基于H2DCFDA荧光探针的植物活性氧检测方法

活性氧(reactive oxygen species, ROS)是植物体内的一把“双刃剑”。ROS作为信号分子在植物生命活动中发挥关键作用,但ROS过量积累会对生物大分子造成氧化损伤。准确测定ROS含量对于评估植物细胞内的氧化还原状态至关重要。由于植物体内ROS各组分半衰期短且反应活性强,定性定量检测较为困难。因此,选择合适的检测方法以提高检测的时空准确性非常重要。目前,荧光分析法因其具有灵敏度高、选择性好、检出限低和直观性强等优点,受到研究人员的广泛关注。该文详细描述基于流式细胞仪和激光共聚焦显微镜,利用2′,7′-二氯二氢荧光素二乙酸酯(H2DCFDA)荧光探针检测水稻(Oryzasativa)体内ROS水平和时空分布的操作流程及注意事项。该技术也可用于直接检测拟南芥(Arabidopsis thaliana)、玉米(Zea mays)和大豆(Glycine max)等模式植物组织中ROS的水平和分布。     

On-Line Detection of Substrate Exhaustion by Using NAD(P)H Fluorescence

In this study we examined the utility of NAD(P)H fluorescence for monitoring aerobic fermentations of the threonine auxotroph Corynebacterium glutamicum ATCC 14296. Instead of attempting complicated mathematical corrections for inner-filter effects, we found that it is possible to use the information contained in the on-line NAD(P)H fluorescence signal to assess culture metabolic activities during fermentation. The first derivative of the filtered fluorescence signal, which approximates the turnover rate of the NAD(P)H pool, can be used to precisely identify the temporal points of threonine and glucose exhaustion.     

Sensitive Detection of Phosphorus Deficiency in Plants Using Chlorophyll a Fluorescence

