Journal of Biological Methods (JBM)
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A mass spectrometric method for quantification of tryptophan-derived uremic solutes in human serum
No full textIn addition to various physiologic roles, emerging evidence strongly points to pathogenic roles of tryptophan and of its metabolites, especially in diseases such as renal failure. Accurate estimation of levels of these metabolites in blood is important to mechanistically probe their contribution to disease pathogenesis, while clinically, such a panel can be used to risk stratify patients for a clinical phenotype. Herein, we describe a comprehensive liquid chromatography-mass spectrometry (LC/MS)-based method to determine the level of tryptophan and its metabolites (kynurenine, kynurenic acid, xanthurenic acid, anthranilic acid, indoxyl sulfate and indoxyl acetate). Human sera samples were processed through a C18 column followed by application of a binary gradient and quantitation by MS/MS. The linearity, lower limit of detection, inter- and intraassay variabilities and recovery were determined, yielding a precise, reproducible method for all the metabolites. Unlike previous studies, we further validated these methods in a well-characterized set of human sera from end stage renal disease patients compared to age-, gender- and ethnic-background matched human controls. Overall, we report an optimized LC/MS-based estimation of a comprehensive panel of tryptophan-derived metabolites with quality features within FDA standards, underscoring their readiness for translational use
Aspiration techniques for bronchoalveolar lavage in translational respiratory research: Paving the way to develop novel therapeutic moieties
No full textBronchoalveolar lavage (BAL) is a simple, yet informative tool in understanding the immunopathology of various lung diseases via quantifying various inflammatory cells, cytokines and growth factors. At present, this traditional method is often blended with several robust and sophisticated molecular and biological techniques sustaining the significance and longevity of this technique. Crucially, the existence of slightly distinct approaches and variables employed at different laboratories around the globe in performing BAL aspiration indeed demands an utmost need to optimize and develop an effective, cost-effective and a reproducible technique. This mini review will be of importance to the biological translational scientist, particularly respiratory researchers in understanding the fundamentals and approaches to apply and consider with BAL aspiration techniques. This will ensure generating a meaningful and clinically relevant data which in turn accelerate the development of new and effective therapeutic moieties for major respiratory conditions
IL-6
No full textThe assignment of cognate odorant/agonist pairs is a prerequisite for an understanding of odorant coding at the receptor level. However, the identification of new ligands for odorant receptors in cell-based assays has been challenging, due to their individual and rather sub-optimal plasma membrane expression, as compared with other G protein-coupled receptors. Accessory proteins, such as the chaperone RTP1S, or Ric8b, have improved the surface expression of at least a portion of odorant receptors. Typically, recombinant odorant receptors carry N-terminal tags, which proved helpful for their functional membrane expression. The most common tag is the ‘Rho-tag’, representing an N-terminal part of rhodopsin, but also ‘Lucy-‘ or ‘Flag-tag’ extensions have been described. Here, we used a bi-functional N-terminal tag, called ‘IL-6-HaloTag®’, with IL-6 facilitating functional cell surface expression of recombinant odorant receptors, and the HaloTag® protein, serving as a highly specific acceptor for cell-impermeant or cell-permeant, fluorophore-coupled ligands, which enable the quantification of odorant receptor expression by live-cell flow cytometry. Our experiments revealed on average an about four-fold increased surface expression, a four-fold higher signaling amplitude, and a significant higher potency of odorant-induced cAMP signaling of six different human IL-6-HaloTag®-odorant receptors across five different receptor families in NxG 108CC15 cells, as compared to their Rho-tag–HaloTag® constructs. We observed similar results in HEK-293 cells. Moreover, screening an IL-6–HaloTag®-odorant receptor library with allyl phenyl acetate, revealed both known receptors as best responders for this compound. In summary, the IL-6–HaloTag® represents a promising tool for the de-orphaning of odorant receptors
Measurement of intracellular calcium of submandibular glands using a high throughput plate reader
