A Lab Assembled Microcontroller-Based Sensor Module for Continuous Oxygen Measurement in Portable Hypoxia Chambers

Background

Hypoxia-based cell culture experiments are routine and essential components of in vitro cancer research. Most laboratories use low-cost portable modular chambers to achieve hypoxic conditions for cell cultures, where the sealed chambers are purged with a gas mixture of preset O₂ concentration. Studies are conducted under the assumption that hypoxia remains unaltered throughout the 48 to 72 hour duration of such experiments. Since these chambers lack any sensor or detection system to monitor gas-phase O₂, the cell-based data tend to be non-uniform due to the ad hoc nature of the experimental setup.

Methodology

With the availability of low-cost open-source microcontroller-based electronic project kits, it is now possible for researchers to program these with easy-to-use software, link them to sensors, and place them in basic scientific apparatus to monitor and record experimental parameters. We report here the design and construction of a small-footprint kit for continuous measurement and recording of O₂ concentration in modular hypoxia chambers. The low-cost assembly (US$135) consists of an Arduino-based microcontroller, data-logging freeware, and a factory pre-calibrated miniature O₂ sensor. A small, intuitive software program was written by the authors to control the data input and output. The basic nature of the kit will enable any student in biology with minimal experience in hobby-electronics to assemble the system and edit the program parameters to suit individual experimental conditions.

Results/Conclusions

We show the kit’s utility and stability of data output via a series of hypoxia experiments. The studies also demonstrated the critical need to monitor and adjust gas-phase O₂ concentration during hypoxia-based experiments to prevent experimental errors or failure due to partial loss of hypoxia. Thus, incorporating the sensor-microcontroller module to a portable hypoxia chamber provides a researcher a capability that was previously available only to labs with access to sophisticated (and expensive) cell culture incubators.     

注射用内给氧对缺血再灌注损伤兔肝脏能量代谢的影响

为了探讨注射用内给氧对肝脏缺血再灌注(I/R)损伤肝脏能量代谢的影响,将48只健康新西兰长耳大白兔随机分为4组:假手术组(A组),缺血再灌注组(B组),缺血再灌注 周围静脉注射用内给氧组(C组),缺血再灌注 肝动脉注射内给氧组(D组),每组12只,采用Pringle氏法建立肝脏I/R模型,比较4组大白兔缺血再灌注后1、2、24h肝组织内三磷酸腺苷(ATP)、二磷酸腺苷(ADP)、一磷酸腺苷(AMP)含量、肝脏的细胞能荷(EC)及肝组织形态学的变化.结果表明,与A组比较,8、C、D三组肝功能损害重,肝组织ATP含量,能荷值(EC)降低(P<0.05);肝组织病理学改变明显(P<0.05),B组以上各项指标差异更为显著,与B组比较,C、D二组肝组织ATP含量,能荷值(EC)高(P<0.05);肝组织病理学改变较轻(P<0.05);C组与D组比较,各参数无显著性差异.以上研究表明.注射用内给氧可通过改善肝细胞的能量代谢而减轻肝缺血再灌注损伤.     

Simultaneous Measurement of Dissolved Oxygen and Oxidation-Reduction Potentials in the Aerobic Culture

The most dominant factor influencing the oxidation-reduction potentials (E) in the cultured system was oxygen tension. H was an useful index to express the degree of oxygen supply in place of dissolved oxygen (P_L) under a limited oxygen supply. The conversion of microbial products caused by the change in oxygen supply was clearly analyzed by the use of E value. Bacillus subtilis excreted lactic acid at the E value ?220 mV, 2,3-butyleneglycol at ?195 mV and acetoin at ?160mV as the main product. E also gave the significant information concerning the changes in cell’s respiration. Cyanide at the concentration of 10^?5m, azide at 10^?3m and 2,4-dinitrophenol (DNP) at 10^?2m inhibited cell respiration causing the decrease in E and the increase in P_L, and DNP at 0.4×10^?3m promoted oxygen uptake of the cells causing the decrease in both E and P_L.     

