We would like to thank Yanmin Wan, Guobin Kang, and Pallabi Kundu for their assistance in generating BLT-humanized mice. We would like to acknowledge the UNMC Genomics Core Facility who receives partial support from the Nebraska Research Network In Functional Genomics NE-INBRE P20GM103427-14, The Molecular Biology of Neurosensory Systems CoBRE P30GM110768, The Fred &#38; Pamela Buffett Cancer Center - P30CA036727, The Center for Root and Rhizobiome Innovation (CRRI) 36-5150-2085-20, and the Nebraska Research Initiative. We would like to thank University of Nebraska - Lincoln Life Sciences Annex and their staff for their assistance. This study is supported in part by the National Institutes of Health (NIH) Grants R01AI124804, R21AI122377-01, P30 MH062261-16A1 Chronic HIV Infection and Aging in NeuroAIDS (CHAIN) Center, 1R01AI111862 to Q Li.&#160; The funders had no role in study design, data collection and analysis, preparation of the manuscript or decision for publication.

All methods described here were conducted in accordance with Institutional Animal Care and Research Committee (IACUC)-approved protocols at the University of Nebraska-Lincoln (UNL). The IACUC at UNL has approved two protocols related to generating and using hu-BLT mice, including double hu-mice. Additionally, the Scientific Research Oversight Committee (SROC) at UNL has also approved the use of human embryonic stem cells and fetal tissues, which are procured from the Advanced Bioscience Resources for humanized mice studies (SROC# 2016-1-002).
1. Mice housing and maintenance
<ol>
	<li>Purchase 6-8-week old NSG mice (NOD.Cg-PrkdcscidIl2rgtm1Wjl/SzJ).</li>
	<li>House the mice under SPF conditions with air exchange, prefilters, and HEPA filters (0.22 &#956;m) in a room with controlled temperature, humidity, and pressure.
	<ol>
		<li>House and maintain the mice in autoclaved individual microisolator cages in a rack system capable of managing air exchange with prefilters and HEPA filters (0.22 &#956;m).</li>
	</ol>
	</li>
	<li>Perform all procedures and manipulations of the mice in a Class II Type A2 biological safety fume hood that has been pre-treated with 70% ethanol. Before working in the animal room, shower and change into clean scrubs, tyvek suit, boot covers, hair cap, face mask, and gloves. Wear a surgery gown over the tyvek suit during surgical procedures. 
	NOTE: Ultraviolet (UV) light decontamination is also preferred prior to procedures.
	<ol>
		<li>Autoclave all instruments and reagents if possible and then disinfect prior to transferring to the fume hood.</li>
		<li>During procedures, remove the animals&#39;&#160;individually-ventilated cages from the rack and disinfect prior to transferring to the fume hood.</li>
	</ol>
	</li>
	<li>Feed the mice an irradiated diet and provide autoclaved water. Provide irradiated food ab libitum and supplement with additional irradiated food as needed. Change autoclaved water weekly or as needed. 
	NOTE: Autoclaved acidified water can also be used. The irradiated diet provided had a shelf-life of 6 months after manufacture.</li>
</ol>
2. Generation of humanized BLT mice
NOTE: Generation of hu-BLT mice has been described previously22,23,24.
<ol>
	<li>On the day of surgery, give the mice whole-body irradiation at a dose of 12 cGy/g of body weight.</li>
	<li>Prepare the mice for surgery.
	<ol>
		<li>Give each irradiated mouse a mixture of Ketamine at the working concentration of 100 mg/kg and Xylazine at the concentration of 12 mg/kg, which ranged from 130-170 &#956;L&#160;per mouse based on body weight by intraperitoneal (IP) injection for anesthesia. To prepare 3 ml of the Ketamine and Xylazine mixture, add 0.27 mL of Ketamine at the stock concentration of 100 mg/mL and 0.03 mL of Xylazine at the concentration of 100 mg/mL to 2.7 mL of sterile saline and mix well.&#160;Disinfect the skin with 70% isopropanol prior to injection.</li>
		<li>Give each irradiated mouse Buprenorphine 1 mg/kg of body weight (half-live 72 h, SR-LAB) by subcutaneous injection for long lasting pain management.</li>
		<li>Give each irradiated mouse 100 &#956;L (858 &#956;g) of Cefazolin by IP injection for preoperative antibiotic prophylaxis.</li>
		<li>Shave the hair of the mice using electric hair clippers around the left lateral and medial side of the mouse at the later surgical site used to expose the left kidney.</li>
		<li>Verify proper level of anesthesia by pedal reflex (firm toe pinch).</li>
		<li>Give isoflurane gas at 3-5% if additional anesthesia is needed at any point during surgery.</li>
		<li>Apply ophthalmic ointment to both eyes to prevent corneal desiccation. Apply ear tag if needed.</li>
	</ol>
	</li>
	<li>Perform surgery to implant the liver and thymus tissues into the left kidney capsule.
	<ol>
		<li>Disinfect the skin at the surgical site by applying iodine scrub starting from the center of the surgical site and moving towards the outside in a circular manner. Repeat this process with 70% isopropanol and a third time with iodine.</li>
		<li>Using forceps, first load one human fetal liver tissue fragment into a trocar. Then using forceps, load one human fetal thymus tissue fragment into the trocar. Then using forceps, load another human fetal liver tissue fragment into the trocar. Tissues should be cut to 1-1.6 mm3 to fit the inner dimensions of the trocar.</li>
		<li>Use forceps to lift the skin and use scissors to make a small cut longitudinally. Extend the cut to 1.5-2 cm in the left side of the mouse.</li>
		<li>Use forceps to lift the muscle layer and use scissors to make a small cut longitudinally. Extend the cut as needed to expose the kidney.</li>
		<li>Expose the kidney by gently grasping the fatty tissue surrounding the kidney. Do not directly touch the kidney parenchyma.</li>
		<li>Make a 1-2 mm incision at the posterior end of the kidney capsule using a scalpel.</li>
		<li>Slowly insert the preloaded trocar through the incision parallel to the long axis of the kidney and release the tissues between the kidney capsule and kidney.</li>
		<li>Carefully return the kidney and bowel to their normal position. Use absorbable 5/0 P-3 (P-13) sutures with 13mm 3/8 circle needle to close the muscle layer and surgical staples to close the skin.</li>
	</ol>
	</li>
	<li>After surgery, put the mouse into a clean autoclaved microisolator cage for recovery.