Phosphorus (P) is a finite natural resource and an essential plant macronutrient with major impact on crop productivity and global food security. Here, we demonstrate that time-resolved chlorophyll a fluorescence is a unique tool to monitor bioactive P in plants and can be used to detect latent P deficiency. When plants suffer from P deficiency, the shape of the time-dependent fluorescence transients is altered distinctively, as the so-called I step gradually straightens and eventually disappears. This effect is shown to be fully reversible, as P resupply leads to a rapid restoration of the I step. The fading I step suggests that the electron transport at photosystem I (PSI) is affected in P-deficient plants. This is corroborated by the observation that differences at the I step in chlorophyll a fluorescence transients from healthy and P-deficient plants can be completely eliminated through prior reduction of PSI by far-red illumination. Moreover, it is observed that the barley (Hordeum vulgare) mutant Viridis-zb⁶³, which is devoid of PSI activity, similarly does not display the I step. Among the essential plant nutrients, the effect of P deficiency is shown to be specific and sufficiently sensitive to enable rapid in situ determination of latent P deficiency across different plant species, thereby providing a unique tool for timely remediation of P deficiency in agriculture.The world population is estimated to exceed 9 billion people by 2050. This means that agriculture on a global scale has to increase food production by 70% to 100%, and, at the same time, handle the consequences of global climate changes and reduce its environmental footprint (Food and Agriculture Organization of the United Nations, 2009; Godfray et al., 2010; Foley et al., 2011). A major challenge related to this is the supply and use of phosphorus (P) to support future plant production (Cordell et al., 2009; Gilbert, 2009; MacDonald et al., 2011).P is an essential plant nutrient, which means that plants require P in adequate amounts to fulfill a complete lifecycle. It has been estimated that 30% of the world’s agricultural soils are P deficient and need fertilizer addition to ensure yield and quality (MacDonald et al., 2011). However, phosphate rock, the main source of P fertilizers, is a finite natural resource, and the known rock phosphate reserves are estimated to last as little as 50 years in the gloomiest forecasts (Gilbert, 2009; Edixhoven et al., 2013). This makes P a potential strategic natural resource similar to oil, as very few countries control the vast majority of the known reserves (Gilbert, 2009; Elser and Bennett, 2011; Edixhoven et al., 2013). Presently, an immense overuse of P is found in some parts of the world, causing eutrophication of lakes and seas, while P depletion results in severe yield limitations elsewhere (MacDonald et al., 2011; Obersteiner et al., 2013). An essential aspect of solving both of these problems is to increase P use efficiency in agriculture, thus reducing the negative environmental impact of agriculture and helping to ensure a sustainable use of P resources while increasing the worldwide food production (Schröder et al., 2011; Veneklaas et al., 2012).Here, we present a unique analytical principle based on chlorophyll a fluorescence that allows rapid, nondestructive, onsite assessment of plant P status by recording the so-called OJIP transient of a dark-adapted leaf.When a chlorophyll molecule absorbs light, one of three events will occur: The light may be used to drive photosynthesis, it can be dissipated as heat, or it can be reemitted as fluorescence. Less than 10% of light absorbed by the plant causes emission of chlorophyll a fluorescence (Govindjee, 2004; Stirbet and Govindjee, 2011). When a dark-adapted leaf is exposed to saturating actinic light, the resulting time-dependent fluorescence forms a so-called Kautsky curve (Kautsky and Hirsch, 1931; McAlister and Myers, 1940). Within 300 ms, the fluorescence increases from a minimum level (F₀) to the maximum level. If measured with a sufficiently high time resolution, a polyphasic transient with four distinct steps, designated as O, J, I, and P, is observed. After reaching maximum intensity at the P step, the fluorescence intensity declines until it reaches a steady state within a few minutes (Harbinson and Rosenqvist, 2003; Govindjee, 2004).The physiological mechanisms underlying the polyphasic OJIP transient are still not clarified, but it is believed that the J and I steps represent dynamic bottlenecks in the photosynthetic electron transport chain. The first rise (2 ms) from O to J is referred to as the photochemical phase due to its dependence on the intensity of the incoming light. This phase is assumed to reflect the reduction of the primary quinone electron acceptor in PSII (Stirbet and Govindjee, 2011). The reduction of the primary quinone electron acceptor results in a decreased electron trapping efficiency and therefore an increase in the dissipation of absorbed light energy by fluorescence and heat. The second part, from J over I to P, is called the thermal phase due to its temperature sensitivity. This phase is much slower than the first, and it is believed that the J-I phase primarily reflects a sequential reduction of the remaining plastoquinone pool of PSII and that the I-P phase reflects the subsequent electron flow through cytochrome b₆f to electron sinks at the PSI acceptor side (Stirbet and Govindjee, 2011). Thus, the OJIP transient resembles a titration of the photochemical quantum yield and reflects the complex electron transport properties of PSII and PSI.Consistent with their known influence on photosynthesis, deficiencies of essential plant nutrients such as Fe, Cu, Mg, Mn, and S have previously been shown to affect OJIP transients (Kastori et al., 2000; Mallick and Mohn, 2003; Larbi et al., 2004; Husted et al., 2009; Tang et al., 2012; Yang et al., 2012). As a consequence, several attempts have been made to identify nutrient imbalances and disorders using one or several parameters derived from the transients, but apart from Mn (Husted et al., 2009; Schmidt et al., 2013), attempts have not been successful in terms of sensitivity and specificity. This includes P, which previously has been reported to have an effect on OJIP transients, yet the reported effects seem mutually contradictory and nonspecific to P (Ripley et al., 2004; Weng et al., 2008; Jiang et al., 2009; Lin et al., 2009).Here, we present the unique finding that increasing levels of P deficiency affect the shape of the OJIP transient around the I step at 20 to 50 ms and causes the I step to gradually straighten and disappear. It is demonstrated that this effect is fully reversible and, among the essential plant nutrients, specific for P deficiency using both monocotyledons (barley [Hordeum vulgare]) and dicotyledons (tomato [Solanum lycopersicum]) plant species. Furthermore, it is shown that it is possible to determine whether a plant is P sufficient or deficient and to quantitatively predict the P concentration in leaf tissue using multivariate analysis of the OJIP transients.     