No full textCalcium ions (Ca2+) impact nearly every aspect of cellular life and intracellular calcium [Ca2+]i is a critical factor in the regulation of a plethora of physiological functions, including: muscle contraction, saliva secretion, metabolism, gene expression, cell survival and death. By measuring the changes of [Ca2+]i levels, critical physiologic functions can be characterized and aberrant pathologic conditions or drug responses can be efficiently monitored. We developed a protocol for assessment of Ca2+ signaling in the acinar units of submandibular glands isolated from C57BL/6 mice, using benchtop, multi-mode, high throughput plate reader (FlexStation 3). This method represents a powerful tool for unlimited in vitro studies to monitor changes in receptor-mediated Ca2+ responses while retaining functional and morphological features of a native setting
A multiplex PCR strategy to screen for known mutations in families with sudden cardiac death burden
No full textVentricular tachyarrythmia occurring in ischemic heart disease, dilated/hypertrophic cardiomyopathies or rare monogenic mutations of cardiac ion channels or associated proteins belong to the most frequent causes of sudden cardiac death (SCD). In further decades, next generation sequencing and bioinformatic analysis will become the gold standard of SCD risk stratification. At the moment, Sanger-sequencing is still obligatory in genetic diagnosis. A multiplex polymerase chain reaction (PCR) assay detecting eight SCD mutations in one reaction-tube was developed. To test the general validity of the assay, it was used with 12 patients, who had one or two of the eight mutations (LMNA, p.V256V; SCN5A, p.R1583C; RYR2, p.G1885E; MYH7, V606M; DSG2, p.T335A; KCNJ8, p.S422L; MYBPC, p.E441K; TNNT2, A38V). Thereafter, we tested the multiplex assay in a real diagnostic environment within a high risk family of several past SCD cases. This method allows efficient discrimination of multiple mutations by allele-specific PCR with standard PCR conditions. It relies on obtaining a PCR product specific to the mutation or wildtype—using primers that have the 3´end base complementary to the DNA template site, i.e. a specific primer only permits amplification to take place when its 3´terminal nucleotide matches with its target sequence. The PCR products are further analyzed by length, with Tape Station®(Agilent Technologies, Germany), a high-fidelity capillary chromatography test. The novel multiplex PCR assay strategy could be a good additional test used for SCD risk stratification. Advantages of the test are high velocity and ease of implementation, low price and flexibility of application within cardiomyopathy families for screening purposes
A fast and reproducible cell- and 96-well plate-based method for the evaluation of P2X7 receptor activation using YO-PRO-1 fluorescent dye
No full textThe YO-PRO-1 assay provides a quantitative estimation of P2X7 receptor activation. P2X7 receptor is associated to pathological conditions including infectious, inflammatory, neurological, musculoskeletal disorders, pain and cancer. Most primary cells and cell lines from diverse origin may be used thanks to the ubiquitous distribution of P2X7 receptor. To study the activation of P2X7 receptor by chemicals or biological agents, we established a microplate-based cytometry protocol to accurately and rapidly quantify the activation of P2X7 receptor that leads to the formation of large pores in cell membranes. The YO-PRO-1 assay is based on the ability of cells to incorporate and bind YO-PRO-1 dye to DNA after activation of P2X7 receptor through pore formation. Cells are seeded in 96-well plates and incubated with the compound being tested for the appropriate time. The microplate is then incubated for 10 min with YO-PRO-1 staining solution. After the 10 min staining time, fluorescence signal is read using a microplate reader in 1 min. This procedure is easier and requires less handling steps than flow cytometry. 96-well plate based YO-PRO-1 assay is a reproducible and fast method to study both P2X7 receptor activation by toxic agents at subnecrotic concentrations and P2X7 receptor inhibition by antagonists
Elucidating endotoxin-biomolecule interactions with FRET: extending the frontiers of their supramolecular complexation
