This work was supported by a pilot project grant from CREH center sponsored by NIEHS and by National Natural Science Foundation of China (No. 31572626).

All animal experiments were executed in compliance with all relevant guidelines, regulations and regulatory agencies. The protocol being demonstrated was performed under the guidance and approval of the Institutional Animal Care and Use Committee (IACUC) of the Texas A&#38;M Institute for Genomic Medicine.
1. Solution Preparation
<ol>
	<li>Dilute 10x Hank&#39;s balanced salt solution to 1x with double distilled H2O, and store at 4 &#176;C.</li>
	<li>Prepare the isolation solution ...

<ol>
	<li>Maruthur, N. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+growing+prevalence+of+type+2+diabetes:+increased+incidence+or+improved+survival?.">The growing prevalence of type 2 diabetes: increased incidence or improved survival?.</a> Current diabetes reports. 13 (6), 786-794 (2013).</li><li>Houstis, N., Rosen, E. D., Lander, E. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reactive+oxygen+species+have+a+causal+role+in+multiple+forms+of+insulin+resistance.">Reactive oxygen species have a causal role in multiple forms of insulin resistance.</a> Nature. 440 (7086), 944-948 (2006).</li><li>Ma, Z. A., Zhao, Z., Turk, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mitochondrial+dysfunction+and+beta-cell+failure+in+type+2+diabetes+mellitus.">Mitochondrial dysfunction and beta-cell failure in type 2 diabetes mellitus.</a> Exp Diabetes Res. , 703538 (2012).</li><li>Valavanidis, A., Vlahogianni, T., Dassenakis, M., Scoullos, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+biomarkers+of+oxidative+stress+in+aquatic+organisms+in+relation+to+toxic+environmental+pollutants.">Molecular biomarkers of oxidative stress in aquatic organisms in relation to toxic environmental pollutants.</a> Ecotoxicology and environmental safety. 64 (2), 178-189 (2006).</li><li>Robertson, R. P., Harmon, J., Tran, P. O., Tanaka, Y., Takahashi, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Glucose+toxicity+in+&#946;-cells:+type+2+diabetes,+good+radicals+gone+bad,+and+the+glutathione+connection.">Glucose toxicity in &#946;-cells: type 2 diabetes, good radicals gone bad, and the glutathione connection.</a> Diabetes. 52 (3), 581-587 (2003).</li><li>Kaneto, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Oxidative+stress+induces+p21+expression+in+pancreatic+islet+cells:+possible+implication+in+beta-cell+dysfunction.">Oxidative stress induces p21 expression in pancreatic islet cells: possible implication in beta-cell dysfunction.</a> Diabetologia. 42 (9), 1093-1097 (1999).</li><li>Maechler, P., Jornot, L., Wollheim, C. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hydrogen+peroxide+alters+mitochondrial+activation+and+insulin+secretion+in+pancreatic+beta+cells.">Hydrogen peroxide alters mitochondrial activation and insulin secretion in pancreatic beta cells.</a> Journal of Biological Chemistry. 274 (39), 27905-27913 (1999).</li><li>Gao, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+effects+of+palmitate+on+hepatic+insulin+resistance+are+mediated+by+NADPH+Oxidase+3-derived+reactive+oxygen+species+through+JNK+and+p38MAPK+pathways.">The effects of palmitate on hepatic insulin resistance are mediated by NADPH Oxidase 3-derived reactive oxygen species through JNK and p38MAPK pathways.</a> Journal of Biological Chemistry. 285 (39), 29965-29973 (2010).</li><li>Efendi&#263;, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pancreastatin+and+islet+hormone+release.">Pancreastatin and islet hormone release.</a> Proceedings of the National Academy of Sciences. 84 (20), 7257-7260 (1987).</li><li>Tian, Y., Ke, S., Denison, M. S., Rabson, A. B., Gallo, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ah+receptor+and+NF-&#954;B+interactions,+a+potential+mechanism+for+dioxin+toxicity.">Ah receptor and NF-&#954;B interactions, a potential mechanism for dioxin toxicity.</a> Journal of Biological Chemistry. 274 (1), 510-515 (1999).</li><li>Cui, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pregnane+X+receptor+regulates+the+AhR/Cyp1A1+pathway+and+protects+liver+cells+from+benzo-[&#945;]-pyrene-induced+DNA+damage.">Pregnane X receptor regulates the AhR/Cyp1A1 pathway and protects liver cells from benzo-[&#945;]-pyrene-induced DNA damage.</a> Toxicology Letters. 275, 67-76 (2017).</li><li>Li, L. A., Wang, P. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=PCB126+induces+differential+changes+in+androgen,+cortisol,+and+aldosterone+biosynthesis+in+human+adrenocortical+H295R+cells.">PCB126 induces differential changes in androgen, cortisol, and aldosterone biosynthesis in human adrenocortical H295R cells.</a> Toxicological Sciences. 85 (1), 530-540 (2005).</li><li>Asahi, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bisphenol+A+induces+endoplasmic+reticulum+stress-associated+apoptosis+in+mouse+non-parenchymal+hepatocytes.">Bisphenol A induces endoplasmic reticulum stress-associated apoptosis in mouse non-parenchymal hepatocytes.</a> Life sciences. 87 (13), 431-438 (2010).</li><li>Szot, G. L., Koudria, P., Bluestone, J. 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(7), e255 (2007).</li><li>Kirstetter, P., Lagneau, F., Lucas, O., Krupa, Y., Marty, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Role+of+endothelium+in+the+modulation+of+isoflurane-induced+vasodilatation+in+rat+thoracic+aorta.">Role of endothelium in the modulation of isoflurane-induced vasodilatation in rat thoracic aorta.</a> British journal of anaesthesia. 79 (1), 84-87 (1997).</li><li>Brown, E., Umino, Y., Loi, T., Solessio, E., Barlow, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Anesthesia+can+cause+sustained+hyperglycemia+in+C57/BL6J+mice.">Anesthesia can cause sustained hyperglycemia in C57/BL6J mice.</a> Visual neuroscience. 22 (5), 615-618 (2005).</li><li>Vaupel, D., McCoun, D., Cone, E. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Phencyclidine+analogs+and+precursors:+rotarod+and+lethal+dose+studies+in+the+mouse.">Phencyclidine analogs and precursors: rotarod and lethal dose studies in the mouse.</a> Journal of Pharmacology and Experimental Therapeutics. 230 (1), 20-27 (1984).</li><li>Neuman, J. C., Truchan, N. A., Joseph, J. W., Kimple, M. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+method+for+mouse+pancreatic+islet+isolation+and+intracellular+cAMP+determination.">A method for mouse pancreatic islet isolation and intracellular cAMP determination.</a> Journal of visualized experiments: JoVE. (88), (2014).</li></ol>

