Flow-Encoded Oxygen Control to Track the Time-Dependence of Molecular Changes Induced by Static or Cycling Hypoxia.
Ontology highlight
ABSTRACT: Detecting the effects of low oxygen on cell function is often dependent on monitoring the expression of a number of hypoxia markers. The time dependence of the appearance and stability of these markers varies between cell lines. Assessing cellular marker dynamics is also critical to determining how quickly cells respond to transient changes in oxygen levels that occurs with cycling hypoxia. We fabricated a manifold designed to use flow-encoding to produce sequential changes in gas mixtures delivered to a permeable-bottom 96-well plate. We show how this manifold and plate design can be used to expose cells to either static or cycling hypoxic conditions for eight different time periods thereby facilitating the study of the time-response of cells to altered oxygen environments. Using this device, we monitored the time-dependence of molecular changes in human PANC-1 pancreatic carcinoma and Caco-2 colon adenocarcinoma cells exposed to increasing periods of static or cycling hypoxia. Using immunohistochemistry, both cell lines show detectable levels of the marker protein hypoxia-inducible factor-1α (HIF-1α) after 3 h of exposure to static hypoxia. Cycling hypoxia increased the expression level of HIF-1α compared to static hypoxia. Both static and cycling hypoxia also increased glucose uptake and aldehyde dehydrogenase activity. This new device offers a facile screening approach to determine the kinetics of cellular alterations under varying oxygen conditions.
Project description:Tumor tissue contains a continuous distribution of static and dynamically changing oxygen environments with levels ranging from physiologically normal oxygen down to anoxia. However, in vitro studies are often performed under oxygen levels that are far higher than those found in vivo. A number of devices are available to alter the oxygen environment in cell culture, including designs from our laboratory. However, in our devices and most other designs, changing the media in order to feed or dose cells remains a disruptive factor in maintaining a consistent hypoxic environment. This report presents a novel 96-well plate design that recirculates the local oxygen environment to shield cells during media changes and facilitates toxicity studies of cells cultured under varying oxygen levels. The principle behind the design is presented and the response of human pancreatic cancer PANC-1 cells treated with tirapazamine and doxorubicin under eight different static or cycling oxygen levels was measured. As expected, tirapazamine is progressively more toxic as oxygen levels decrease but retains some toxicity as oxygen is cycled between hypoxic and normoxic levels. Doxorubicin sensitivity is largely unaffected by changing oxygen levels. This technology is ideal for assessing the effects of oxygen as a variable in toxicity screens.
Project description:Electroosmotic flow that involves one fluid displacing another fluid is commonly encountered in various microfludic applications and experiments, for example, current monitoring technique to determine zeta potential of microchannel. There is experimentally observed anomaly in such flow, namely, the displacement time is flow direction dependent, i.e., it depends if it is a high concentration fluid displacing a low concentration fluid, or vice versa. Thus, this investigation focuses on the displacement flow of two fluids with various concentration differences. The displacement time was determined experimentally with current monitoring method. It is concluded that the time required for a high concentration solution to displace a low concentration solution is smaller than the time required for a low concentration solution to displace a high concentration solution. The percentage displacement time difference increases with increasing concentration difference and independent of the length or width of the channel and the voltage applied. Hitherto, no theoretical analysis or numerical simulation has been conducted to explain this phenomenon. A numerical model based on finite element method was developed to explain the experimental observations. Simulations showed that the velocity profile and ion distribution deviate significantly from a single fluid electroosmotic flow. The distortion of ion distribution near the electrical double layer is responsible for the displacement time difference for the two different flow directions. The trends obtained from simulations agree with the experimental findings.
Project description:This study was conducted to analyze phenotypic and proteomic differences of two Lactiplantibacillus plantarum strains (WCFS1, model strain from human saliva, and CIP104448, stool isolate) when a biofilm was produced under static conditions (well researched), or with the addition of flow (novel).
Project description:Accurate radio frequency (RF)-based indoor localization systems are more and more applied during sports. The most accurate RF-based localization systems use ultra-wideband (UWB) technology; this is why this technology is the most prevalent. UWB positioning systems allow for an in-depth analysis of the performance of athletes during training and competition. There is no research available that investigates the feasibility of UWB technology for indoor track cycling. In this paper, we investigate the optimal position to mount the UWB hardware for that specific use case. Different positions on the bicycle and cyclist were evaluated based on accuracy, received power level, line-of-sight, maximum communication range, and comfort. Next to this, the energy consumption of our UWB system was evaluated. We found that the optimal hardware position was the lower back, with a median ranging error of 22 cm (infrastructure hardware placed at 2.3 m). The energy consumption of our UWB system is also taken into account. Applied to our setup with the hardware mounted at the lower back, the maximum communication range varies between 32.6 m and 43.8 m. This shows that UWB localization systems are suitable for indoor positioning of track cyclists.