Towards Fluorescence In Vivo Hybridization (FIVH) Detection of H. pylori in Gastric Mucosa Using Advanced LNA Probes

In recent years, there have been several attempts to improve the diagnosis of infection caused by Helicobacter pylori. Fluorescence in situ hybridization (FISH) is a commonly used technique to detect H. pylori infection but it requires biopsies from the stomach. Thus, the development of an in vivo FISH-based method (FIVH) that directly detects and allows the visualization of the bacterium within the human body would significantly reduce the time of analysis, allowing the diagnosis to be performed during endoscopy. In a previous study we designed and synthesized a phosphorothioate locked nucleic acid (LNA)/ 2’ O-methyl RNA (2’OMe) probe using standard phosphoramidite chemistry and FISH hybridization was then successfully performed both on adhered and suspended bacteria at 37°C. In this work we simplified, shortened and adapted FISH to work at gastric pH values, meaning that the hybridization step now takes only 30 minutes and, in addition to the buffer, uses only urea and probe at non-toxic concentrations. Importantly, the sensitivity and specificity of the FISH method was maintained in the range of conditions tested, even at low stringency conditions (e.g., low pH). In conclusion, this methodology is a promising approach that might be used in vivo in the future in combination with a confocal laser endomicroscope for H. pylori visualization.     

Creation of Circularly Permutated Yellow Fluorescent Proteins Using Fluorescence Screening and a Tandem Fusion Template

By experimenting with many different circularly permutated yellow fluorescent protein (cpYFP) variants as acceptors in fluorescence resonance energy transfer based biosensors, the optimal dynamic range can be discovered by sampling the possibilities of relative fluorophore orientations before and after bioactivity. Hence, to facilitate the sampling process, we introduced a new approach to construct a library of cpYFP variants using fluorescence screening and a tandem fusion template. This new approach is rapid because it does not require creating intermediate N- and C-terminal fragments and it allows quick screening for positive colonies by fluorescence. As a demonstration, eleven cpYFP variants were created and eight showed fluorescence. The emission and excitation spectra of these cpYFP variants showed strong similarity to YFP and therefore can be used in replacement. Revisions requested 27 October 2005; Revisions received 23 December 2005     

Enzyme-Free Detection of Mutations in Cancer DNA Using Synthetic Oligonucleotide Probes and Fluorescence Microscopy

Background

Rapid reliable diagnostics of DNA mutations are highly desirable in research and clinical assays. Current development in this field goes simultaneously in two directions: 1) high-throughput methods, and 2) portable assays. Non-enzymatic approaches are attractive for both types of methods since they would allow rapid and relatively inexpensive detection of nucleic acids. Modern fluorescence microscopy is having a huge impact on detection of biomolecules at previously unachievable resolution. However, no straightforward methods to detect DNA in a non-enzymatic way using fluorescence microscopy and nucleic acid analogues have been proposed so far.

Methods and Results

Here we report a novel enzyme-free approach to efficiently detect cancer mutations. This assay includes gene-specific target enrichment followed by annealing to oligonucleotides containing locked nucleic acids (LNAs) and finally, detection by fluorescence microscopy. The LNA containing probes display high binding affinity and specificity to DNA containing mutations, which allows for the detection of mutation abundance with an intercalating EvaGreen dye. We used a second probe, which increases the overall number of base pairs in order to produce a higher fluorescence signal by incorporating more dye molecules. Indeed we show here that using EvaGreen dye and LNA probes, genomic DNA containing BRAF V600E mutation could be detected by fluorescence microscopy at low femtomolar concentrations. Notably, this was at least 1000-fold above the potential detection limit.

