Sensitive detection of LiveEscherichia coliby bacteriophage amplification-coupled immunoassay on the Luminex®MAGPIX instrument

AbstractPhages are natural predators of bacteria and have been exploited in bacterial detection because of their exquisite specificity to their cognate bacterial hosts. In this study, we present a bacteriophage amplification-coupled assay as a surrogate for detecting a bacterium present in a sample. The assay entails detection of progeny phage resulting from infection and subsequent growth inside the bacterium present in suspected samples. This approach reduces testing time and enhances sensitivity to identify pathogens compared to traditional overnight plaque assay. Further, the assay has the ability to discriminate between live and dead cells since phages require live host cells to infect and replicate. To demonstrate its utility, phage MS2 amplification-coupled, bead-based sandwich type immunoassay on the Luminex®MAGPIX instrument forEscherichia colidetection was performed. The assay not only showed live cell discrimination ability but also a limit ofE. colidetection of 1×102cells/mL of live cells after a 3-hour incubation. In addition, the sensitivity of the assay was not impaired in the presence of dead cells. These results demonstrate that bacteriophage amplification-coupled assay can be a rapid live cell detection assay compared to traditional culture methods and a promising tool for quick validation of bacterial inactivation. Combined with the unique multiplex bead chemistry afforded by Luminex®MAGPIX platform, the phage assay can be expanded to be an ultra-deep multiplex assay for the simultaneous detection of multiple pathogens using specific phages directed against the target pathogens.

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Characterization of a Replicating Mammalian Orthoreovirus with Tetracysteine-Tagged μNS for Live-Cell Visualization of Viral Factories

Journal of Virology ◽

10.1128/jvi.01371-17 ◽

2017 ◽

Vol 91 (22) ◽

Cited By ~ 9

Author(s):

Luke D. Bussiere ◽

Promisree Choudhury ◽

Bryan Bellaire ◽

Cathy L. Miller

Keyword(s):

Nonstructural Protein ◽

Critical Role ◽

Viral Proteins ◽

Live Cell ◽

Host Cells ◽

Live Cells ◽

Virus Protein ◽

Mammalian Orthoreovirus ◽

Virus Proteins

ABSTRACT Within infected host cells, mammalian orthoreovirus (MRV) forms viral factories (VFs), which are sites of viral transcription, translation, assembly, and replication. The MRV nonstructural protein μNS comprises the structural matrix of VFs and is involved in recruiting other viral proteins to VF structures. Previous attempts have been made to visualize VF dynamics in live cells, but due to current limitations in recovery of replicating reoviruses carrying large fluorescent protein tags, researchers have been unable to directly assess VF dynamics from virus-produced μNS. We set out to develop a method to overcome this obstacle by utilizing the 6-amino-acid (CCPGCC) tetracysteine (TC) tag and FlAsH-EDT2 reagent. The TC tag was introduced into eight sites throughout μNS, and the capacity of the TC-μNS fusion proteins to form virus factory-like (VFL) structures and colocalize with virus proteins was characterized. Insertion of the TC tag interfered with recombinant virus rescue in six of the eight mutants, likely as a result of loss of VF formation or important virus protein interactions. However, two recombinant (r)TC-μNS viruses were rescued and VF formation, colocalization with associating virus proteins, and characterization of virus replication were subsequently examined. Furthermore, the rTC-μNS viruses were utilized to infect cells and examine VF dynamics using live-cell microscopy. These experiments demonstrate active VF movement with fusion events as well as transient interactions between individual VFs and demonstrate the importance of microtubule stability for VF fusion during MRV infection. This work provides important groundwork for future in-depth studies of VF dynamics and host cell interactions. IMPORTANCE MRV has historically been used as a model to study the double-stranded RNA (dsRNA) Reoviridae family, the members of which infect and cause disease in humans, animals, and plants. During infection, MRV forms VFs that play a critical role in virus infection but remain to be fully characterized. To study VFs, researchers have focused on visualizing the nonstructural protein μNS, which forms the VF matrix. This work provides the first evidence of recovery of replicating reoviruses in which VFs can be labeled in live cells via introduction of a TC tag into the μNS open reading frame. Characterization of each recombinant reovirus sheds light on μNS interactions with viral proteins. Moreover, utilizing the TC-labeling FlAsH-EDT2 biarsenical reagent to visualize VFs, evidence is provided of dynamic VF movement and interactions at least partially dependent on intact microtubules.

