Rapid direct methods for enumeration of specific, active bacteria in water and biofilms

Conventional methods for detecting indicator and pathogenic bacteria in water may underestimate the actual population due to sublethal environmental injury, inability of the target bacteria to take up nutrients and other physiological factors which reduce bacterial culturability. Rapid and direct methods are needed to more accurately detect and enumerate active bacteria. Such a methodological advance would provide greater sensitivity in assessing the microbiological safety of water and food. The principle goal of this presentation is to describe novel approaches we have formulated for the rapid and simultaneous detection of bacteria plus the determination of their physiological activity in water and other environmental samples. The present version of our method involves the concentration of organisms by membrane filtration or immunomagnetic separation and combines an intracellular fluorochrome (CTC) for assessment of respiratory activity plus fluorescent-labelled antibody detection of specific bacteria. This approach has also been successfully used to demonstrate spatial and temporal heterogeneities of physiological activities in biofilms when coupled with cryosectioning. Candidate physiological stains include those capable of determining respiratory activity, membrane potential, membrane integrity, growth rate and cellular enzymatic activities. Results obtained thus far indicate that immunomagnetic separation can provide a high degree of sensitivity in the recovery of seeded target bacteria (Escherichia coli O157:H7) in water and hamburger. The captured and stained target bacteria are then enumerated by either conventional fluorescence microscopy or ChemScan(R), a new instrument that is very sensitive and rapid. The ChemScan(R) laser scanning instrument (Chemunex, Paris, France) provides the detection of individual fluorescently labelled bacterial cells using three emission channels in less than 5 min. A high degree of correlation has been demonstrated between results obtained with the ChemScan and traditional plate counts of mixed natural bacterial populations in water. The continuing evolution of these methods will be valuable in the rapid and accurate analysis of environmental samples.     

Simple and rapid determination of thiol compounds by HPLC and fluorescence detection with 1,3,5,7-tetramethyl-8-phenyl-(2-maleimide) difluoroboradiaza-s-indacene

A rapid and simple background-free high-performance liquid chromatographic (HPLC) approach has been developed for simultaneously determining free thiol compounds including coenzyme A (CoA), cysteine (Cys), glutathione (GSH) and N-acetyl-cysteine (NAC) in biological samples by using 1,3,5,7-tetramethyl-8-phenyl-(2-maleimide) difluoroboradiaza-s-indacene (TMPAB-o-M) as fluorogenic reagent. After derivatization under physiological conditions within 6 min, baseline separation was finished in just 6 min using isocratic elution with reversed-phase HPLC and fluorescence detection. Excellent linearity was observed for all analytes over their concentration ranges of 1-500 nM and detection limits ranging 0.13 nM for CoA to 0.25 nM for Cys (S/N=3) were achieved. The utility of the proposed method has been validated by measuring thiol compounds mentioned above in tissue, fluid and cell samples. The results indicated that this approach was well suited for high-throughput quantitative determination of thiols and study of the physiological role of them.     

Application of electrochemical properties of ordered mesoporous carbon to the determination of glutathione and cysteine

In this study, the electrochemical activity of ordered mesoporous carbon (OMC) was investigated and applied to the determination of glutathione (GSH) and cysteine (CySH). It has been demonstrated that the ordered mesostructure of OMC has an important role in the electrocatalytic activity towards thiols, and the destruction of this structure results in the decrease of such properties. The electrochemical behavior of GSH at an OMC electrode was also investigated. The results showed that the process of oxidation of GSH at the OMC electrode is differs from that of CySH at the same electrode by the peak at 0.47 V associated with CySH. This difference helped to reduce the interference of GSH during the determination of CySH in the presence of GSH. A sensor for the two thiols was developed with acceptable sensitivity and detection limits in a large determination range. These results obtained in the physiological medium and in the physiological levels of GSH and CySH, suggest that OMC is a promising material in the detection of thiols in biologically relevant experimental conditions (in terms of pH).     

Visceral perception versus visceral detection: disentangling methods and assumptions

A within-subject experiment compared three paradigms commonly used in visceral perception: self-report, heartbeat tracking, and signal detection. Eighteen undergraduates estimated heart rate using each technique while engaging in a number of separate tasks conducted a week apart. Although all three techniques significantly tapped accuracy of heart rate perception, only the self-report and signal detection methods were reliable over time. Most important, there was no relationship involving any of the methods in measuring accuracy. The findings suggest some fundamental differences in the assumptions and perceptual properties of the various paradigms. A distinction is made between visceral perception and detection. Perception implies the subject's use of both internal physiological and external environmental information in the perception of visceral state. Detection connotes the subject's use of only physiological information--to the exclusion of all other factors. The relevance of these approaches for biofeedback and real-world symptom perception is discussed.     