Accumulating evidence suggests that exposure to environmental chemicals plays an important role in the development of T2DM. Xenobiotics-induced ROS has been recognized as a potential etiological factor contributing to the development of T2DM. Humans are exposed to a wide range of xenobiotic chemicals and there is a great need for novel research techniques to effectively identify the pancreatic toxicants and to investigate the mechanism of toxicity specific to the pancreatic cells.
In this stud...

Increases in the prevalence of T2DM have become a global health crisis in recent years posing a serious threat to public health1. Many factors have been found to be causally linked to the development of T2DM, among which, recurring findings suggest that one common convergent point for these factors is the induction of oxidative stress which leads to the generation of excessive ROS2,3. 
A wide spectrum of environmental chemicals including PCBs, dioxins, and BaP has been found to induce oxidative stress, which may impair the function of pancreatic &#946; cells and lead to insulin resistance and T2DM4. Although the physiological level of ROS plays an important role in cellular functions, exposure to ROS that exceeds the capacity of the antioxidant system results in the damage to cells/tissues and leads to diseases5. Pancreatic &#946; cells express a low level of antioxidant enzyme, and thus are a sensitive target for the oxidative stress-mediated damage6,7. Chronic exposure to high levels of ROS has been shown to cause stress-induced pancreatic cell dysfunction5 as well as insulin resistance in the liver and adipose tissue8. 
The overall goal of this project is to develop a cell-based assay to screen chemicals for their diabetogenic potentials based on their induction of ROS in pancreatic cells. The pancreas lacks metabolic detoxification and is a sensitive target for xenobiotic-induced damage6,7. Therefore, by directly measuring the ROS generated in the pancreatic cells, this assay should provide a direct approximation of the chemical-induced injury in the pancreas. To develop this method, we isolated mouse pancreatic islets, cultured the isolated islet under cell culture condition with chemicals, and utilized the chemical-induced ROS generation as the readout. This procedure is simple and effective in identifying ROS-inducing chemicals in the isolated islet; it can be further developed for investigation of the mechanisms of toxicity that are specific to the pancreas in vitro.