Conclusion

Overall, the novel assay we describe could become a new approach to rapid, reliable and enzyme-free diagnostics of cancer or other associated DNA targets. Importantly, stoichiometry of wild type and mutant targets is conserved in our assay, which allows for an accurate estimation of mutant abundance when the detection limit requirement is met. Using fluorescence microscopy, this approach presents the opportunity to detect DNA at single-molecule resolution and directly in the biological sample of choice.     

Visualization of Differential Gene Expression by Improved Cyan Fluorescent Protein and Yellow Fluorescent Protein Production in Bacillus subtilis

The distinguishable cyan and yellow fluorescent proteins (CFP and YFP) enable the simultaneous in vivo visualization of different promoter activities. Here, we report new cloning vectors for the construction of cfp and yfp fusions in Bacillus subtilis. By extending the N-terminal portions of previously described CFP and YFP variants, 20- to 70-fold-improved fluorescent-protein production was achieved. Probably, the addition of sequences encoding the first eight amino acids of the N-terminal part of ComGA of B. subtilis overcomes the slow translation initiation that is provoked by the eukaryotic codon bias present in the original cfp and yfp genes. Using these new vectors, we demonstrate that, within an isogenic population of sporulating B. subtilis cells, expression of the abrB and spoIIA genes is distinct in individual cells.     

生物大分子相互作用检测技术新进展———三色荧光级联荧光共振能量转移技术

荧光共振能量转移(fluorescenceresonanceenergytransfer,FRET),是指能量从一种受激发的荧光基团(fluorophore)以非辐射的方式转移到另一种荧光基团的物理现象.FRET的能量转移效率是两个荧光基团间距离的函数,并对此距离十分敏感,它的有效响应距离一般在1～10nm之间,因而可被用于测定原子间及分子间的距离.这一特点使FRET技术在大分子构象变化、大分子之间相互作用、细胞信号通路等研究中发挥重要作用,成为生物医学研究中的重要方法.但细胞内的生物学过程常常涉及多于两个的大分子间相互作用,二色荧光基团的FRET技术不能满足这种生物学研究的需求.最近,两个研究小组在这方面取得突破,建立了分别基于共聚焦显微镜和流式细胞仪的三色荧光级联FRET技术.这一技术的出现将会极大地促进生物学及相关研究领域的发展.     

PCR-Free Detection of Genetically Modified Organisms Using Magnetic Capture Technology and Fluorescence Cross-Correlation Spectroscopy

The safety of genetically modified organisms (GMOs) has attracted much attention recently. Polymerase chain reaction (PCR) amplification is a common method used in the identification of GMOs. However, a major disadvantage of PCR is the potential amplification of non-target DNA, causing false-positive identification. Thus, there remains a need for a simple, reliable and ultrasensitive method to identify and quantify GMO in crops. This report is to introduce a magnetic bead-based PCR-free method for rapid detection of GMOs using dual-color fluorescence cross-correlation spectroscopy (FCCS). The cauliflower mosaic virus 35S (CaMV35S) promoter commonly used in transgenic products was targeted. CaMV35S target was captured by a biotin-labeled nucleic acid probe and then purified using streptavidin-coated magnetic beads through biotin-streptavidin linkage. The purified target DNA fragment was hybridized with two nucleic acid probes labeled respectively by Rhodamine Green and Cy5 dyes. Finally, FCCS was used to detect and quantify the target DNA fragment through simultaneously detecting the fluorescence emissions from the two dyes. In our study, GMOs in genetically engineered soybeans and tomatoes were detected, using the magnetic bead-based PCR-free FCCS method. A detection limit of 50 pM GMOs target was achieved and PCR-free detection of GMOs from 5 µg genomic DNA with magnetic capture technology was accomplished. Also, the accuracy of GMO determination by the FCCS method is verified by spectrophotometry at 260 nm using PCR amplified target DNA fragment from GM tomato. The new method is rapid and effective as demonstrated in our experiments and can be easily extended to high-throughput and automatic screening format. We believe that the new magnetic bead-assisted FCCS detection technique will be a useful tool for PCR-free GMOs identification and other specific nucleic acids.     

