evaluated the effectiveness of level of 1-MCP on delaying the ripening of Kandula banana. Banana hands were exposed to three levels of 1-MCP concentrations (0.5, 1.0 and 2.0 ppm) for 6 h and stored at 14±10C and 85-90% Relative Humidity (RH). The untreated samples (control) were stored under similar conditions. Peel colour, pulp firmness, total soluble solids (TSS), physiological weight loss and pulp to peel ratio were measured at initial stage and during storage at 4-day intervals. Peel colour of banana samples treated with different levels of 1-MCP remained light green in colour even on day 29 in storage whereas untreated samples fully ripened on day 21 in storage. Further, samples treated with different levels of 1-MCP (0.5, 1.0 and 2.0 ppm) resulted in a least change in a* and b* values compared to the control. Initial a* value (19.63±0.95) of the samples treated with 0.5, 1.0 and 2.0 ppm and the control reduced to -17.63±0.32, -17.30±0.53, -18.47±0.31 and -4.16±0.76 respectively on day 21 in storage. Initial b* value (36.36±1.15) of the samples treated 0.5, 1.0 and 2.0 ppm and the control changed to 37.13±0.32, 34.80±0.00, 36.40±0.35 and 49.26±1.61 respectively on day 21 in storage. Initial pulp firmness (29.20±1.68) of the samples treated with 0.5, 1.0 and 2.0 ppm and the control reduced to 28.10±2.91, 23.90±2.50, 25.20±2.05 and 5.00±0.70 N respectively on day 21 in storage. The TSS of the samples treated with 0.5, 1.0 and 2.0 ppm and the control were 4.80±0.50, 5.20±0.25, and 5.10±0.35 and 15.10±1.00 brix respectively on day 21 in storage. Percentage weight loss of the samples treated with 0.5, 1.0 and 2.0 ppm and the control were 2.08±0.05, 1.93±0.24, 2.04±0.14 and 2.56±1.70% respectively on day 21 in storage. Pulp to peel ratio of the treated samples and the control was not significantly (p˃0.05) different on day 21 in storage. Treating banana hands with 1- MCP was found to be successful in delaying of ripening. However, as no significant effect of different levelsof 1-MCP on delaying of ripening was evident, 0.5 ppm 1- MCP can be recommended to extend the postharvest life of Kandula banana beyond 29 days when stored at 14±10C

Hacia una plataforma de orquestación de procesos para la localización de código abierto.

Aquest article descriu el disseny i la implementació d'un nou entorn per a la interconnectivitat de components de localització distribuïts, tant de codi obert com a propietari, donant així resposta a l'escassetat d'estudis d'investigació rellevants en aquesta àrea, ja que considerem que la interoperabilitat és clau per aconseguir la integració completa de diferents entitats. La solució que proposem permet la interoperabilitat mitjançant un esquema d'arquitectura orientada a serveis (SOA) que es basa en els estàndards de localització habituals. Descriurem un escenari d'ús genèric i l'arquitectura de l'entorn que ens permetrà estudiar qüestions d'interoperabilitat en els processos de localització. Aquest entorn va ser presentat amb èxit en el CNGL Public Showcase que es va celebrar a la seu de Microsoft, Irlanda, el novembre de 2010.This paper describes the design and implementation of a novel environment for the inter-connectivity of distributed localisation components - both open source and proprietary - in response to the scarcity of relevant research in this area, as we view interoperability as being the key to the seamless integration of different entities. The proposed solution promotes interoperability through the adoption of a Service Oriented Architecture (SOA) framework based on established localisation standards. We describe a generic use scenario and the architecture of the environment that allows us to study interoperability issues in localisation processes. This environment was successfully demonstrated at the CNGL Public Showcase in Microsoft, Ireland, November 2010.Este artículo describe el diseño y la implementación de un nuevo entorno para la interconectividad de componentes de localización distribuidos, tanto de código abierto como propietario, con el propósito de paliar la escasez de estudios de investigación relevantes en esta área. A nuestro entender, la interoperabilidad es clave para conseguir la integración completa de diferentes entidades. La solución que proponemos permite la interoperabilidad mediante un esquema de arquitectura orientada a servicios (SOA) que se basa en los estándares de localización habituales. Describiremos un escenario de uso genérico y la arquitectura del entorno que nos permitirá estudiar cuestiones de interoperabilidad en los procesos de localización. Este entorno fue presentado con éxito en el CNGL Public Showcase que se celebró en la sede de Microsoft en Irlanda en noviembre de 2010

Detection-segmentation convolutional neural network for autonomous vehicle perception