<table border="0" cellpadding="0" cellspacing="0" width="858">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:289px;">10&#215;Hank&#8217;s balanced salt solution&#160;</td>
			<td style="width:283px;">GIBCO</td>
			<td style="width:119px;">14185-052</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Collagenase Type 4</td>
			<td>Worthington Biochemical Corporation</td>
			<td>CLS-4</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">polysucrose/sodium diatrizoate solution&#160;</td>
			<td>Sigma</td>
			<td>10771</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">2&#8217;,7&#8217;-dichlorofluorescein (DCFH-DA)</td>
			<td>Sigma</td>
			<td>D6883-50MG</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">fluorescence microplate reader&#160;</td>
			<td>Biotek</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">L-glutamine</td>
			<td>Sigma</td>
			<td>G8540-25G</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">streptomycin</td>
			<td>GIBCO</td>
			<td>15140148</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">FBS</td>
			<td>GIBCO</td>
			<td>26140079</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">RPMI 1640</td>
			<td>GIBCO</td>
			<td>11875-085</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">avertin</td>
			<td>Sigma</td>
			<td>T48402-25G</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Rat/Mouse Insulin ELISA Kit</td>
			<td>Millipore</td>
			<td>EZRMI-13K</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Centrifuge</td>
			<td>Sorval</td>
			<td>Sorval RT7</td>
			<td>for 96-well plate centrifuge</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Microplate reader</td>
			<td>Biotek</td>
			<td>Epoch 2</td>
			<td>for fluorescence reading</td>
		</tr>
	</tbody>
</table>

a murine pancreatic islet cell-based screening for diabetogenic environmental chemicals

Exposure to certain environmental chemicals in human and animals has been found to cause cellular damage of the pancreatic &#946; cells which will lead to the development of type 2 diabetes mellitus (T2DM). Although the mechanisms for the chemical-induced &#946; cell damage were unclear and likely to be complex, one recurring finding is that these chemicals induce oxidative stress leading to the generation of excessive reactive oxygen species (ROS) which induce damage to the &#946; cell. To identify potential diabetogenic environmental chemicals, we isolated pancreatic islet cells from C57BL/6 mice and cultured islet cells in 96-well cell culture plates; then, the islet cells were dosed with chemicals and the ROS generation was detected by 2',7'-dichlorofluorescein (DCFH-DA) fluorescent dye. Using this method, we found that bisphenol A (BPA), Benzo[a]pyrene (BaP), and polychlorinated biphenyls (PCBs), could induce high levels of ROS, suggesting that they may potentially induce damage in islet cells. This method should be useful for screening diabetogenic xenobiotics. In addition, the cultured islet cells may also be adapted for in vitro analysis of chemical-induced toxicity in pancreatic cells.

A micrograph of the healthy Isolated islet is shown in Figure 2, in which islets have a round or oval&#160;shape with relatively uniform size (although size uniformity can vary from strain to strain). We next investigated the pancreatic islet functions in an in vitro assay by isolating the islet and stimulating the insulin secretion in the culture islets. Figure 3 shows our typical analysis of the GSIS assay from C57BL/6...

Watch this Scientific Journal Video about A Murine Pancreatic Islet Cell-based Screening for Diabetogenic Environmental Chemicals at JoVE.com

A Murine Pancreatic Islet Cell-based Screening for Diabetogenic Environmental Chemicals

Here we present a protocol to isolate mouse pancreatic islet cells for screening the ROS inductions by the xenobiotics in order to identify the potential diabetogenic xenobiotic chemicals.

Exposure to certain environmental chemicals in human and animals has been found to cause cellular damage of the pancreatic &#946; cells which will lead to ...

