Neuromodulation and Mitochondrial Transport: Live Imaging in Hippocampal Neurons over Long Durations

To understand the relationship between mitochondrial transport and neuronal function, it is critical to observe mitochondrial behavior in live cultured neurons for extended durations^1-3. This is now possible through the use of vital dyes and fluorescent proteins with which cytoskeletal components, organelles, and other structures in living cells can be labeled and then visualized via dynamic fluorescence microscopy. For example, in embryonic chicken sympathetic neurons, mitochondrial movement was characterized using the vital dye rhodamine 123⁴. In another study, mitochondria were visualized in rat forebrain neurons by transfection of mitochondrially targeted eYFP⁵. However, imaging of primary neurons over minutes, hours, or even days presents a number of issues. Foremost among these are: 1) maintenance of culture conditions such as temperature, humidity, and pH during long imaging sessions; 2) a strong, stable fluorescent signal to assure both the quality of acquired images and accurate measurement of signal intensity during image analysis; and 3) limiting exposure times during image acquisition to minimize photobleaching and avoid phototoxicity.Here, we describe a protocol that permits the observation, visualization, and analysis of mitochondrial movement in cultured hippocampal neurons with high temporal resolution and under optimal life support conditions. We have constructed an affordable stage-top incubator that provides good temperature regulation and atmospheric gas flow, and also limits the degree of media evaporation, assuring stable pH and osmolarity. This incubator is connected, via inlet and outlet hoses, to a standard tissue culture incubator, which provides constant humidity levels and an atmosphere of 5-10% CO₂/air. This design offers a cost-effective alternative to significantly more expensive microscope incubators that don''t necessarily assure the viability of cells over many hours or even days. To visualize mitochondria, we infect cells with a lentivirus encoding a red fluorescent protein that is targeted to the mitochondrion. This assures a strong and persistent signal, which, in conjunction with the use of a stable xenon light source, allows us to limit exposure times during image acquisition and all but precludes photobleaching and phototoxicity. Two injection ports on the top of the stage-top incubator allow the acute administration of neurotransmitters and other reagents intended to modulate mitochondrial movement. In sum, lentivirus-mediated expression of an organelle-targeted red fluorescent protein and the combination of our stage-top incubator, a conventional inverted fluorescence microscope, CCD camera, and xenon light source allow us to acquire time-lapse images of mitochondrial transport in living neurons over longer durations than those possible in studies deploying conventional vital dyes and off-the-shelf life support systems.     

Improved Temperature-Gradient Incubator and the Maximal Growth Temperature and Heat Resistance of Salmonella

An improved all-metal temperature-gradient incubator produces its gradient by means of a bar permanently installed in a near-vertical position with its lower end in a cool constant-temperature water bath and with thermostatically controlled heaters near its top. Bolts hold the incubator in contact with the temperature-gradient bar, and polyurethane foam insulates the entire assemblage during use. Maximal growth temperatures of 34 representative strains of Salmonella were found to be between 43.2 and 46.2 C. In an agar medium with an initial level of 10⁶ cells per milliliter, no strain survived 50 C for 48 hr. S. senftenberg 775W showed no greater heat resistance at or near 48 C than did other species or other S. senftenberg strains. However, it was considerably more resistant than other strains at 55 C.     

Vat incubator with immersion core illumination — a new,inexpensive setup for mass phytoplankton culture

One of the shortcomings in studies of bivalve grazing has been the difficulty of culturing and making available sufficient quantities of algae. This was overcome using a 2501 capacity vat incubator with immersion core illumination (VIICI) in connection with experiments involving the diatom Nitzschia pungens f. multiseries, which produces domoic acid, the cause of amnesic shellfish poisoning. Nitzschia cultures grown in this incubator yielded maximum cell concentrations of 158–166 × 10⁶ cells 1⁻¹, a peak intracellular domoic acid level of 2.0 pg cell⁻¹ and a maximum division rate of 0.3 d ⁻¹. The VIICI design is ideally suited for laboratory mass culture of phytoplankton, and has potential for wide application in phycotoxin, toxicological and environmental research, as well as for aquaculture.     

