A novel follicle culture system markedly increases follicle volume, cell number and oestradiol secretion

This study reports a novel, simple method for culture of mouse follicles which results in follicles with cell numbers similar to in vivo fully grown follicles. Using this method, follicles (180-240 microm in diameter) were cultured in a 100 microl inverted drop of medium without oil and compared with culture in upright drops with and without a mineral oil overlay. Follicles, isolated from C57BL/6 x CBA/ca crossbred and MF1 inbred mice, were cultured individually at 37 degrees C in 96-well round-bottomed suspension cell tissue culture plates for 6 days. Follicles grown in the inverted drop culture system reached a markedly higher final diameter (means+/-s.e.m.; 471 +/- 6.0 microm) as compared with the upright with oil (363 +/- 2.7 microm) and without oil (358 +/- 4.0) systems. There was no significant effect of mouse strain on follicle diameter. Follicular secretion of oestradiol and lactate into the medium was measured on days 2, 4 and 6 of culture. Secretion of oestradiol per follicle on day 6 was 2.49 +/- 0.45 ng in the inverted and 0.90 +/- 0.17 ng in the upright without oil system (P < 0.001). Follicular secretion of lactate on a per unit of follicle volume basis remained constant in the inverted system over days 2, 4 and 6 and was less (P < 0.001) than secretion in both the upright with and without oil systems. Follicle cell proliferation was markedly increased in the inverted as compared with the upright with oil system; the increases in cell numbers were significant on day 3 (P < 0.01) and on all subsequent days (P < 0.001). These results are discussed in relation to the supply of oxygen to the follicle in culture.     

Effects of aluminum and pH on the early life stages of smallmouth bass (Micropterus dolomieui)

The sensitivity of smallmouth bass Micropterus dolomieui to acidified conditions was examined by exposing recently-hatched fish to pH levels ranging from 5.1 to 7.5 and aluminum concentrations ranging from 32 to 1000 μg l⁻¹. The range of pH and aluminum concentrations included those found in the northern part of the species' range. Acute bioassays (96 h) conducted at a pH of 5.1 and aluminum concentrations 180 μgl⁻¹ resulted in total mortality. The LC₅₀ calculated for this species was 130 μg l⁻¹. At pH values of 6.1 and 7.5, mortality was low ( 20%) regardless of aluminum concentrations. A 30-day chronic toxicity test was conducted at three pH levels (low 5.1, intermediate 5.5–5.7 and high 7.3), each with two aluminum concentrations (approx. 0 and 200 μg l⁻¹). Survival was significantly lower in the test at pH 5.1 with aluminum, and at pH 5.7 with aluminum treatments than in the other treatments. Fish in the pH 5.1 without aluminum treatment had intermediate survival, while fish exposed to pH 5.7 without aluminum, pH 7.3 without aluminum and pH 7.3 with aluminum had high, and similar, survival. Sublethal effects on fish exposed to low pH and aluminum included deformities, reduced activity and abnormal swimming behavior. We conclude that the sensitivity of smallmouth bass to low pH and aluminum concentrations corroborates field investigations linking acidification and aluminum mobilization with depletion of smallmouth bass populations.     

Quantitative profiling of endogenous retinoic acid in vivo and in vitro by tandem mass spectrometry

We report an improved tandem mass spectrometric assay for retinoic acid (RA) applicable to in vitro and in vivo biological samples. This liquid chromatography tandem mass spectrometric (LC/MS/MS) assay for direct RA quantification is the most sensitive to date, with a 62.5 attomol lower limit of detection and a linear range spanning greater than 4 orders of magnitude (from 250 attomol to 10 pmol). This assay resolves all-trans-RA (atRA) from its endogenous geometric isomers, is applicable to samples of limited size (10-20 mg of tissue), and functions with complex biological matrixes. Coefficients of variation are as follows: instrumental, < or =2.6%; intraday, 5.2% +/- 0.7%; interday, 6.7% +/- 0.9%. In vitro capabilities are demonstrated by quantification of endogenous RA and RA production (from retinol) in primary cultured astrocytes. Quantification of endogenous atRA and its geometric isomers in 129SV mouse serum and tissues (liver, kidney, adipose, muscle, spleen, testis, and brain) reveals in vivo utility of the assay. The ability to discriminate spatial concentrations of RA in vivo is illustrated with C57BL/6 mouse brain loci (hippocampus, cortex, olfactory bulb, thalamus, cerebellum, and striatum), as well as with Lewis rat proximal/distal mammary gland regions during various morphological stages: virgin, early pregnancy (e7), late pregnancy (e20), lactating (day 4), involuting day 1, and involuting day 11. This assay provides the sensitivity necessary for direct, endogenous RA quantification necessary to elucidate RA function, e.g., in neurogenesis, morphogenesis, and the contribution of altered RA homeostasis to diseases, such as Alzheimer's disease, type 2 diabetes, obesity, and cancer.     

