This work was supported by the National Natural Science Foundation of China (31670845, 31870832, 32000811) and the Program of Distinguished Professor of Liaoning Province (LJH2018-35).

Peripheral blood from volunteers applied in this research was approved by the Institutional Review Board of the first Hospital of China Medical University (#2020-2020-237-2).
1. Preparation of reagents and culture medium
<ol>
	<li>Prepare the red blood cell lysis buffer by adding 8.29 g of NH4Cl, 1 g of KHCO3, 37.2 mg of Na2EDTA into 1 L of double distilled water and adjust the pH to 7.2-7.4. Remove the bacteria by filtration using 0.22 &#181;m filters.</li>
	<li>Prepare experimental culture medium for neutrophils by adding 5% fetal bovine serum to RPMI 1640 medium and store at 4 &#176;C. Equilibrate to room temperature before use. 
	NOTE: Use the RPMI 1640 medium without phenol red.</li>
	<li>Prepare phosphate buffer saline (PBS) by adding 8 g of NaCl, 0.2 g of KCl, 1.44 g of Na2HPO4&#183;2H2O and 0.2 g of KH2PO4 into 1 L of double distilled water in a 1 L glass flask. Adjust the pH to 7.2-7.4. Autoclave it for 15 min at 121 &#176;C.</li>
	<li>Prepare rifampicin solution by dissolving 0.5 g of rifampicin powder in 10 mL of dimethyl sulfoxide (DMSO) to yield a 50 mg/mL rifampicin solution.</li>
	<li>Prepare LB agar solution by adding 10 g of NaCl, 10 g of tryptone, 5 g of yeast extract and 15 g of agar powder into 1 L of double distilled water and autoclave the mixture. Fill the Petri dishes to half of the volume by pouring warm LB agar solution containing 100 &#181;g/mL rifampicin. Store the cooled solid plate at 4 &#176;C.</li>
	<li>Prepare brain heart infusion (BHI) broth appropriate for bacterial strains by dissolving 37 g of BHI powder into 1 L of double distilled water. Adjust the pH to 7.2 and autoclave it.</li>
	<li>Dissolve the fluorescent probe dihydroethidium (DHE) in DMSO solvent to yield a 10 mM stock solution. Gently mix before use. 
	&#8203;NOTE: Aliquot the stock solution immediately into light-proof vials. The shelf life of the stock solution is 6 months at -20 &#176;C.</li>
</ol>
2. Preparation of E44 bacteria strain
NOTE: E44 is a mutant strain of meningitic&#160;E. coli with rifampicin resistance.
<ol>
	<li>Dip the cryopreserved E44 colony with a sterile pipette tip, inoculate the E44 strain on the LB agar plate containing 100 &#181;g/mL rifampicin by drawing lines. Put the plate upside down in the incubator at 37 &#176;C overnight.</li>
	<li>One day before the experiment, pick one E44 colony from the plate with a sterile pipette tip and put it in 5 mL of BHI broth containing 100 &#181;g/mL rifampicin in a 50 mL flask. Incubate the bacterial culture at 37 &#176;C with 90 rpm for 17 h in an incubation shaker.</li>
</ol>
3. Isolation of neutrophils from human peripheral blood
<ol>
	<li>Draw 5 mL of blood sample from volunteers intravenously to the vacuum blood collection tube containing EDTA for anticoagulation.</li>
	<li>Centrifuge the peripheral blood samples at 500&#160;x g for 5 min. The blood samples are divided into three layers by centrifugation, which from bottom to top are the red blood cell (RBC) layer, the white blood cell (WBC) layer and the plasma layer, sequentially.</li>
	<li>Aspirate the white blood cell layer with a pipet to a new tube with 3x RBC lysis buffer. Blend the mixture thoroughly and place at room temperature for 5 min.</li>