	<ol>
		<li>To minimize heat loss during post-surgical recovery, put the cage containing the mice on a heating pad that is connected to a water pump that warms and circulates the water.</li>
		<li>Monitor the mice until they have regained sufficient consciousness to maintain sternal recumbency.</li>
	</ol>
	</li>
	<li>Within 6 h of surgery completion, via the tail-vein, inject CD34+ hematopoietic stem cells isolated from human fetal liver tissue.
	<ol>
		<li>Warm up the mice with a heat-lamp. Disinfect the tail with 70% isopropanol and then inject 1.5 to 5 &#215; 105 stem cells/200 &#956;L into the tail vein.</li>
		<li>Stop any bleeding from the injection and return mice to the microisolator cage and the microisolator cage to the cage rack. 
		NOTE: Post-surgery mice are typically housed together, five per microisolator cage, during and after recovery. However, mice are only housed together if they all received surgery on the same day.</li>
	</ol>
	</li>
	<li>Check the mice daily. Carefully monitor the surgical staples and replace them as needed. Closely monitor the mice for any sign of infection or discomfort. Supply autoclaved food on the floor of the microisolator cage for a few days post surgery.</li>
	<li>Remove the surgical staples 7-10 days after surgery. Give isoflurane gas at 3-5% to anesthetize the mice. Carefully remove the staples and then apply antibiotic and pain-relieving ointment onto the site.</li>
	<li>Allow 9-12 weeks for reconstitution of human immune cells, then collect peripheral blood from the medial saphenous vein from each of the humanized mice.
	<ol>
		<li>Restrain conscious mice using an appropriately sized plastic cone restraint with an opening near the head of the mouse for breathing and an opening near the back of the mouse to isolate one leg. Put the mice into the restraint cone head first and then gently pull one leg through the leg opening.</li>
		<li>Spray the medial side of the isolated leg with 70% isopropanol, then spread antibiotic and pain-relieving ointment onto the same site. 
		NOTE: The ointment helps to reveal the location of the vein without the need for hair removal and also assists in blood droplet formation.</li>
		<li>Using a 25-gauge needle at a 90&#176; angle, puncture the vein and collect 50-100 &#956;L of blood using an EDTA coated blood collection tube. Stop the bleeding by applying pressure to the site with sterile gauze. Once the bleeding has stopped, return the mice to their cage. 
		NOTE: The maximum blood volumes collected are typically 50 &#956;L per week or 100 &#956;L every two weeks.</li>
		<li>Use the collected peripheral blood to test the level of human immune cell reconstitution using flow cytometry with antibodies for hCD45, mCD45, hCD3, hCD4, hCD8, hCD19.</li>
	</ol>
	</li>
</ol>
3. Antibiotic treatment
<ol>
	<li>Prior to antibiotic treatment, collect pre-treatment fecal samples. Move the mice to a new autoclaved microisolator cage.</li>
	<li>Prepare a fresh cocktail of broad-spectrum antibiotics daily.
	<ol>
		<li>Prepare 250 mL of water containing freshly prepared Metronidazole (1 g/L), Neomycin (1 g/L), Vancomycin (0.5 g/L), and Ampicillin (1 g/L) for each microisolator cage of mice. 
		NOTE: Use autoclaved or sterile water for the antibiotic supplemented drinking water.</li>
		<li>Add 9.2 g of grape sugar sweetened drink mix to 250 mL of antibiotic supplemented water. 
		NOTE: The use of grape sugar sweetened drink mix masks the bitter taste of the antibiotics and helps prevent dehydration in the mice.</li>
		<li>Change the antibiotic and grape sugar sweetened drink mix supplemented water and place the mice in a new autoclaved cage daily. 
		NOTE: Mice are coprophagic and changing the cages daily prevents the mice from re-inoculating themselves with gut bacteria.</li>
		<li>For maximal engraftment of the human-like gut microbiome, provide antibiotics for 14 days. During antibiotic treatment, monitor the mice for weight loss and dehydration. Weight loss is expected during the first 3-4 days and plateaus after that for the duration of the treatment. If dehydration occurs, give the mice saline or Ringers Solution via intraperitoneal injection. 
		NOTE: Other gut microbiome humanization protocols call for oral gavage of antibiotics. While effective at depleting the murine gut bacteria, we found that the less invasive method of adding antibiotics to the drinking water put less stress on our humanized mice and led to better outcomes.</li>
	</ol>
	</li>
</ol>
4. Donor samples and fecal transplant
<ol>
	<li>Prepare human donor fecal samples.
	<ol>
		<li>Use properly prepared sources of fecal microbiota transplant (FMT) material for fecal transplant into the humanized mice.</li>
		<li>Thaw FMT preparations and aliquot them under anaerobic conditions in an anaerobic chamber.</li>
		<li>If desired, at this step mix equal parts of the fecal samples together to create an unbiased &#34;human&#34;&#160;sample.</li>
		<li>Keep freezing and thawing of the FMT material to a minimum, if aliquoting or mixing of samples is not needed, only thaw immediately before the procedure.</li>
	</ol>
	</li>
	<li>Human fecal transplant
	<ol>
		<li>After completion of 14 days antibiotic treatment, change the drinking water to autoclaved water and move the mice into a new autoclaved cage. Stop daily cage changes and implement a once every 1-2 weeks cage changing schedule.</li>
		<li>Give two fecal transplants at 24 and 48 h post cessation of antibiotics.</li>
		<li>24 h after antibiotics treatment, thaw the needed amount of FMT material, give each mouse 200 &#956;L of FMT material via oral gavage. Repeat procedure again at 48 h post antibiotics.</li>
		<li>Spread any remaining or leftover thawed FMT material on the fur of the humanized mice or onto the cage bedding.</li>
	</ol>
	</li>
</ol>
5. Fresh fecal sample collection
<ol>
	<li>Autoclave individual paper bags prior to transferring to the fume hood.</li>
	<li>In the fume hood, put one mouse into each individual paper bag and allow the mouse to defecate.</li>
	<li>Using sterile forceps, collect the fecal sample into 1.5 mL plastic tubes and freeze at -80 &#176;C. Return the mice to microisolator cages. 
	NOTE: This method of collecting fecal samples allows for fresh fecal sample collection from individual mice without any stress inducing manipulations.</li>
</ol>