Fluorescence Lifetime Readouts of Troponin-C-Based Calcium FRET Sensors: A Quantitative Comparison of CFP and mTFP1 as Donor Fluorophores

We have compared the performance of two Troponin-C-based calcium FRET sensors using fluorescence lifetime read-outs. The first sensor, TN-L15, consists of a Troponin-C fragment inserted between CFP and Citrine while the second sensor, called mTFP-TnC-Cit, was realized by replacing CFP in TN-L15 with monomeric Teal Fluorescent Protein (mTFP1). Using cytosol preparations of transiently transfected mammalian cells, we have measured the fluorescence decay profiles of these sensors at controlled concentrations of calcium using time-correlated single photon counting. These data were fitted to discrete exponential decay models using global analysis to determine the FRET efficiency, fraction of donor molecules undergoing FRET and calcium affinity of these sensors. We have also studied the decay profiles of the donor fluorescent proteins alone and determined the sensitivity of the donor lifetime to temperature and emission wavelength. Live-cell fluorescence lifetime imaging (FLIM) of HEK293T cells expressing each of these sensors was also undertaken. We confirmed that donor fluorescence of mTFP-TnC-Cit fits well to a two-component decay model, while the TN-L15 lifetime data was best fitted to a constrained four-component model, which was supported by phasor analysis of the measured lifetime data. If the constrained global fitting is employed, the TN-L15 sensor can provide a larger dynamic range of lifetime readout than the mTFP-TnC-Cit sensor but the CFP donor is significantly more sensitive to changes in temperature and emission wavelength compared to mTFP and, while the mTFP-TnC-Cit solution phase data broadly agreed with measurements in live cells, this was not the case for the TN-L15 sensor. Our titration experiment also indicates that a similar precision in determination of calcium concentration can be achieved with both FRET biosensors when fitting a single exponential donor fluorescence decay model to the fluorescence decay profiles. We therefore suggest that mTFP-based probes are more suitable for FLIM experiments than CFP-based probes.     

Detection of Change to SSVEPs Using Analysis of Phase Space Topological Features: A Novel Approach

Neurophysiology - A novel method based on EEG nonlinear analysis and analysis of steady-state visual evoked potentials (SSVEPs) has been processed. The EEG phase space is reconstructed, and some...     

Intrinsic Protein Fluorescence Interferes with Detection of Tear Glycoproteins in SDS-Polyacrylamide Gels Using Extrinsic Fluorescent Dyes

Intrinsic protein fluorescence may interfere with the visualization of proteins after SDS-polyacrylamide electrophoresis. In an attempt to analyze tear glycoproteins in gels, we ran tear samples and stained the proteins with a glycoprotein-specific fluorescent dye. The fluorescence detected was not limited to glycoproteins. There was strong intrinsic fluorescence of proteins normally found in tears after soaking the gels in 40% methanol plus 1-10% acetic acid and, to a lesser extent, in methanol or acetic acid alone. Nanograms of proteins gave visible native fluorescence and interfere with extrinsic fluorescent dye detection. Poly-L-lysine, which does not contain intrinsically fluorescent amino acids, did not fluoresce.     

Characterization of Interactions Between and Among Components of the Meiotic Silencing by Unpaired DNA Machinery in Neurospora crassa Using Bimolecular Fluorescence Complementation

Bimolecular fluorescence complementation (BiFC) is based on the complementation between two nonfluorescent fragments of the yellow fluorescent protein (YFP) when they are united by interactions between proteins covalently linked to them. We have successfully applied BiFC in Neurospora crassa using two genes involved in meiotic silencing by unpaired DNA (MSUD) and observed macromolecular complex formation involving only SAD-1 proteins, only SAD-2 proteins, and mixtures of SAD-1 and SAD-2 proteins.     

