Academic literature on the topic 'Pathway redirection'

We have previously reported that squalene overproducing yeast self-downregulate the expression of the ethanol pathway (non-essential pathway) to divert the metabolic flux to the squalene pathway. In this study, the effect of co-production of squalene and ethanol on other non-essential pathways (fusel alcohol pathway, FA) of Saccharomyces cerevisiae was evaluated. However, before that, 13 constitutive promoters, like IRA1p, PET9p, RHO1p, CMD1p, ATP16p, USA3p, RER2p, COQ1p, RIM1p, GRS1p, MAK5p, and BRN1p, were engineered using transcription factor bindings sites from strong promoters HHF2p (−300 to −669 bp) and TEF1p (−300 to −579 bp), and employed to co-overexpress squalene and ethanol pathways in S. cerevisiae. The FSE strain overexpressing the key genes of the squalene pathway accumulated 56.20 mg/L squalene, a 16.43-fold higher than wild type strain (WS). The biogenesis of lipid droplets was stimulated by overexpressing DGA1 and produced 106 mg/L squalene in the FSE strain. AFT1p and CTR1p repressible promoters were also characterized and employed to downregulate the expression of ERG1, which also enhanced the production of squalene in FSE strain up to 42.85- (148.67 mg/L) and 73.49-fold (255.11 mg/L) respectively. The FSE strain was further engineered by overexpressing the key genes of the ethanol pathway and produced 40.2 mg/mL ethanol in the FSE1 strain, 3.23-fold higher than the WS strain. The FSE1 strain also self-downregulated the expression of the FA pathway up to 73.9%, perhaps by downregulating the expression of GCN4 by 2.24-fold. We demonstrate the successful tuning of the strength of yeast promoters and highest coproduction of squalene and ethanol in yeast, and present GCN4 as a novel metabolic regulator that can be manipulated to divert the metabolic flux from the non-essential pathway to engineered pathways.

Abstract Introduction: Breast cancer research has advanced understanding of breast cancer development and progression greatly over the past few decades. Despite progress in research, breast cancer is the second leading cause of cancer death in North America and is the most frequent type of cancer for women. Better treatments and diagnostic tools have increased breast cancer survival rates, but there is still a fundamental lack of understanding in tumor progression. Previous studies show the normal mammary microenvironment can influence non-mammary cells and tumor-derived cancer cells to participate in normal mammary gland development. The tumorigenic cells lose their tumor-forming capabilities and are “redirected” into phenotypically normal, non-tumorigenic cells. The purpose of this study is to obtain knowledge of the mechanisms that play a role in cancer cell redirection and therefore be able manipulate those mechanisms for therapeutic treatment in a clinical setting. Our hypothesis is that microenvironmental elements control whether a tumorigenic cell will enter a redirected state. We have developed and validated an in vitro model to mimic the mammary microenvironment to study cancer cell redirection. We found that when cancer cells that overexpress HER2+ are redirected in our cancer redirection model, phospho-HER2+ is silenced. We use HER2+ phosphorylation as a marker for cancer cell redirection though not a mechanism for cancer cell redirection. Materials and Methods: HER2+ breast cancer cells and normal breast epithelial cells (BECs) were co-cultured in ratios of 1:1 or 1:50. Monocultures of breast cancer cells and BECs were used as controls. Western analysis and immunostaining was used to assess attenuation of HER2+ and phospho-HER2+ receptors and RNAseq was used for pathway analyses. Images were taken using a Leica confocal microscope. For comparison of 2 or more groups, a one-way analysis of variance was performed. A p-value of less than 0.05 was considered significant. Results and Discussion: We found that the redirected cells underwent a phenotype shift in which the redirected cells adopted a normal mammary epithelial phenotype based on gene expression profiles. Furthermore, when HER2+ breast cancer cells were redirected in vitro they lost their tumor-forming potential in vivo. Signal pathway analyses revealed that redirected cancer cells are adopting a normal phenotype compared to breast cancer cells. Conclusions: These results indicate that epithelial cells provide signals that influence HER2+ breast cancer cells to undergo a shift in phenotype. The phenotypic shift in cancer cell redirection includes multiple intracellular signaling pathways that may be the key towards effective cancer treatment. Acknowledgements: This research was supported by South Carolina Idea Networks of Biomedical Research Excellence (SC INBRE). Citation Format: Caroline J. Campbell, Brian W. Booth. Investigating the mechanisms of HER2+ breast cancer cell redirection [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2536.