<ol>
	<li>Simpson-Abelson, M. R., 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=Long-term+engraftment+and+expansion+of+tumor-derived+memory+T+cells+following+the+implantation+of+non-disrupted+pieces+of+human+lung+tumor+into+NOD-scid+IL2R+gamma(null)+mice.">Long-term engraftment and expansion of tumor-derived memory T cells following the implantation of non-disrupted pieces of human lung tumor into NOD-scid IL2R gamma(null) mice.</a> Journal of Immunology. 180 (10), 7009-7018 (2008).</li><li>Bankert, R. B., 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=Humanized+Mouse+Model+of+Ovarian+Cancer+Recapitulates+Patient+Solid+Tumor+Progression,+Ascites+Formation,+and+Metastasis.">Humanized Mouse Model of Ovarian Cancer Recapitulates Patient Solid Tumor Progression, Ascites Formation, and Metastasis.</a> PLoS One. 6 (9), (2011).</li><li>Vudattu, N. K., 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=Humanized+Mice+as+a+Model+for+Aberrant+Responses+in+Human+T+Cell+Immunotherapy.">Humanized Mice as a Model for Aberrant Responses in Human T Cell Immunotherapy.</a> Journal of Immunology. 193 (2), 587-596 (2014).</li><li>Whitfield-Larry, F., 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=HLA-A2+Matched+Peripheral+Blood+Mononuclear+Cells+From+Type+1+Diabetic+Patients,+but+Not+Nondiabetic+Donors,+Transfer+Insulitis+to+NOD-scid/gamma+c(null)/HLA-A2+Transgenic+Mice+Concurrent+With+the+Expansion+of+Islet-Specific+CD8(+)+T+cells.">HLA-A2 Matched Peripheral Blood Mononuclear Cells From Type 1 Diabetic Patients, but Not Nondiabetic Donors, Transfer Insulitis to NOD-scid/gamma c(null)/HLA-A2 Transgenic Mice Concurrent With the Expansion of Islet-Specific CD8(+) T cells.</a> Diabetes. 60 (6), 1726-1733 (2011).</li><li>Yi, G. 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=A+DNA+Vaccine+Protects+Human+Immune+Cells+against+Zika+Virus+Infection+in+Humanized+Mice.">A DNA Vaccine Protects Human Immune Cells against Zika Virus Infection in Humanized Mice.</a> EBioMedicine. 25, 87-94 (2017).</li><li>Stary, G., 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=A+mucosal+vaccine+against+Chlamydia+trachomatis+generates+two+waves+of+protective+memory+T+cells.">A mucosal vaccine against Chlamydia trachomatis generates two waves of protective memory T cells.</a> Science. 348 (6241), (2015).</li><li>Sun, Z. F., 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=Intrarectal+transmission,+systemic+infection,+and+CD4(+)+T+cell+depletion+in+humanized+mice+infected+with+HIV-1.">Intrarectal transmission, systemic infection, and CD4(+) T cell depletion in humanized mice infected with HIV-1.</a> Journal of Experimental Medicine. 204 (4), 705-714 (2007).</li><li>Wang, L. X., 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=Humanized-BLT+mouse+model+of+Kaposi's+sarcoma-associated+herpesvirus+infection.">Humanized-BLT mouse model of Kaposi's sarcoma-associated herpesvirus infection.</a> Proceedings of the National Academy of Sciences of the United States of America. 111 (8), 3146-3151 (2014).</li><li>Ernst, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Humanized+mice+in+infectious+diseases.">Humanized mice in infectious diseases.</a> Comparative Immunology Microbiology and Infectious Diseases. 49, 29-38 (2016).</li><li>Turnbaugh, P. 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=An+obesity-associated+gut+microbiome+with+increased+capacity+for+energy+harvest.">An obesity-associated gut microbiome with increased capacity for energy harvest.</a> Nature. 444 (7122), 1027-1031 (2006).</li><li>Gopalakrishnan, V., 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=Gut+microbiome+modulates+response+to+anti-PD-1+immunotherapy+in+melanoma+patients.">Gut microbiome modulates response to anti-PD-1 immunotherapy in melanoma patients.</a> Science. 359 (6371), 97-103 (2018).</li><li>Routy, B., 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=Gut+microbiome+influences+efficacy+of+PD-1-based+immunotherapy+against+epithelial+tumors.">Gut microbiome influences efficacy of PD-1-based immunotherapy against epithelial tumors.</a> Science. 359 (6371), (2018).</li><li>Clemente, J. C., Manasson, J., Scher, J. U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+role+of+the+gut+microbiome+in+systemic+inflammatory+disease.">The role of the gut microbiome in systemic inflammatory disease.</a> Bmj-British Medical Journal. 360, (2018).</li><li>Kau, A. L., Ahern, P. P., Griffin, N. W., Goodman, A. L., Gordon, J. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+nutrition,+the+gut+microbiome+and+the+immune+system.">Human nutrition, the gut microbiome and the immune system.</a> Nature. 474 (7351), 327-336 (2011).</li><li>Hooper, L. V., Littman, D. R., Macpherson, A. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Interactions+Between+the+Microbiota+and+the+Immune+System.">Interactions Between the Microbiota and the Immune System.</a> Science. 336 (6086), 1268-1273 (2012).</li><li>Maynard, C. L., Elson, C. O., Hatton, R. D., Weaver, C. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reciprocal+interactions+of+the+intestinal+microbiota+and+immune+system.">Reciprocal interactions of the intestinal microbiota and immune system.</a> Nature. 489 (7415), 231-241 (2012).