Effect of Oxygen on Lactose Metabolism in Lactic Streptococci

Three strains of Streptococcus lactis, two of Streptococcus cremoris, and one of Streptococcus thermophilus metabolized oxygen in the presence of added carbohydrate primarily via a closely coupled NADH oxidase/NADH peroxidase system. No buildup of the toxic intermediate H₂O₂ was detected with the three S. lactis strains. All six strains contained significant superoxide dismutase activity and are clearly aerotolerant. Lactose- or glucose-driven oxygen consumption was biphasic, with a rapid initial rate followed by a slower secondary rate which correlated with factors affecting the in vivo activation of lactate dehydrogenase. The rate of oxygen consumption was rapid under conditions that led to a reduction in lactate dehydrogenase activity (low intracellular fructose 1,6-bisphosphate concentration). These conditions could be achieved with nongrowing cells by adding lactose at a constant but limiting rate. When the rate of lactose fermentation was limited to 5% of its maximum, nongrowing cells of S. lactis strains ML3 and ML8 carried out an essentially homoacetic fermentation under aerobic conditions. These same cells carried out the expected homolactic fermentation when presented with excess lactose under anaerobic conditions. Homoacetic fermentation leads to the generation of more energy, by substrate-level phosphorylation via acetate kinase, than the homolactic fermentation. However, it was not observed in growing cells and was restricted to slow fermentation rates with nongrowing cells.     

An Apparatus (Georgiou-Petri Dish) for Growing Fungi and other Microorganisms on Liquid Media in a Petri Dish

This work describes a new apparatus for growing fungi and other microorganisms on liquid nutrient media in a Petri dish. The apparatus is composed of a net supporting a cellophane membrane stretched between an outer and an inner ring that is placed inside a Petri dish. This modification of the standard Petri dish offers many advantages for studying growth, metabolism, differentiation, and other aspects of fungi in liquid cultures with minimal waste of expensive chemicals. Monitoring of excreted or absorbed substances by the fungi, the aseptic transfer of undisturbed fungal colonies from dish to dish, and harvesting are made easier, using this apparatus.     

基于光学相干层析成像技术的小鼠表皮厚度的无损测量及分析

光学相干层析成像技术(optical coherence tomography,OCT)在研究活体皮肤的表皮和真皮上层方面是一种很有潜力的手段.本文以小白鼠为研究对象,脱毛后,采用OCT系统对同一部位的小鼠皮肤在制作切片过程中(活体、离体、固定24 h后)进行成像跟踪,分别获得OCT强度图中第一强度峰到第一个波谷,以及第一、二强度峰之间的距离,比较两种结果和组织学的对应情况,并分析其原因,同时关注切片过程中各种物理、化学因素对皮肤各层厚度的影响,研究了OCT图像的各个反射峰与实际皮肤的结构之间联系.     

Radiation Effects on Cellular Metabolism and Disease Resistance of Jute

Air dried seeds of seven jute (Corchorus capsularis L.) cultivars were irradiated with different doses of X-rays. JRC 1108 and JRC 412 were most and least sensitive to X-rays, respectively. JRC 412 was susceptible to Colletotrichum corchorum both before and after X-irradiation while JRC1108 became more resistant after treatment. Leaf diffusates but not leaf exudates of both non-irradiated and X-irradiated (40,000 r) cvs. exhibited antifungal activity which was more pronounced in case of irradiated JRC 1108. JRC 412 contained greater amounts of amino acids than JRC 1108 both before and after X-irradiation. Signifinon-irradiated, infected ones. The physiological implications of the results are discussed in relation to greater resistance of X-irradiated JRC 1108.     

Effects of Colicins E1 and K on Cellular Metabolism

Colicins E1 and K inhibited a whole series of energy-dependent reactions in Escherichia coli cells, including motility, biosynthesis of nucleic acids, proteins and polysaccharides, and the conversion of ornithine to citrulline. Respiration was only partially affected, and substrates such as glucose continued to be catabolized through the normal pathways, albeit with reduced CO(2) production. The soluble products of aerobic glucose catabolism by colicin-treated cells were analyzed. Pyruvate replaced acetate as the major excreted product, and the following intermediates of glycolysis were excreted in significant amounts: glucose-6-phosphate, fructose-1,6-diphosphate, dihydroxyacetone phosphate, and 3-phosphoglycerate. Anaerobically growing cells manifested a somewhat enhanced tolerance to the colicins. This protection by anaerobiosis appeared to depend on the exclusion of oxygen more than on the extent of fermentative catabolism versus catabolism of the respiratory type. These results are interpreted in terms of possible functions of colicin in lowering the adenosine triphosphate (ATP) content of the cells and in terms of the role of lowered ATP levels in inhibiting many of the energy-requiring reactions.     