黄河流域农业生产活性氮排放及减排对策

人口增长和城市化进程促使粮食和肉禽奶类食品需求不断增加,由此带来的农业生产活性氮(Nr)大量排放对生态环境及人类健康的影响日益加剧。黄河流域作为中国的粮食主产区,农业生产活动强度高,为研究其Nr排放规律,采用排放因子法估算2000、2005及2010年黄河流域内9省(区)农业生产不同形态Nr的排放源。结果表明：(1)黄河流域9省(区)中,农业生产Nr排放量最大的为河南省,最小的为四川省,河南省Nr排放量是四川的8倍。(2)4种形态Nr排放量从大到小依次为Nr-wp(排放到水体的Nr)、NH₃、N₂O和NO_x。化学氮肥施用和畜禽散养是NH₃排放的最主要贡献源,其次是规模化养殖和放牧饲养,四者贡献率达85%以上。农田作物系统径流、淋洗以及畜禽养殖流失淋洗对Nr-wp排放的贡献率各占1/3左右。四季非蔬菜旱地和畜禽养殖是N₂O排放的主要来源,其贡献率之和大于66%。(3)黄河流域内9省(区)单位农业GDP、单位耕地面积、单位农村人口Nr排放强度最大的均为青海省,单位农业GDP和单位农...     

应用SYBR实时荧光定量Real—TimePCR法检测人宫颈癌MCPH1 mRNA表达

目的 应用SYBR实时荧光定量RT-PCR法检测MCPH1/BRIT1 mRNA的表达.方法 提取人宫颈癌总RNA,经逆转录PCR获得靶基因（MCPH1）及管家基因（GAPDH）的CDNA,采用SYBR Green 荧光实时定量法检测,以GAPDH基因作为内参,计算各组MCPH1 mRNA的相对表达量.结果 在31例宫颈癌标本中,MCPH1基因mRNA的表达,19例癌比正常低,1 例癌比正常高,其余标本无统计学意义;6例癌比癌旁低,1例癌比癌旁高,其余无统计学意义.结论 利用SYBR实时荧光定量RT-PCR检测出人宫颈癌中MCPH1基因mRNA的表达下调,为进一步研究MCPH1在宫颈癌中的作用及功能奠定了基础.     

Extraction and Sensitive Detection of Toxins A and B from the Human Pathogen Clostridium difficile in 40 Seconds Using Microwave-Accelerated Metal-Enhanced Fluorescence

Clostridium difficile is the primary cause of antibiotic associated diarrhea in humans and is a significant cause of morbidity and mortality. Thus the rapid and accurate identification of this pathogen in clinical samples, such as feces, is a key step in reducing the devastating impact of this disease. The bacterium produces two toxins, A and B, which are thought to be responsible for the majority of the pathology associated with the disease, although the relative contribution of each is currently a subject of debate. For this reason we have developed a rapid detection assay based on microwave-accelerated metal-enhanced fluorescence which is capable of detecting the presence of 10 bacteria in unprocessed human feces within 40 seconds. These promising results suggest that this prototype biosensor has the potential to be developed into a rapid, point of care, real time diagnostic assay for C. difficile.     

Recovery and Detection of Escherichia coli O157:H7 in Surface Water,Using Ultrafiltration and Real-Time PCR