	<li>Centrifuge the tube at 500 x g for 5 min. Aspirate the supernatant completely and discard.
	<ol>
		<li>Repeat the lysis procedure with RBC lysis buffer 1-2 times, until the precipitate turns white.</li>
	</ol>
	</li>
	<li>Wash the cells by resuspending the precipitate with 2 mL of PBS. Then centrifuge at 300 x g for 5 min to let the cells settle down to the bottom of the tube.</li>
	<li>Label the neutrophils with CD16 microbeads by resuspending the sediment with 50 &#181;L of precooled magnetic cell sorting buffer. Then mix with 50 &#181;L of human CD16 microbeads thoroughly. Incubate the mixture at 4 &#176;C for 30 min. 
	NOTE: Solutions should be pre-cooled to prevent capping of antibodies on the cell surface and non-specific labeling. Most adults have about 4,000 to 10,000 white blood cells per microliter of blood, among which, neutrophils account for approximately 50-70%. By estimate, the counts of total white blood cells in 5 mL of human peripheral blood are usually up to 2-5 x 107.</li>
	<li>Wash the cells by adding 2 mL of magnetic cell sorting buffer and centrifuge at 4 &#176;C, 300 x g for 10 min. Discard the supernatant completely and resuspend the precipitate with 500 &#181;L of sorting buffer.</li>
	<li>Assemble the magnetic column and separating shelf. Move the separator to the shelf with the magnetic column and rinse the column with 3 mL of sorting buffer.</li>
	<li>Drop the cell suspension into the column to allow the neutrophils labeled by the magnetic beads to attach to the magnetic column.</li>
	<li>Wash off the non-labeled cells by adding 3 mL of magnetic cell sorting buffer 3 times, making sure that the column reservoir is empty each time.</li>
	<li>Remove the column from the magnetic separator and put it on a 15 mL tube. Add 5 mL of magnetic cell sorting buffer to the column. Push out the magnetic labeled cells using a plunger. 
	NOTE: To improve the purity of the neutrophils, the sorting steps may be repeated using a new column.</li>
	<li>Centrifuge the tube at 300 x g for 5 min, discard the supernatant completely and resuspend the precipitate with 1 mL of culture medium. Determine the cell number with a cell counter and prepare the cells for further experiments.</li>
</ol>
4. Measurement of ROS
<ol>
	<li>Centrifuge the isolated neutrophils at 300 x g for 5 min, resuspend the precipitate, and adjust the cell concentration to 2 x 106/mL with culture medium containing 5 &#181;M DHE fluorescence probe.</li>
	<li>Incubate the neutrophils at 37 &#176;C for 30 min to load the DHE probe, and then allocate the cell suspension to a 96-well black polystyrene microplate with 200 &#181;L per well.</li>
	<li>Turn on the microplate reader and open the detection software. Choose opaque 96-wells plate format and determine the reading area.
	<ol>
		<li>Set the fluorescence (Ex/Em = 518/605 nm) in kinetic mode every 5 min for 60 min at 37 &#176;C. Make sure to shake the plate for 3 s before each reading.</li>
	</ol>
	</li>
	<li>Take out the microplate from the incubator, add the cultured E44 (MOI=100) or phorbol 12-myristate 13-acetate (PMA) (100 ng/mL) to each well containing preloaded neutrophils with 3 replicates. Use PMA as a positive control.</li>
	<li>Place the plate in the microplate reader and start the assay immediately.</li>
</ol>