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Characterization of a replicating mammalian orthoreovirus with tetracysteine tagged μNS for live cell visualization of viral factories

10.1101/174235 ◽

2017 ◽

Cited By ~ 1

Author(s):

Luke D. Bussiere ◽

Promisree Choudhury ◽

Bryan Bellaire ◽

Cathy L. Miller

Keyword(s):

Critical Role ◽

Viral Proteins ◽

Live Cell ◽

Host Cells ◽

Live Cells ◽

Virus Protein ◽

Structural Protein ◽

Mammalian Orthoreovirus ◽

Virus Proteins

AbstractWithin infected host cells, mammalian orthoreovirus (MRV) forms viral factories (VFs) which are sites of viral transcription, translation, assembly, and replication. MRV non-structural protein, μNS, comprises the structural matrix of VFs and is involved in recruiting other viral proteins to VF structures. Previous attempts have been made to visualize VF dynamics in live cells but due to current limitations in recovery of replicating reoviruses carrying large fluorescent protein tags, researchers have been unable to directly assess VF dynamics from virus-produced μNS. We set out to develop a method to overcome this obstacle by utilizing the 6 amino-acid (CCPGCC) tetracysteine (TC)-tag and FlAsH-EDT2 reagent. The TC-tag was introduced into eight sites throughout μNS, and the capacity of the TC-μNS fusion proteins to form virus factory-like (VFL) structures and colocalize with virus proteins was characterized. Insertion of the TC-tag interfered with recombinant virus rescue in six of the eight mutants, likely as a result of loss of VF formation or important virus protein interactions. However, two recombinant (r)TC-μNS viruses were rescued and VF formation, colocalization with associating virus proteins, and characterization of virus replication were subsequently examined. Furthermore the rTC-μNS viruses were utilized to infect cells and examine VF dynamics using live cell microscopy. These experiments demonstrate active VF movement with fusion events as well as transient interactions between individual VFs, and demonstrate the importance of microtubule stability for VF fusion during MRV infection. This work provides important groundwork for future in depth studies of VF dynamics and host cell interactions.ImportanceMRV has historically been used as a model to study the double-stranded RNA (dsRNA)Reoviridaefamily, which infect and cause disease in humans, animals, and plants. During infection, MRV forms VFs that play a critical role in virus infection, but remain to be fully characterized. To study VFs, researchers have focused on visualizing the non-structural protein μNS which forms the VF matrix. This work provides the first evidence of recovery of replicating reoviruses in which VFs can be labeled in live cells via introduction of a TC-tag into the μNS open reading frame. Characterization of each recombinant reovirus sheds light on μNS interactions with viral proteins. Moreover, utilizing the TC labeling FlAsH-EDT2 biarsenical reagent to visualize VFs, evidence is provided of dynamic VF movement and interactions at least partially dependent on intact microtubules.

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α-Methylene-β-Lactone Probe for Measuring Live-Cell Reactions of Small Molecules

10.26434/chemrxiv.12078582.v2 ◽

2020 ◽

Author(s):

Lei Wang ◽

Louis Riel ◽

Bekim Bajrami ◽

Bin Deng ◽

Amy Howell ◽

...

Keyword(s):

Mass Spectra ◽

Live Cell ◽

Live Cells ◽

The Novel ◽

Proteomics Analysis ◽

Chemical Proteomics ◽

Tandem Mass Spectra ◽

Modified Peptides ◽

Covalent Probes ◽

Ht 29

The novel use of the α-methylene-β-lactone (MeLac) moiety as a warhead of multiple electrophilic sites is reported. In this study, we demonstrate that a MeLac-alkyne is a competent covalent probe and reacts with diverse proteins in live cells. Proteomics analysis of affinity-enriched samples identifies probe-reacted proteins, resolves their modified peptides/residues, and thus characterizes probe-protein reactions. Unique methods are developed to evaluate confidence in the identification of the reacted proteins and modified peptides. Tandem mass spectra of the peptides reveal that MeLac reacts with nucleophilic cysteine, serine, lysine, threonine, and tyrosine residues, through either Michael addition or acyl addition. A peptide-centric proteomics platform, using MeLac-alkyne as the measurement probe, successfully analyzes the Orlistat selectivity in live HT-29 cells. MeLac is a versatile warhead demonstrating enormous potential to expedite the development of covalent probes and inhibitors in interrogating protein (re)activity. MeLac-empowered platforms in chemical proteomics are widely adaptable for measuring the live-cell action of reactive molecules.