</li><li>Xiao, L., 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=A+catalog+of+the+mouse+gut+metagenome.">A catalog of the mouse gut metagenome.</a> Nature Biotechnology. 33 (10), 1103 (2015).</li><li>Nguyen, T. L. A., Vieira-Silva, S., Liston, A., Raes, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=How+informative+is+the+mouse+for+human+gut+microbiota+research.">How informative is the mouse for human gut microbiota research.</a> Disease Models &amp; Mechanisms. 8 (1), 1-16 (2015).</li><li>Turnbaugh, P. 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=The+Effect+of+Diet+on+the+Human+Gut+Microbiome:+A+Metagenomic+Analysis+in+Humanized+Gnotobiotic+Mice.">The Effect of Diet on the Human Gut Microbiome: A Metagenomic Analysis in Humanized Gnotobiotic Mice.</a> Science Translational Medicine. 1 (6), (2009).</li><li>Hazenberg, M. P., Bakker, M., Verschoor-Burggraaf, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+the+human+intestinal+flora+on+germ-free+mice.">Effects of the human intestinal flora on germ-free mice.</a> Journal of Applied Bacteriology. 50 (1), 95-106 (1981).</li><li>Hansen, C. H. F., 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=Patterns+of+Early+Gut+Colonization+Shape+Future+Immune+Responses+of+the+Host.">Patterns of Early Gut Colonization Shape Future Immune Responses of the Host.</a> PLoS One. 7 (3), (2012).</li><li>Lan, P., Tonomura, N., Shimizu, A., Wang, S. M., Yang, Y. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reconstitution+of+a+functional+human+immune+system+in+immunodeficient+mice+through+combined+human+fetal+thymus/liver+and+CD34(+)+cell+transplantation.">Reconstitution of a functional human immune system in immunodeficient mice through combined human fetal thymus/liver and CD34(+) cell transplantation.</a> Blood. 108 (2), 487-492 (2006).</li><li>Li, Q. 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=Early+Initiation+of+Antiretroviral+Therapy+Can+Functionally+Control+Productive+HIV-1+Infection+in+Humanized-BLT+Mice.">Early Initiation of Antiretroviral Therapy Can Functionally Control Productive HIV-1 Infection in Humanized-BLT Mice.</a> Jaids-Journal of Acquired Immune Deficiency Syndromes. 69 (5), 519-527 (2015).</li><li>Brainard, D. M., 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=Induction+of+Robust+Cellular+and+Humoral+Virus-Specific+Adaptive+Immune+Responses+in+Human+Immunodeficiency+Virus-Infected+Humanized+BLT+Mice.">Induction of Robust Cellular and Humoral Virus-Specific Adaptive Immune Responses in Human Immunodeficiency Virus-Infected Humanized BLT Mice.</a> Journal of Virology. 83 (14), 7305-7321 (2009).</li><li>Greenblatt, M. B., 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=Graft+versus+Host+Disease+in+the+Bone+Marrow,+Liver+and+Thymus+Humanized+Mouse+Model.">Graft versus Host Disease in the Bone Marrow, Liver and Thymus Humanized Mouse Model.</a> PLoS One. 7 (9), (2012).</li><li>Hintze, K. 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=Broad+scope+method+for+creating+humanized+animal+models+for+animal+health+and+disease+research+through+antibiotic+treatment+and+human+fecal+transfer.">Broad scope method for creating humanized animal models for animal health and disease research through antibiotic treatment and human fecal transfer.</a> Gut Microbes. 5 (2), 183-191 (2014).</li><li>Ericsson, A. C., Personett, A. R., Turner, G., Dorfmeyer, R. A., Franklin, C. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Variable+Colonization+after+Reciprocal+Fecal+Microbiota+Transfer+between+Mice+with+Low+and+High+Richness+Microbiota.">Variable Colonization after Reciprocal Fecal Microbiota Transfer between Mice with Low and High Richness Microbiota.</a> Frontiers in Microbiology. 8, 1-13 (2017).</li><li>Ellekilde, M., 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=Transfer+of+gut+microbiota+from+lean+and+obese+mice+to+antibiotic-treated+mice.">Transfer of gut microbiota from lean and obese mice to antibiotic-treated mice.</a> Scientific Reports. 4, (2014).</li><li>Staley, C., 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=Stable+engraftment+of+human+microbiota+into+mice+with+a+single+oral+gavage+following+antibiotic+conditioning.">Stable engraftment of human microbiota into mice with a single oral gavage following antibiotic conditioning.</a> Microbiome. 5, (2017).</li><li>Zhou, W., Chow, K. H., Fleming, E., Oh, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Selective+colonization+ability+of+human+fecal+microbes+in+different+mouse+gut+environments.">Selective colonization ability of human fecal microbes in different mouse gut environments.</a> ISME J. , (2018).</li><li>Lundberg, R., Toft, M. F., August, B., Hansen, A. K., Hansen, C. H. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Antibiotic-treated+versus+germ-free+rodents+for+microbiota+transplantation+studies.">Antibiotic-treated versus germ-free rodents for microbiota transplantation studies.</a> Gut Microbes. 7 (1), 68-74 (2016).</li><li>Wos-Oxley, M., 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=Comparative+evaluation+of+establishing+a+human+gut+microbial+community+within+rodent+models.">Comparative evaluation of establishing a human gut microbial community within rodent models.</a> Gut Microbes. 3 (3), 234-249 (2012).</li></ol>