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The authors declare no competing financial interests or other conflicts of interest.

Neutrophils act as the most abundant component of white blood cells in human blood circulation. They are important effector cells in the innate human immune system, which builds the first line of defense against the invasion of pathogens11. The generation of ROS represents one of the major bactericidal mechanisms of neutrophils following phagocytosis11. Recent studies have shown that a net-like structure released by a neutrophil called neutrophil extracellular trap (NET) is...

Neonatal bacterial meningitis is a common pediatric infectious disease. Escherichia coli (E. coli) with a K1 capsule is the most common Gram-negative pathogen causing neonatal bacterial meningitis, accounting for about 80% of the total incidence1,2,3. Despite the advances in the antimicrobial chemotherapy and supportive care, bacterial meningitis is still one of the most devastating conditions with high morbidity and mortality4.
The occurrence of neonatal bacterial meningitis usually begins with bacteremia caused by the entry of pathogenic bacteria into the peripheral circulation from the local lesions of the newborns, followed by penetration through the blood-brain barrier (BBB) into the brain, resulting in the inflammation of the meninges4. The onset of bacteremia depends on the interaction between bacteria and host immune cells including neutrophils and macrophages, etc. Neutrophils, which account for ~50-70% of white blood cells, are the first line of defense against bacterial infections5,6. During the invasion of bacteria, the activated neutrophils are recruited to the infectious sites and release reactive oxygen species (ROS) including the superoxide anion, hydrogen peroxide, hydroxyl radicals, and singlet oxygen7. The ROS undergo redox reactions with the cell membrane, nucleic acid molecules and proteins of the bacteria, resulting in the injury and death of the invading bacteria8. The mitochondria is the main site of ROS production in eukaryotic cells, and various oxidases (e.g., nicotinamide adenine dinucleotide phosphate (NADPH) oxidase complex, lipoxygenase system, protein kinase C and cyclooxygenase system) mediate the production of ROS9,10. The real-time measurement of the production of ROS, representing the primary antimicrobial mechanism in neutrophils, is a useful method for studying host defense during the bacteria-host interaction.
In this protocol, the time-dependent ROS production in neutrophils infected with meningitic&#160;E. coli was quantified with a fluorescent ROS probe DHE, detected by a real-time fluorescence microplate reader. This method may also be applied to the assessment of ROS production in other mammalian cells during the pathogen-host interaction.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>15 mL polypropylene conical centrifuge tubes</td>
			<td>KIRGEN</td>
			<td>KG2611</td>
			<td></td>
		</tr>
		<tr>
			<td>96-well plate</td>
			<td>Corning</td>
			<td>3025</td>
			<td></td>
		</tr>
		<tr>
			<td>Agar</td>
			<td>DINGGUO</td>
			<td>DH010-1.1</td>
			<td></td>
		</tr>
		<tr>
			<td>Autuomated cell counter</td>
			<td>Bio-rad</td>
			<td>508BR03397</td>
			<td></td>
		</tr>
		<tr>
			<td>Biological Safety Carbinet</td>
			<td>Shanghai Lishen</td>
			<td>Hfsafe-1200Lcb2</td>
			<td></td>
		</tr>
		<tr>
			<td>Brain heart infusion</td>
			<td>BD</td>
			<td>237500</td>
			<td></td>
		</tr>
		<tr>
			<td>CD16 Microbeads, human</td>
			<td>Miltenyi Biotec</td>
			<td>130-045-701</td>
			<td></td>
		</tr>
		<tr>
			<td>Centrifuge</td>
			<td>Changsha Xiangyi</td>
			<td>TDZ5-WS</td>
			<td></td>
		</tr>
		<tr>
			<td>Columns</td>
			<td>Miltenyi Biotec</td>
			<td>130-042-401</td>
			<td></td>
		</tr>
		<tr>
			<td>Dihydroethidium (DHE)</td>
			<td>MedChemExpress</td>
			<td>104821-25-2</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal bovine serum</td>
			<td>Cellmax</td>
			<td>SA211.02</td>
			<td></td>
		</tr>
		<tr>
			<td>Incubator</td>
			<td>Heraeus</td>
			<td>Hera Cell</td>
			<td></td>
		</tr>
		<tr>
			<td>MACS separation buffer</td>
			<td>Miltenyi Biotec</td>
			<td>130-091-221</td>
			<td></td>
		</tr>
		<tr>
			<td>Microplate Reader</td>
			<td>Molecular Devices</td>
			<td>SpectraMax M5</td>
			<td></td>
		</tr>
		<tr>
			<td>Phorbol 12-myristate 13-acetate (PMA)</td>
			<td>Beyoitme</td>
			<td>S1819-1mg</td>
			<td></td>
		</tr>
		<tr>
			<td>QuadroMACS separation Unit</td>
			<td>Miltenyi Biotec</td>
			<td>130-090-976</td>
			<td></td>
		</tr>
		<tr>
			<td>Rifampicin</td>
			<td>Solarbio</td>
			<td>13292-46-1</td>
			<td></td>
		</tr>
		<tr>
			<td>RPMI1640 medium</td>
			<td>Sangon Biotech</td>
			<td>E600027-0500</td>
			<td></td>
		</tr>
		<tr>
			<td>Thermostatic shaker</td>
			<td>Shanghai Zhicheng</td>
			<td>ZWY-100D</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypton</td>
			<td>OXOID</td>
			<td>LP0042</td>
			<td></td>
		</tr>
		<tr>
			<td>Yeast extract</td>
			<td>OXOID</td>
			<td>LP0021</td>
			<td></td>
		</tr>
	</tbody>
</table>

real-time quantification of reactive oxygen species in neutrophils infected with meningitic escherichia coli