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ACIS, A Novel KepTide™, Binds to ACE-2 Receptor and Inhibits the Infection of SARS-CoV2 Virus in vitro in Primate Kidney Cells: Therapeutic Implications for COVID-19 (Preprint)

10.2196/preprints.25089 ◽

2020 ◽

Author(s):

Avik Sotira Scientific

Keyword(s):

Social Life ◽

Plaque Assay ◽

Host Cells ◽

Surface Glycoprotein ◽

Crystallographic Structure ◽

Therapeutic Implications ◽

Biochemical Assays ◽

Targeted Medicine

UNSTRUCTURED Coronavirus disease 2019 (COVID-19) is a severe acute respiratory syndrome (SARS) caused by a virus known as SARS-Coronavirus 2 (SARS-CoV2). Without a targeted-medicine, this disease has been causing a massive humanitarian crisis not only in terms of mortality, but also imposing a lasting damage to social life and economic progress of humankind. Therefore, an immediate therapeutic strategy needs to be intervened to mitigate this global crisis. Here, we report a novel KepTide™ (Knock-End Peptide) therapy that nullifies SARS-CoV2 infection. SARS-CoV2 employs its surface glycoprotein “spike” (S-glycoprotein) to interact with angiotensin converting enzyme-2 (ACE-2) receptor for its infection in host cells. Based on our in-silico-based homology modeling study validated with a recent X-ray crystallographic structure (PDB ID:6M0J), we have identified that a conserved motif of S-glycoprotein that intimately engages multiple hydrogen-bond (H-bond) interactions with ACE-2 enzyme. Accordingly, we designed a peptide, termed as ACIS (ACE-2 Inhibitory motif of Spike), that displayed significant affinity towards ACE-2 enzyme as confirmed by biochemical assays such as BLItz and fluorescence polarization assays. Interestingly, more than one biochemical modifications were adopted in ACIS in order to enhance the inhibitory action of ACIS and hence called as KEpTide™. Consequently, a monolayer invasion assay, plaque assay and dual immunofluorescence analysis further revealed that KEpTide™ efficiently mitigated the infection of SARS-CoV2 in vitro in VERO E6 cells. Finally, evaluating the relative abundance of ACIS in lungs and the potential side-effects in vivo in mice, our current study discovers a novel KepTide™ therapy that is safe, stable, and robust to attenuate the infection of SARS-CoV2 virus if administered intranasally. INTERNATIONAL REGISTERED REPORT RR2-https://doi.org/10.1101/2020.10.13.337584

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Fluorescent Probes for Live Cell Thiol Detection

Molecules ◽

10.3390/molecules26123575 ◽

2021 ◽

Vol 26 (12) ◽

pp. 3575

Author(s):

Shenggang Wang ◽

Yue Huang ◽

Xiangming Guan

Keyword(s):

Fluorescent Probes ◽

Biological System ◽

High Sensitivity ◽

Intracellular Distribution ◽

Live Cell ◽

Live Cells ◽

High Demand ◽

Non Invasive

Thiols play vital and irreplaceable roles in the biological system. Abnormality of thiol levels has been linked with various diseases and biological disorders. Thiols are known to distribute unevenly and change dynamically in the biological system. Methods that can determine thiols’ concentration and distribution in live cells are in high demand. In the last two decades, fluorescent probes have emerged as a powerful tool for achieving that goal for the simplicity, high sensitivity, and capability of visualizing the analytes in live cells in a non-invasive way. They also enable the determination of intracellular distribution and dynamitic movement of thiols in the intact native environments. This review focuses on some of the major strategies/mechanisms being used for detecting GSH, Cys/Hcy, and other thiols in live cells via fluorescent probes, and how they are applied at the cellular and subcellular levels. The sensing mechanisms (for GSH and Cys/Hcy) and bio-applications of the probes are illustrated followed by a summary of probes for selectively detecting cellular and subcellular thiols.

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Simultaneous detection of apoptosis and mitochondrial superoxide production in live cells by flow cytometry and confocal microscopy

Nature Protocols ◽

10.1038/nprot.2007.327 ◽

2007 ◽

Vol 2 (9) ◽

pp. 2295-2301 ◽

Cited By ~ 256

Author(s):

Partha Mukhopadhyay ◽

Mohanraj Rajesh ◽

György Haskó ◽

Brian J Hawkins ◽

Muniswamy Madesh ◽

...

Keyword(s):

Flow Cytometry ◽

Confocal Microscopy ◽

Simultaneous Detection ◽

Superoxide Production ◽

Live Cells ◽

Mitochondrial Superoxide

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Establishment of live-cell-based coupled assay system for identification of compounds to modulate metabolic activities of cells

Cell Reports ◽

10.1016/j.celrep.2021.109311 ◽

2021 ◽

Vol 36 (1) ◽

pp. 109311

Author(s):

Kouichi Yanagi ◽

Toru Komatsu ◽

Shusuke Ogihara ◽

Takayoshi Okabe ◽

Hirotatsu Kojima ◽

...