Investigating the Effects of Statins on Cellular Lipid Metabolism Using a Yeast Expression System

In humans, defects in lipid metabolism are associated with a number of severe diseases such as atherosclerosis, obesity and type II diabetes. Hypercholesterolemia is a primary risk factor for coronary artery disease, the major cause of premature deaths in developed countries. Statins are inhibitors of 3-hydroxy-3-methylglutaryl-CoA reductase (HMGR), the key enzyme of the sterol synthesis pathway. Since yeast Saccharomyces cerevisiae harbours many counterparts of mammalian enzymes involved in lipid-synthesizing pathways, conclusions drawn from research with this single cell eukaryotic organism can be readily applied to higher eukaryotes. Using a yeast strain with deletions of both HMG1 and HMG2 genes (i.e. completely devoid of HMGR activity) with introduced wild-type or mutant form of human HMGR (hHMGR) gene we investigated the effects of statins on the lipid metabolism of the cell. The relative quantification of mRNA demonstrated a different effect of simvastatin on the expression of the wild-type and mutated hHMGR gene. GC/MS analyses showed a significant decrease of sterols and enhanced conversion of squalene and sterol precursors into ergosterol. This was accompanied by the mobilization of ergosterol precursors localized in lipid particles in the form of steryl esters visualized by confocal microscopy. Changes in the level of ergosterol and its precursors in cells treated with simvastatin depend on the mutation in the hHMGR gene. HPLC/MS analyses indicated a reduced level of phospholipids not connected with the mevalonic acid pathway. We detected two significant phenomena. First, cells treated with simvastatin develop an adaptive response compensating the lower activity of HMGR. This includes enhanced conversion of sterol precursors into ergosterol, mobilization of steryl esters and increased expression of the hHMGR gene. Second, statins cause a substantial drop in the level of glycerophospholipids.     

Fabrication and Operation of an Oxygen Insert for Adherent Cellular Cultures

Oxygen is a key modulator of many cellular pathways, but current devices permitting in vitro oxygen modulation fail to meet the needs of biomedical research. The hypoxic chamber offers a simple system to control oxygenation in standard culture vessels, but lacks precise temporal and spatial control over the oxygen concentration at the cell surface, preventing its application in studying a variety of physiological phenomena. Other systems have improved upon the hypoxic chamber, but require specialized knowledge and equipment for their operation, making them intimidating for the average researcher. A microfabricated insert for multiwell plates has been developed to more effectively control the temporal and spatial oxygen concentration to better model physiological phenomena found in vivo . The platform consists of a polydimethylsiloxane insert that nests into a standard multiwell plate and serves as a passive microfluidic gas network with a gas-permeable membrane aimed to modulate oxygen delivery to adherent cells. The device is simple to use and is connected to gas cylinders that provide the pressure to introduce the desired oxygen concentration into the platform. Fabrication involves a combination of standard SU-8 photolithography, replica molding, and defined PDMS spinning on a silicon wafer. The components of the device are bonded after surface treatment using a hand-held plasma system. Validation is accomplished with a planar fluorescent oxygen sensor. Equilibration time is on the order of minutes and a wide variety of oxygen profiles can be attained based on the device design, such as the cyclic profile achieved in this study, and even oxygen gradients to mimic those found in vivo . The device can be sterilized for cell culture using common methods without loss of function. The device''s applicability to studying the in vitro wound healing response will be demonstrated.Open in a separate windowClick here to view.^{(69M, flv)}     

Noninvasive Monitoring of Small Intestinal Oxygen in a Rat Model of Chronic Mesenteric Ischemia

We noninvasively monitored the partial pressure of oxygen (pO₂) in rat’s small intestine using a model of chronic mesenteric ischemia by electron paramagnetic resonance oximetry over a 7-day period. The particulate probe lithium octa-n-butoxynaphthalocyanine (LiNc-BuO) was embedded into the oxygen permeable material polydimethyl siloxane by cast-molding and polymerization (Oxy-Chip). A one-time surgical procedure was performed to place the Oxy-Chip on the outer wall of the small intestine (SI). The superior mesenteric artery (SMA) was banded to ~30 % of blood flow for experimental rats. Noninvasive measurement of pO₂ was performed at the baseline for control rats or immediate post-banding and on days 1, 3, and 7. The SI pO₂ for control rats remained stable over the 7-day period. The pO₂ on day-7 was 54.5 ± 0.9 mmHg (mean ± SE). SMA-banded rats were significantly different from controls with a noted reduction in pO₂ post banding with a progressive decline to a final pO₂ of 20.9 ± 4.5 mmHg (mean ± SE; p = 0.02). All SMA-banded rats developed adhesions around the Oxy-Chip, yet remained asymptomatic. The hypoxia marker Hypoxyprobe? was used to validate the low tissue pO₂. Brown cytoplasmic staining was consistent with hypoxia. Mild brown staining was noted predominantly on the villus tips in control animals. SMA-banded rats had an extended region of hypoxic involvement in the villus with a higher intensity of cytoplasmic staining. Deep brown stainings of the enteric nervous system neurons and connective tissue both within layers and in the mesentery were noted. SMA-banded rats with lower pO₂ values had a higher intensity of staining. Thus, monitoring SI pO₂ using the probe Oxy-Chip provides a valid measure of tissue oxygenation. Tracking pO₂ in conditions that produce chronic mesenteric ischemia will contribute to our understanding of intestinal tissue oxygenation and how changes impact symptom evolution and the trajectory of chronic disease.     