Escherichia coli (E. coli) is the most common Gram-negative bacteria causing neonatal meningitis. The occurrence of bacteremia and bacterial penetration through the blood-brain barrier are indispensable steps for the development of E. coli meningitis. Reactive oxygen species (ROS) represent the major bactericidal mechanisms of neutrophils to destroy the invaded pathogens. In this protocol, the time-dependent intracellular ROS production in neutrophils infected with meningitic E. coli was quantified using fluorescent ROS probes detected by a real-time fluorescence microplate reader. This method may also be applied to the assessment of ROS production in mammalian cells during pathogen-host interactions.

Using the protocol outlined in this article, the neutrophils were isolated from human peripheral blood and loaded with fluorescence probe DHE to detect the changes of ROS levels in response to E44 infection. Here, we provide representative data demonstrating the ROS production evoked by E44 strain determined by a microplate reader in real-time. By adding E44 strains at a MOI of 100, the ROS levels increased immediately and showed a continuous upward trend with a time-dependent manner (Figure 1</stron...

Watch this Scientific Journal Video about Real-Time Quantification of Reactive Oxygen Species in Neutrophils Infected with Meningitic Escherichia Coli at JoVE.com

Real-Time Quantification of Reactive Oxygen Species in Neutrophils Infected with Meningitic Escherichia Coli

Escherichia coli is the leading cause of neonatal Gram-negative bacterial meningitis. During the bacterial infection, reactive oxygen species produced by neutrophils play a major bactericidal role. Here we introduce a method to detect the reactive oxygen species in neutrophils in response to meningitis E. coli.

Real-Time Quantification of Reactive Oxygen Species in Neutrophils Infected with Meningitic Escherichia Coli

Escherichia coli (E. coli) is the most common Gram-negative bacteria causing neonatal meningitis. The occurrence of bacteremia and bacterial penetration ...

real-time-quantification-reactive-oxygen-species-neutrophils-infected

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Immunology and Infection

4.3K Views.  Ministry of Education, China Medical University. Escherichia coli is the leading cause of neonatal Gram-negative bacterial meningitis. During the bacterial infection, reactive oxygen species produced by neutrophils play a major bactericidal role. Here we introduce a method to detect the reactive oxygen species in neutrophils in response to meningitis E. coli.

Video: Real-Time Quantification of Reactive Oxygen Species in Neutrophils Infected with Meningitic Escherichia Coli

Real-time Imaging of Leukotriene B4 Mediated Cell Migration and BLT1 Interactions with &beta;-arrestin

G-protein coupled receptors (GPCRs) belong to the seven transmembrane protein family and mediate the transduction of extracellular signals to intracellular responses. GPCRs control diverse biological functions such as chemotaxis, intracellular calcium release, gene regulation in a ligand dependent manner via heterotrimeric G-proteins1-2. Ligand binding induces a series of conformational changes leading to activation of heterotrimeric G-proteins that modulate levels of second messengers such as cyclic adenosine monophosphate (cAMP), inositol triphosphate (IP3) and diacyl glycerol (DG). Concomitant with activation of the receptor ligand binding also initiates a series of events to attenuate the receptor signaling via desensitization, sequestration and/or internalization. The desensitization process of GPCRs occurs via receptor phosphorylation by G-protein receptor kinases (GRKs) and subsequent binding of &beta;-arrestins3. &beta;-arrestins are cytosolic proteins and translocate to membrane upon GPCR activation, binding to phosphorylated receptors (most cases) there by facilitating receptor internalization 4-6.
Leukotriene B4 (LTB4) is a pro-inflammatory lipid molecule derived from arachidonic acid pathway and mediates its actions via GPCRs, LTB4 receptor 1 (BLT1; a high affinity receptor) and LTB4 receptor 2 (BLT2; a low affinity receptor)7-9. The LTB4-BLT1 pathway has been shown to be critical in several inflammatory diseases including, asthma, arthritis and atherosclerosis10-17. The current paper describes the methodologies developed to monitor LTB4-induced leukocyte migration and the interactions of BLT1 with &beta;-arrestin and , receptor translocation in live cells using microscopy imaging techniques18-19.
Bone marrow derived dendritic cells from C57BL/6 mice were isolated and cultured as previously described 20-21. These cells were tested in live cell imaging methods to demonstrate LTB4 induced cell migration. The human BLT1 was tagged with red fluorescent protein (BLT1-RFP) at C-terminus and &beta;-arrestin1 tagged with green fluorescent protein (&beta;-arr-GFP) and transfected the both plasmids into Rat Basophilic Leukomia (RBL-2H3) cell lines18-19. The kinetics of interaction between these proteins and localization were monitored using live cell video microscopy. The methodologies in the current paper describe the use of microscopic techniques to investigate the functional responses of G-protein coupled receptors in live cells. The current paper also describes the use of Metamorph software to quantify the fluorescence intensities to determine the kinetics of receptor and cytosolic protein interactions.

