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Aerobic Cometabolism of 1,4-Dioxane and cis-Dichloroethylene by Gellan Gum and Polyvinyl Alcohol/Sodium Alginate Hydrogel Beads Co-Encapsulated with Rhodococcus rhodochrous Strain ATCC 21198 and Slow-Release Growth Substrates: Batch, Column, and Toxicity Studies
1,4-Dioxane (dioxane) and cis-dichloroethylene (cDCE) are compounds commonly found in industrial cleaning and degreasing agents that are frequently present as groundwater contaminants. In an effort to develop a more effective treatment method for these compounds, hydrogel beads were fabricated with either gellan gum or a combination of polyvinyl alcohol (PVA) and sodium alginate (SA) co-encapsulated with Rhodococcus rhodochrous Strain ATCC 21198 (21198) and a slow-release growth substrate (SRS). The hydrogel beads were investigated in batch reactors and column systems to determine the long-term ability of these beads to successfully transform dioxane and cDCE through aerobic cometabolism. Tetra-butylorthosilicate (TBOS) and tetra-s-butylorthosilicate (T2BOS) were used to slowly release 1-butanol and 2-butanol, respectively, as growth substrates for 21198.
The batch reactor studies conducted using gellan gum hydrogel beads co-encapsulated with 21198 and either TBOS or T2BOS demonstrated an ability to transform essentially all (>99%) of the added dioxane at a range of ~250-1000 µg/L and the added cDCE at ~250 µg/L throughout several additions. The beads containing TBOS showed significantly faster rates of transformation for both contaminants. An embryonic zebrafish toxicity study completed using the TBOS gellan gum beads showed no significant toxicity was generated after the complete transformation of cDCE.
A column study was completed by packing a glass column with gellan gum beads co-encapsulated with 21198 and T2BOS. The influent media was composed of 10x diluted phosphate buffer mineral salt media (MSM) and added contaminants. Hydrogen peroxide (H2O2) was added to the influent media at concentrations of 25-100 mg/L to meet oxygen demands. The column had a hydraulic retention time (HRT) of 26 hours. This column initially demonstrated the ability to transform essentially all (>99%) influent cDCE (~300 µg/L) from 52 to 75 pore volumes (PV), while showing no evidence of transforming influent dioxane (~200 µg/L). A high-flow study was completed in which the influent flow rate was increased from 1 mL/hr to 90 mL/hr. This study provided evidence that cometabolism of cDCE was occurring due to the presence of cDCE epoxide in the effluent, but it also exposed the entire bead pack to the epoxide. This epoxide likely damaged the health of the column, and the column was unable to recover from this high-flow transient test and a subsequent stop-flow study. This was evidenced by a decrease in cDCE transformation. By the end of the column’s operation at 118 PV, a mass balance determined that a total of 71.5% of the influent cDCE was transformed. However, none of the influent dioxane was transformed.
A column study was also completed by packing a glass column with side sampling ports with PVA/SA beads co-encapsulated with 21198 and TBOS. The influent media was made using a synthetic groundwater formulation and 200x diluted phosphate buffer MSM for additional nutrients. H2O2 was added at increasing concentrations (50-250 mg/L) over the operating period to meet oxygen demands. The influent pH was increased from 7.0 to 7.5 to 7.9 during the operating period in an effort to lower the rate of TBOS hydrolysis to 1-butanol. The column initially had an HRT of 28 hours from 0 to 7 PV. The flow rate was then doubled and the column had an HRT of 14 hours for the final 90 PV. The column demonstrated the ability to transform >95% of influent cDCE (~200 µg/L) when the influent media contained 250 mg/L H2O2 and was at a pH of 7.5. Side port sampling showed the presence of cDCE epoxide throughout the column, signifying the aerobic cometabolism of cDCE. There was evidence that nearly all of the transformation occurred directly at the influent end of the column. A visual orange tint, representing significant 21198 biostimulation in the bead, was evident mainly in that end of the column. By the end of the column’s operating period, samples from the lower side sampling port located near the influent also measured nearly equal in cDCE concentration to the column effluent, indicating that the majority of cometabolic transformation was occurring where visual biomass growth was observed. 1-butanol remained >50 mg/L in the effluent throughout the column’s operating period, indicating biostimulation did not occur throughout the column. Embryonic zebrafish toxicity studies demonstrated that the column effluent was significantly toxic when 1-butanol levels were ~120 mg/L and not significantly toxic when ~60 mg/L, suggesting that 1-butanol was the source of toxicity. More detailed studies are needed to establish factors causing the observed toxicity in the column effluent
Electrical coupling between ventricular myocytes and myofibroblasts in the infarcted mouse heart
Aims:
Recent studies have demonstrated electrotonic coupling between scar tissue and the surrounding myocardium in cryoinjured hearts. However, the electrical dynamics occurring at the myocyte-nonmyocyte interface in the fibrotic heart remain undefined. Here, we sought to develop an assay to interrogate the nonmyocyte cell type contributing to heterocellular coupling and to characterize, on a cellular scale, its voltage response in the infarct border zone of living hearts.