Visceral perception versus visceral detection: Disentangling methods and assumptions

A within-subject experiment compared three paradigms commonly used in visceral perception: self-report, heartbeat tracking, and signal detection. Eighteen undergraduates estimated heart rate using each technique while engaging in a number of separate tasks conducted a week apart. Although all three techniques significantly tapped accuracy of heart rate perception, only the self-report and signal detection methods were reliable over time. Most important, there was no relationship involving any of the methods in measuring accuracy. The findings suggest some fundamental differences in the assumptions and perceptual properties of the various paradigms. A distinction is made between visceral perception and detection. Perception implies the subject's use of both internal physiological and external environmental information in the perception of visceral state. Detection connotes the subject's use of only physiological information — to the exclusion of all other factors. The relevance of these approaches for biofeedback and real-world symptom perception is discussed.     

Physiological stress induces the metastasis marker AGR2 in breast cancer cells

As an approach to understanding the factors that activate expression of tumor progression genes, the role of physiological stress in the activation of a panel of tumor cell markers was investigated. These studies identify the developmental gene product, anterior gradient 2 (AGR2) as a cancer cell marker specifically up-regulated in response to depletion of serum and oxygen. AGR2 has been identified as a tumor marker in primary and secondary cancer lesions, and as a marker for detection of circulating tumor cells (CTCs). Elevated levels of AGR2 are known to increase the metastatic potential of cancer cells, but conditions leading to increased expression of AGR2 are not well understood. The present results identify novel physiological parameters likely to contribute to AGR2 induction in situ. Daniel R. Zweitzig and Denis A. Smirnov contributed equally to this work.     

蛋白质O-GlcNAc修饰的检测技术

O-连接的N-乙酰葡糖胺（O-GlcNAc）修饰是位于细胞浆和细胞核蛋白质的丝氨酸或苏氨酸上的一种翻译后修饰,在高等真核生物细胞中广泛存在.越来越多的研究表明,O-GlcNAc修饰在代谢调控、压力应激、细胞周期、凋亡、糖尿病、心血管疾病和癌症等多种生理和病理过程中发挥重要作用,因此, O-GlcNAc修饰已受到众多生命科学领域研究人员的关注.然而,由于O-GlcNAc修饰与传统的N聚糖和O聚糖修饰有所不同,常规糖基化修饰的检测方法并不适用于O-GlcNAc.本文对O-GlcNAc修饰的检测及其修饰位点的确定方法进行了综述,并分析了各种方法的优缺点.     

Determination of cefotaxime and desacetylcefotaxime in plasma and urine by high-performance liquid chromatography

A high-performance liquid chromatographic method is described for the analysis of the anti-bacterial agent cefotaxime and desacetylcefotaxime in physiological fluids. Plasma or serum samples were mixed with chloroform—acetone to remove proteins and most lipid material. The aqueous phase was then freeze-dried, reconstituted in mobile phase and chromatographed on a reversed-phase column using UV detection at 262 nm. Urine was analysed directly after centrifugation to remove particulate matter. The detection limit was 0.5–1.0 μg/ml for serum and 5 μg/ml for urine. The method has been applied to the analyses of cefotaxime and desacetylcefotaxime in plasma, serum, urine, cerebrospinal fluid, saliva, and pus from infected wound secretions. Two additional metabolites, which are lactones, in which the β-lactam ring has been opened, could be separated by this method.     

Studying the mechanisms of genomic and synaptic plasticity in neuronal cultures with microelectrode arrays

The plasticity of neural networks is a complex process determined by changes in physiological status, gene expression and phenotype of a cell. A detailed study of this process dynamics requires the simultaneous recording of electrical and genomic activities in networks of neurons. This sets up one of the tasks for modern neuroscience as development of integration of electrophysiology and molecular biology methods. In the paper we review the current approaches to such integration, as well as the choice of molecular markers for detection of genomic and synaptic plasticity of neurons by use of physiological micro-sensorial system based on neuronal cells cultured on the micro-electrode arrays.     

Acoustic detection and communication by decapod crustaceans

This paper reviews behavioral, physiological, anatomical, and ecological aspects of sound and vibration detection by decapod crustaceans. Our intent is to demonstrate that despite very limited work in this area in the past 20 years, evidence suggests that at least some decapod crustaceans are able to detect and use sounds in ways that parallel detection and processing mechanisms in aquatic and terrestrial vertebrates. Some aquatic decapod crustaceans produce sounds, and many are able to detect substrate vibration at sensitivities sufficient to tell of the proximity of mates, competitors, or predators. Some semi-terrestrial crabs produce and use sounds for communication. These species detect acoustic stimuli as either air- or substrate-borne energies, socially interact in acoustic "choruses," and probably use "calls" to attract mates.     