Real-time Imaging of Leukotriene B4 Mediated Cell Migration and BLT1 Interactions with &#946;-arrestin

This paper describes the methodology to determine the chemotactic response of leukocytes to specific ligands and identify interactions between the cell surface receptors and cytosolic proteins using live cell imaging techniques.

G-protein coupled receptors (GPCRs) belong to the seven transmembrane protein family and mediate the transduction of extracellular signals to ...

Direct Observation of Phagocytosis and NET-formation by Neutrophils in Infected Lungs using 2-photon Microscopy

After the gastrointestinal tract, the lung is the second largest surface for interaction between the vertebrate body and the 
environment. Here, an effective gas exchange must be maintained, while at the same time avoiding infection by the multiple pathogens that are inhaled 
during normal breathing. To achieve this, a superb set of defense strategies combining humoral and cellular immune mechanisms exists. One of the most 
effective measures for acute defense of the lung is the recruitment of neutrophils, which either phagocytose the inhaled pathogens or kill them by 
releasing cytotoxic chemicals. A recent addition to the arsenal of neutrophils is their explosive release of extracellular DNA-NETs by which bacteria 
or fungi can be caught or inactivated even after the NET releasing cells have died. We present here a method that allows one to directly observe neutrophils, 
migrating within a recently infected lung, phagocytosing fungal pathogens as well as visualize the extensive NETs that they have produced throughout the 
infected tissue. The method describes the preparation of thick viable lung slices 7 hours after intratracheal infection of mice with conidia of the mold 
Aspergillus fumigatus and their examination by multicolor time-lapse 2-photon microscopy. This approach allows one to directly investigate antifungal 
defense in native lung tissue and thus opens a new avenue for the detailed investigation of pulmonary immunity.

We show, how to use 2-photon microscopy for the observation of the dynamics of neutrophil granulocytes in infected lungs while they phagocytose pathogens or produce neutrophil extracellular traps (NETs).

After the gastrointestinal tract, the lung is the second largest surface for interaction between the vertebrate body and the 
environment. Here, an ...

Non-Invasive Model of Neuropathogenic Escherichia coli Infection in the Neonatal Rat

Investigation of the interactions between animal host and bacterial pathogen is only meaningful if the infection model employed replicates the principal features of the natural infection. This protocol describes procedures for the establishment and evaluation of systemic infection due to neuropathogenic Escherichia coli K1 in the neonatal rat. Colonization of the gastrointestinal tract leads to dissemination of the pathogen along the gut-lymph-blood-brain course of infection and the model displays strong age dependency. A strain of E. coli O18:K1 with enhanced virulence for the neonatal rat produces exceptionally high rates of colonization, translocation to the blood compartment and invasion of the meninges following transit through the choroid plexus. As in the human host, penetration of the central nervous system is accompanied by local inflammation and an invariably lethal outcome. The model is of proven utility for studies of the mechanism of pathogenesis, for evaluation of therapeutic interventions and for assessment of bacterial virulence.