Horizontal transfer of mobile genetic elements (MGEs) is a key aspect of the evolution of bacterial pathogens. Transduction by bacteriophages is especially important in this process. Bacteriophages—which assemble a machinery for efficient encapsidation and transfer of genetic material—often transfer MGEs and other chromosomal DNA in a more-or-less nonspecific low-frequency process known as generalized transduction. However, some MGEs have evolved highly specific mechanisms to take advantage of bacteriophages for their own propagation and high-frequency transfer while strongly interfering with phage production—“molecular piracy”. These mechanisms include the ability to sense the presence of a phage entering lytic growth, specific recognition and packaging of MGE genomes into phage capsids, and the redirection of the phage assembly pathway to form capsids with a size more appropriate for the size of the MGE. This review focuses on the process of assembly redirection, which has evolved convergently in many different MGEs from across the bacterial universe. The diverse mechanisms that exist suggest that size redirection is an evolutionarily advantageous strategy for many MGEs.

Transferrin receptor 2 (TfR2) is a homologue of transferrin receptor 1 (TfR1), the protein that delivers iron to cells through receptor-mediated endocytosis of diferric transferrin (Fe2Tf). TfR2 also binds Fe2Tf, but it seems to function primarily in the regulation of systemic iron homeostasis. In contrast to TfR1, the trafficking of TfR2 within the cell has not been extensively characterized. Previously, we showed that Fe2Tf increases TfR2 stability, suggesting that trafficking of TfR2 may be regulated by interaction with its ligand. In the present study, therefore, we sought to identify the mode of TfR2 degradation, to characterize TfR2 trafficking, and to determine how Fe2Tf stabilizes TfR2. Stabilization of TfR2 by bafilomycin implies that TfR2 traffics to the lysosome for degradation. Confocal microscopy reveals that treatment of cells with Fe2Tf increases the fraction of TfR2 localizing to recycling endosomes and decreases the fraction of TfR2 localizing to late endosomes. Mutational analysis of TfR2 shows that the mutation G679A, which blocks TfR2 binding to Fe2Tf, increases the rate of receptor turnover and prevents stabilization by Fe2Tf, indicating a direct role of Fe2Tf in TfR2 stabilization. The mutation Y23A in the cytoplasmic domain of TfR2 inhibits its internalization and degradation, implicating YQRV as an endocytic motif.