Methods and results:
We used two-photon laser scanning microscopy in conjunction with a voltage-sensitive dye to record transmembrane voltage changes simultaneously from cardiomyocytes and adjoined nonmyocytes in Langendorff-perfused mouse hearts with healing myocardial infarction. Transgenic mice with cardiomyocyte-restricted expression of a green fluorescent reporter protein underwent permanent coronary artery ligation and their hearts were subjected to voltage imaging 7-10 days later. Reporter-negative cells, i.e. nonmyocytes, in the infarct border zone exhibited depolarizing transients at a 1:1 coupling ratio with action potentials recorded simultaneously from adjacent, reporter-positive ventricular myocytes. The electrotonic responses in the nonmyocytes exhibited slower rates of de- and repolarization compared to the action potential waveform of juxtaposed myocytes. Voltage imaging in infarcted hearts expressing a fluorescent reporter specifically in myofibroblasts revealed that the latter were electrically coupled to border zone myocytes. Their voltage transient properties were indistinguishable from those of nonmyocytes in hearts with cardiomyocyte-restricted reporter expression. The density of connexin43 expression at myofibroblast-cardiomyocyte junctions was ∼5% of that in the intercalated disc regions of paired ventricular myocytes in the remote, uninjured myocardium, whereas the ratio of connexin45 to connexin43 expression levels at heterocellular contacts was ∼1%.
Conclusion:
Myofibroblasts contribute to the population of electrically coupled nonmyocytes in the infarct border zone. The slower kinetics of myofibroblast voltage responses may reflect low electrical conductivity across heterocellular junctions, in accordance with the paucity of connexin expression at myofibroblast-cardiomyocyte contacts
Intravital FRAP imaging using an E-cadherin-GFP mouse reveals disease- and drug-dependent dynamic regulation of cell-cell junctions in live tissue
E-cadherin-mediated cell-cell junctions play a prominent role in maintaining the epithelial architecture. The disruption or deregulation of these adhesions in cancer can lead to the collapse of tumor epithelia that precedes invasion and subsequent metastasis. Here we generated an E-cadherin-GFP mouse that enables intravital photobleaching and
quantification of E-cadherin mobility in live tissue without affecting normal biology. We demonstrate the broad applications of this mouse by examining
E-cadherin regulation in multiple tissues, including mammary, brain, liver, and kidney tissue, while specifically monitoring E-cadherin mobility during
disease progression in the pancreas. We assess E-cadherin stability in native pancreatic tissue upon genetic manipulation involving Kras and p53
or in response to anti-invasive drug treatment and gain insights into the dynamic remodeling of E-cadherin during in situ cancer progression. FRAP in the E-cadherin-GFP mouse, therefore, promises to be a valuable tool to fundamentally expand our understanding of E-cadherin-mediated events in native microenvironments
Three-dimensional organotypic matrices from alternative collagen sources as pre-clinical models for cell biology.
Organotypic co-cultures bridge the gap between standard two-dimensional culture and mouse models. Such assays increase the fidelity of pre-clinical studies, to better inform lead compound development and address the increasing attrition rates of lead compounds within the pharmaceutical industry, which are often a result of screening in less faithful two-dimensional models. Using large-scale acid-extraction techniques, we demonstrate a step-by-step process to isolate collagen I from commercially available animal byproducts. Using the well-established rat tail tendon collagen as a benchmark, we apply our novel kangaroo tail tendon collagen as an alternative collagen source for our screening-ready three-dimensional organotypic co-culture platform. Both collagen sources showed equal applicability for invasive, proliferative or survival assessment of well-established cancer models and clinically relevant patient-derived cancer cell lines. Additional readouts were also demonstrated when comparing these alternative collagen sources for stromal contributions to stiffness, organization and ultrastructure via atomic force microscopy, second harmonic generation imaging and scanning electron microscopy, among other vital biological readouts, where only minor differences were found between the preparations. Organotypic co-cultures represent an easy, affordable and scalable model to investigate drug responses within a physiologically relevant 3D platform
The Effects of Two Types of Sleep Deprivation on Visual Working Memory Capacity and Filtering Efficiency
