Co-regulation of primary mouse hepatocyte viability and function by oxygen and matrix

Although oxygen and extracellular matrix cues both influence differentiation state and metabolic function of primary rat and human hepatocytes, relatively little is known about how these factors together regulate behaviors of primary mouse hepatocytes in culture. To determine the effects of pericellular oxygen tension on hepatocellular function, we employed two methods of altering oxygen concentration in the local cellular microenvironment of cells cultured in the presence or absence of an extracellular matrix (Matrigel) supplement. By systematically altering medium depth and gas phase oxygen tension, we created multiple oxygen regimes (hypoxic, normoxic, and hyperoxic) and measured the local oxygen concentrations in the pericellular environment using custom-designed oxygen microprobes. From these measurements of oxygen concentrations, we derived values of oxygen consumption rates under a spectrum of environmental contexts, thus providing the first reported estimates of these values for primary mouse hepatocytes. Oxygen tension and matrix microenvironment were found to synergistically regulate hepatocellular survival and function as assessed using quantitative image analysis for cells stained with vital dyes, and assessment of secretion of albumin. Hepatocellular viability was affected only at strongly hypoxic conditions. Surprisingly, albumin secretion rates were greatest at a moderately supra-physiological oxygen concentration, and this effect was mitigated at still greater supra-physiological concentrations. Matrigel enhanced the effects of oxygen on retention of function. This study underscores the importance of carefully controlling cell density, medium depth, and gas phase oxygen, as the effects of these parameters on local pericellular oxygen tension and subsequent hepatocellular function are profound.National Institutes of Health (U.S.) (Grant P50-GM068762-08)National Institutes of Health (U.S.) (Grant R01-EB010246-04)National Institutes of Health (U.S.) (Grant R01-ES015241)National Institutes of Health (U.S.) (Grant P30-ES002109

Perfused multiwell plate for 3D liver tissue engineering

In vitro models that capture the complexity of in vivo tissue and organ behaviors in a scalable and easy-to-use format are desirable for drug discovery. To address this, we have developed a bioreactor that fosters maintenance of 3D tissue cultures under constant perfusion and we have integrated multiple bioreactors into an array in a multiwell plate format. All bioreactors are fluidically isolated from each other. Each bioreactor in the array contains a scaffold that supports formation of hundreds of 3D microscale tissue units. The tissue units are perfused with cell culture medium circulated within the bioreactor by integrated pneumatic diaphragm micropumps. Electronic controls for the pumps are kept outside the incubator and connected to the perfused multiwell by pneumatic lines. The docking design and open-well bioreactor layout make handling perfused multiwell plates similar to using standard multiwell tissue culture plates. A model of oxygen consumption and transport in the circulating culture medium was used to predict appropriate operating parameters for primary liver cultures. Oxygen concentrations at key locations in the system were then measured as a function of flow rate and time after initiation of culture to determine oxygen consumption rates. After seven days of culture, tissue formed from cells seeded in the perfused multiwell reactor remained functionally viable as assessed by immunostaining for hepatocyte and liver sinusoidal endothelial cell (LSEC) phenotypic markers.National Institute of Environmental Health Sciences (grant number 5P30ES002109-30)National Institutes of Health (U.S.) (NIH grant number 5R01ES015241)DuPont MIT AlliancePfizer Inc.National Science Foundation (U.S.) (NSF grant number EEC-9843342

RR Lyrae-based calibration of the Globular Cluster Luminosity Function

We test whether the peak absolute magnitude Mv(TO) of the Globular Cluster Luminosity Function (GCLF) can be used for reliable extragalactic distance determinations. Starting with the luminosity function of the Galactic Globular Clusters listed in Harris catalog, we determine Mv(TO) either using current calibrations of the absolute magnitude Mv(RR) of RR Lyrae stars as a function of the cluster metal content [Fe/H] and adopting selected cluster samples. We show that the peak magnitude is slightly affected by the adopted Mv(RR)-[Fe/H] relation, while it depends on the criteria to select the cluster sample. As for the GCLFs in other external galaxies, using Surface Brightness Fluctuations (SBF) measurements we give evidence that the luminosity functions of the blue (metal-poor) Globular Clusters peak at the same luminosity within ~0.2 mag, whereas for the red (metal-rich) samples the agreement is within ~0.5 mag even accounting for the theoretical metallicity correction expected for clusters with similar ages and mass distributions. Then, using the SBF absolute magnitudes provided by a Cepheid distance scale calibrated on a fiducial distance to LMC (m(LMC)=18.50 mag), we show that the Mv(TO) value of the metal-poor clusters in external galaxies(-7.67+/-0.23 mag) is in excellent agreement with the value of both Galactic (-7.66+/-0.11 mag) and M31 (-7.65+/-0.19 mag)ones.Comment: 13 pages, 8 figures, 8 tables, accepted for publication on MNRA