Non-Invasive Model of Neuropathogenic Escherichia coli Infection in the Neonatal Rat

Here, a procedure is described for the establishment of systemic infection in the neonatal rat with cultures of Escherichia coli K1. This non-invasive procedure permits colonization of the gastrointestinal tract, translocation of the pathogen to the systemic circulation, and invasion of the central nervous system at the choroid plexus.

Investigation of the interactions between animal host and bacterial pathogen is only meaningful if the infection model employed replicates the principal ...

Imaging Neutrophils and Monocytes in Mesenteric Veins by Intravital Microscopy on Anaesthetized Mice in Real Time

Efficient immune response is dependent on rapid mobilization of blood leukocytes to the site of infection or injury. Investigating leukocyte migration in vivo is crucial for understanding the molecular basis of leukocyte transendothelial migration and interaction with vascular endothelium. One powerful approach involves intravital microscopy on transgenic mice expressing fluorescent proteins in cells of interest.
Here we present a protocol for imaging monocytes and neutrophils in the CX3CR1gfp/wt mouse i.v. injected with orange dye-labeled neutrophils with an inverted confocal microscope. Time-lapse movies gathered from 30 min&#160;to several hours of imaging allow the analysis of leukocyte behavior in mesenteric veins under both steady state and inflammatory conditions. We also describe the steps to locally induce blood vessel inflammation with TLR2/TLR1 agonist Pam3SK4 and monitor the subsequent recruitment of neutrophils and monocytes.
The presented technique can also be used to monitor other populations of leukocytes and investigate molecules implicated in leukocyte recruitment or trafficking using other stimuli or transgenic mice.

We detail a protocol to monitor the behavior of neutrophils and monocytes in mesenteric veins under steady state and inflammatory conditions using intravital confocal microscopy on anaesthetized mice.

Efficient immune response is dependent on rapid mobilization of blood leukocytes to the site of infection or injury. Investigating leukocyte migration in ...

One-step Negative Chromatographic Purification of Helicobacter pylori Neutrophil-activating Protein Overexpressed in Escherichia coli in Batch Mode

Helicobacter pylori neutrophil-activating protein (HP-NAP) is a major virulence factor of Helicobacter pylori (H. pylori). It plays a critical role in H. pylori-induced gastric inflammation by activating several innate leukocytes including neutrophils, monocytes, and mast cells. The immunogenic and immunomodulatory properties of HP-NAP make it a potential diagnostic and vaccine candidate for H. pylori and a new drug candidate for cancer therapy. In order to obtain substantial quantities of purified HP-NAP used for its clinical applications, an efficient method to purify this protein with high yield and purity needs to be established.
In this protocol, we have described a method for one-step negative chromatographic purification of recombinant HP-NAP overexpressed in Escherichia coli (E. coli) by using diethylaminoethyl (DEAE) ion-exchange resins (e.g., Sephadex) in batch mode. Recombinant HP-NAP constitutes nearly 70% of the total protein in E. coli and is almost fully recovered in the soluble fraction upon cell lysis at pH 9.0. Under the optimal condition at pH 8.0, the majority of HP-NAP is recovered in the unbound fraction while the endogenous proteins from E. coli are efficiently removed by the resin.
This purification method using negative mode batch chromatography with DEAE ion-exchange resins yields functional HP-NAP from E. coli in its native form with high yield and purity. The purified HP-NAP could be further utilized for the prevention, treatment, and prognosis of H. pylori-associated diseases as well as cancer therapy.

One-step Negative Chromatographic Purification of Helicobacter pylori Neutrophil-activating Protein Overexpressed in Escherichia coli in Batch Mode

A high yield method for one-step negative purification of recombinant Helicobacter pylori neutrophil-activating protein (HP-NAP) overexpressed in Escherichia coli by using diethylaminoethyl resins in batch mode is described. HP-NAP purified by this method is beneficial for the development of vaccines, drugs, or diagnostics for H. pylori-associated diseases.

Helicobacter pylori neutrophil-activating protein (HP-NAP) is a major virulence factor of Helicobacter pylori (H. pylori). It plays a critical role in H. ...