The authors have nothing to disclose.

The protocol described here is for the creation of double hu-BLT mice that feature both a functional human immune system and a stable human-like gut microbiome. This protocol can be adapted to other humanized or non-humanized mice models without the need for GF animals and gnotobiotic facilities. While the methods described here are relatively simple, there are several critical details that are important for the successful creation of double hu-BLT mice. NSG mice are extremely immunodeficient and preventing infections is...

Humanized mice (hu-mice) have transformed the study of many aspects of human health and disease including hematopoiesis, immunity, cancer, autoimmune disease, and infectious disease1,2,3,4,5,6,7,8,9. These hu-mice have the distinct advantage over other mouse models in that they have a functional human immune system and can be infected with human specific pathogens. Nevertheless, the importance of the gut microbiome has been demonstrated by its role in many human diseases such as obesity, metabolic syndrome, inflammatory diseases, and cancer10,11,12,13. The mucosal immune system and gut microbiome are reciprocally regulated to maintain gut and systemic homeostasis. The immune system is shaped by antigens presented by the gut microbiome and reciprocally the immune system plays an important regulatory role in promoting commensal gut bacteria and eliminating pathogens14,15,16. However, the gut microbiome of hu-mice has not been well characterized and the murine gut microbiome differs substantially in composition and function from humans17. This is due to evolutionary, physiological, and anatomical differences between the murine and human gut as well as other important factors such as diet, which may influence the experimental results of hu-mice disease models18. Therefore, beyond classification of murine gut microbiome of hu-mice, an animal model featuring both a human immune system and human gut microbiome is needed to study the complex interactions of human disease in vivo.
The study of human diseases directly in human subjects is often impractical or unethical. Many animal models cannot be used to study human pathogens like human immunodeficiency virus type 1 (HIV-1). Non-human primate models are genetically outbred, very expensive, and are not susceptible to many human pathogens. Mice that have been derived as germ free (GF) and reconstituted with human-like gut microbiomes have been widely used to study human health and disease19,20. However, these animals do not have a human immune system and working with GF animals requires specialized facilities, procedures, and expertise. Therefore, there is a need for improved pre-clinical models to study the complex relationship of the gut microbiome and the human immune system. Many strains of mice, such as NOD.Cg-PrkdcscidIl2rgtm1Wjl/SzJ (NSG), are not commercially available as GF. GF animals also may suffer from long-lasting immune deficiencies that are not completely reversed by the engraftment of microbes21. Therefore, we created a double hu-mice featuring both a functional human immune system and stable human-like gut microbiome under specific pathogen free (SPF) conditions. To generate double hu-mice, surgery was performed on NSG mice to create bone-marrow, liver, thymus humanized mice (hu-BLT). The hu-BLT mice were then treated with broad spectrum antibiotics and then given fecal transplants with a healthy human donor sample. We characterized the bacterial gut microbiome of 173 fecal samples from 45 double hu-BLT mice and 4 human fecal donor samples. Double hu-BLT mice have unique 16S rRNA gene profiles based on the individual human donor sample that is transplanted. Importantly, the transplanted human-like microbiome was stable in the mice for the duration of the study up to 14.5 weeks post-transplant. In addition, the predicted metagenomes showed that double hu-BLT mice have different predicted functional capacity than hu-mice that is more similar to the human donor samples.