Le transfert adoptif de cellules T exprimant un récepteur chimérique reconnaissant un antigène (CAR), est un traitement qui génère des réponses impressionnantes dans les cancers hématologiques mais est beaucoup moins efficace pour le traitement de tumeurs solides. Les tumeurs solides modulent leur microenvironnement induisant des formes multiples d’immunosuppression qui inhibent l’efficacité des fonctions effectrices des cellules T ayant infiltrées la tumeur. Au cours de ma thèse, j’ai évalué le potentiel de deux stratégies pour améliorer les réponses anti-tumorales des cellules T CAR. La première se focalise sur l’étude du rôle potentiel des cellules immunes non T, exprimant un CAR sur la stimulation des fonctions et de la persistance de cellules T CAR+ dans le microenvironnement tumoral. Afin d’étudier la fonction des cellules CAR non T, nous avons généré un modèle de souris transgénique (vav-CAR) dans lequel les cellules immunes expriment un CAR reconnaissant l’antigène tumoral Her2 (ErbB2). Comme attendu, les cellules T CAR+ possèdent des fonctions anti-tumorales, mais nous avons aussi mis en évidence que les macrophages et les cellules NK exprimant le CAR montraient une réponse cytokinique, cytotoxique et phagocytiques spécifiques de l’antigène. De plus, en utilisant le modèle vav-CAR, nous avons démontré le potentiel des cellules immunes CAR+ dans le rejet des tumeurs et cela indépendamment des cellules T CD8+. Les cellules T CD4+ sont essentielles puisque leur élimination réduit considérablement les réponses anti-tumorales dans notre modèle vav-CAR. Il a été démontré que certaines sous-populations de cellules T auxiliaires participent aux réponses anti-tumorales avec les cellules Th1 et Th17 démontrant une efficacité plus robuste que les autres sous-populations. Notre deuxième stratégie s’est focalisée sur l’étude de l’impact du métabolisme au cours de la polarisation des cellules T CD4+ et plus particulièrement lors de la différenciation des cellules T CAR+ en cellules Th1. En effet, l’activation et différenciation des cellules T sont fortement associées à une augmentation des besoins métaboliques. Dans le microenvironnement tumoral, en raison de la forte demande en ressources de la tumeur, la déprivation en nutriments ainsi générée peut limiter l’accès aux nutriments d’autres types cellulaires et ainsi altérer le devenir et les fonctions des cellules immunes greffés infiltrant la tumeur. En conséquence, modifier les cellules immunes CAR+ afin qu’elles puissent résister à la compétition métabolique du microenvironnement tumoral pourrait leur permettre de conserver leurs fonctions effectrices. En étudiant l’impact de la déprivation en nutriments sur la différenciation des cellules T, nous avons trouvé que des concentrations limitantes en glutamine, l’acide aminé le plus abondant du plasma, inhibaient le potentiel des cellules T à se différencier vers la voie Th1 associée à la production d’IFNγ. Au contraire, cette condition favorisait la conversion de cellules T CD4 naïves en cellules régulatrices Foxp3+ ayant des fonctions suppressives (Tregs). De plus, nous avons montré que la présence d’un seul métabolite dérivé de la glutamine, l’α-ketoglutarate (αKG), suffisait à augmenter les fonctions effectrices anti-tumorales de plusieurs sous-types de cellules T auxiliaires CAR+, augmentant la production d’IFNγ et diminuant l’expression de FOXP3. Ainsi, durant ma thèse, j’ai développé un modèle murin vav-CAR, générant un outil permettant d’étudier et manipuler les fonctions de multiples populations de cellules immunitaires exprimant un CAR. Ce modèle permettra de promouvoir l’utilisation de cellules immunes optimisées exprimant un CAR dans le cadre d’immunothérapies dirigées contre des tumeurs solides. De plus, en utilisant ce modèle, nous avons identifié un métabolite de la glutamine, qui orchestre les réponses immunitaires au moyen d’une reprogrammation métabolique des cellules T CD4
The adoptive transfer of T cells expressing a chimeric antigen receptor (CAR) as a treatment for cancer has achieved impressive responses in haematological malignancies, but has been less successful in the treatment of solid tumors. The tumor microenvironment of solid tumors presents multiple forms of immunosuppression, inhibiting the efficient effector function of infiltrating anti-tumor T cells. During my PhD, we assessed the potential of two strategies to enhance the anti-tumor function of CAR T cells. The first focuses on the potential of other CAR-expressing immune subsets to stimulate CAR T cell function and persistence in the tumor microenvironment. To elucidate the function of CAR-expressing non-T lymphocytes, we generated a transgenic mouse model (vav-CAR) in which immune cells express a CAR against the Her2 (ErbB2) tumor antigen. As expected, CAR T cells harboured anti-tumor function but we also found that CAR-modified macrophages and natural killer cells (NKs) exhibited significant antigen specific cytokine secretion, cytotoxicity and phagocytosis. Moreover, using the vav-CAR model, we demonstrated the potential of CAR immune cells to mediate tumor rejection independently of CD8+ T cells. CD4+ T cells were critical for this response as their deletion severely abrogated the anti-tumor responses in our vav-CAR model. Distinct T helper subsets have been shown to participate to anti-tumor responses, with Th1 and Th17 cells demonstrating a more robust efficacy as compared to other T helper subsets. Our second strategy was focused on the impact of metabolism in the polarisation of CD4+ T cells, in particular the differentiation of CAR T cells to Th1 lineage. T cell activation and polarisation is highly associated with increased metabolic needs. Given that nutrient deprivation in the tumor microenvironment, due to a high demand of the tumor for resources, can limit the nutrients available for other cell types, the fate and function of adoptively transferred immune cells may be altered upon entering the tumor. Therefore, modifying CAR immune cells to resist metabolic suppression in the tumor microenvironment may help retain their effector functions. Upon assessing the effects of nutrient deprivation on T cell differentiation, we found that limiting concentrations of glutamine, the most abundant amino acid in the plasma, inhibited the potential of T cells to undergo Th1 differentiation with associated IFNγ secretion. Rather, this condition resulted in the conversion of naïve CD4+ T cells into suppressive Foxp3+ regulatory T cells (Tregs). Furthermore, we determined that a single glutamine-derived metabolite, α-ketoglutarate (αKG), enhanced the anti-tumor effector functions of multiple CAR T helper subsets, increasing the production of IFNγ and reducing FOXP3 expression.Thus, during my PhD, I generated a vav-CAR model, providing a platform in which the function of multiple CAR-bearing immune subsets can be studied and manipulated. This model will promote the utilisation of optimized CAR-bearing immune cells in adoptive immunotherapy for solid tumors. Furthermore, using the CAR model, we have identified a glutamine metabolite that orchestrates immune responses through the metabolic reprogramming of CD4 T cells