Object detection and segmentation are two core modules of an autonomous vehicle perception system. They should have high efficiency and low latency while reducing computational complexity. Currently, the most commonly used algorithms are based on deep neural networks, which guarantee high efficiency but require high-performance computing platforms. In the case of autonomous vehicles, i.e. cars, but also drones, it is necessary to use embedded platforms with limited computing power, which makes it difficult to meet the requirements described above. A reduction in the complexity of the network can be achieved by using an appropriate: architecture, representation (reduced numerical precision, quantisation, pruning), and computing platform. In this paper, we focus on the first factor - the use of so-called detection-segmentation networks as a component of a perception system. We considered the task of segmenting the drivable area and road markings in combination with the detection of selected objects (pedestrians, traffic lights, and obstacles). We compared the performance of three different architectures described in the literature: MultiTask V3, HybridNets, and YOLOP. We conducted the experiments on a custom dataset consisting of approximately 500 images of the drivable area and lane markings, and 250 images of detected objects. Of the three methods analysed, MultiTask V3 proved to be the best, achieving 99% mAP_50 for detection, 97% MIoU for drivable area segmentation, and 91% MIoU for lane segmentation, as well as 124 fps on the RTX 3060 graphics card. This architecture is a good solution for embedded perception systems for autonomous vehicles. The code is available at: https://github.com/vision-agh/MMAR_2023.Comment: The paper was accepted for the MMAR 2023 conference (27th International Conference on Methods and Models in Automation and Robotics

Identification and Characterization of Macrophage Migration Inhibitory Factor from the American Dog Tick, Dermacentor Variabilis (Say)

This study identified and characterized macrophage migration inhibitory factor (MIF) from the American dog tick, Dermacentor variabilis. The gene expression, protein level changes and function of MIF was studied using a number of biochemical and molecular biology methods. The study further identified the phylogenetic relationship of MIF from different species using high throughput bioinformatics tools. The current study has identified and characterized the full length cDNA sequence of the macrophage migration inhibitory factor (MIF) from the American dog tick, D. variabilis and an up-regulation of the MIF gene expression during 0-96 hrs fed female D. variabilis ticks in both salivary glands and midgut tissues. The highest level of MIF expression was observed in midgut compared to the salivary glands. In contrast, the change in protein levels found in salivary glands and midgut tissues declined as the feeding progressed from 0-96 hrs. RNAi mediated gene silencing of D. variabilis MIF was shown to down-regulate the expression approximately by 90% compared to the buffer-injected controls in the salivary gland and midgut but gene silencing has not shown a negative impact on the ticks' ability to uptake blood meal. In both salivary gland and midgut, subolesin dsRNA injection down-regulated the MIF expression approximately by 50% compared to the buffer-injected control. The bioinformatics analysis showed the across species conservation of the MIF amino acid sequence in ticks, nematodes and insects. The phylogenetic analysis revealed that tick MIFs share a close evolutionary proximity to some parasitic nematode MIFs rather than insect MIF. The homology modeling identified the closest D. variabilis MIF homolog available by the analysis was performed was from T. spiralis and the identified model and D. variabilis shared the same highly conserved amino acids.Department of Entomology and Plant Patholog

Dissemination of Escherichia coli O157:h7 to the Spinach, Spinacea oleraceae Phylloplane by House Flies, Musca domestica L.

Insects may be involved in the dissemination of human pathogens to fresh produce. This study aimed to determine if E. coli O157:H7 could colonize the spinach phylloplane via regurgitation spots deposited by house flies after exposure to E. coli O157:H7 acquisition sources. Attachment and colonization of E. coli O157:H7 on the external body surfaces of the house flies was also studied. Flies were exposed to different acquisition sources with and without E. coli O157:H7. Exposed flies were transferred to spinach plants and the regurgitation spots on days 0, 4 and 8 were analyzed by relative quantitative PCR. Also bacteria were spotted onto spinach leaves subjected to relative qPCR to understand the fate of the E. coli O157:H7 without the insect involvement on the phylloplane from 0-12 days post inoculation. Exposed fly legs and heads were dissected and E. coli O157:H7 were enumerated by microbiological methods from 0-13 days post exposure. Mouthparts and legs of bacteria-exposed flies were examined by scanning electron microscopy. The relative qPCR of the regurgitation spots showed that the E. coli O157:H7 DNA concentration increased on day 4 which suggested that the bacteria multiplied within the regurgitation spots when flies acquired the bacteria from inoculated manure. The relative qPCR of the artificial spotting did not show any significant change in the E. coli O157:H7 DNA levels on the phylloplane. E. coli O157:H7 persisted on the fly external body surfaces for 13 days and a colonization period was suggested from days 2-6. Overall the research data suggested that fly regurgitation is an important mode of human pathogen dissemination under laboratory conditions. Regurgitant may be a potential nutrition source for the bacteria. Body hairs and pseudotracheae may be potential niches for the bacteria to survive until they reach a more suitable environment. These data show the potential of house flies to contaminate spinach under laboratory conditions. Future studies will be aimed on to study the bacterial survival after regurgitation under field conditions.Department of Entomology and Plant Patholog