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			<td height="21" style="height:21px;width:337px;">NSG mice (NOD.Cg-PrkdcscidIl2rgtm1Wjl/SzJ)</td>
			<td style="width:225px;">The Jackson Laboratory</td>
			<td style="width:103px;">005557</td>
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a double humanized blt-mice model featuring a stable human-like gut microbiome and human immune system

Humanized mice (hu-mice) that feature a functional human immune system have fundamentally changed the study of human pathogens and disease. They can be used to model diseases that are otherwise difficult or impossible to study in humans or other animal models. The gut microbiome can have a profound impact on human health and disease. However, the murine gut microbiome is very different than the one found in humans. &#160;There is a need for improved pre-clinical hu-mice models that have an engrafted human gut microbiome. Therefore, we created double hu-mice that feature both a human immune system and stable human-like gut microbiome. NOD.Cg-PrkdcscidIl2rgtm1Wjl/SzJ (NSG) mice are one of the best animals for humanization due to their high level of immunodeficiency. However, germ-free NSG mice, and various other important germ-free mice models are not currently commercially available. Further, many research settings do not have access to gnotobiotic facilities, and working under gnotobiotic conditions can often be expensive and time consuming. Importantly, germ-free mice have several immune deficiencies that exist even after the engraftment of microbes. Therefore, we developed a protocol that does not require germ-free animals or gnotobiotic facilities. To generate double hu-mice, NSG mice were treated with radiation prior to surgery to create bone-marrow, liver, thymus-humanized (hu-BLT) mice. The mice were then treated with broad spectrum antibiotics to deplete the pre-existing murine gut microbiome. After antibiotic treatment, the mice were given fecal transplants with healthy human donor samples via oral gavage. Double hu-BLT mice had unique 16S rRNA gene profiles based on the individual human donor sample that was transplanted. Importantly, the transplanted human-like microbiome was stable in the double hu-BLT mice for the duration of the study up to 14.5 weeks post-transplant.

Figure 1 shows an outline of the methods used to create double hu-BLT mice and briefly describes the process of adding a functional human immune system and stable human-like gut microbiome to the NSG mice. Figure 2 shows an example of flow cytometry analysis of peripheral blood from a humanized BLT-mouse 10 weeks post-surgery. Figure 3 shows the relative abundance of the human fecal donor samples used to transfer a gut microbiome to...

Watch this Scientific Journal Video about A Double Humanized BLT-mice Model Featuring a Stable Human-Like Gut Microbiome and Human Immune System at JoVE.com

A Double Humanized BLT-mice Model Featuring a Stable Human-Like Gut Microbiome and Human Immune System

We describe a novel method for generating double humanized BLT-mice that feature a functional human immune system and a stable engrafted human-like gut microbiome. This protocol can be followed without the need for germ-free mice or gnotobiotic facilities.

Humanized mice (hu-mice) that feature a functional human immune system have fundamentally changed the study of human pathogens and disease. They can be ...

a-double-humanized-blt-mice-model-featuring-stable-human-like-gut

Research

JoVE Journal

Biochemistry

8.6K Views.  Nebraska Center for Virology. We describe a novel method for generating double humanized BLT-mice that feature a functional human immune system and a stable engrafted human-like gut microbiome. This protocol can be followed without the need for germ-free mice or gnotobiotic facilities.

Video: A Double Humanized BLT-mice Model Featuring a Stable Human-Like Gut Microbiome and Human Immune System

Thermostabilization, Expression, Purification, and Crystallization of the Human Serotonin Transporter Bound to S-citalopram

The serotonin transporter is a sodium and chloride-coupled transporter that &#34;pumps&#34; extracellular serotonin into cells. S-citalopram is a drug used to treat depression and anxiety by binding to the serotonin transporter with high-affinity, blocking serotonin reuptake. Here we report an efficient procedure and a set of tools to stabilize, express, purify, and crystallize serotonin transporter-antibody complexes bound to S-citalopram and other antidepressants. Mutations which stabilize the serotonin transporter were identified using an S-citalopram binding assay. Serotonin transporter expressed in baculovirus-transduced HEK293S GnTI- cells, was reconstituted into proteoliposomes and used to raise high-affinity antibodies. We have developed a strategy to discover antibodies that are useful for structural studies. A straightforward approach for the expression of antibody fragments in Sf9 cells has also been established. Transporter-antibody complexes purified using this procedure are well-behaved and readily crystallize, producing complexes with S-citalopram that diffract X-rays to 3-4 &#197; resolution. The strategies developed here can be utilized to determine the structure of other challenging membrane proteins.

Thermostabilization, Expression, Purification, and Crystallization of the Human Serotonin Transporter Bound to S-citalopram

This manuscript describes how to screen for thermostabilizing mutations, purify the human serotonin transporter, generate high affinity antibodies, and crystallize the serotonin transporter-antibody complex bound to the antidepressant drug S-citalopram. This protocol can be adapted to the study of other challenging membrane transporters, receptors, and channels.

The serotonin transporter is a sodium and chloride-coupled transporter that &#34;pumps&#34; extracellular serotonin into cells. S-citalopram is a drug ...

A Method for Measuring Metabolism in Sorted Subpopulations of Complex Cell Communities Using Stable Isotope Tracing

Mammalian cell types exhibit specialized metabolism, and there is ample evidence that various co-existing cell types engage in metabolic cooperation. Moreover, even cultures of a single cell type may contain cells in distinct metabolic states, such as resting or cycling cells. Methods for measuring metabolic activities of such subpopulations are valuable tools for understanding cellular metabolism. Complex cell populations are most commonly separated using a cell sorter, and subpopulations isolated by this method can be analyzed by metabolomics methods. However, a problem with this approach is that the cell sorting procedure subjects cells to stresses that may distort their metabolism.
To overcome these issues, we reasoned that the mass isotopomer distributions (MIDs) of metabolites from cells cultured with stable isotope-labeled nutrients are likely to be more stable than absolute metabolite concentrations, because MIDs are formed over longer time scales and should be less affected by short-term exposure to cell sorting conditions. Here, we describe a method based on this principle, combining cell sorting with liquid chromatography-high resolution mass spectrometry (LC-HRMS). The procedure involves analyzing three types of samples: (1) metabolite extracts obtained directly from the complex population; (2) extracts of "mock sorted" cells passed through the cell sorter instrument without gating any specific population; and (3) extracts of the actual sorted populations. The mock sorted cells are compared against direct extraction to verify that MIDs are indeed not altered by the cell sorting procedure itself, prior to analyzing the actual sorted populations. We show example results from HeLa cells sorted according to cell cycle phase, revealing changes in nucleotide metabolism.