Sleep deprivation has adverse consequences for a variety of cognitive functions. The exact effects of sleep deprivation, though, are dependent upon the cognitive process examined. Within working memory, for example, some component processes are more vulnerable to sleep deprivation than others. Additionally, the differential impacts on cognition of different types of sleep deprivation have not been well studied. The aim of this study was to examine the effects of one night of total sleep deprivation and 4 nights of partial sleep deprivation (4 hours in bed/night) on two components of visual working memory: capacity and filtering efficiency. Forty-four healthy young adults were randomly assigned to one of the two sleep deprivation conditions. All participants were studied: 1) in a well-rested condition (following 6 nights of 9 hours in bed/night); and 2) following sleep deprivation, in a counter-balanced order. Visual working memory testing consisted of two related tasks. The first measured visual working memory capacity and the second measured the ability to ignore distractor stimuli in a visual scene (filtering efficiency). Results showed neither type of sleep deprivation reduced visual working memory capacity. Partial sleep deprivation also generally did not change filtering efficiency. Total sleep deprivation, on the other hand, did impair performance in the filtering task. These results suggest components of visual working memory are differentially vulnerable to the effects of sleep deprivation, and different types of sleep deprivation impact visual working memory to different degrees. Such findings have implications for operational settings where individuals may need to perform with inadequate sleep and whose jobs involve receiving an array of visual information and discriminating the relevant from the irrelevant prior to making decisions or taking actions (e.g., baggage screeners, air traffic controllers, military personnel, health care providers)
Air-sea CO2 exchange in the equatorial Pacific
Author Posting. © American Geophysical Union, 2004. This article is posted here by permission of American Geophysical Union for personal use, not for redistribution. The definitive version was published in Journal of Geophysical Research 109 (2004): C08S02, doi:10.1029/2003JC002256.GasEx-2001, a 15-day air-sea carbon dioxide (CO2) exchange study conducted in the equatorial Pacific, used a combination of ships, buoys, and drifters equipped with ocean and atmospheric sensors to assess variability and surface mechanisms controlling air-sea CO2 fluxes. Direct covariance and profile method air-sea CO2 fluxes were measured together with the surface ocean and marine boundary layer processes. The study took place in February 2001 near 125°W, 3°S in a region of high CO2. The diurnal variation in the air-sea CO2 difference was 2.5%, driven predominantly by temperature effects on surface solubility. The wind speed was 6.0 ± 1.3 m s−1, and the atmospheric boundary layer was unstable with conditions over the range −1 < z/L < 0. Diurnal heat fluxes generated daytime surface ocean stratification and subsequent large nighttime buoyancy fluxes. The average CO2 flux from the ocean to the atmosphere was determined to be 3.9 mol m−2 yr−1, with nighttime CO2 fluxes increasing by 40% over daytime values because of a strong nighttime increase in (vertical) convective velocities. The 15 days of air-sea flux measurements taken during GasEx-2001 demonstrate some of the systematic environmental trends of the eastern equatorial Pacific Ocean. The fact that other physical processes, in addition to wind, were observed to control the rate of CO2 transfer from the ocean to the atmosphere indicates that these processes need to be taken into account in local and global biogeochemical models. These local processes can vary on regional and global scales. The GasEx-2001 results show a weak wind dependence but a strong variability in processes governed by the diurnal heating cycle. This implies that any changes in the incident radiation, including atmospheric cloud dynamics, phytoplankton biomass, and surface ocean stratification may have significant feedbacks on the amount and variability of air-sea gas exchange. This is in sharp contrast with previous field studies of air-sea gas exchange, which showed that wind was the dominating forcing function. The results suggest that gas transfer parameterizations that rely solely on wind will be insufficient for regions with low to intermediate winds and strong insolation.This work was performed with the support
of the National Science Foundation Grant OCE-9986724 and the NOAA
Global Carbon Cycle Program Grants NA06GP048, NA17RJ1223, and
NA87RJ0445 in the Office of Global Programs
Transient tissue priming via ROCK inhibition uncouples pancreatic cancer progression, sensitivity to chemotherapy, and metastasis
The emerging standard of care for patients with inoperable pancreatic cancer is a combination of cytotoxic drugs gemcitabine and Abraxane, but patient response remains moderate. Pancreatic cancer development and metastasis occur in complex settings, with reciprocal feedback from microenvironmental cues influencing both disease progression and drug response. Little is known about how sequential dual targeting of tumor tissue tension and vasculature before chemotherapy can affect tumor response. We used intravital imaging to assess how transient manipulation of the tumor tissue, or "priming," using the pharmaceutical Rho kinase inhibitor Fasudil affects response to chemotherapy. Intravital Förster resonance energy transfer imaging of a cyclin-dependent kinase 1 biosensor to monitor the efficacy of cytotoxic drugs revealed that priming improves pancreatic cancer response to gemcitabine/Abraxane at both primary and secondary sites. Transient priming also sensitized cells to shear stress and impaired colonization efficiency and fibrotic niche remodeling within the liver, three important features of cancer spread. Last, we demonstrate a graded response to priming in stratified patient-derived tumors, indicating that fine-tuned tissue manipulation before chemotherapy may offer opportunities in both primary and metastatic targeting of pancreatic cancer
First Sagittarius A* Event Horizon Telescope Results. I. The Shadow of the Supermassive Black Hole in the Center of the Milky Way