Detection of Enterohemorrhagic Escherichia Coli Colonization in Murine Host by Non-invasive In Vivo Bioluminescence System

Enterohemorrhagic E. coli (EHEC) O157:H7, which is a foodborne pathogen that causesdiarrhea, hemorrhagic colitis (HS), and hemolytic uremic syndrome (HUS), colonize to the intestinal tract of humans. To study the detailed mechanism of EHEC colonization in vivo, it is essential to have animal models to monitor and quantify EHEC colonization. We demonstrate here a mouse-EHEC colonization model by transforming the bioluminescent expressing plasmid to EHEC to monitor and quantify EHEC colonization in living hosts. Animals inoculated with bioluminescence-labeled EHEC show intense bioluminescent signals in mice by detection with a non-invasive in vivo imaging system. After 1 and 2 days post infection, bioluminescent signals could still be detected in infected animals, which suggests that EHEC colonize in hosts for at least 2 days. We also demonstrate that these bioluminescent EHEC locate to mouse intestine, specifically in the cecum and colon, from ex vivo images. This mouse-EHEC colonization model may serve as a tool to advance the current knowledge of the EHEC colonization mechanism.

Detection of Enterohemorrhagic Escherichia Coli Colonization in Murine Host by Non-invasive In Vivo Bioluminescence System

A detailed protocol of a mouse model for enterohemorrhagic E. coli (EHEC) colonization by using bioluminescence-labeled bacteria is presented. The detection of these bioluminescent bacteria by a non-invasive in vivo imaging system in live animals can advance our current understanding of EHEC colonization.

Enterohemorrhagic E. coli (EHEC) O157:H7, which is a foodborne pathogen that causesdiarrhea, hemorrhagic colitis (HS), and hemolytic uremic syndrome ...

Overexpression and Purification of Human Cis-prenyltransferase in Escherichia coli

Prenyltransferases (PT) are a group of enzymes that catalyze chain elongation of allylic diphosphate using isopentenyl diphosphate (IPP) via multiple condensation reactions. DHDDS (dehydrodolichyl diphosphate synthase) is a eukaryotic long-chain cis-PT (forming cis double bonds from the condensation reaction) that catalyzes chain elongation of farnesyl diphosphate (FPP, an allylic diphosphate) via multiple condensations with isopentenyl diphosphate (IPP). DHDDS is of biomedical importance, as a non-conservative mutation (K42E) in the enzyme results in retinitis pigmentosa, ultimately leading to blindness. Therefore, the present protocol was developed in order to acquire large quantities of purified DHDDS, suitable for mechanistic studies. Here, the usage of protein fusion, optimized culture conditions and codon-optimization were used to allow the overexpression and purification of functionally active human DHDDS in E. coli. The described protocol is simple, cost-effective and time sparing. The homology of cis-PT among different species suggests that this protocol may be applied for other eukaryotic cis-PT as well, such as those involved in natural rubber synthesis.

Overexpression and Purification of Human Cis-prenyltransferase in Escherichia coli

A simple protocol for overexpression and purification of codon-optimized, human cis-prenyltransferase, under non-denaturing conditions, from Escherichia coli, is described, along with an enzymatic activity assay. This protocol can be generalized for production of other cis- prenyltransferase proteins in quantity and quality suitable for mechanistic studies.

Prenyltransferases (PT) are a group of enzymes that catalyze chain elongation of allylic diphosphate using isopentenyl diphosphate (IPP) via multiple ...

A Facile Protocol to Generate Site-Specifically Acetylated Proteins in Escherichia Coli

Post-translational modifications that occur at specific positions of proteins have been shown to play important roles in a variety of cellular processes. Among them, reversible lysine acetylation is one of the most widely distributed in all domains of life. Although numerous mass spectrometry-based acetylome studies have been performed, further characterization of these putative acetylation targets has been limited. One possible reason is that it is difficult to generate purely acetylated proteins at desired positions by most classic biochemical approaches. To overcome this challenge, the genetic code expansion technique has been applied to use the pair of an engineered pyrrolysyl-tRNA synthetase variant, and its cognate tRNA from Methanosarcinaceae species, to direct the cotranslational incorporation of acetyllysine at the specific site in the protein of interest. After first application in the study of histone acetylation, this approach has facilitated acetylation studies on a variety of proteins. In this work, we demonstrated a facile protocol to produce site-specifically acetylated proteins by using the model bacterium Escherichia coli as the host. Malate dehydrogenase was used as a demonstration example in this work.