Enterohemorrhagic Escherichia coli O157:H7 (EHEC O157:H7) outbreaks have revealed the need for improved analytical techniques for environmental samples. Ultrafiltration (UF) is increasingly recognized as an effective procedure for concentrating and recovering microbes from large volumes of water and treated wastewater. This study describes the application of hollow-fiber UF as the primary step for concentrating EHEC O157:H7 seeded into 40-liter samples of surface water, followed by an established culture/immunomagnetic-separation (IMS) method and a suite of real-time PCR assays. Three TaqMan assays were used to detect the stx1, stx2, and rfbE gene targets. The results from this study indicate that approximately 50 EHEC O157:H7 cells can be consistently recovered from a 40-liter surface water sample and detected by culture and real-time PCR. Centrifugation was investigated and shown to be a viable alternative to membrane filtration in the secondary culture/IMS step when water quality limits the volume of water that can be processed by a filter. Using multiple PCR assay sets to detect rfbE, stx1, and stx2 genes allowed for specific detection of EHEC O157:H7 from strains that do not possess all three genes. The reported sample collection and analysis procedure should be a sensitive and effective tool for detecting EHEC O157:H7 in response to outbreaks of disease associated with contaminated water.Several highly publicized outbreaks of gastrointestinal diseases caused by enterohemorrhagic Escherichia coli O157:H7 (EHEC O157:H7) have highlighted the threat this pathogen poses to public health (1, 2, 3, 14). Although the predominant mode of transmission to humans appears to be contaminated meat or meat products, there have been a number of outbreaks associated with contaminated water (18). Microbiological, epidemiological, and environmental studies have found an association between EHEC O157:H7 outbreaks and recreational water, drinking water, crop irrigation, and wastewater (1, 2, 14). These investigations have also revealed that enhanced rapid analytical techniques are needed to improve the speed and effectiveness of these types of investigations.Hollow-fiber ultrafiltration (UF) is a sampling technique that is emerging as an option for recovering diverse microbes from large-volume water samples (8, 9, 12, 13, 15). There have been reports of the successful application of UF for surface water as well as for other E. coli strains (8, 13), but additional data are needed to evaluate the robustness of UF for surface water and its ability to effectively concentrate EHEC O157:H7 in the presence of background microbes. The presence of competitive microbes has been shown to significantly alter the growth rate and maximal density of EHEC O157:H7 in broth culture (5).EHEC O157:H7 is generally detected in water samples by using membrane filtration, selective broth enrichment, immunomagnetic-separation (IMS), and isolation on selective agar culture plates, followed by confirmatory tests such as PCR or serological tests (6, 7). However, sensitive detection of EHEC O157:H7 in surface waters can be difficult due to high levels of competing background microorganisms (7). Membrane filtration can also limit the volume processed for turbid surface waters due to filter clogging. Centrifugation is an alternative to membrane filtration and has an advantage of not being subject to potential sample volume processing constraints for turbid water samples, so the technique could potentially increase the sensitivity of detection. A number of PCR assays have been developed for detection of EHEC O157:H7 that target a variety of virulence genes (17). Testing multiple gene targets is necessary for accurate detection because certain non-EHEC O157:H7 serotypes and other bacterial species are known to possess the target genes; therefore, the isolate cannot be determined to be EHEC O157:H7 unless multiple assays show a positive signal (19).The goals of this study were to evaluate (i) the effectiveness of a previously reported UF method (8) for application to recovering EHEC O157:H7, (ii) the effectiveness of the culture/IMS technique performed in conjunction with primary UF concentration, (iii) the effectiveness of centrifugation as an alternative for membrane filtration in the culture/IMS method, and (iv) the ability of three previously reported real-time PCR assays to accurately detect EHEC O157:H7 in surface waters (16, 17).     

Reciprocal Modulation Between Microglia and Astrocyte in Reactive Gliosis Following the CNS Injury

Reactive gliosis, also known as glial scar formation, is an inflammatory response characterized by the proliferation of microglia and astrocytes as well as astrocytic hypertrophy following injury in the central nervous system (CNS). The glial scar forms a physical and molecular barrier to isolate the injured area from adjacent normal nervous tissue for re-establishing the integrity of the CNS. It prevents the further spread of cellular damage but represents an obstacle to regrowing axons. In this review, we integrated the current findings to elucidate the tightly reciprocal modulation between activated microglia and astrocytes in reactive gliosis and proposed that modification of cellular response to the injury or cellular reprogramming in the glial scar could lead advances in axon regeneration and functional recovery after the CNS injury.