Picoplankton photosynthesis and diurnal variations in photosynthesis-irradiance relationship in a eutrophic and meso-oligotrophic lake

The abundance and relative importance of autotrophic picoplankton were investigated in two lakes of different trophic status. In the eutrophic lake, measurements of primary production were performed on water samples in situ and in a light incubator three times during the day whereas for the oligotrophic lake, only one measurement of primary production was performed on water samples in the incubator. Dark-carbon losses of phytoplankton from Lake Loosdrecht were investigated in time series. Cell numbers of autotrophic picoplankton in eutrophic Lake Loosdrecht (3.2 × 10⁴ cells ml^–1) were lower than in meso-oligotrophic Lake Maarsseveen (9.8 and 11.4 × 10⁴ cells ml^–1 at the surface and bottom respectively). In the phytoplankton of both lakes the ratio of picoplankton production increased with decreasing light intensity. In Lake Loosdrecht depth-integrated contribution of picoplankton to total photosynthesis was less than 4%. The P-I-relationship showed diurnal variations in light saturated photosynthesis, while light limited carbon uptake remained constant during the day. Dark carbon losses from short-term labelled phytoplankton during the first 12 hours of the night period accounted for 10–25% of material fixed during the preceeding light period.     

Phytoplankton primary production in a eutrophic cooling water pond

The seasonal variation of phytoplankton photosynthesis was measured with ¹⁴C-method in a warmed ice-free pond in central Finland. Simultaneously with in situ measurements the photosynthesis was also measured in an incubator with different water temperatures and constant light (ca. 16 W m^–2). The total annual photosynthesis was 57.2 C m^–2 a^–1. The portion of the winter and spring production of the annual photosynthesis was 18.4%, that of the autumn production ws 17.4%. Thus 64.3% of the total annual phytoplankton photosynthesis occurred in the three summer months. The range of the daily integrated photosynthesis per unit area was 1.9—563 mg C m^–2d^–1. The photosynthetic rate per unit chlorophyll a varied in situ from 0.94 to 33.1 mg C (mg chl. a)^–1 d^–1. The highest value was measured in the beginning of July and the lowest in mid-January. The photosynthetic rate increased in situ exponentially with increasing water temperature. In the incubator the highest photosynthetic rate values were also found in July and August (at+20 °C) when the phytoplankton population was increasing and the minimum values occurred after every diatom maximum both in spring and autumn. Light was a limiting factor for photosynthesis from September to Mid-January, low water temperature was a limiting factor from late January through May. The efficiency of the photosynthesis varied between 0.1 and 0.7% of P.A.R. According to the incubator experiments the Q₁₀ values for the photosynthesis were 2.45 and 2.44 for the winter population between 1 and 10° C and for the summer population between 5 and 15° C, respectively, but the Q₁₀ values decrease at the higher temperatures. The main effect of the warm effluents on the yearly photosynthesis was the increase of production in spring months due to the lack of ice cover. However, the increase of total annual phytoplankton photosynthesis was only ca. 10–15%, because the water temperature was during the spring months below 10° C.     

Conception d’un logiciel de calcul de la thermoneutralité dans les incubateurs fermés pour nouveau-nés prématurés (projet Pretherm®)

ObjectivePreterm neonates should be nursed in a closed incubator and at appropriate incubator air temperatures and humidities, in order to increase their chances of survival and reduce their length of stay in the intensive care unit. At present, these “thermoneutral” conditions do not take account of all the thermal parameters involved in neonate's cooling. The project is led under the ANR TecSan call. It aims to create and validate a software for assessing thermoneutrality in closed incubators for preterm neonates completely.MethodsWe have written software that models the neonate's thermal balance. It considers all the pathways for heat losses from the neonate by accounting for physiological and morphological criteria that are highly specific for this population.ResultsPretherm^® software evaluates the optimal incubator air temperature and humidity for each preterm neonate on a daily basis. It also takes account of the neonate's state of clothing (i.e. a diaper in the presence or absence of a hat and nest), birthweight and postnatal age. The software also defines the critical air incubator temperatures above which the neonate runs a risk of hyper- or hypothermia within the coming hour.ConclusionOur software serves as a decision support tool for physicians and should improve the standard of care provided to neonates at a high risk of functional impairments after birth. Clinical validation of the software is in progress, with an analysis of the medical and physiological criteria that reflect the neonate's vital functions.     