This article describes a method for studying cellular metabolism in complex communities of multiple cell types, using a combination of stable isotope tracing, cell sorting to isolate specific cell types, and mass spectrometry.

Mammalian cell types exhibit specialized metabolism, and there is ample evidence that various co-existing cell types engage in metabolic cooperation. ...

A Modified Yeast-one Hybrid System for Heteromeric Protein Complex-DNA Interaction Studies

Over the years, the yeast one-hybrid assay has proven to be an important technique for the identification and validation of physical interactions between proteins such as transcription factors (TFs) and their DNA target. The method presented here utilizes the underlying concept of the Y1H but is modified further to study and validate protein complexes binding to their target DNA. Hence, it is referred to as the modified yeast one-hybrid (Y1.5H) assay. This assay is cost effective and can be easily performed in a regular laboratory setting. Albeit using a heterologous system, the described method could be a valuable tool to test and validate the heteromeric protein complex binding to their DNA target(s) for functional genomics in any system of study, especially plant genomics.

The modified yeast one-hybrid assay described here is an extension of the classical yeast one-hybrid (Y1H) assay to study and validate the heteromeric protein complex-DNA interaction in a heterologous system for any functional genomics study.

Over the years, the yeast one-hybrid assay has proven to be an important technique for the identification and validation of physical interactions between ...

Assessing the Viability of a Synthetic Bacterial Consortium on the In Vitro Gut Host-microbe Interface

The interplay between host and microbiota has been long recognized and extensively described. The mouth is similar to other sections of the gastrointestinal tract, as resident microbiota occurs and prevents colonisation by exogenous bacteria. Indeed, more than 600 species of bacteria are found in the oral cavity, and a single individual may carry around 100 different at any time. Oral bacteria possess the ability to adhere to the various niches in the oral ecosystem, thus becoming integrated within the resident microbial communities, and favouring growth and survival. However, the flow of bacteria into the gut during swallowing has been proposed to disturb the balance of the gut microbiota. In fact, oral administration of P. gingivalis shifted bacterial composition in the ileal microflora. We used a synthetic community as a simplified representation of the natural oral ecosystem, to elucidate the survival and viability of oral bacteria subjected to simulated gastrointestinal transit conditions. Fourteen species were selected, subjected to in vitro salivary, gastric, and intestinal digestion processes, and presented to a multicompartment cell model containing Caco-2 and HT29-MTX cells to simulate the gut mucosal epithelium. This model served to unravel the impact of swallowed bacteria on cells involved in the enterohepatic circulation. Using synthetic communities allows for controllability and reproducibility. Thus, this methodology can be adapted to assess pathogen viability and subsequent inflammation-associated changes, colonization capacity of probiotic mixtures, and ultimately, potential bacterial impact on the presystemic circulation.

Assessing the Viability of a Synthetic Bacterial Consortium on the In Vitro Gut Host-microbe Interface

Gut host-microbe interactions were assessed using a novel approach combining a synthetic oral community, in vitro gastrointestinal digestion, and a model of the small intestine epithelium. We present a method that can be adapted to evaluate cell invasion of pathogens and multi-species biofilms, or even to test probiotic formulations&#39; survivability.

The interplay between host and microbiota has been long recognized and extensively described. The mouth is similar to other sections of the ...

Sampling and Pretreatment of Tooth Enamel Carbonate for Stable Carbon and Oxygen Isotope Analysis

Stable carbon and oxygen isotope analysis of human and animal tooth enamel carbonate has been applied in paleodietary, paleoecological, and paleoenvironmental research from recent historical periods back to over 10 million years ago. Bulk approaches provide a representative sample for the period of enamel mineralization, while sequential samples within a tooth can track dietary and environmental changes during this period. While these methodologies have been widely applied and described in archaeology, ecology, and paleontology, there have been no explicit guidelines to aid in the selection of necessary lab equipment and to thoroughly describe detailed laboratory sampling and protocols. In this article, we document textually and visually, the entire process from sampling through pretreatment and diagenetic screening to make the methodology more widely available to researchers considering its application in a variety of laboratory settings.

Stable carbon and oxygen isotope analysis of human and animal tooth enamel carbonate has been used as a proxy for individual diet and environmental reconstruction. Here, we provide a detailed description and visual documentation of bulk and sequential tooth enamel sampling as well as pretreatment of archaeological and paleontological samples.

Stable carbon and oxygen isotope analysis of human and animal tooth enamel carbonate has been applied in paleodietary, paleoecological, and ...