We present the first Event Horizon Telescope (EHT) observations of Sagittarius A* (Sgr A*), the Galactic center source associated with a supermassive black hole. These observations were conducted in 2017 using a global interferometric array of eight telescopes operating at a wavelength of λ = 1.3 mm. The EHT data resolve a compact emission region with intrahour variability. A variety of imaging and modeling analyses all support an image that is dominated by a bright, thick ring with a diameter of 51.8 \ub1 2.3 μas (68% credible interval). The ring has modest azimuthal brightness asymmetry and a comparatively dim interior. Using a large suite of numerical simulations, we demonstrate that the EHT images of Sgr A* are consistent with the expected appearance of a Kerr black hole with mass ∼4
7 106 M☉, which is inferred to exist at this location based on previous infrared observations of individual stellar orbits, as well as maser proper-motion studies. Our model comparisons disfavor scenarios where the black hole is viewed at high inclination (i > 50\ub0), as well as nonspinning black holes and those with retrograde accretion disks. Our results provide direct evidence for the presence of a supermassive black hole at the center of the Milky Way, and for the first time we connect the predictions from dynamical measurements of stellar orbits on scales of 103-105 gravitational radii to event-horizon-scale images and variability. Furthermore, a comparison with the EHT results for the supermassive black hole M87* shows consistency with the predictions of general relativity spanning over three orders of magnitude in central mass
Loss of Metal Ions, Disulfide Reduction and Mutations Related to Familial ALS Promote Formation of Amyloid-Like Aggregates from Superoxide Dismutase
Mutations in the gene encoding Cu-Zn superoxide dismutase (SOD1) are one of the causes of familial amyotrophic lateral sclerosis (FALS). Fibrillar inclusions containing SOD1 and SOD1 inclusions that bind the amyloid-specific dye thioflavin S have been found in neurons of transgenic mice expressing mutant SOD1. Therefore, the formation of amyloid fibrils from human SOD1 was investigated. When agitated at acidic pH in the presence of low concentrations of guanidine or acetonitrile, metalated SOD1 formed fibrillar material which bound both thioflavin T and Congo red and had circular dichroism and infrared spectra characteristic of amyloid. While metalated SOD1 did not form amyloid-like aggregates at neutral pH, either removing metals from SOD1 with its intramolecular disulfide bond intact or reducing the intramolecular disulfide bond of metalated SOD1 was sufficient to promote formation of these aggregates. SOD1 formed amyloid-like aggregates both with and without intermolecular disulfide bonds, depending on the incubation conditions, and a mutant SOD1 lacking free sulfhydryl groups (AS-SOD1) formed amyloid-like aggregates at neutral pH under reducing conditions. ALS mutations enhanced the ability of disulfide-reduced SOD1 to form amyloid-like aggregates, and apo-AS-SOD1 formed amyloid-like aggregates at pH 7 only when an ALS mutation was also present. These results indicate that some mutations related to ALS promote formation of amyloid-like aggregates by facilitating the loss of metals and/or by making the intramolecular disulfide bond more susceptible to reduction, thus allowing the conversion of SOD1 to a form that aggregates to form resembling amyloid. Furthermore, the occurrence of amyloid-like aggregates per se does not depend on forming intermolecular disulfide bonds, and multiple forms of such aggregates can be produced from SOD1
A Universal Power-law Prescription for Variability from Synthetic Images of Black Hole Accretion Flows
We present a framework for characterizing the spatiotemporal power spectrum of the variability expected from the horizon-scale emission structure around supermassive black holes, and we apply this framework to a library of general relativistic magnetohydrodynamic (GRMHD) simulations and associated general relativistic ray-traced images relevant for Event Horizon Telescope (EHT) observations of Sgr A*. We find that the variability power spectrum is generically a red-noise process in both the temporal and spatial dimensions, with the peak in power occurring on the longest timescales and largest spatial scales. When both the time-averaged source structure and the spatially integrated light-curve variability are removed, the residual power spectrum exhibits a universal broken power-law behavior. On small spatial frequencies, the residual power spectrum rises as the square of the spatial frequency and is proportional to the variance in the centroid of emission. Beyond some peak in variability power, the residual power spectrum falls as that of the time-averaged source structure, which is similar across simulations; this behavior can be naturally explained if the variability arises from a multiplicative random field that has a steeper high-frequency power-law index than that of the time-averaged source structure. We briefly explore the ability of power spectral variability studies to constrain physical parameters relevant for the GRMHD simulations, which can be scaled to provide predictions for black holes in a range of systems in the optically thin regime. We present specific expectations for the behavior of the M87* and Sgr A* accretion flows as observed by the EHT
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