A Facile Protocol to Generate Site-Specifically Acetylated Proteins in Escherichia Coli

Genetic code expansion serves as a powerful tool to study a wide range of biological processes, including protein acetylation. Here we demonstrate a facile protocol to exploit this technique for generating homogeneously acetylated proteins at specific sites in Escherichia coli cells.

Post-translational modifications that occur at specific positions of proteins have been shown to play important roles in a variety of cellular processes. ...

Measuring Dengue Virus RNA in the Culture Supernatant of Infected Cells by Real-time Quantitative Polymerase Chain Reaction

At present, real-time polymerase chain reaction (PCR) technology is an indispensable tool for the detection and quantification of viral genomes in research laboratories, as well as for molecular diagnosis, because of its sensitivity, specificity, and convenience. However, in most cases, the quantitative PCR (qPCR) assay generally used to detect virus infection has relied on the purification of viral nucleic acid prior to the PCR step. In this study, the fluorescence-based reverse transcription qPCR (RT-qPCR) assay is developed through the combination of a processing buffer and a one-step RT-PCR reagent so that the whole process, from the harvest of the culture supernatant of virus-infected cells until real-time detection, can be performed without viral RNA purification. The established protocol enables the quantification of a wide range of RNA concentrations of dengue virus (DENV) within 90 min. In addition, the adaptability of the direct RT-qPCR assay to the evaluation of an antiviral agent is demonstrated by an in vitro experiment using a previously reported DENV inhibitor, mycophenolic acid (MPA). Moreover, other RNA viruses, including yellow fever virus (YFV), Chikungunya virus (CHIKV), and measles virus (MeV), can be quantified by direct RT-qPCR with the same protocol. Therefore, the direct RT-qPCR assay described in this report is useful for monitoring RNA virus replication in a simple and rapid manner, which will be further developed into a promising platform for a high-throughput screening study and clinical diagnosis.

Real-time quantitative polymerase chain reaction analysis combined with reverse transcription (RT-qPCR) has been widely used to measure the level of RNA virus infections. Here we present a direct RT-qPCR assay, which does not require an RNA purification step, developed for the quantification of several RNA viruses, including dengue virus.

At present, real-time polymerase chain reaction (PCR) technology is an indispensable tool for the detection and quantification of viral genomes in ...

In Vivo Imaging of Reactive Oxygen Species in a Murine Wound Model

The generation of reactive oxygen species (ROS) is a hallmark of inflammatory processes, but in excess, oxidative stress is widely implicated in various pathologies such as cancer, atherosclerosis and diabetes. We have previously shown that dysfunction of the Nuclear factor (erythroid-derived 2)-like 2 (Nrf2)/ Kelch-like erythroid cell-derived protein 1 (Keap1) signaling pathway leads to extreme ROS imbalance during cutaneous wound healing in diabetes. Since ROS levels are an important indicator of progression of wound healing, specific and accurate quantification techniques are valuable. Several in vitro assays to measure ROS in cells and tissues have been described; however, they only provide a single cumulative measurement per sample. More recently, the development of protein-based indicators and imaging modalities have allowed for unique spatiotemporal analyses. L-012 (C13H8ClN4NaO2) is a luminol derivative that can be used for both in vivo and in vitro chemiluminescent detection of ROS generated by NAPDH oxidase. L-012 emits a stronger signal than other fluorescent probes and has been shown to be both sensitive and reliable for detecting ROS. The time lapse applicability of L-012-facilitated imaging provides valuable information about inflammatory processes while reducing the need for sacrifice and overall reducing the number of study animals. Here, we describe a protocol utilizing L-012-facilitated in vivo imaging to quantify oxidative stress in a model of excisional wound healing using diabetic mice with locally dysfunctional Nrf2/Keap1.

In Vivo Imaging of Reactive Oxygen Species in a Murine Wound Model

We describe a non-invasive in vivo imaging protocol that is streamlined and cost-effective, utilizing L-012, a chemiluminescent luminol-analog, to visualize and quantify reactive oxygen species (ROS) generated in a mouse excisional wound model.

The generation of reactive oxygen species (ROS) is a hallmark of inflammatory processes, but in excess, oxidative stress is widely implicated in various ...