Pancreas cells are sensitive target for ROS induced damages, which is associated with development of type-2 diabetes. A pancreas cell-based assays will help identify ROS inducing chemicals. The main advantage of this technique is to use isolated pancreas islet cells for simple and effective identification of the ROS inducing chemicals which may have the added diabetogenic potentials.This method can also be used for an in vitro analysis on the effects of chemicals on a physiological function of the pancreas such as a glucose-stimulated insulin secretion. Use a pair of curved forceps to clamp the duodenum close to the position of the sphincter of Oddi. With another pair of curved forceps, clamp the duodenum wall to block the bile pathway.Find the common bile duct and with a 30 gauge needle and a five milliliter syringe, slowly inject three milliliters of collagenase solution. Then, reposition the mouse with the head proximal and the feet distal. Ensure that the common bile duct and pancreas are inflated after the injection.Using the curved forceps and fine scissors, remove the distended pancreas to separate it from the descending colon, intestines, stomach and spleen. Then, place the organ in a 50 milliliter tube containing three milliliters of collagenase solution and keep it on ice. Place each 50 milliliter tube into a 37 degrees Celsius water bath and incubate them for 15 to 20 minutes.Vortex the tubes, then pool four to five digested organs in a large sterile sink strainer and use a scoop to thoroughly break apart the pancreas. With a 23 gauge needle and ten milliliter syringe containing wash solution, vigorously pipet the digested cells off the screen. Then, collect the wash solution in a 50 milliliter tube.Centrifuge the tubes at 97g and four degrees Celsius for one minute and decant the supernatant. Add 25 milliliters of wash solution to the tubes, and resuspend the tissue by gently vortexing. Then, spin down the tissue, and decant the supernatant.Repeat the wash three times, taking care not to dislodge the pelleted tissue. To purify the islets, add ten milliliters of purification solution to the washed tissue pellet and gently resuspend the contents. Gently overlay five milliliters of isolation solution onto the purification solution.Ensure that a sharp liquid interface is maintained between the two solutions. Centrifuge the tube at 560g at four degrees Celsius for 15 minutes, with slow acceleration and no break. Then, insert the pipet into the interface and collect the islet layer.Transfer the solution into new 50 milliliter tubes before washing the islet as just demonstrated. After removing the final wash, add ten milliliters of islet culture solution and gently and thoroughly mix to resuspend the islets. Then, using a multi-channel pipet transfer 100 microliters per well to the wells of a 96 well plate.Incubate the plate in a tissue culture incubator for 12 hours. Then, centrifuge the plate at 112g and remove the culture medium. Dilute the environmental chemicals in islet culture medium.Add 100 micro liters of each of the chemical diluents into separate wells of the plate. Resuspend the islets in each well and incubate the plate at 37 degrees Celsius for 12 hours. Following chemical treatment of the islets, use 1x PBS to wash the samples.Then, add five millimolar N-Acetyl Cysteine or NAC to the wells, as an antioxidant to quench ROS and incubate the plates at 37 degrees Celsius for 12 hours. Centrifuge the 96 well plate at 112g then, add five micromolar solution of fluorescent probe and incubate the plate at 37 degrees Celsius for 60 minutes. Use PBS to wash the treated islet cells three times and use a microplate reader at 480 nanometers to measure the fluorescence.Finally, perform the glucose-stimulated insulin secretion, or GSIS assay, on the ROS inducing xenobiotic treated and untreated islets, to measure the xenobiotic effects on the physiological function of the pancreatic islets. Shown here, are healthy pancreatic islets, isolated from C57 Black/6 mice, that are round or oval in shape and are relatively uniformed in size. In this GSIS assay, using isolated islets stimulated with glucose to secrete insulin, a dose-dependent response was observed.When isolated islets were treated with the xenobiotics listed here, several chemicals including Atrazine, BaP, BPA and PCB caused a significant induction of reactive oxygen species. However, the addition of NAC was able to quench the production of ROS by the chemical treatments. In addition, pancreatic islets pretreated with PCB126 and BPA showed a significant decrease in insulin secretion compared to the control group, confirming the important role of ROS in causing damage to the physiological function of pancreatic beta cells which lead to T2DM.Once mastered, the isolation of the pancreas islets can be done in two hours if it is performed properly. While attempting this procedure it's important to use gradient centrifugation to purify the islets away from the other pancreatic debris. Following this procedure, other methods like glucose-stimulated insulin secretion can be performed in order to answer additional questions like effects of the chemicals on the physiological functions of the pancreas islets.After its development, this technique paved the way for researchers in the field of toxicology to explore diabetogenic potentials of the environmental chemicals in isolated mouse pancreas islet cells. After watching this video, you should have a good understanding of how to isolate the mouse pancreas islets and use these islet cells for identification.

a-murine-pancreatic-islet-cell-based-screening-for-diabetogenic

Research

JoVE Journal

Immunology and Infection

7.2K vistas.  Hunan Agriculture University. Pancreas cells are sensitive target for ROS induced damages, which is associated with development of type-2 diabetes. A pancreas cell-based assays will help identify ROS inducing chemicals. The main advantage of this technique is to use isolated pancreas islet cells for simple and effective identification of the ROS inducing chemicals which may have the added diabetogenic potentials.This method can also be used for an in vitro analysis on the effects of chemicals on a physiological fun...

Video: A Murine Pancreatic Islet Cell-based Screening for Diabetogenic Environmental Chemicals

A New Screening Method for the Directed Evolution of Thermostable Bacteriolytic Enzymes