Inositol transport in mouse oocytes and preimplantation embryos: effects of mouse strain,embryo stage,sodium and the hexose transport inhibitor,phloridzin

The uptake of myo-inositol by mouse oocytes and preimplantation embryos of a crossbred (DBA x C57BL/6) and a purebred outbred strain (MF1) was measured using [2-(3)H]myo-inositol. Uptake in crossbred embryos increased about 15-fold between the one- and two-cell stages and increased again by about sixfold at the blastocyst stage compared with the morula stage. Uptake in purebred embryos increased about 42-fold between the one- and two-cell stages and increased more than threefold at the blastocyst stage compared with the morula stage. In all stages examined, except two-cell crossbred embryos, inositol uptake was, depending on the stage, either largely or partly sodium dependent and could be inhibited by the sodium-dependent hexose transport inhibitor, phloridzin. This is consistent with the hypothesis that transport occurs via a sodium myo-inositol transporter (SMIT) protein. In addition, there was strong evidence that a sodium-independent mechanism of uptake, possibly a channel, was switched on at the two-cell stage coincident with zygotic gene activation which resulted in 141-fold and 71-fold increases in sodium-independent uptake from the one-cell to two-cell stages in crossbred and purebred embryos, respectively. This mechanism was either abolished or drastically downregulated at the blastocyst stage, whereas sodium-dependent uptake was markedly upregulated. In two-cell crossbred embryos, there was a complete abolition of sodium-dependent uptake, again possibly regulated by zygotic gene activation. The hypothesis that the changes in mechanism of inositol uptake at about the two-cell stage are due to zygotic gene activation was supported by the finding that these changes did not occur in parthenogenetic two-cell embryos.     

Burrowing mayfly (Ephemeroptera: Ephemeridae: Hexagenia spp.) bioturbation and bioirrigation: A source of internal phosphorus loading in Lake Erie

Traditional lake eutrophication models predict lower phosphorus concentrations with decreased external loads. However, in lakes where decreased external phosphorus loads are accompanied by increasing phosphorus concentrations, a seeming “trophic paradox” exists. Western Lake Erie is an example of such a paradox. Internal phosphorus loads may help explain this paradox. We examined bioturbation and bioirrigation created from burrowing mayfly, Hexagenia spp., as a possible source of internal phosphorus loading. Phosphorus concentrations of experimental microcosms containing lake sediments, filtered lake water, and nymphs (417/m²) collected from western Lake Erie were compared to control microcosms containing sediments and lake water over a 7-day period. Phosphorus concentrations in microcosms containing Hexagenia were significantly greater than microcosms without nymphs. Further, we estimate the soluble reactive phosphorus flux from the sediments due to Hexagenia is 1.03 mg/m²/day. Thus, Hexagenia are a source of internal phosphorus loading. High densities of Hexagenia nymphs in western Lake Erie may help explain the “trophic paradox.” Furthermore, Hexagenia may be a neglected source of internal phosphorus loading in any lake in which they are abundant. Future studies of phosphorus dynamics in lakes with Hexagenia must account for the ability of these organisms to increase lake internal phosphorus loading.     

