Low-Cost Motility Tracking System (LOCOMOTIS) for time-lapse microscopy applications and cell visualisation

This article has been made available through the Brunel Open Access Publishing Fund.Direct visualisation of cells for the purpose of studying their motility has typically required expensive microscopy equipment. However, recent advances in digital sensors mean that it is now possible to image cells for a fraction of the price of a standard microscope. Along with low-cost imaging there has also been a large increase in the availability of high quality, open-source analysis programs. In this study we describe the development and performance of an expandable cell motility system employing inexpensive, commercially available digital USB microscopes to image various cell types using time-lapse and perform tracking assays in proof-of-concept experiments. With this system we were able to measure and record three separate assays simultaneously on one personal computer using identical microscopes, and obtained tracking results comparable in quality to those from other studies that used standard, more expensive, equipment. The microscopes used in our system were capable of a maximum magnification of 413.6x. Although resolution was lower than that of a standard inverted microscope we found this difference to be indistinguishable at the magnification chosen for cell tracking experiments (206.8x). In preliminary cell culture experiments using our system, velocities (mean mm/min ± SE) of 0.81±0.01 (Biomphalaria glabrata hemocytes on uncoated plates), 1.17±0.004 (MDA-MB-231 breast cancer cells), 1.24±0.006 (SC5 mouse Sertoli cells) and 2.21±0.01 (B. glabrata hemocytes on Poly-L-Lysine coated plates), were measured and are consistent with previous reports. We believe that this system, coupled with open-source analysis software, demonstrates that higher throughput time-lapse imaging of cells for the purpose of studying motility can be an affordable option for all researchers. © 2014 Lynch et al

Endocrine disrupting effects on the nesting behaviour of male three-spined stickleback Gasterosteus aculeatus L

The analysis of patterns of temporal variability in the nesting behaviour of male threespined stickleback (Gasterosteus aculeatus) exposed to the synthetic oestrogen, 17β-ethinylestradiol, revealed immediate, but transient, treatment-related effects. Gluing frequency and time spent near nest were significantly reduced in exposed fish at the beginning of the experiment. The expression of these behaviours subsequently recovered and there was no effect of treatment on nest building success. The potential causes and implications of these findings are discussed

The rodent uterotrophic assay: Critical protocol features, studies with nonyl phenols, and comparison with a yeast estrogenicity assay

The major protocol features of the immature rat uterotrophic assay have been evaluated using a range of reference chemicals. The protocol variables considered include the selection of the test species and route of chemical administration, the age of the test animals, the maintenance diet used, and the specificity of the assay for estrogens. It is concluded that three daily oral administrations of test chemicals to 21- to 22-day-old rats, followed by determination of absolute uterus weights on the fourth day, provide a sensitive and toxicologically relevant in vivo estrogenicity assay. Rats are favored over mice for reasons of toxicological practice, but the choice of test species is probably not a critical protocol variable, as evidenced by the similar sensitivity of rats and mice to the uterotrophic activity of methoxychlor. Vaginal opening is shown to be a useful, but nondefinitive, adjunct to the uterotrophic assay. The ability of test chemicals to reduce or abolish the uterotrophic response of estradiol is suggested to provide a useful extension of the uterotrophic assay for the purpose of detecting antiestrogens. The results of a series of studies on the environmental estrogen nonyl phenol (NP), and its linear isomer n -nonyl phenol, confirm that branching of the aliphatic side chain is important for activity. 17beta-Desoxyestradiol is shown to be of similar activity to estradiol in the uterotrophic assay and is suggested to represent the "parent" estrogen of NP. Benzoylation of NP and 17-desoxyestradiol did not affect their uterotrophic activity, in contrast to the enhancing effect of benzoylation on estradiol. Selected chemicals shown to be active in the immature rat uterotrophic assay were also evaluated in an in vitro yeast human estrogen receptor transactivation assay. Most of the chemicals gave similar qualitative responses to those seen in the uterotrophic assay, and the detection of the estrogen methoxychlor by the yeast assay evidenced a degree of intrinsic metabolic competence. However, the assay had a reduced ability (compared to rodents) to hydrolyze the benzoate ester of estradiol, and the estrogenic benzoate derivative of NP was not active in the yeast assay. These last results indicate that current metabolic deficiencies of in vitro estrogenicity assays will limit the value of negative data for the immediate future. The results described illustrate the intrinsic complexity of evaluating chemicals for estrogenic activities and confirm the need for rigorous attention to experimental design and criteria for assessing estrogenic activity