Keyword(s):

Live Cell ◽

Assay System ◽

Metabolic Activities ◽

Coupled Assay

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Novel anthracene- and pyridine-containing Schiff base probe for selective “off–on” fluorescent determination of Cu2+ ions towards live cell application

New Journal of Chemistry ◽

10.1039/c5nj03168k ◽

2016 ◽

Vol 40 (7) ◽

pp. 6101-6108 ◽

Cited By ~ 14

Author(s):

Turibius Simon ◽

Muthaiah Shellaiah ◽

Venkatesan Srinivasadesikan ◽

Ching-Chang Lin ◽

Fu-Hsiang Ko ◽

...

Keyword(s):

Electron Transfer ◽

Schiff Base ◽

Photoinduced Electron Transfer ◽

Transfer Mechanism ◽

Live Cell ◽

Live Cells ◽

Electron Transfer Mechanism

A simple anthracene-based AP probe was synthesized to detect Cu2+ ions, via the photoinduced electron transfer mechanism, in live cells.

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tdLanYFP, a yellow, bright, photostable and pH insensitive fluorescent protein for live cell imaging and FRET-based sensing strategies

10.1101/2021.04.27.441613 ◽

2021 ◽

Author(s):

Y. Bousmah ◽

H. Valenta ◽

G. Bertolin ◽

U. Singh ◽

V. Nicolas ◽

...

Keyword(s):

Single Molecule ◽

Live Cell Imaging ◽

Cell Imaging ◽

Fluorescent Protein ◽

Fluorescent Proteins ◽

Yellow Fluorescent Protein ◽

Resonance Energy ◽

Live Cell ◽

Live Cells

AbstractYellow fluorescent proteins (YFP) are widely used as optical reporters in Förster Resonance Energy Transfer (FRET) based biosensors. Although great improvements have been done, the sensitivity of the biosensors is still limited by the low photostability and the poor fluorescence performances of YFPs at acidic pHs. In fact, today, there is no yellow variant derived from the EYFP with a pK1/2 below ∼5.5. Here, we characterize a new yellow fluorescent protein, tdLanYFP, derived from the tetrameric protein from the cephalochordate B. lanceolatum, LanYFP. With a quantum yield of 0.92 and an extinction coefficient of 133 000 mol−1.L.cm−1, it is, to our knowledge, the brightest dimeric fluorescent protein available, and brighter than most of the monomeric YFPs. Contrasting with EYFP and its derivatives, tdLanYFP has a very high photostability in vitro and preserves this property in live cells. As a consequence, tdLanYFP allows the imaging of cellular structures with sub-diffraction resolution with STED nanoscopy. We also demonstrate that the combination of high brightness and strong photostability is compatible with the use of spectro-microscopies in single molecule regimes. Its very low pK1/2 of 3.9 makes tdLanYFP an excellent tag even at acidic pHs. Finally, we show that tdLanYFP can be a FRET partner either as donor or acceptor in different biosensing modalities. Altogether, these assets make tdLanYFPa very attractive yellow fluorescent protein for long-term or single-molecule live-cell imaging that is also suitable for FRET experiment including at acidic pH.

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CRISPR-mediated Multiplexed Live Cell Imaging of Nonrepetitive Genomic Loci

10.1101/2020.03.03.974923 ◽

2020 ◽

Author(s):

Patricia A. Clow ◽

Nathaniel Jillette ◽

Jacqueline J. Zhu ◽

Albert W. Cheng

Keyword(s):

Live Cell Imaging ◽

Cell Imaging ◽

Repetitive Sequences ◽

Binary Sequence ◽

Three Dimensional ◽

Live Cell ◽

Live Cells ◽

Imaging Method ◽

Genomic Locus ◽

Time Dynamics

AbstractThree-dimensional (3D) structures of the genome are dynamic, heterogeneous and functionally important. Live cell imaging has become the leading method for chromatin dynamics tracking. However, existing CRISPR- and TALE-based genomic labeling techniques have been hampered by laborious protocols and low signal-to-noise ratios (SNRs), and are thus mostly applicable to repetitive sequences. Here, we report a versatile CRISPR/Casilio-based imaging method, with an enhanced SNR, that allows for one nonrepetitive genomic locus to be labeled using a single sgRNA. We constructed Casilio dual-color probes to visualize the dynamic interactions of cohesin-bound elements in single live cells. By forming a binary sequence of multiple Casilio probes (PISCES) across a continuous stretch of DNA, we track the dynamic 3D folding of a 74kb genomic region over time. This method offers unprecedented resolution and scalability for delineating the dynamic 4D nucleome.One Sentence SummaryCasilio enables multiplexed live cell imaging of nonrepetitive DNA loci for illuminating the real-time dynamics of genome structures.

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