Liver-specific functional studies in a microfluidic array of primary mammalian hepatocytes

Nearly half a billion dollars in resources are lost each time a drug candidate is withdrawn from the market by the Food and Drug Administration (FDA) for reasons of liver toxicity. The number of late-phase drug developmental failures due to liver toxicity could potentially be reduced through the use of hepatocyte-based systems capable of modeling the response of in vivo liver tissue to toxic insults. With this article, we report progress toward the goal of realizing an array of primary hepatocytes for use in high-throughput liver toxicity studies. Described herein is the development of a 64 (8 x 8) element array of microfluidic wells capable of supporting micropatterned primary rat hepatocytes in coculture with 3T3-J2 fibroblasts. Each of the wells within the array was continuously perfused with medium and oxygen in a nonaddressable format. The key features of the system design and fabrication are described, including the use of two microfluidic perfusion networks to provide the coculture with an independent and continuous supply of cell culture medium and oxygen. Also described are the fabrication techniques used to selectively pattern hepatocytes and 3T3-J2 fibroblasts within the wells of the array. The functional studies used to demonstrate the synthetic and metabolic capacity of the array are outlined in this article. These studies demonstrate that the hepatocytes contained within the array are capable of continuous, steady-state albumin synthesis (78.4 microg/day, sigma = 3.98 microg/day, N = 8) and urea production (109.8 microg/day, sigma = 11.9 microg/day, N = 8). In the final section of the article, these results are discussed as they relate to the final goal of this research effort, the development of an array of primary hepatocytes for use in physiologically relevant toxicology studies.     

Developmental change in judging important and critical elements of stories.

Conducted 2 experiments with 54 2nd, 4th, and 6th graders and 15 undergraduates (Exp I) and 45 2nd and 4th graders (Exp II) to examine children's understanding (metacognitive awareness) that in a simple story the following parts are most important or essential for comprehending it: what precipitates the character's action (initiating event), what the character did (action), and what follows the character's action (consequence). Ss' judgments of simple stories showed that 2nd graders seldom selected this sequence, but 4th, 5th, and 6th graders and adults did so under a variety of conditions. In addition there was a modest relation between recall of the stories and older children's (5th graders) judgments of them. (9 ref) (PsycINFO Database Record (c) 2010 APA, all rights reserved)     

Raman spectroscopic characterization of SiO2 phase transformation and Si substrate stress relevant to EBC performance

To accurately model the long-term durability of environmental barrier coatings (EBCs), a more complete understanding of the phase composition and transformations of the thermally grown oxide SiO₂ (TGO) is desired. For the TGO formed during thermal cycling in steam, cristobalite formation and the subsequent β- to α-cristobalite transformation has been identified as a potentially life-limiting mechanism. In this study, Raman micro-spectroscopy was used to quantify the cristobalite transformation on a polycrystalline Si coupon that was exposed to steam at 1350°C for 100 h. The phase transformation was mapped at 200–260°C on the TGO surface at different ramp rates using a heating stage and a micro-positioning stage. The stress in the Si substrate was also determined using Raman spectroscopy by measuring the stress induced peak shift. The α→β phase transformation produced a 300–500 MPa tensile stress in the Si substrate, which compared well to the stress predicted from the volumetric expansion of the cristobalite. Quantifying the phase transformation and residual stress are critical tools in developing the next generation of high performance EBCs.     

A New Class of Proton Conductors with Dramatically Enhanced Stability and High Conductivity for Reversible Solid Oxide Cells

Reversible solid oxide cells based on proton conductors (P-ReSOCs) have potential to be the most efficient and low-cost option for large-scale energy storage and power generation, holding promise as an enabler for the implementation of intermittent renewable energy technologies and the widespread utilization of hydrogen. Here, the rational design of a new class of hexavalent Mo/W-doped proton-conducting electrolytes with excellent durability while maintaining high conductivity is reported. Specifically, BaMo(W)_0.03Ce_0.71Yb_0.26O_3-δ exhibits dramatically enhanced chemical stability against high concentrations of steam and carbon dioxide than the state-of-the-art electrolyte materials while retaining similar ionic conductivity. In addition, P-ReSOCs based on BaW_0.03Ce_0.71Yb_0.26O_3-δ demonstrate high peak power densities of 1.54, 1.03, 0.72, and 0.48 W cm⁻² at 650, 600, 550, and 500 °C, respectively, in the fuel cell mode. During steam electrolysis, a high current density of 2.28 A cm⁻² is achieved at a cell voltage of 1.3 V at 600 °C, and the electrolysis cell can operate stably with no noticeable degradation when exposed to high humidity of 30% H₂O at −0.5 A cm⁻² and 600 °C for over 300 h. Overall, this work demonstrates the promise of donor doping for obtaining proton conductors with both high conductivity and chemical stability for P-ReSOCs.