No substantial changes in estrogen receptor and estrogen-related receptor orthologue gene transcription in Marisa cornuarietis exposed to estrogenic chemicals

This article is made available through the Brunel Open Access Publishing Fund. This is an open-access article distributed under the terms of the Creative Commons Attribution-NonCommercial-No Derivative Works License, which permits non-commercial use, distribution, and reproduction in any medium, provided the original author and source are credited.Estrogen receptor orthologues in molluscs may be targets for endocrine disruptors, although mechanistic evidence is lacking. Molluscs are reported to be highly susceptible to effects caused by very low concentrations of environmental estrogens which, if substantiated, would have a major impact on the risk assessment of many chemicals. The present paper describes the most thorough evaluation to-date of the susceptibility of Marisa cornuarietis ER and ERR gene transcription to modulation by vertebrate estrogens in vivo and in vitro. We investigated the effects of estradiol-17β and 4-tert-Octylphenol exposure on in vivo estrogen receptor (ER) and estrogen-related receptor (ERR) gene transcription in the reproductive and neural tissues of the gastropod snail M. cornuarietis over a 12-week period. There was no significant effect (p > 0.05) of treatment on gene transcription levels between exposed and non-exposed snails. Absence of a direct interaction of estradiol-17β and 4-tert-Octylphenol with mollusc ER and ERR protein was also supported by in vitro studies in transfected HEK-293 cells. Additional in vitro studies with a selection of other potential ligands (including methyl-testosterone, 17α-ethinylestradiol, 4-hydroxytamoxifen, diethylstilbestrol, cyproterone acetate and ICI182780) showed no interaction when tested using this assay. In repeated in vitro tests, however, genistein (with mcER-like) and bisphenol-A (with mcERR) increased reporter gene expression at high concentrations only (>10−6 M for Gen and >10−5 M for BPA, respectively). Like vertebrate estrogen receptors, the mollusc ER protein bound to the consensus vertebrate estrogen-response element (ERE). Together, these data provide no substantial evidence that mcER-like and mcERR activation and transcript levels in tissues are modulated by the vertebrate estrogen estradiol-17β or 4-tert-Octylphenol in vivo, or that other ligands of vertebrate ERs and ERRs (with the possible exception of genistein and bisphenol A, respectively) would do otherwise.BBSR

Antifoams:the overlooked additive?

Present research has found that antifoams can have a broad range of effects upon bioprocesses, both on the culture environment and upon the cells themselves

Antifoam addition to shake flask cultures of recombinant Pichia pastoris increases yield