Analysis of the shape of P vs I curves of a natural phytoplankton assemblage during the year in the mesotrophic Lake Maarsseveen (I), The Netherlands

Production versus light response curves were made of natural phytoplankton assemblages during the year in an incubator at 8 different light intensities ranging from 60 to 1500 Einstein.m^–2.sec^–1. The shape of these curves is analyzed in relation to the sensitivity for photo-inhibition. At the end of the month of April, there is a sudden shift in this sensitivity at higher light intensities. The algal assemblage becomes less sensitive for photo-inhibition. The influence of light-adaptation, temperature, nutrient limitation and species composition is discussed.     

Longevity and food consumption of microwave-treated (2.45 GHz Cw) honeybees in the laboratory

Adult honeybees, confined singly or in small clusters, were exposed for 0.5, 6, and 24 hours to 2.45-GHz continuous wave microwave radiation at power densities of 3, 6, 12, 25, and 50 mW/cm². Following exposure, bees were held in the incubator for 21 days to determine the consumption of sucrose syrup and to observe mortality. No significant differences were found between microwave-treated and sham-treated or control bees.     

Bioprocessing in space: Human cells attach to beads in microgravity

Attachment to a substrate and survival of human embryonic kidney (HEK) cells have been tested in an incubator installed in the flight-deck of the Space Shuttle ‘Challenger’ during its eighth mission.HEK cells are producing the enzyme urokinase and are presently investigated as candidates for electrophoretic separation in an apparatus developed and manufactured by McDonnell Douglas.Attachment of HEK cells to a substrate is mandatory for survival and production of urokinase after electrophoretic separation.Analysis of the samples shows that cells adhere, spread and survive in microgravity (< 10⁻³ ×g) conditions as well as the ground controls at 1 × g. This result represents an important step towards further bioprocessing in space.     

A Strain of Pseudomonas sp. Isolated from Piggery Wastewater Treatment Systems with Heterotrophic Nitrification Capability in Taiwan

A high concentration of NH₄⁺ in piggery wastewater is major problem in Taiwan. Therefore, in our study, we isolated native heterotrophic nitrifiers for piggery wastewater treatment. Heterotrophic nitrifier AS-1 was isolated and characterized from the activated sludge of a piggery wastewater system. Sets of triplicate crimp-sealed serum bottles were used to demonstrate the heterotrophic nitrifying capability of strain AS-1 in an incubator at 30°C. All serum bottles contained 80 mL medium, and the remainder of the bottle headspace was filled with pure oxygen. The experimental results showed that 2.5 ± 0.2 mmol L⁻¹ NH₄⁺ was removed by 58 hours, and, eventually, 1.5 ± 0.5 mmol L⁻¹ N₂ and 0.2 ± 0.0 mmol L⁻¹ N₂O were produced. The removal rate of NH₄⁺ by the strain AS-1 was 1.75 mmol NH₄⁺ g cell⁻¹ h⁻¹. This strain was then identified as Pseudomonas alcaligenes (97% identity) by sequencing its 16S rDNA and comparing it with other microorganisms. Thus, strain AS-1 displays high promise for future application for in situ NH₄⁺ removal from piggery wastewater.     

Deciphering Axonal Pathways of Genetically Defined Groups of Neurons in the Chick Neural Tube Utilizing in ovo Electroporation

Employment of enhancer elements to drive expression of reporter genes in neurons is a widely used paradigm for tracking axonal projection. For tracking axonal projection of spinal interneurons in vertebrates, germ line-targeted reporter genes yield bilaterally symmetric labeling. Therefore, it is hard to distinguish between the ipsi- and contra-laterally projecting axons. Unilateral electroporation into the chick neural tube provides a useful means to restrict expression of a reporter gene to one side of the central nervous system, and to follow axonal projection on both sides ^{1 ,2-5}. This video demonstrates first how to handle the eggs prior to injection. At HH stage 18-20, DNA is injected into the sacral level of the neural tube, then tungsten electrodes are placed parallel to the embryo and short electrical pulses are administered with a pulse generator. The egg is sealed with tape and placed back into an incubator for further development. Three days later (E6) the spinal cord is removed as an open book preparation from embryo, fixed, and processed for whole mount antibody staining. The stained spinal cord is mounted on slide and visualized using confocal microscopy.     