Measurement of Reactive Oxygen Species in the Culture Media Using Acridan Lumigen PS-3 Assay

Reactive oxygen species (ROS) are generated continuously during aerobic metabolism. ROS are highly reactive molecules and in excessive amounts, can lead to protein and DNA oxidation, protein cross-linking, and cell death. Cell-culture models provide a valuable tool in understanding the mechanisms that lead to cell death. Accumulation of ROS within cells and/or their release into the culture media are highly cell type-specific. The ability to estimate ROS levels in the culture media is an important step in understanding the mechanisms contributing to disease processes. In this paper, we describe the optimization of a simple method to estimate ROS levels in the culture media using the Acridan Lumigen PS-3 reagent provided in the Amersham ECL Plus kit (GE Healthcare, UK). We have shown that the Acridan Lumigen PS-3 assay generates ROS-specific chemiluminescence in fresh as well as media stored at −20°C, in as little as 10–20 μl of samples. The method was able to detect the dose (of stimulants)- and time (acute and chronic)-dependent changes in ROS levels in media collected from various cell types. Our results suggest that the kit reagents, PBS buffer, and various media did not contribute significantly to the overall chemiluminescence generated in the assay; however, we suggest that the unused medium specific for each cell type should be used as blanks and final readings of test samples normalized against these readings. As this method uses commonly available laboratory equipment and commercially available reagents, we believe this assay is convenient, economical, and specific in estimating ROS released extracellularly into the culture media.     

Effects of Oxygen on Metabolism by the Glycollate Pathway in Leaves

Segments of wheat leaves were supplied in the light with ¹⁴C-labelledserine or glucose in atmospheres containing different concentrationsof O₂ and zero or 350 parts/10⁶ CO₂. Some O₂ was necessary forsucrose synthesis from either serine or glucose but sucrosesynthesis from glucose depended on reactions with a high affinityfor O₂ whereas sucrose synthesis from serine depended both onreactions with high and low affinities for O₂. In the presenceof CO₂ sucrose synthesis from serine was decreased when theO₂ concentration was increased from 20 to 80% by volume andCO₂ was liberated; sucrose synthesis from glucose was almostunaffected by the same change in conditions. Also, in an atmospherecontaining 80% O₂ and 350 parts/10⁶ CO₂, radioactivity from[¹⁴C]serine, was incorporated into glycine. This was not truefor glucose feeding. Hence glucose provides a substrate forsucrose synthesis but not for photorespiration whereas serineis used for both processes in the presence of CO₂; in the absenceof CO₂ glucose provides substrate for both sucrose synthesisand photorespiration and serine metabolism to sucrose is restricted.     

A New Functional MRI Approach for Investigating Modulations of Brain Oxygen Metabolism

Functional MRI (fMRI) using the blood oxygenation level dependent (BOLD) signal is a common technique in the study of brain function. The BOLD signal is sensitive to the complex interaction of physiological changes including cerebral blood flow (CBF), cerebral blood volume (CBV), and cerebral oxygen metabolism (CMRO₂). A primary goal of quantitative fMRI methods is to combine BOLD imaging with other measurements (such as CBF measured with arterial spin labeling) to derive information about CMRO₂. This requires an accurate mathematical model to relate the BOLD signal to the physiological and hemodynamic changes; the most commonly used of these is the Davis model. Here, we propose a new nonlinear model that is straightforward and shows heuristic value in clearly relating the BOLD signal to blood flow, blood volume and the blood flow-oxygen metabolism coupling ratio. The model was tested for accuracy against a more detailed model adapted for magnetic fields of 1.5, 3 and 7T. The mathematical form of the heuristic model suggests a new ratio method for comparing combined BOLD and CBF data from two different stimulus responses to determine whether CBF and CMRO₂ coupling differs. The method does not require a calibration experiment or knowledge of parameter values as long as the exponential parameter describing the CBF-CBV relationship remains constant between stimuli. The method was found to work well for 1.5 and 3T but is prone to systematic error at 7T. If more specific information regarding changes in CMRO₂ is required, then with accuracy similar to that of the Davis model, the heuristic model can be applied to calibrated BOLD data at 1.5T, 3T and 7T. Both models work well over a reasonable range of blood flow and oxygen metabolism changes but are less accurate when applied to a simulated caffeine experiment in which CBF decreases and CMRO₂ increases.     

