128 research outputs found
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Novel Approaches for the Application of Mitochondrial Respirometry in Human Studies
No full textThough mitochondrial research has become more prominent in the last forty years, most methodologies to study mitochondria have been focused on cell and animal models. Respirometry, the assessment of mitochondrial function measured as oxygen consumption by Complex IV of the electron transport chain, is the “gold standard” for mitochondrial assessment, yet it is rarely utilized in human samples. This is due to two fundamental methodological challenges: (1) samples for respirometry must be processed and ran within hours of acquisition on specialized and complex equipment, and (2) human sampling for respirometry must be acquired using invasive tissue biopsies. While we as a field realize the importance of oxygen consumption as a measurement of mitochondrial function, the current methodologies are now limiting the types of biological and clinical questions we can ask and answer. Addressing the first challenge preventing widespread adoption of respirometry, the recently developed RIFS (Respirometry In previously-Frozen Specimen) assay allows for the measurement of maximal respiratory capacity from cells or tissues which have been previously frozen. This allows samples to be processed and then stored or biobanked for later respirometry assessment, ultimately making respirometry methodologies significantly easier, cheaper, and more accessible. To the second challenge though, there has been minimal breakthrough.In this thesis we will cover novel methodologies overcoming challenges which are paramount to the expansion of respirometry methodologies for clinical applications. We will discuss the RIFS protocol in detail, describe the methodologies developed to perform a first-of-its-kind high-throughput and high-resolution respirometry study of the human brain, and will detail the development of a technique to measure mitochondrial respiration in cells human salivary leukocytes. Together, these chapters present novel methodologies designed to overcome the primary limitations of performing respirometry in human studies
Characterization of a bacteriocin with effectivity against Klebsiella
No full textWithin the last century, antibiotics have been discovered and proved to be successful in killing or inhibiting growth of bacteria. However, in its short history, antibiotics have been overprescribed and misused, which has caused many species of bacteria to become resistant to its effects. This has led to a dramatic increase in infections that cannot be resolved with antibiotics. Therefore, antimicrobial research has turned to finding new ways to counter bacterial infections such as understanding and utilizing polymicrobial interactions. Previous research in our laboratory has found that Klebsiella pneumoniae, a Gram-negative, rod-shaped bacterium that can cause pneumonia, possess inhibitory properties that reduce the growth of certain bacterial genus including Enterobacter, Citrobacter, and even other Klebsiella species. In the current research, our lab has demonstrated that supernatant from a specific strain of K. pneumoniae, which includes the bacteria’s metabolites and proteins without cells, have antimicrobial properties that inhibit growth of other strains of K. pneumoniae including K. Pneumoniae 9997 in both planktonic and biofilm growth. Development of a treatment that delivers the antimicrobial compound from K. pneumoniae to those with drug resistant infections could become an alternative treatment to antibiotics that have failed in treating infections
Architectural Components to Contribute to Social Interaction
No full textThe amount of people with feelings of loneliness has increased dramatically in recent years. Loneliness is an undesirable and horrible situation where one can feel completely abandoned. Solo dwellers and elderly are found to be the most vulnerable.Besides the devastating personal situation of people with feelings of loneliness, consequences of this problem also touches society.The main question of this research comprises the issue what architectural requirements a residential building should meet, in order to create a living environment that is designed to contribute to interaction between solo dwellers of different generations.From the problem statement, the target group emerged: single and isolated elderly and young adult and adult solo dwellers. The needs of this target group are examined into detail.The research thoughtfully examines how to stimulate interaction among this intended target group. This is considered on the level of spatial design as well as a psychological approach.In order to achieve a complete and accurate analysis, multiple theories and ideas from researchers, scientists, sociologists, and architects are consulted.In addition, existing projects and buildings were selected to obtain knowledge from existing buildings projects. During the research process, these contributed to gain information about the research subject as well as visualizing the knowledge gained from the literature and existing studies.Additionally, one of the selected building projects has been visited to conduct fieldwork research. This in order to experience the building physically and obtain first-hand experiences, views and opinions from residents.The study revealed that concerning social interaction, several factors are involved.BleijburchtAdvanced Dwelling: Ecology of InclusionArchitecture, Urbanism and Building Sciences | Advanced Housing Desig
Measuring CPT-1-mediated respiration in permeabilized cells and isolated mitochondria
No full textSummary: Carnitine palmitoyltransferase-1 (CPT-1) is a rate-controlling enzyme for long-chain fatty acid oxidation. This manuscript provides protocols for measuring CPT-1-mediated respiration in permeabilized, adherent cell monolayers and mitochondria freshly isolated from tissue, along with examples to assess the potency and specificity of interventions targeting CPT-1. Strengths of the approach include ease, speed, and breadth of analysis, whereas drawbacks include loss of physiological regulation in reductionist systems and indirect assessment of CPT-1 enzymatic activity.For complete details on the use and execution of this protocol, please refer to Divakaruni et al. (2018)
Abstract B37: Targeting the energy generation triangle to achieve synthetic lethality for treatment of hepatocellular carcinoma
No full textAbstract
