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.     

Discovery of a Redox Thiol Switch: Implications for Cellular Energy Metabolism

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Highlights

• •Develop a TMT-based proteomics tool to profile cysteine persulfides in the cellular proteomes.
• •Discover a Redox Thiol Switch from protein S-glutathioinylation to S-persulfidation (RTS^GS) with implications in the regulation of cellular energy metabolism under oxidative stress.

     

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.     

钙对根际低氧胁迫下黄瓜幼苗活性氧代谢的影响

采用营养液栽培系统,以黄瓜品种中农8号为材料,研究了Ca2 对根际低氧胁迫下黄瓜幼苗体内超氧阴离子(O2?-)、过氧化氢(H2O2)、丙二醛(MDA)含量和超氧化物歧化酶(SOD)、过氧化物酶(POD)、过氧化氢酶(CAT)、抗坏血酸过氧化物酶(APX)、谷胱苷肽还原酶(GR)活性的影响.结果表明:低氧胁迫下黄瓜体内活性氧含量和保护酶活性均高于对照;低氧缺钙处理的活性氧含量最高,而保护酶活性却较低;营养液Ca2 浓度提高到8mmol/L后,显著降低了低氧胁迫下黄瓜幼苗体内MDA、H2O2含量和O?2-产生速率,提高了SOD、POD、CAT、APX、GR活性,说明Ca2 可减少低氧胁迫下黄瓜幼苗体内活性氧的产生,提高抗氧化酶的活性、降低膜脂过氧化水平,减缓低氧胁迫对植株的伤害,增强黄瓜幼苗对低氧逆境的适应性.     

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.     

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.     

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.     

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.     

Immobilization Methods for NMR Studies of Cellular Metabolism - A Practical Guide

Nuclear magnetic resonance (NMR) can be used in a nondestructive manner to study cellular metabolism in intact cell samples such as a suspension of cells. However, many different cell types require a well-regulated medium that includes a buffered pH, as well as a continuous supply of oxygen. A series of methods that have been used for the maintenance of the extracellular conditions involves the immobilization of cells, followed by perfusion of the immobilized cell sample. NMR studies can then be performed for extended periods of time, as well as under sterile conditions. We discuss methodology, with perfused erythrocytes and thymocytes as specific examples.     

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.     

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.