Project description:IntroductionCerebral blood flow (CBF) is reduced in cognitively impaired (CI) Alzheimer's disease (AD) patients. We checked the sensitivity of time-encoded arterial spin labeling (te-ASL) in measuring CBF alterations in individuals with positive AD biomarkers and associations with relevant biomarkers in cognitively unimpaired (CU) individuals.MethodsWe compared te-ASL with single-postlabel delay (PLD) ASL in measuring CBF in 59 adults across the AD continuum, classified as CU amyloid beta (Aβ) negative (-), CU Aβ positive (+), and CI Aβ+. We sought associations of CBF with biomarkers of AD, cerebrovascular disease, synaptic dysfunction, neurodegeneration, and cognition in CU participants.Resultste-ASL was more sensitive at detecting CBF reduction in the CU Aβ+ and CI Aβ+ groups. In CU participants, lower CBF was associated with altered biomarkers of Aβ, tau, synaptic dysfunction, and neurodegeneration.DiscussionCBF reduction occurs early in the AD continuum. te-ASL is more sensitive than single-PLD ASL at detecting CBF changes in AD.HighlightsLower CBF can be detected in CU subjects in the early AD continuum. te-ASL is more sensitive than single-PLD ASL at detecting CBF alterations in AD. CBF is linked to biomarkers of AD, synaptic dysfunction, and neurodegeneration.
Project description:Hypoxia occurs in pathological conditions, such as cancer, as a result of the imbalance between oxygen supply and consumption by proliferating cells. HIFs are critical molecular mediators of the physiological response to hypoxia but also regulate multiple steps of carcinogenesis including tumor progression and metastasis. Recent data support that sumoylation, the covalent attachment of the Small Ubiquitin-related MOdifier (SUMO) to proteins, is involved in the activation of the hypoxic response and the ensuing signaling cascade. To gain insights into differences of the SUMO1 and SUMO2/3 proteome of HeLa cells under normoxia and cells grown for 48 h under hypoxic conditions, we employed endogenous SUMO-immunoprecipitation in combination with quantitative mass spectrometry (SILAC). The group of proteins whose abundance was increased both in the total proteome and in the SUMO IPs from hypoxic conditions was enriched in enzymes linked to the hypoxic response. In contrast, proteins whose SUMOylation status changed without concomitant change in abundance were predominantly transcriptions factors or transcription regulators. Particularly interesting was transcription factor TFAP2A (Activating enhancer binding Protein 2 alpha), whose sumoylation decreased on hypoxia. TFAP2A is known to interact with HIF-1 and we provide evidence that deSUMOylation of TFAP2A enhances the transcriptional activity of HIF-1 under hypoxic conditions. Overall, these results support the notion that SUMO-regulated signaling pathways contribute at many distinct levels to the cellular response to low oxygen.
Project description:Hypoxia inducible factor-1 (HIF1) is a stress-responsive nuclear transcription factor that is activated with a decrease in oxygen availability. HIF1 regulates the expression of genes involved in a cell's adaptation to hypoxic stress, including those with mitochondrial specific function. To gain a more comprehensive understanding of the role of HIF1 in mitochondrial homeostasis, we studied the link between hypoxia, HIF1 transactivation, and electron transport chain (ETC) function. We established immortalized mouse embryonic fibroblasts (MEFs) for HIF1α wild-type (WT) and null cells and tested whether HIF1α regulates mitochondrial respiration by modulating gene expressions of nuclear-encoded ETC components. High-throughput quantitative real-time polymerase chain reaction was performed to screen nuclear-encoded mitochondrial genes related to the ETC to identify those whose regulation was HIF1α-dependent. Our data suggest that HIF1α regulates transcription of cytochrome c oxidase (CcO) heart/muscle isoform 7a1 (Cox7a1) under hypoxia, where it is induced 1.5-2.5-fold, whereas Cox4i2 hypoxic induction was HIF1α-independent. We propose that adaptation to hypoxic stress of CcO as the main cellular oxygen consumer is mediated by induction of hypoxia-sensitive tissue-specific isoforms. We suggest that HIF1 plays a central role in maintaining homeostasis in cellular respiration during hypoxic stress via regulation of CcO activity.