The influence of the spectral composition of irradiance on primary production in the Eastern Scheldt (The Netherlands)

The error inin vivo ¹⁴C incubator measurements of primary production in the Eastern Scheldt when neutral density filters were used and the error obtained when no account was taken of the spectral changes in submarine irradiance that occur with increasing depth, were evaluated theoretically. By multiplying the photosynthetic action spectra of two marine algae by calculated irradiance in the euphotic layer using Kd and Kd() respectively, the gross primary production P[Ed(400–700)] and P[Ed()] was computed. In the green-brown waters of the Eastern Scheldt estuary the use of neutral density filters was sufficient to simulate the underwater light conditions. In clear waters it can cause an overestimation of the gross production.     

Mechanical stimulation-induced chilling tolerance in tobacco suspension cultured cells and its relation to proline

Mechanical stimulation (MS), widely existing but usually ignored in nature, is one of the major environmental stress factors. MS by increasing the rotational speed of shaker incubator could alleviate a decrease in vitality of tobacco (Nicotiana tabacum L.) suspension cultured cells and reduce the accumulation of MDA under chilling stress at 1°C, which in turn improved survival percentage under chilling stress and regrowth ability of tobacco suspension cells after chilling stress. In addition, MS could increase the activity of Δ¹-pyrroline-5-carboxylate synthetase (P5CS) and induce the accumulation of endogenous proline in tobacco cells; exogenously applied proline also could enhance its endogenous level under normal culture conditions and survival percent-age of the cells under chilling stress. These results suggest that MS could improve chilling tolerance of tobacco suspension cells and the acquisition of this chilling tolerance was related to proline.     

Temperature profiles of growth and ethanol tolerance of the xylose-fermenting yeasts Candida shehatae and Pichia stipitis

Summary The effect of different ethanol concentrations on the growth of Candida shehatae and Pichia stipitis with xylose as substrate was evaluated in a temperature gradient incubator. The upper limit of the temperature profiles of ethanol tolerance of both yeast strains were similar, although P. stipitis appeared to have a slightly higher ethanol tolerance in the higher temperature range. An increase in the ethanol concentration severely depressed the maximum growth temperature, and also increased the minimum growth temperature slightly. The ethanol tolerance limit of 46–48 g·l^-1 occurred within a narrow temperature plateau of 11 to 22° C. The low ethanol tolerance of these pentose fermenting yeasts is detrimental for commercial ethanol production from hemicellulose hydrolysates.     

Lactobacillus salivarius CTC2197 Prevents Salmonella enteritidis Colonization in Chickens

A rifampin-resistant Lactobacillus salivarius strain, CTC2197, was assessed as a probiotic in poultry, by studying its ability to prevent Salmonella enteritidis C-114 colonization in chickens. When the probiotic strain was dosed by oral gavage together with S. enteritidis C-114 directly into the proventriculus in 1-day-old Leghorn chickens, the pathogen was completely removed from the birds after 21 days. The same results were obtained when the probiotic strain was also administered through the feed and the drinking water apart from direct inoculation into the proventriculus. The inclusion of L. salivarius CTC2197 in the first day chicken feed revealed that a concentration of 10⁵ CFU g⁻¹ was enough to ensure the colonization of the gastrointestinal tract of the birds after 1 week. However, between 21 and 28 days, L. salivarius CTC2197 was undetectable in the gastrointestinal tract of some birds, showing that more than one dose would be necessary to ensure its presence till the end of the rearing time. Freeze-drying and freezing with glycerol or skim milk as cryoprotective agents, appeared to be suitable methods to preserve the probiotic strain. The inclusion of the L. salivarius CTC2197 in a commercial feed mixture seemed to be a good way to supply it on the farm, although the strain showed sensitivity to the temperatures used during the feed mixture storage and in the chicken incubator rooms. Moreover, survival had been improved after several reinoculations in chicken feed mixture.     