Despite increasing prevalence and lethality for hepatocellular carcinoma (HCC), the need for effective treatment remains unmet. Metabolic reprogramming during hepatocarcinogenesis promotes cancer cell growth and survival, but also suggests the possibility of identifying as yet unexplored drug targets. In this study, we developed a synthetic lethal drug combination to treat HCC through the inhibition of the energy generation triangle. Most cancers, including HCC, increase glycolysis (the Warburg effect) to ensure sufficient supplies of energy (ATP), reducing equivalents (NADPH), and biochemical building blocks for cell growth and proliferation. Unlike most tumors, which express both hexokinase 1 (HK1) and HK2, many HCC tumors express only HK2, whereas normal hepatocytes only express HK4. We investigated the role of HK2 in HCC using a doxycycline (DOX)-inducible shRNA knockdown system. The HK4-to-HK2 isoform switch during hepatocarcinogenesis results in high sensitivity to HK2 silencing-induced glycolysis inhibition and cytostasis in HCC cells, in contrast to cancer cells expressing both HK1 and HK2. We performed a high throughput screen (HTS) of 3,205 drug-like small molecules and 119 FDA-approved oncology drugs to identify synthetic lethal partners of HK2 knockdown, using the CellTiter-Glo assay for primary HTS and confirmed with the alamarBlue assay. Diphenyleneiodonium (DPI), a mitochondrial complex-I inhibitor, is the best compound in our HTS that shows synthetic lethality in combination with HK2 silencing/inhibition in HCC cells that express only HK2. Cell proliferation was measured by the MTT assay and cell counting. Cytotoxicity was measured using Trypan blue to distinguish live and dead cells. HK2 activity in xenograft tumors was monitored non-invasively using 18F-fludeoxyglucose PET/CT scans. Perhexiline (PER), a fatty acid oxidation inhibitor, further sensitizes HCC cells to the complex-I/HK2-targeted combination. Cellular ATP levels were examined by a luciferase activity assay, oxygen consumption was examined by a Seahorse respirometry assay, and cell metabolism was examined by a metabolomics analysis. The inclusion PER significantly enhances the reduction of HCC cellular energy production caused by the combination of HK2 inhibition and DPI, and the efficacy of the complex-I/HK2-targeted combination in treating HCC tumors in vivo. Our findings indicate that selective inhibition of the energy generation triangle (glycolysis, oxidative phosphorylation, and fatty acid oxidation), due to the unique presence of only the HK2 isoform, appears promising for HCC therapy.
Citation Format: Shili Xu, Arthur Catapang, Daniel Braas, Linsey Stiles, Jason T. Lee, Thomas G. Graeber, Robert Damoiseaux, Orian Shirihai, Harvey R. Herschman. Targeting the energy generation triangle to achieve synthetic lethality for treatment of hepatocellular carcinoma [abstract]. In: Proceedings of the AACR Precision Medicine Series: Opportunities and Challenges of Exploiting Synthetic Lethality in Cancer; Jan 4-7, 2017; San Diego, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2017;16(10 Suppl):Abstract nr B37.</jats:p
Sex differences in the neuronal transcriptome and synaptic mitochondrial function in the cerebral cortex of a multiple sclerosis model
No full textIntroductionMultiple sclerosis (MS) affects the cerebral cortex, inducing cortical atrophy and neuronal and synaptic pathology. Despite the fact that women are more susceptible to getting MS, men with MS have worse disability progression. Here, sex differences in neurodegenerative mechanisms are determined in the cerebral cortex using the MS model, chronic experimental autoimmune encephalomyelitis (EAE).MethodsNeurons from cerebral cortex tissues of chronic EAE, as well as age-matched healthy control, male and female mice underwent RNA sequencing and gene expression analyses using RiboTag technology. The morphology of mitochondria in neurons of cerebral cortex was assessed using Thy1-CFP-MitoS mice. Oxygen consumption rates were determined using mitochondrial respirometry assays from intact as well as permeabilized synaptosomes.ResultsRNA sequencing of neurons in cerebral cortex during chronic EAE in C57BL/6 mice showed robust differential gene expression in male EAE compared to male healthy controls. In contrast, there were few differences in female EAE compared to female healthy controls. The most enriched differential gene expression pathways in male mice during EAE were mitochondrial dysfunction and oxidative phosphorylation. Mitochondrial morphology in neurons showed significant abnormalities in the cerebral cortex of EAE males, but not EAE females. Regarding function, synaptosomes isolated from cerebral cortex of male, but not female, EAE mice demonstrated significantly decreased oxygen consumption rates during respirometry assays.DiscussionCortical neuronal transcriptomics, mitochondrial morphology, and functional respirometry assays in synaptosomes revealed worse neurodegeneration in male EAE mice. This is consistent with worse neurodegeneration in MS men and reveals a model and a target to develop treatments to prevent cortical neurodegeneration and mitigate disability progression in MS men
The Effect of Natural Language Processing in Bioinspired Design
No full textBioinspired design methods are a new and evolving collection of techniques used to extract biological principles from nature to solve engineering problems. The application of bioinspired design methods is typically confined to existing problems encountered in new product design or redesign. A primary goal of this research is to utilize existing bioinspired design methods to solve a complex engineering problem to examine the versatility of the method in solving new problems. Here, current bioinspired design methods are applied to seek a biologically inspired solution to geoengineering. Bioinspired solutions developed in the case study include droplet density shields, phosphorescent mineral injection, and reflective orbiting satellites. The success of the methods in the case study indicates that bioinspired design methods have the potential to solve new problems and provide a platform of innovation for old problems.
A secondary goal of this research is to help engineers use bioinspired design methods more efficiently by reducing post-processing time and eliminating the need for extensive knowledge of biological terminology by applying natural language processing techniques. Using the complex problem of geoengineering, a hypothesis is developed that asserts the usefulness of nouns in creating higher quality solutions. A designation is made between the types of nouns in a sentence, primary and spatial, and the hypothesis is refined to state that primary nouns are the most influential part of speech in providing biological inspiration for high quality ideas. Through three design experiments, the author determines that engineers are more likely to develop a higher quality solution using the primary noun in a given passage of biological text.
The identification of primary nouns through part of speech tagging will provide engineers an analogous biological system without extensive analysis of the results. The use of noun identification to improve the efficiency of bioinspired design method applications is a new concept and is the primary contribution of this research
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