Project description:Post-translational histone modifications play key roles in gene regulation, development, and differentiation, but their dynamics in living organisms remain almost completely unknown. To address this problem, we developed a genetically encoded system for tracking histone modifications by generating fluorescent modification-specific intracellular antibodies (mintbodies) that can be expressed in vivo. To demonstrate, an H3 lysine 9 acetylation specific mintbody (H3K9ac-mintbody) was engineered and stably expressed in human cells. In good agreement with the localization of its target acetylation, H3K9ac-mintbody was enriched in euchromatin, and its kinetics measurably changed upon treatment with a histone deacetylase inhibitor. We also generated transgenic fruit fly and zebrafish stably expressing H3K9ac-mintbody for in vivo tracking. Dramatic changes in H3K9ac-mintbody localization during Drosophila embryogenesis could highlight enhanced acetylation at the start of zygotic transcription around mitotic cycle 7. Together, this work demonstrates the broad potential of mintbody and lays the foundation for epigenetic analysis in vivo.
Project description:d9 and d12 Mks were either cultured statically or subjected to shear flow for 30 min; at d9, half the Mks were placed back in culture for 30 min (60 min time point) Megakaryocytes (Mks) are exposed to shear flow as they migrate from the bone marrow hematopoietic compartment into circulation thus releasing platelets and pro/preplatelets directly into the blood stream. Shear forces have been now established as promoting Mk maturation and platelet biogenesis. In order to understand the underlying mechanisms that modulate the response of Mks to shear forces, we carried out transcriptional analysis on immature and mature stem cell-derived Mks that were exposed to physiologically-relevant shear (2.5 dyn/cm2). In immature (d9) Mks, shear exposure upregulated genes related to growth and Mk maturation, while in mature (d12) Mks, it upregulated genes involved in apoptosis and intracellular transport. Following shear-flow exposure, 6 AP-1 transcripts (ATF4, JUNB, JUN, FOSB, FOS, and JUND) were upregulated at d9 and two AP-1 proteins (JunD and c-Fos) were upregulated both at d9 and d12. Our data show that MAPK signaling is linked to both the shear-stress response and AP-1 upregulation. JNK phosphorylation increased significantly following shear stimulation, while JNK inhibition reduced shear-induced JunD protein expression. Although p38 phosphorylation did not increase following shear flow, its inhibition reduced shear-induced JunD and c-Fos protein expression. JNK inhibition reduced fibrinogen binding of d9 and d12 platelet-like particle s (PLPs) and P-selectin expression at d12 PLPs, while p38 inhibition reduced fibrinogen binding of d12 PLPs. Here we show that mechanotransduction of shear forces in Mks results in JNK activation, AP-1 upregulation, and downstream transcriptional changes that promote maturation of immature Mks and platelet biogenesis in mature Mks.
Project description:d9 and d12 Mks were either cultured statically or subjected to shear flow for 30 min; at d9, half the Mks were placed back in culture for 30 min (60 min time point) Megakaryocytes (Mks) are exposed to shear flow as they migrate from the bone marrow hematopoietic compartment into circulation thus releasing platelets and pro/preplatelets directly into the blood stream. Shear forces have been now established as promoting Mk maturation and platelet biogenesis. In order to understand the underlying mechanisms that modulate the response of Mks to shear forces, we carried out transcriptional analysis on immature and mature stem cell-derived Mks that were exposed to physiologically-relevant shear (2.5 dyn/cm2). In immature (d9) Mks, shear exposure upregulated genes related to growth and Mk maturation, while in mature (d12) Mks, it upregulated genes involved in apoptosis and intracellular transport. Following shear-flow exposure, 6 AP-1 transcripts (ATF4, JUNB, JUN, FOSB, FOS, and JUND) were upregulated at d9 and two AP-1 proteins (JunD and c-Fos) were upregulated both at d9 and d12. Our data show that MAPK signaling is linked to both the shear-stress response and AP-1 upregulation. JNK phosphorylation increased significantly following shear stimulation, while JNK inhibition reduced shear-induced JunD protein expression. Although p38 phosphorylation did not increase following shear flow, its inhibition reduced shear-induced JunD and c-Fos protein expression. JNK inhibition reduced fibrinogen binding of d9 and d12 platelet-like particle s (PLPs) and P-selectin expression at d12 PLPs, while p38 inhibition reduced fibrinogen binding of d12 PLPs. Here we show that mechanotransduction of shear forces in Mks results in JNK activation, AP-1 upregulation, and downstream transcriptional changes that promote maturation of immature Mks and platelet biogenesis in mature Mks. Two- and Three-condition experiment (flow vs. static culture condition, d9 vs. d12, and 30 min vs. 60 min at d9); Biological replicates: 3; Technical replicates: 1 (dye-swap)