Directed evolution is defined as a method to harness natural selection in order to engineer proteins to acquire particular properties that are not associated with the protein in nature. Literature has provided numerous examples regarding the implementation of directed evolution to successfully alter molecular specificity and catalysis1. The primary advantage of utilizing directed evolution instead of more rational-based approaches for molecular engineering relates to the volume and diversity of variants that can be screened2. One possible application of directed evolution involves improving structural stability of bacteriolytic enzymes, such as endolysins. Bacteriophage encode and express endolysins to hydrolyze a critical covalent bond in the peptidoglycan (i.e. cell wall) of bacteria, resulting in host cell lysis and liberation of progeny virions. Notably, these enzymes possess the ability to extrinsically induce lysis to susceptible bacteria in the absence of phage and furthermore have been validated both in vitro and in vivo for their therapeutic potential3-5. The subject of our directed evolution study involves the PlyC endolysin, which is composed of PlyCA and PlyCB subunits6. When purified and added extrinsically, the PlyC holoenzyme lyses group A streptococci (GAS) as well as other streptococcal groups in a matter of seconds and furthermore has been validated in vivo against GAS7. Significantly, monitoring residual enzyme kinetics after elevated temperature incubation provides distinct evidence that PlyC loses lytic activity abruptly at 45 &deg;C, suggesting a short therapeutic shelf life, which may limit additional development of this enzyme. Further studies reveal the lack of thermal stability is only observed for the PlyCA subunit, whereas the PlyCB subunit is stable up to ~90 &deg;C (unpublished observation). In addition to PlyC, there are several examples in literature that describe the thermolabile nature of endolysins. For example, the Staphylococcus aureus endolysin LysK and Streptococcus pneumoniae endolysins Cpl-1 and Pal lose activity spontaneously at 42 &deg;C, 43.5 &deg;C and 50.2 &deg;C, respectively8-10. According to the Arrhenius equation, which relates the rate of a chemical reaction to the temperature present in the particular system, an increase in thermostability will correlate with an increase in shelf life expectancy11. Toward this end, directed evolution has been shown to be a useful tool for altering the thermal activity of various molecules in nature, but never has this particular technology been exploited successfully for the study of bacteriolytic enzymes. Likewise, successful accounts of progressing the structural stability of this particular class of antimicrobials altogether are nonexistent. In this video, we employ a novel methodology that uses an error-prone DNA polymerase followed by an optimized screening process using a 96 well microtiter plate format to identify mutations to the PlyCA subunit of the PlyC streptococcal endolysin that correlate to an increase in enzyme kinetic stability (Figure 1). Results after just one round of random mutagenesis suggest the methodology is generating PlyC variants that retain more than twice the residual activity when compared to wild-type (WT) PlyC after elevated temperature treatment.

A novel directed evolution method specific to the field of thermostability engineering was developed and consequently validated for bacteriolytic enzymes. After only one round of random mutagenesis, an evolved bacteriolytic enzyme, PlyC 29C3, displayed greater than twice the residual activity when compared to the wild-type protein after elevated temperature incubation.

Directed evolution is defined as a method to harness natural selection in order to engineer proteins to acquire particular properties that are not ...

Isolation, Identification, and Purification of Murine Thymic Epithelial Cells

The thymus is a vital organ for T lymphocyte development. Of thymic stromal cells, thymic epithelial cells (TECs) are particularly crucial at multiple stages of T cell development: T cell commitment, positive selection and negative selection. However, the function of TECs in the thymus remains incompletely understood. In the article, we provide a method to isolate TEC subsets from fresh mouse thymus using a combination of mechanical disruption and enzymatic digestion. The method allows thymic stromal cells and thymocytes to be efficiently released from cell-cell and cell-extracellular matrix connections and to form a single-cell suspension. Using the isolated cells, multiparameter flow cytometry can be applied to identification and characterization of TECs and dendritic cells. Because TECs are a rare cell population in the thymus, we also describe an effective way to enrich and purify TECs by depleting thymocytes, the most abundant cell type in the thymus. Following the enrichment, cell sorting time can be decreased so that loss of cell viability can be minimized during purification of TECs. Purified cells are suitable for various downstream analyses like Real Time-PCR, Western blot and gene expression profiling. The protocol will promote research of TEC function and as well as the development of in vitro T cell reconstitution.

Here we describe an efficient method for isolation, identification, and purification of mouse thymic epithelial cells (TECs). The protocol can be utilized for studies of thymus function for normal T cell development, thymus dysfunction, and T cell reconstitution.

The thymus is a vital organ for T lymphocyte development. Of thymic stromal cells, thymic epithelial cells (TECs) are particularly crucial at multiple ...