Genome landscapes and bacteriophage codon usage

Across all kingdoms of biological life, protein-coding genes exhibit unequal usage of synonmous codons. Although alternative theories abound, translational selection has been accepted as an important mechanism that shapes the patterns of codon usage in prokaryotes and simple eukaryotes. Here we analyze patterns of codon usage across 74 diverse bacteriophages that infect E. coli, P. aeruginosa and L. lactis as their primary host. We introduce the concept of a `genome landscape,' which helps reveal non-trivial, long-range patterns in codon usage across a genome. We develop a series of randomization tests that allow us to interrogate the significance of one aspect of codon usage, such a GC content, while controlling for another aspect, such as adaptation to host-preferred codons. We find that 33 phage genomes exhibit highly non-random patterns in their GC3-content, use of host-preferred codons, or both. We show that the head and tail proteins of these phages exhibit significant bias towards host-preferred codons, relative to the non-structural phage proteins. Our results support the hypothesis of translational selection on viral genes for host-preferred codons, over a broad range of bacteriophages.Comment: 9 Color Figures, 5 Tables, 53 Reference

Development of a high throughput 3D perfused liver tissue bioreactor

Thesis (S.M.)--Massachusetts Institute of Technology, Dept. of Mechanical Engineering, 2006.Includes bibliographical references (p. 125-127).This thesis describes the development of a device designed for culturing liver tissue in a 3D perfused environment. Cells form tissue inside miniature channels of a scaffold, and the tissue is perfused with culture medium to create a culture microenvironment that has previously been described by the Griffith lab. In order to support this microenvironment, the reactor needs a pumping system, reservoirs and a controller. Previously, these have all been stand-alone components. This work focuses on the development of a new, integrated culture system. This system integrates 12 reactor microenvironments, reservoirs and pumping systems onto a single plate with a configuration modeled after standard multi-well plates. Each of the 12 bioreactor units utilize pneumatic pumps driven by a single external controller. This design offers substantial advantages over previous systems as it is far more user-friendly and can be used in a higher throughput capacity. The thesis describes the design and fabrication of the reactor and controller, including several models that were used during the development process. It also offers mechanical and biological characterizations of the device.by Samuel Walker Inman.S.M

Integration of real time oxygen measurements with a 3D perfused tissue culture system

Thesis (Ph. D.)--Massachusetts Institute of Technology, Dept. of Mechanical Engineering, 2011.Cataloged from PDF version of thesis.Includes bibliographical references (p. 115-117).In vitro models that capture the complexity of human tissue and organ behaviors in a scalable and easy-to- use format are of increasing interest for both technological applications in drug development and in basic biology research. Tissues and organs are perfused continuously with blood, which delivers nutrients, oxygen, and macromolecular regulatory molecules. In vitro culture models that incorporate local micro-perfusion in a format that allows accesses to cells and their microenvironment are desirable to a broad research community. This thesis describes a platform that features an array of bioreactors that foster three dimensional tissue organization under continuous perfusion. Each bioreactor contains a scaffold that supports formation of hundreds of 3D microscale tissue units. Perfusion through the tissue is achieved using integrated pneumatic diaphragm micropumps. Pumps continuously circulate cell culture medium within each of the fluidically isolated bioreactors in the array. Pulsatile flow from the pumps is filtered using integrated fluidic capacitors such that the flow rate through the scaffold is constant. The format of the device mimics the familiar multiwell tissue culture plate and is easily integrated into existing laboratory facilities. One desirable feature for both parsing metabolic function and assessing response to treatments is a real time read out of oxygen tension at key points in the bioreactor. Such added dimension of real time measurement significantly enhances the value of a cue-response experiment such as a liver drug toxicology study. The thesis describes optical oxygen sensors that measure the florescence decay time of a ruthenium complex, which varies predictably in different oxygen environments. The sensors excite a layer of ruthenium glued to the end of an optical fiber using a stochastic signal from a light emitting diode (LED). The response is then measured on a photodiode. System identification techniques are used to determine the relevant time constants which are subsequently converted to oxygen measurements. Application to real time monitoring of liver tissue function is used for illustration of the utility of the measurements.by Samuel Walker Inman.Ph.D

New physical characterization of the Fontana Lapilli basaltic Plinian eruption, Nicaragua

The Fontana Lapilli deposit was erupted in the late Pleistocene from a vent, or multiple vents, located near Masaya volcano (Nicaragua) and is the product of one of the largest basaltic Plinian eruptions studied so far. This eruption evolved from an initial sequence of fluctuating fountain-like events and moderately explosive pulses to a sustained Plinian episode depositing fall beds of highly vesicular basaltic-andesite scoria (SiO2 > 53 wt%). Samples show unimodal grain size distribution and a moderate sorting that are uniform in time. The juvenile component predominates (> 96 wt%) and consists of vesicular clasts with both sub-angular and fluidal, elongated shapes. We obtain a maximum plume height of 32 km and an associated mass eruption rate of 1.4 × 108 kg s−1 for the Plinian phase. Estimates of erupted volume are strongly sensitive to the technique used for the calculation and to the distribution of field data. Our best estimate for the erupted volume of the majority of the climactic Plinian phase is between 2.9 and 3.8 km3 and was obtained by applying a power-law fitting technique with different integration limits. The estimated eruption duration varies between 4 and 6 h. Marine-core data confirm that the tephra thinning is better fitted by a power-law than by an exponential trend