Effects of Oxygen and Glucose on Energy Metabolism and Dimorphism of Mucor genevensis Grown in Continuous Culture: Reversibility of Yeast-Mycelium Conversion

Mucor genevensis was grown in both glucose-limited and glucose-excess continuous cultures over a range of dissolved oxygen concentrations (<0.1 to 25 muM) to determine the effects of glucose and the influence of metabolic mode (fermentative versus oxidative) on dimorphic transformations in this organism. The extent of differentiation between yeast and mycelial phases has been correlated with physiological and biochemical parameters of the cultures. Under glucose limitation, oxidative metabolism increased as the dissolved oxygen concentration increased, and this paralleled the increase in the proportion of the mycelial phase in the cultures. Filamentous growth and oxidative metabolism were both inhibited by glucose even though mitochondrial development was only slightly repressed. However, the presence of chloramphenicol in glucose-limited aerobic cultures inhibited mitochondrial respiratory development but did not induce yeast-like growth, indicating that oxidative metabolism is not essential for mycelial development. Once mycelial cultures had been established under aerobic, glucose-limited conditions, subsequent reversal to anaerobic conditions or treatment with chloramphenicol caused only a limited reversal (<35%) to the yeast-like form. Glucose, however, induced a complete reversion to yeast-like form. It is concluded that glucose is the most important single culture factor determining the morphological status of M. genevensis; mitochondrial development and the functional oxidative capacities of the cell appear to be less important factors in the differentiation process.     

Measurement of Cellular Immunity in Human Coccidioidomycosis

Measurement of cellular immune response in human coccidioidomycosis, a disease of the Western Hemisphere caused by the soil-dwelling fungus Coccidioides, began more than 75 years ago. Numerous studies have shown that measurement of coccidioidal cellular immunity is a useful epidemiologic and clinical tool. The first widely used coccidioidal skin-test reagent, coccidioidin, was derived from a filtrate of cultured mycelia of Coccidioides by Smith and colleagues in the 1940's. It remained the standard until the 1970's, when spherulin, obtained from the spherule form of the fungus, was found to be more sensitive. Both reagents are specific indicators of coccidioidal cellular immunity. Since then, other coccidioidal antigens have been identified and in vitro tests of cellular immunity have been shown to yield comparable results to skin testing. In vitro assays have also begun to open a window on the specific immunologic events of coccidioidal cellular immunity. Persistent expression of coccidioidal cellular immune response appears to augur an improved clinical outcome. Despite this, a study of a coccidioidal vaccine failed to demonstrate efficacy. Current and future studies are focused on modulating the coccidioidal immune response in vitro and in vivo and on developing an improved vaccine.     

汞对慈姑活性氧代谢和染色体的影响

研究了不同浓度Hg2 以及处理时间对慈姑叶和根的活性氧(reactive oxygen species,ROS)和染色体的影响。结果显示：汞能诱导慈姑叶和根大量产生ROS,其中H2O2的突发早于O2。0．5-5mg／L处理组染色体畸变率(Chromosomal aberrant frequency,CAF)和微核率(micronucleus frequency,MCNF)在72h内与处理浓度、时间呈正相关。CAF、MCNF所反映的细胞遗传毒性的大小主要取决于汞处理浓度,而ROS则起次要作用。H2O2很可能在慈姑抵御汞胁迫中起信号传导作用;慈姑通过促进根部部分细胞死亡提高抗汞能力。     

逆境胁迫下植物体内活性氧代谢及调控机理研究进展

活性氧(reactive oxygen species, ROS)在植物生长发育中扮演着十分重要的角色。适当浓度的ROS是植物所必需的,而在逆境胁迫下ROS会大量积累,从而抑制植物的生长发育甚至杀死植物。为了维持体内ROS的动态平衡,植物进化出了一系列的ROS产生及清除机制。本文对近年来植物在逆境下的ROS产生、清除及其调节机制的研究进展予以综述,重点介绍转录及翻译后水平的ROS清除及其调节机制,并对植物ROS代谢及调控机理的研究提出了进一步展望。