A Rapid Strategy for the Isolation of New Faustoviruses from Environmental Samples Using Vermamoeba vermiformis

The isolation of giant viruses is of great interest in this new era of virology, especially since these giant viruses are related to protists. Giant viruses may be potentially pathogenic for many species of protists. They belong to the recently described order of Megavirales. The new lineage Faustovirus that has been isolated from sewage samples is distantly related to the mammalian pathogen African swine fever virus. This virus is also specific to its amoebal host, Vermamoeba vermiformis, a protist common in health care water systems. It is crucial to continue isolating new Faustovirus genotypes in order to enlarge its genotype collection and study its pan-genome. We developed new strategies for the isolation of additional strains by improving the use of antibiotic and antifungal combinations in order to avoid bacterial and fungal contaminations of the amoeba co-culture and favoring the virus multiplication. We also implemented a new starvation medium to maintain V. vermiformis in optimal conditions for viruses co-culture. Finally, we used flow cytometry rather than microscopic observation, which is time-consuming, to detect the cytopathogenic effect. We obtained two isolates from sewage samples, proving the efficiency of this method and thus widening the collection of Faustoviruses, to better understand their environment, host specificity and genetic content.

A Rapid Strategy for the Isolation of New Faustoviruses from Environmental Samples Using Vermamoeba vermiformis

We describe here the latest advances in viral isolation for the characterization of new genotypes of Faustovirus, a new asfarvirus-related lineage of giant viruses. This protocol can be applied to the high throughput isolation of viruses, especially giant viruses infecting amoeba.

The isolation of giant viruses is of great interest in this new era of virology, especially since these giant viruses are related to protists. Giant ...

Developing a Salivary Antibody Multiplex Immunoassay to Measure Human Exposure to Environmental Pathogens

The etiology and impacts of human exposure to environmental pathogens are of major concern worldwide and, thus, the ability to assess exposure and infections using cost effective, high-throughput approaches would be indispensable. This manuscript describes the development and analysis of a bead-based multiplex immunoassay capable of measuring the presence of antibodies in human saliva to multiple pathogens simultaneously. Saliva is particularly attractive in this application because it is noninvasive, cheaper and easier to collect than serum. Antigens from environmental pathogens were coupled to carboxylated microspheres (beads) and used to measure antibodies in very small volumes of human saliva samples using a bead-based, solution-phase assay. Beads were coupled with antigens from Campylobacter jejuni, Helicobacter pylori, Toxoplasma gondii, noroviruses (G I.1 and G II.4) and hepatitis A virus. To ensure that the antigens were sufficiently coupled to the beads, coupling was confirmed using species-specific, animal-derived primary capture antibodies, followed by incubation with biotinylated anti-species secondary detection antibodies and streptavidin-R-phycoerythrin reporter (SAPE). As a control to measure non-specific binding, one bead set was treated identically to the others except it was not coupled to any antigen. The antigen-coupled and control beads were then incubated with prospectively-collected human saliva samples, measured on a high throughput analyzer based on the principles of flow cytometry, and the presence of antibodies to each antigen was measured in Median Fluorescence Intensity units (MFI). This multiplex immunoassay has a number of advantages, including more data with less sample; reduced costs and labor; and the ability to customize the assay to many targets of interest. Results indicate that the salivary multiplex immunoassay may be capable of identifying previous exposures and infections, which can be especially useful in surveillance studies involving large human populations.

In the current climate of scarce resources, new technologies are emerging that allow researchers to conduct studies cheaper, faster and with more precision. Here we describe the development of a bead-based salivary antibody multiplex immunoassay to measure human exposure to multiple environmental pathogens simultaneously.

The etiology and impacts of human exposure to environmental pathogens are of major concern worldwide and, thus, the ability to assess exposure and ...

A Murine Model of Group B Streptococcus Vaginal Colonization

Streptococcus agalactiae (group B Streptococcus, GBS), is a Gram-positive, asymptomatic colonizer of the human gastrointestinal tract and vaginal tract of 10 - 30% of adults. In immune-compromised individuals, including neonates, pregnant women, and the elderly, GBS may switch to an invasive pathogen causing sepsis, arthritis, pneumonia, and meningitis. Because GBS is a leading bacterial pathogen of neonates, current prophylaxis is comprised of late gestation screening for GBS vaginal colonization and subsequent peripartum antibiotic treatment of GBS-positive mothers. Heavy GBS vaginal burden is a risk factor for both neonatal disease and colonization. Unfortunately, little is known about the host and bacterial factors that promote or permit GBS vaginal colonization. This protocol describes a technique for establishing persistent GBS vaginal colonization using a single &#946;-estradiol pre-treatment and daily sampling to determine bacterial load. It further details methods to administer additional therapies or reagents of interest and to collect vaginal lavage fluid and reproductive tract tissues. This mouse model will further the understanding of the GBS-host interaction within the vaginal environment, which will lead to potential therapeutic targets to control maternal vaginal colonization during pregnancy and to prevent transmission to the vulnerable newborn. It will also be of interest to increase our understanding of general bacterial-host interactions in the female vaginal tract.