视黄醇结合蛋白4对肾脏疾病早期诊断的评估

视黄醇结合蛋白4(RBP4)是一类合成于肝脏且广泛分布于人体血液、尿液等其它体液中的维生素A运载蛋白,它在协助维生素A发挥生理功能中起着决定性的作用。近年来的研究表明:当近端肾小管受损时,人体血液或尿液中视黄醇结合蛋白4的含量会出现不同程度的升高。同时它与糖尿病肾病、营养性疾病的发展等其它疾病都在临床上存在着一定的相关性。临床上已将RBP4的含量测定作为判定肾功能有效且成熟的指标之一。目前临床上对视黄醇结合蛋白4的检测主要是传统的酶联免疫法、免疫比浊法等,近年来随着研究水平的不断提高,对RBP4的检测方法已有逐渐转向新型的快速检测方法的趋势。本文在对视黄醇结合蛋白4的理化性质、在肾脏等各类疾病方面的应用以及新型临床检测方法等方面作一综述,随着研究的不断完善,视黄醇结合蛋白4的更多临床意义将逐渐显现。     

Identification of low abundant secreted proteins and peptides from primary culture supernatants of human T-cells

Live cells continually communicate with their surroundings by the secretion of biomolecules, among which proteins and/or peptides are an important class. As such, these protein/peptide signals which end up in the extracellular medium, reflect the state of a cell in a certain condition, and as by definition are potential biomarkers indicative for specific physiological/pathological processes. We here report on a mass spectrometry based method for the detection and analysis of peptides and proteins secreted in a highly complex background, such as cell culture supernatant. Our method, which combines chromatography, high duty cycle tandem mass spectrometry and bio-informatics, enables the detection of interleukin-2 (IL-2), a cytokine secreted by activated T-cells, present in cell supernatant while representing only 0.006‰ of the total protein content. Moreover, the method allows the mass spectrometric analysis of signaling proteins in a non-targeted way and without any prior immunodepletion of the highest abundant cell culture medium proteins. In this study this is exemplified by the detection of yet two other secretory peptides, i.e., the granulins A and B, in the primary culture supernatant of non-activated T-cells.     

Clinical chemistry of serotonin and metabolites

Analyses of serotonin and other 5-hydroxyindoles, such as its precursor 5-hydroxytryptophan and major metabolite 5-hydroxyindoleacetic acid (5-HIAA), are indispensable for the elucidation of their (patho)physiological roles. In clinical chemistry attention is mainly focused on the diagnosis and follow-up of carcinoid tumours. For this most laboratories routinely measure urinary 5-HIAA. More recently, measurements of serotonin in platelets and urine have been advocated. Platelet serotonin may be the most sensitive indole marker for the detection of carcinoid tumours that secrete only small amounts of serotonin and/or its precursor 5-hydroxytryptophan. Although several chromatographic techniques have emerged for the analysis of tryptophan-related indoles, HPLC with either electrochemical or fluorometric detection have become the methods of choice for their quantification. HPLC-based methods combine selectivity, sensitivity and high precision, and enable the simultaneous investigation of several metabolically related indoles. This review aims to place the analysis of indoles in biological matrices in a biochemical, physiological and clinical perspective and highlights several important steps in their chromatographic analysis and quantification.     

Surface plasmon resonance detection of blood coagulation and platelet adhesion under venous and arterial shear conditions

A surface plasmon resonance (SPR) based flow chamber device was designed for real time detection of blood coagulation and platelet adhesion in platelet rich plasma (PRP) and whole blood. The system allowed the detection of surface interactions throughout the 6mm length of the flow chamber. After deposition of thromboplastin onto a section of the sensor surface near the inlet of the flow chamber, coagulation was detected downstream of this position corresponding to a SPR signal of 7 to 8 mRIU (7 to 8 ng/mm2). A nonmodified control surface induced coagulation 3.5 times slower. Platelet adhesion to gold and fibrinogen coated surfaces in the magnitude of 1.25 and 1.66 mRIU was also shown with platelets in buffer, respectively. SPR responses obtained with PRP and whole blood on surfaces that were methylated or coated with von Willebrand factor (vWF), fibrinogen, or collagen, coincided well with platelet adhesion as observed with fluorescence microscopy in parallel experiments. The present SPR detection equipped flow chamber system is a promising tool for studies on coagulation events and blood cell adhesion under physiological flow conditions, and allows monitoring of short-range surface processes in whole blood.     

Simultaneous high-performance liquid chromatographic determination of ascorbic acid and dehydroascorbic acid in biological samples

The ascorbic acid (AA)—dehydroascorbic acid redox couple is an important component of many biological systems, and various physiological roles have been described for this vitamin. Simultaneous measurement of both AA and dehydroascorbate using high-performance liquid chromatography (HPLC) has proven difficult owing to detection problems. A simple, single-step HPLC assay for the simultaneous detection of both AA and dehydroascorbate was developed without the burden of derivatization of either compounds. This has proven to be a reliable technique and should be applicable to a wide variety of biological samples.     