<p>Abstract</p> <p>Background</p> <p><it>Pichia pastoris </it>is a widely-used host for recombinant protein production. Initial screening for both suitable clones and optimum culture conditions is typically carried out in multi-well plates. This is followed by up-scaling either to shake-flasks or continuously stirred tank bioreactors. A particular problem in these formats is foaming, which is commonly prevented by the addition of chemical antifoaming agents. Intriguingly, antifoams are often added without prior consideration of their effect on the yeast cells, the protein product or the influence on downstream processes such as protein purification. In this study we characterised, for the first time, the effects of five commonly-used antifoaming agents on the total amount of recombinant green fluorescent protein (GFP) secreted from shake-flask cultures of this industrially-relevant yeast.</p> <p>Results</p> <p>Addition of defined concentrations of Antifoam A (Sigma), Antifoam C (Sigma), J673A (Struktol), P2000 (Fluka) or SB2121 (Struktol) to shake-flask cultures of <it>P. pastoris </it>increased the total amount of recombinant GFP in the culture medium (the total yield) and in the case of P2000, SB2121 and J673A almost doubled it. When normalized to the culture density, the GFP specific yield (μg OD₅₉₅^-1) was only increased for Antifoam A, Antifoam C and J673A. Whilst none of the antifoams affected the growth rate of the cells, addition of P2000 or SB2121 was found to increase culture density. There was no correlation between total yield, specific yield or specific growth rate and the volumetric oxygen mass transfer coefficient (<it>k_La</it>) in the presence of antifoam. Moreover, the antifoams did not affect the dissolved oxygen concentration of the cultures. A comparison of the amount of GFP retained in the cell by flow cytometry with that in the culture medium by fluorimetry suggested that addition of Antifoam A, Antifoam C or J673A increased the specific yield of GFP by increasing the proportion secreted into the medium.</p> <p>Conclusions</p> <p>We show that addition of a range of antifoaming agents to shake flask cultures of <it>P. pastoris </it>increases the total yield of the recombinant protein being produced. This is not only a simple method to increase the amount of protein in the culture, but our study also provides insight into how antifoams interact with microbial cell factories. Two mechanisms are apparent: one group of antifoams (Antifoam A, Antifoam C and J673A) increases the specific yield of GFP by increasing the total amount of protein produced and secreted per cell, whilst the second (P2000 or SB2121) increases the total yield by increasing the density of the culture.</p

Os avanços da aqüicultura.

A produção global da aqüicultura tem apresentado elevados níveis de crescimento nas últimas duas décadas, e no Brasil, esta expansão está expressa no aumento do consumo per capita de pescados, de 6 para 8kg/ano. Este aumento na produção tem gerado excedentes exportáveis que tornaram a balança comercial brasileira superavitária desde 2001 (MDIC/SECEX)

Ultrasonic Spray Coating for the Fabrication of Polymeric Organic Light-Emitting Diodes