A Murine Model of Group B Streptococcus Vaginal Colonization

The purpose of this protocol is to imitate human group B Streptococcus (GBS) vaginal colonization in a murine model. This method may be used to investigate host immune responses and bacterial factors contributing to GBS vaginal persistence, as well as to test therapeutic strategies.

Streptococcus agalactiae (group B Streptococcus, GBS), is a Gram-positive, asymptomatic colonizer of the human gastrointestinal tract and vaginal tract of ...

A Fluorescence-based Lymphocyte Assay Suitable for High-throughput Screening of Small Molecules

High-throughput screening (HTS) is currently the mainstay for the identification of chemical entities capable of modulating biochemical reactions or cellular processes. With the advancement of biotechnologies and the high translational potential of small molecules, a number of innovative approaches in drug discovery have evolved, which explains the resurgent interest in the use of HTS. The oncology field is currently the most active research area for drug screening, with no major breakthrough made for the identification of new immunomodulatory compounds targeting transplantation-related complications or autoimmune ailments. Here, we present a novel in vitro murine fluorescent-based lymphocyte assay easily adapted for the identification of new immunomodulatory compounds. This assay uses T or B cells derived from a transgenic mouse, in which the Nur77 promoter drives GFP expression upon T- or B-cell receptor stimulation. As the GFP intensity reflects the activation/transcriptional activity of the target cell, our assay defines a novel tool to study the effect of given compound(s) on cellular/biological responses. For instance, a primary screening was performed using 4,398 compounds in the absence of a &#34;target hypothesis&#34;, which led to the identification of 160 potential hits displaying immunomodulatory activities. Thus, the use of this assay is suitable for drug discovery programs exploring large chemical libraries prior to further in vitro/in vivo validation studies.

We present in the current study a novel fluorescence-based assay using lymphocytes derived from a transgenic mouse. This assay is suitable for high-throughput screening (HTS) of small molecules endowed with the capacity of either inhibiting or promoting lymphocyte activation.

High-throughput screening (HTS) is currently the mainstay for the identification of chemical entities capable of modulating biochemical reactions or ...

A Method to Test the Efficacy of Handwashing for the Removal of Emerging Infectious Pathogens

Handwashing is widely recommended to prevent infectious disease transmission. However, little comparable evidence exists on the efficacy of handwashing methods in general. Additionally, little evidence exists comparing handwashing methods to determine which are most efficacious at removing infectious pathogens. Research is needed to provide evidence for the different approaches to handwashing that may be employed during infectious disease outbreaks. Here, a laboratory method to assess the efficacy of handwashing methods at removing microorganisms from hands and their persistence in rinse water is described. Volunteers' hands are first spiked with the test organism and then washed with each handwashing method of interest. Generally, surrogate microorganisms are used to protect human subjects from disease. The number of organisms remaining on volunteers' hands after washing is tested using a modified "glove juice" method: the hands are placed in gloves with an eluent and are scrubbed to suspend the microorganisms and make them available for analysis by membrane filtration (bacteria) or plaque assay (viruses/bacteriophages). Rinse water produced from the handwashing is directly collected for analysis. Handwashing efficacy is quantified by comparing the log reduction value between samples taken after handwashing to samples with no handwashing. Rinse water persistence is quantified by comparing rinse water samples from various handwashing methods to samples collected after handwashing with just water. While this method is limited by the need to use surrogate organisms to preserve the safety of human volunteers, it captures aspects of handwashing that are difficult to replicate in an in vitro study and fills research gaps on handwashing efficacy and the persistence of infectious organisms in rinse water.

Handwashing is widely recommended to prevent infectious disease transmission. However, there is little evidence on which handwashing methods are most efficacious at removing infectious disease pathogens. We developed a method to assess the efficacy of handwashing methods at removing microorganisms.

Handwashing is widely recommended to prevent infectious disease transmission. However, little comparable evidence exists on the efficacy of handwashing ...