Hydrogen is an environmentally friendly and high energy caffier that could be a potential replacement for depleting fossil fuels. Fermentative hydrogen production (FHP) has received great interest in recent decades, as it offers a potential means of producing H₂ from a variety of renewable and waste resources via a low energy process. However, the commercial application of the FHP process has been hampered by its low yields. It becomes important to obtain a better understanding of metabolic pathways and their regulation in H₂-producing microorganisms. The aim of this work was to improve fundamental knowledge of the central metabolic flux network associated with FHP using Clostridium butyricum, and to restructure metabolic pathways to enhance hydrogen production yield. The metabolic network model was firstly constructed for C. butyricum and metabolic flux analysis (MFA) using this model was applied to predict metabolic flux distribution under varying initial glucose concentrations and operational pHs when the specific growth rate was chosen as the objective function. MFA results suggested that pH has a more significant effect on hydrogen production yield compare to the initial glucose concentration. These results also suggested that the phosphoenolpymvate (PEP), pyruvate and Acetyl CoA (AcCoA) nodes are not rigid nodes. MFA was found to be a useful tool to provide valuable information for optimization of the fermentative hydrogen production process and for future design of metabolic engineering strategies. The butyrate formation pathway was blocked using a shuttle plasmid pMTL007 containing a group II intron designed for targeting hbd, which encodes β-hydroxybuffil-CoA dehydrogenase in the C. butyricum 'W5 genome. A method for transforming the plasmid into C. butyricum was developed. Fermentation studies showed that the resulting hbd-deficient strain M3-1 performed less H₂ production with a substantial increase in ethanol production compared.to the wild type strain W5; while under nitrogen sparging conditions, M3-1 exhibited increased H₂ production with the simultaneouS decrease of ethanol production. These results indicated that H₂ production by C. butyricum may compete for reduced nicotinamide adenine dinucleotide (NADH) with the ethanol formation pathway. Homologs of all three subunits of a bifurcating hydrogenase from Thermotoga maritime were amplified from the strain W5, indicating that W5 may possess a potential bifurcating hydrogenase which utilized reduced ferredoxin and NADH simultaneously to produce H₂. The ethanol formation pathway was blocked by disrupting the acetaldehyde CoA dehydrogenase (ACDH) domain on aad (encoding a bifunctional aldehyde-alcohol dehydrogenase) using pMTL007C-E2. Fermentation studies showed that the aad-deficient strain M6 produced 484% more lactate, 32% more acetate, 9% less butyrate and 78% less H₂ than the wild type strain W5; while with the addition of sodium acetate (NaAc) to the culture of M6, carbon flux to the lactate formation pathway was redirected to the butyrate formation pathway, resulting in the increase of final H₂ yield from 0.94 mol/mol glucose to 1.65 mol/mol glucose. The results from this study have provided a better understanding of the metabolic flux network associated with hydrogen production by C. butyricum, and developed a genetic and metabolic approach to the enhancement of hydrogen production yield.
Thesis (Ph.D.) -- University of Adelaide, School of Earth and Environmental Sciences, 2012

Recently, calls have been made for an increased focus on successful development in young people and how optimal developmental pathways can be promoted. The concept of healthy functioning or positive development is particularly relevant to the emerging-adulthood period because of the significant potential for positive change and redirection of life pathways observed during this time. This chapter focuses on one empirically tested model of positive development in emerging adulthood developed with data from the Australian Temperament Project. Positive development is conceptualized as comprising the dimensions of civic action and engagement, trust and tolerance of others, trust in authorities and organizations, social competence, and life satisfaction. A growing body of research suggests that positive development in emerging adulthood is an important asset for young people, with distinct developmental antecedents and consequences for later functioning. The findings provide possible targets for interventions to promote healthy developmental pathways into adulthood.