Assessments of the chemistry and vasodilatory activity of nitrite with hemoglobin under physiologically relevant conditions

Hypoxic vasodilation involves detection of the oxygen content of blood by a sensor, which rapidly transduces this signal into vasodilatory bioactivity. Current perspectives on the molecular mechanism of this function hold that hemoglobin (Hb) operates as both oxygen sensor and a condition-responsive NO reactor that regulates the dispensing of bioactivity through release of the NO group from the beta-cys93 S-nitroso derivative of Hb, SNO-Hb. A common path to the formation of SNO-Hb involves oxidative transfer of the NO-group from heme to thiol. We have previously reported that the reaction of nitrite with deoxy-Hb, which furnishes heme-Fe(II)NO, represents one attractive route for the formation of SNO-Hb. Recent literature, however, posits that the nitrite-reductase reaction of Hb might produce physiological vasodilatory effects through NO that evades trapping on heme-Fe(II) and may be stored before release as Fe(III)NO. In this article, we briefly review current perspectives in NO biology on the nitrite-reductase reaction of Hb. We report in vitro spectroscopic (UV/Vis, EPR) studies that are difficult to reconcile with suggestions that this reaction either generates a heme-Fe(III)NO reservoir or significantly liberates NO. We further show in bioassay experiments that combinations of nitrite and deoxy-Hb--under conditions that suppress SNO-Hb formation--exhibit no direct vasodilatory activity. These results help underscore the differences between physiological, RBC-regulated, hypoxic vasodilation versus pharmacological effects of exogenous nitrite.     

A “fluorescence switch” technique increases the sensitivity of proteomic detection and identification of S‐nitrosylated proteins

Protein S‐nitrosylation is a reversible post‐translational modification of protein cysteines that is increasingly being considered as a signal transduction mechanism. The “biotin switch” technique marked the beginning of the study of the S‐nitrosoproteome, based on the specific replacement of the labile S‐nitrosylation by a more stable biotinylation that allowed further detection and purification. However, its application for proteomic studies is limited by its relatively low sensitivity. Thus, typical proteomic experiments require high quantities of protein extracts, which precludes the use of this method in a number of biological settings. We have developed a “fluorescence switch” technique that, when coupled to 2‐DE proteomic methodologies, allows the detection and identification of S‐nitrosylated proteins by using limited amounts of starting material, thus significantly improving the sensitivity. We have applied this methodology to detect proteins that become S‐nitrosylated in endothelial cells when exposed to S‐nitroso‐L ‐cysteine, a physiological S‐nitrosothiol, identifying already known S‐nitrosylation targets, as well as proteins that are novel targets. This “fluorescence switch” approach also allowed us to identify several proteins that are denitrosylated by thioredoxin in cytokine‐activated RAW264.7 (murine macrophage) cells. We believe that this method represents an improvement in order to approach the identification of S‐nitrosylated proteins in physiological conditions.     

Development of a matrix-assisted laser desorption/ionization-mass spectrometry screening test to evidence reversible and irreversible inhibitors of CDC25 phosphatases

The cell division cycle 25 phosphatases (CDC25s) are key regulators of the physiological cell cycle progression. Their overexpression has been reported in a significant number of cancers, and their inhibition appears to be an interesting strategy for treatments. We propose here a rapid screening test allowing the detection of reversible and irreversible CDC25A and -C inhibitors. The test is based on the incubation of the candidate molecules with the human CDC25 proteins followed by an ultrafiltration step. The retentate is then directly analyzed by matrix-assisted laser desorption/ionization-time-of-flight mass spectrometry (MALDI-TOFMS) to detect reversible inhibitors or submitted to peptide mass fingerprint (PMF) analysis to reveal irreversible inhibitors covalently bound to the protein active site. After its validation, the protocol is applied to the detection of a novel candidate inhibitor of CDC25s named SV37. The screening procedure, as well as the preliminary biological results, demonstrates that this compound behaves as a reversible inhibitor.     

Conformational impurity of disulfide proteins: detection, quantification, and properties

The conformations of native proteins are in principle, and in most cases, dictated by the law of thermodynamics. Accordingly, a native protein must always exist in equilibrium with a minor concentration of nonnative (denatured) conformational isomers even at nondenaturing conditions. The presence of an infinitesimal quantity of nonnative conformational isomers at physiological conditions is biologically relevant due to their propensity to aggregate, which is an underlying cause of many neurodegenerative diseases. However, their detection and quantification are inherently difficult. In this article, we describe a simple strategy using the technique of disulfide scrambling to identify and quantify such minute concentrations of nonnative isomers. It is demonstrated that even for small stable proteins such as epidermal growth factor and hirudin, approximately 1% of heterogeneous nonnative isomers coexist with the native proteins under physiological conditions.