Microbial technology with major potentials for the urgent environmental needs of the next decades

Several needs in the context of the water-energy-food nexus will become more prominent in the next decades. It is crucial to delineate these challenges and to find opportunities for innovative microbial technologies in the framework of sustainability and climate change. Here, we focus on four key issues, that is the imbalance in the nitrogen cycle, the diffuse emission of methane, the necessity for carbon capture and the deterioration of freshwater reserves. We suggest a set of microbial technologies to deal with each of these issues, such as (i) the production of microbial protein as food and feed, (ii) the control of methanogenic archaea and better use of methanotrophic consortia, (iii) the avoidance of nitrification and (iv) the upgrading of CO2 to microbial bioproducts. The central message is that instead of using crude methods to exploit microorganisms for degradations, the potentials of the microbiomes should be used to create processes and products that fit the demands of the cyclic market economy

Challenges in Using Cultured Primary Rodent Hepatocytes or Cell Lines to Study Hepatic HDL Receptor SR-BI Regulation by Its Cytoplasmic Adaptor PDZK1

Background: PDZK1 is a four PDZ-domain containing cytoplasmic protein that binds to a variety of membrane proteins via their C-termini and can influence the abundance, localization and/or function of its target proteins. One of these targets in hepatocytes in vivo is the HDL receptor SR-BI. Normal hepatic expression of SR-BI protein requires PDZK1 - <5% of normal hepatic SR-BI is seen in the livers of PDZK1 knockout mice. Progress has been made in identifying features of PDZK1 required to control hepatic SR-BI in vivo using hepatic expression of wild-type and mutant forms of PDZK1 in wild-type and PDZK1 KO transgenic mice. Such in vivo studies are time consuming and expensive, and cannot readily be used to explore many features of the underlying molecular and cellular mechanisms. Methodology/Principal Findings: Here we have explored the potential to use either primary rodent hepatocytes in culture using 2D collagen gels with newly developed optimized conditions or PDZK1/SR-BI co-transfected cultured cell lines (COS, HEK293) for such studies. SR-BI and PDZK1 protein and mRNA expression levels fell rapidly in primary hepatocyte cultures, indicating this system does not adequately mimic hepatocytes in vivo for analysis of the PDZK1 dependence of SR-BI. Although PDZK1 did alter SR-BI protein expression in the cell lines, its influence was independent of SR-BI’s C-terminus, and thus is not likely to occur via the same mechanism as that which occurs in hepatocytes in vivo. Conclusions/Significance: Caution must be exercised in using primary hepatocytes or cultured cell lines when studying the mechanism underlying the regulation of hepatic SR-BI by PDZK1. It may be possible to use SR-BI and PDZK1 expression as sensitive markers for the in vivo-like state of hepatocytes to further improve primary hepatocyte cell culture conditions.National Institutes of Health (U.S.) (Grant HL052212)National Institutes of Health (U.S.) (Grant HL066105)National Institutes of Health (U.S.) (Grant ES015241)National Institutes of Health (U.S.) (Grant GM068762

Scale-dependent perspectives on the geomorphology and evolution of beachdune systems

Despite widespread recognition that landforms are complex Earth systems with process-response linkages that span temporal scales from seconds to millennia and spatial scales from sand grains to landscapes, research that integrates knowledge across these scales is fairly uncommon. As a result, understanding of geomorphic systems is often scale-constrained due to a host of methodological, logistical, and theoretical factors that limit the scope of how Earth scientists study landforms and broader landscapes. This paper reviews recent advances in understanding of the geomorphology of beach-dune systems derived from over a decade of collaborative research from Prince Edward Island (PEI), Canada. A comprehensive summary of key findings is provided from short-term experiments embedded within a decade-long monitoring program and a multi-decadal reconstruction of coastal landscape change. Specific attention is paid to the challenges of scale integration and the contextual limitations research at specific spatial and/or temporal scales imposes. A conceptual framework is presented that integrates across key scales of investigation in geomorphology and is grounded in classic ideas in Earth surface sciences on the effectiveness of formative events at different scales. The paper uses this framework to organize the review of this body of research in a 'scale aware' way and, thereby, identifies many new advances in knowledge on the form and function of subaerial beach-dune systems. Finally, the paper offers a synopsis of how greater understanding of the complexities at different scales can be used to inform the development of predictive models, especially those at a temporal scale of decades to centuries, which are most relevant to coastal management issues. Models at this (landform) scale require an understanding of controls that exist at both ‘landscape’ and ‘plot’ scales. Landscape scale controls such as sea level change, regional climate, and the underlying geologic framework essentially provide bounding conditions for independent variables such as winds, waves, water levels, and littoral sediment supply. Similarly, an holistic understanding of the range of processes, feedbacks, and linkages at the finer plot scale is required to inform and verify the assumptions that underly the physical modelling of beach-dune interaction at the landform scale