An Anaerobic, Intrachamber Incubator for Growth of Methanosarcina spp. on Methanol-Containing Solid Media

To simplify the incubation of Methanosarcina spp. on solid agar medium, a two-port, manual, rectangular air lock was modified to serve as an anaerobic incubator. In one operation, it is possible to incubate 153 petri plates, the equivalent of 11 standard anaerobic jars, with plating efficiencies identical to those of traditional protocols.     

茉莉酸甲酯对马尾松萜类合成酶活性及其细胞化学定位的影响

马尾松(Pinus massoniana)是我国南方生态建设与造林用材的主要树种,为了揭示马尾松抗虫机理尤其是诱导抗虫性的分子机制,该研究以马尾松幼苗为材料,通过外源喷施茉利酸甲酯(Me JA),分析了处理与对照间植株针叶显微结构、萜类合成酶活性及其细胞化学定位的变化。结果表明:在0.2 mmol·L~(-1)Me JA处理下马尾松植株松针中萜类物质,尤其是单萜、二萜的相对含量增加,马尾松毛虫拒食性明显,诱导抗性增强。显微观测中,针叶叶肉细胞内树脂道分泌物增加,叶绿体数目减少,但叶绿体体积增大,叶绿体片层结构增加。Me JA处理4周后,针叶中萜类合成酶活性增加,通过电镜酶细胞化学观察,膜系统尤其是叶绿体膜上萜类合成酶活性定位明显增强。这说明Me JA诱导的马尾松诱导抗性可能与改变的叶绿体结构及绿色质体萜类合成酶活性密切相关。     

Fluorescence optical detection in situ for real‐time monitoring of cytochrome P450 enzymatic activity of liver cells in multiple microfluidic devices

We describe an in situ fluorescence optical detection system to demonstrate real‐time and non‐invasive detection of reaction products in a microfluidic device while under perfusion within a standard incubator. The detection system is designed to be compact and robust for operation inside a mammalian cell culture incubator for quantitative detection of fluorescent signal from microfluidic devices. When compared to a standard plate reader, both systems showed similar biphasic response curves with two linear regions. Such a detection system allows real‐time measurements in microfluidic devices with cells without perturbing the culture environment. In a proof‐of‐concept experiment, the cytochrome P450 1A1/1A2 activity of a hepatoma cell line (HepG2/C3A) was monitored by measuring the enzymatic conversion of ethoxyresorufin to resorufin. The hepatoma cell line was embedded in Matrigel^TM construct and cultured in a microfluidic device with medium perfusion. The response of the cells, in terms of P450 1A1/1A2 activity, was significantly different in a plate well system and the microfluidic device. Uninduced cells showed almost no activity in the plate assay, while uninduced cells in Matrigel^TM with perfusion in a microfluidic device showed high activity. Cells in the plate assay showed a significant response to induction with 3‐Methylcholanthrene while cells in the microfluidic device did not respond to the inducer. These results demonstrate that the system is a potentially useful method to measure cell response in a microfluidic system. Biotechnol. Bioeng. 2009; 104: 516–525 © 2009 Wiley Periodicals, Inc.     

Potential of essential oil combinations for surface and air disinfection

In this study, it was aimed to develop a novel disinfectant from various essential oils containing active components with antimicrobial activity. The mixture of oregano, cinnamon and clove oils (1 : 1 : 1) with 10% oil concentration (SOM) was used as potential disinfectant on various areas and showed the highest antimicrobial activity among oil combinations tested. SOM reduced the numbers of total mesophilic aerobic bacteria (TMAB; 2·27 log CFU per 25 cm²) and Escherichia coli (4·60 log CFU per 25 cm²) under the detection limits. Application of SOM (1, 2, 3, 4 and 6%) into incubators reduced TMAB and mould-yeast counts of incubator air by 82·9 and 100% respectively. SOM application (3%) into ambient air also reduced its TMAB and mould-yeast counts by 92 and 84·6% respectively. While ethanol is commonly used for the disinfection of environments, equipment and surfaces, SOM is an important alternative that may also be used for the disinfection of various surfaces as well as air.