No full textEndotoxin has been one of the topical chemical contaminants of major concern to researchers, especially in the field of bioprocessing. This major concern of researchers stems from the fact that the presence of Gram-negative bacterial endotoxin in intracellular products is unavoidable and requires complex downstream purification steps. For instance, endotoxin interacts with recombinant proteins, peptides, antibodies and aptamers and these interactions have formed the foundation for most biosensors for endotoxin detection. It has become imperative for researchers to engineer reliable means/techniques to detect, separate and remove endotoxin, without compromising the quality and quantity of the end-product. However, the underlying mechanism involved during endotoxin-biomolecule interaction is still a gray area. The use of quantitative molecular microscopy that provides high resolution of biomolecules is highly promising, hence, may lead to the development of improved endotoxin detection strategies in biomolecule preparation. Förster resonance energy transfer (FRET) spectroscopy is one of the emerging most powerful tools compatible with most super-resolution techniques for the analysis of molecular interactions. However, the scope of FRET has not been well-exploited in the analysis of endotoxin-biomolecule interaction. This article reviews endotoxin, its pathophysiological consequences and the interaction with biomolecules. Herein, we outline the common potential ways of using FRET to extend the current understanding of endotoxin-biomolecule interaction with the inference that a detailed understanding of the interaction is a prerequisite for the design of strategies for endotoxin identification and removal from protein milieus
A fluorescence assay for detecting amyloid-β using the cytomegalovirus enhancer/promoter
No full textRobust assays for detecting the effects of elevated concentrations of amyloid-β (Aβ) may facilitate Alzheimer’s disease research. An appropriate assay would be high-throughput and enable identification of drugs and genetic mutations that block the effects of Aβ, potentially leading to treatments for Alzheimer’s disease. We discovered that the commonly used cytomegalovirus (CMV) enhancer/promoter is sensitive to the effects of Aβ. By combining the CMV enhancer/promoter with a fluorescent protein, we created a reporter system that produces changes in intracellular fluorescence in response to Aβ. Using hippocampal neurons, we quantified the ability of a CMV-fluorescent protein recombinant reporter to detect both exogenously applied and overexpressed Aβ. This is the first report of a high-throughput enhancer/promoter-based Aβ detection method. The reporter is able to detect the effects of elevated concentrations of Aβ in a high-throughput fashion, providing a new tool for Alzheimer’s disease research and important knowledge about the commonly used CMV enhancer/promoter
A bi-functional IL-6-HaloTag® as a tool to measure the cell-surface expression of recombinant odorant receptors and to facilitate their activity quantification
No full textThe functional cell surface expression of recombinant odorant receptors typically has been investigated by expressing N-terminally extended, “tagged” receptors in test cell systems, using antibody-based immunocytochemistry or flow cytometry, and by measuring odorant/receptor-induced cAMP signaling, mostly by an odorant/receptor-induced and cAMP signaling-dependent transcriptional activation of a luciferase-based luminescence assay. In the present protocol, we explain a method to measure the cell-surface expression and signaling of recombinant odorant receptors carrying a bi-functional, N-terminal ‘IL-6-HaloTag®’. IL-6, being a secreted cytokine, facilitates functional cell surface expression of recombinant HaloTag®-odorant receptors, and the HaloTag® protein serves as a highly specific acceptor for cell-impermeant or cell-permeant, fluorophore-coupled ligands, which enable the quantification of odorant receptor expression by antibody-independent, chemical live-cell staining and flow cytometry. Here, we describe how to measure the cell surface expression of recombinant IL-6-HaloTag®-odorant receptors in HEK-293 cells or NxG 108CC15 cells, by live-cell staining and flow cytometry, and how to measure an odorant-induced activation of these receptors by the fast, real-time, luminescence-based GloSensor® cAMP assay
β-mercaptoethanol assists efficient construction of sperm bacterial artificial chromosome library
No full textBacterial artificial chromosome (BAC) library plays a critical role in the strategic research in genomics. Sperm is known as a good source for BAC library construction. However, preparation of intact DNA from the highly condensed sperm nuclei is not easy. Here we developed and validated an efficient DNA extraction strategy for BAC library construction from sperm embedded in agarose plugs. The protocol used a combination of lauroylsarcosine, proteinase K and β-mercaptoethanol (a reducing agent of nucleus) In comparison with the normal protocol without reducing agents, β-mercaptoethanol released high-molecular-weight DNA from the protamines which permit DNA to be packed very densely within the spermatozoan nucleus, without damaging DNA. Extracted DNA by this method was readily digested by restriction enzymes and ideal for BAC library construction