Electrochemiluminescence Assays for Human Islet Autoantibodies

Pinpointing islet autoantibodies associated with type 1 diabetes (T1D) leads the way to project and deter this disease in the general population. A novel ECL assay is a nonradioactive fluid phase assay for islet autoantibodies with higher sensitivity and specificity than the current 'gold' standard radio-binding assay (RBA). ECL assays can more precisely define the onset of presymptomatic T1D by distinguishing the high-risk, high-affinity autoantibodies from the low-risk, low-affinity autoantibodies generated in RBAs, and conventional enzyme-linked immunosorbent assays (ELISA). The antigen protein used in this ECL assay is labeled with Sulfo-tag and Biotin, respectively. Each ECL autoantibody assay that uses a particular antigen protein needs an optimization step before it can be used for laboratory application. This step is especially vital in determining the requirements for serum acid treatments, concentrations, and ratios of the two different antigens labeled with Sulfo-tag and Biotin. To perform the assay, serum samples are mixed with Sulfo-tag-conjugated and biotinylated capture antigen protein in phosphate buffered solution (PBS), containing 5% Bovine Serum Albumin (BSA). Afterwards, the samples are incubated overnight at 4 &#176;C. The same day, a streptavidin-coated plate is prepared with blocker buffer and incubated overnight at 4 &#176;C. On the second day, wash the streptavidin plate and transfer the serum-antigen mixture onto the plate. Place the plate on the plate shaker, set it at low speed, and incubate at room temperature for 1 h. Subsequently, the plate is washed again, and reader buffer is added. The plate is then counted on the plate reader machine. The results are conveyed through an index, which is generated from internal standard positive and negative control serum samples.

Here, we presented the methods to detect human islet autoantibodies using electrochemiluminescence (ECL) assays. The protocol, used to predict type 1 diabetes, can be expanded upon to detect autoantibodies for other autoimmune diseases.

Pinpointing islet autoantibodies associated with type 1 diabetes (T1D) leads the way to project and deter this disease in the general population. A novel ...

A High-throughput Platform for the Screening of Salmonella spp./Shigella spp.

Fecal-oral transmission of acute gastroenteritis occurs from time to time, especially when people who handled food and water are infected by Salmonella spp./Shigella spp. The gold standard method for the detection of Salmonella spp./Shigella spp. is direct culture but this is labor-intensive and time-consuming. Here, we describe a high-throughput platform for Salmonella spp./Shigella spp. screening, using real-time polymerase chain reaction (PCR) combined with guided culture. There are two major stages: real-time PCR and the guided culture. For the first stage (real-time PCR), we explain each step of the method: sample collection, pre-enrichment, DNA extraction and real-time PCR. If the real-time PCR result is positive, then the second stage (guided culture) is performed: selective culture, biochemical identification and serological characterization. We also illustrate representative results generated from it. The protocol described here would be a valuable platform for the rapid, specific, sensitive and high-throughput screening of Salmonella spp./Shigella spp.

A High-throughput Platform for the Screening of Salmonella spp./Shigella spp.

Salmonella spp./Shigella spp. are common pathogens attributed to diarrhea. Here, we describe a high-throughput platform for the screening of Salmonella spp./Shigella spp. using real-time PCR combined with guided culture.

Fecal-oral transmission of acute gastroenteritis occurs from time to time, especially when people who handled food and water are infected by Salmonella ...

Arbovirus Infections As Screening Tools for the Identification of Viral Immunomodulators and Host Antiviral Factors

RNA interference- and genome editing-based screening platforms have been widely used to identify host cell factors that restrict virus replication. However, these screens are typically conducted in cells that are naturally permissive to the viral pathogen under study. Therefore, the robust replication of viruses in control conditions may limit the dynamic range of these screens. Furthermore, these screens may be unable to easily identify cellular defense pathways that restrict virus replication if the virus is well-adapted to the host and capable of countering antiviral defenses. In this article, we describe a new paradigm for exploring virus-host interactions through the use of screens that center on naturally abortive infections by arboviruses such as vesicular stomatitis virus (VSV). Despite the ability of VSV to replicate in a wide range of dipteran insect and mammalian hosts, VSV undergoes a post-entry, abortive infection in a variety of cell lines derived from lepidopteran insects, such as the gypsy moth (Lymantria dispar). However, these abortive VSV infections can be &#34;rescued&#34; when host cell antiviral defenses are compromised. We describe how VSV strains encoding convenient reporter genes and restrictive L. dispar cell lines can be paired to set-up screens to identify host factors involved in arbovirus restriction. Furthermore, we also show the utility of these screening tools in the identification of virally encoded factors that rescue VSV replication during coinfection or through ectopic expression, including those encoded by mammalian viruses. The natural restriction of VSV replication in L. dispar cells provides a high signal-to-noise ratio when screening for the conditions that promote VSV rescue, thus enabling the use of simplistic luminescence- and fluorescence-based assays to monitor the changes in VSV replication. These methodologies are valuable for understanding the interplay between host antiviral responses and viral immune evasion factors.

Here, we present the protocols to identify 1) virus-encoded immunomodulators that promote arbovirus replication and 2) eukaryotic host factors that restrict arbovirus replication. These fluorescence- and luminescence-based methods allow researchers to rapidly obtain quantitative readouts of arbovirus replication in simplistic assays with low signal-to-noise ratios.

RNA interference- and genome editing-based screening platforms have been widely used to identify host cell factors that restrict virus replication. ...