This thesis investigates the use of the large-area, roll-to-roll compatible deposition technique of ultrasonic spray coating for the fabrication of polymeric organic light-emitting diodes (OLEDs). Firstly, in Chapter 4 a range of different materials are investigated as potential solution processable electron-injection layers, typically this layer is deposited via thermal evaporation in OLEDs but a solution based alternative is required for deposition via ultrasonic spray coating. Solution-processed electron-injection layers: caesium carbonate (Cs2CO3), 8-hydroxy-quinolinato lithium (Liq) and polyethylenimine-ethoxylated (PEIE), are spin cast to tune thickness for optimal device performance and investigated for compatibly with ultrasonic spray coating under ambient conditions. Caesium carbonate deposited in an inert atmosphere was found to improve device performance compared to thermally evaporated lithium fluoride references but when cast in an ambient atmosphere the device performance was very poor due to the hygroscopic nature of Cs2CO3. Devices fabricated with optimal thickness Liq electron-injection layer had low mean peak luminance of 1197 cd m-2. Devices with PEIE layers of 2.7 ± 0.1 nm spin cast under ambient conditions have mean peak performance metrics of 3.18 cd A-1, 1.14 Lm W-1, and 10690 cd m-2. In Chapter 5 the wide parameter space of ultrasonic spray coating is probed in order to determine the processing window and optimisation process for depositing uniform polymer thin films. The parameter space for ultrasonic spray coating has been investigated and an optimisation process for spraying uniform thin polymer films has been developed using hole-transporting polymer poly[(9,9-dioctylfluorenyl-2,7-diyl)-co-(4,4’(N-(4-sec-butylphenyl))) diphenylamine] (TFB). The processing window for casting uniform films of TFB from a 4 mg ml-1 toluene solution cast onto a substrate held at 25 °C with a fluid pressure of 50 mbar have been shown to be a pass height of 40 mm and a range of pass speeds from 125 – 200 mm s-1 to fabricate films between 66 – 97 nm. In Chapter 6 a study is undertaken to determine if there is a fundamental issues with films deposited via ultrasonic spray coating that could limit the electrical performance of devices containing these film compared to those deposited via spin coating. The influence of thin film processing technique and surface roughness on the electrical performance of unipolar polymer OLEDs are studied, and a negligible difference is found between low-roughness spray cast (Ra < 10 nm) and spin cast devices of equivalent thicknesses. However, above 10 nm roughness there is a reduction in injection efficiency, up to an 86 % loss in performance for roughnesses of the order of 40 % of the thickness of the film. As such a processing window of Ra < 10 nm for achieving comparable electrical performance between spin and spray cast devices is demonstrated. Finally, in Chapter 7 the different layers of a White-emitting polymer OLED are deposited via ultrasonic spray coating in separate devices for optimisation, then the layers are sprayed subsequently in the same device and lastly large-area devices are fabricated to demonstrate the scalability of the process. White-emitting polymer OLEDs have been fabricated in which the hole-injection layer, emissive layer and electron-injection layer were deposited via ultrasonic spray coating. Several different device studies have been conducted: Firstly, devices with PEDOT:PSS deposited via ultrasonic spray coating as a hole-injection layer were fabricated and show comparable device performance to those with spin-cast PEDOT:PSS. Secondly, devices with a white-light-emitting polymer (LEP), deposited via ultrasonic spray coating, have mean peak current efficiency of 4.93 cd A-1, 90 % of the value of the spin cast references 5.45 cd A-1. The mean peak power efficiency of the spray cast devices was 2.42 Lm W-1, 82 % of the spin cast references 2.97 Lm W-1. The mean peak luminance of the spray cast devices was 8149 cd m-2, 80% of the reference spin cast value 10189 cd m-2. These results are equivalent to those in literature where devices fabricated by Gilissen et al. in which a yellow-light-emitting polymer was deposited via ultrasonic spray coating achieved 9.71 Lm W-1, 81 % of the spin cast reference. Thirdly, attempts were made to replace the electron-injection layer with an air-stable, non-ionic and non-conjugated polymer, polyethylenimine-ethoxylated (PEIE), layer deposited via ultrasonic spray coating. Devices in which PEIE was deposited via ultrasonic spray coating as an electron-injection layer showed poor device metrics and non-uniform emission. Finally, white-light-emitting devices were then fabricated in which the hole-injection and emissive layers were sequentially deposited via ultrasonic spray coating, a first for polymer OLEDs. The mean peak current efficiency for the spray cast devices was 2.43 cd A-1, 72 % of the value of the spin cast references 3.39 cd A-1. The mean peak power efficiency of the spray cast devices was 0.83 Lm W-1, 71 % of the spin cast references 1.17 Lm W-1. The mean peak luminance of the spray cast devices was 7409 cd m-2, 70 % of the reference spin cast value 10626 cd m-2. The same layers were then spray cast to fabricate large-area devices to demonstrate the possibility of coating over large areas and the potential compatibility of this technique with roll-to-roll processing, a yield of working pixels of 95.8 % was achieved

Common aquatic pollutants modify hemocyte immune responses in Biomphalaria glabrata.

Funding The studentship supporting this research was awarded by the UK Natural Environment Research Council (NERC). Acknowledgments We are grateful to Dr. David Rollinson (Natural History Museum) for providing B. glabrata snails and to Drs. Nuha Mansour and Quentin Bickle (London School of Hygiene and Tropical Medicine) for providing parasite material. We thank the UK Natural Environment Research Council (NERC) for funding.Peer reviewedPublisher PD