Calibration-free In Vitro Quantification of Protein Homo-oligomerization Using Commercial Instrumentation and Free, Open Source Brightness Analysis Software

Number and brightness is a calibration-free fluorescence fluctuation spectroscopy (FFS) technique for detecting protein homo-oligomerization. It can be employed using a conventional confocal microscope equipped with digital detectors. A protocol for the use of the technique in vitro is shown by means of a use case where number and brightness can be seen to accurately quantify the oligomeric state of mVenus-labelled FKBP12F36V before and after the addition of the dimerizing drug AP20187. The importance of using the correct microscope acquisition parameters and the correct data preprocessing and analysis methods are discussed. In particular, the importance of the choice of photobleaching correction is stressed. This inexpensive method can be employed to study protein-protein interactions in many biological contexts.

Calibration-free In Vitro Quantification of Protein Homo-oligomerization Using Commercial Instrumentation and Free, Open Source Brightness Analysis Software

This protocol describes a calibration-free approach for quantifying protein homo-oligomerization in vitro based on fluorescence fluctuation spectroscopy using commercial light scanning microscopy. The correct acquisition settings and analysis methods are shown.

Number and brightness is a calibration-free fluorescence fluctuation spectroscopy (FFS) technique for detecting protein homo-oligomerization. It can be ...

Untargeted Liquid Chromatography-Mass Spectrometry-Based Metabolomics Analysis of Wheat Grain

Understanding the interactions between genes, the environment and management in agricultural practice could allow more accurate prediction and management of product yield and quality. Metabolomics data provides a read-out of these interactions at a given moment in time and is informative of an organism&#39;s biochemical status. Further, individual metabolites or panels of metabolites can be used as precise biomarkers for yield and quality prediction and management. The plant metabolome is predicted to contain thousands of small molecules with varied physicochemical properties that provide an opportunity for a biochemical insight into physiological traits and biomarker discovery. To exploit this, a key aim for metabolomics researchers is to capture as much of the physicochemical diversity as possible within a single analysis. Here we present a liquid chromatography-mass spectrometry-based untargeted metabolomics method for the analysis of field-grown wheat grain. The method uses the liquid chromatograph quaternary solvent manager to introduce a third mobile phase and combines a traditional reversed-phase gradient with a lipid-amenable gradient. Grain preparation, metabolite extraction, instrumental analysis and data processing workflows are described in detail. Good mass accuracy and signal reproducibility were observed, and the method yielded approximately 500 biologically relevant features per ionization mode. Further, significantly different metabolite and lipid feature signals between wheat varieties were determined.

A method for the untargeted analysis of wheat grain metabolites and lipids is presented. The protocol includes an acetonitrile metabolite extraction method and reversed phase liquid chromatography-mass spectrometry methodology, with acquisition in positive and negative electrospray ionization modes.

Understanding the interactions between genes, the environment and management in agricultural practice could allow more accurate prediction and management ...

Analysis and Specification of Starch Granule Size Distributions

Starch from all plant sources are made up of granules in a range of sizes and shapes having different occurrence frequencies, i.e., exhibiting a size and a shape distribution. Starch granule size data determined using several types of particle sizing techniques are often problematic due to poor reproducibility or lack of statistical significance resulting from some insurmountable systematic errors, including sensitivity to granule shapes and limits of granule-sample sizes. We outlined a procedure for reproducible and statistically valid determinations of starch granule size distributions using the electrical sensing zone technique, and for specifying the determined granule lognormal size distributions using an adopted two-parameter multiplicative form with improved accuracy and comparability. It is applicable to all granule sizing analyses of gram-scale starch samples, and, therefore, could facilitate studies on how starch granule sizes are molded by the starch biosynthesis apparatus and mechanisms; and how they impact properties and functionality of starches for food and industrial uses. Representative results are presented from replicate analyses of granule size distributions of sweetpotato starch samples using the outlined procedure. We further discussed several key technical aspects of the procedure, especially, the multiplicative specification of granule lognormal size distributions and some technical means for overcoming frequent aperture blockage by granule aggregates.

Presented here is a procedure for reproducible and statistically valid determinations of starch granule size distributions, and for specifying the determined granule lognormal size distributions using a two-parameter multiplicative form. It is applicable to all granule sizing analyses of gram-scale starch samples for plant and food science research. &#160;

Starch from all plant sources are made up of granules in a range of sizes and shapes having different occurrence frequencies, i.e., exhibiting a size and ...

An Economical and Versatile High-Throughput Protein Purification System Using a Multi-Column Plate Adapter

Protein purification is imperative to the study of protein structure and function and is usually used in combination with biophysical techniques. It is also a key component in the development of new therapeutics. The evolving era of functional proteomics is fueling the demand for high-throughput protein purification and improved techniques to facilitate this. It was hypothesized that a multi column plate adaptor (MCPA) can interface multiple chromatography columns of different resins with multi-well plates for parallel purification. This method offers an economical and versatile method of protein purification that can be used under gravity or vacuum, rivaling the speed of an automated system. The MCPA can be used to recover milligram yields of protein by an affordable and time efficient method for subsequent characterization and analysis. The MCPA has been used for high-throughput affinity purification of SH3 domains. Ion exchange has also been demonstrated via the MCPA to purify protein post Ni-NTA affinity chromatography, indicating how this system can be adapted to other purification types. Due to its setup with multiple columns, individual customization of parameters can be made in the same purification, unachievable by the current plate-based methods.

A multi-column plate adapter allows chromatography columns to be interfaced with multi-well collection plates for parallel affinity or ion exchange purification providing an economical high throughput protein purification method. It can be used under gravity or vacuum yielding milligram quantities of protein via affordable instrumentation.

Protein purification is imperative to the study of protein structure and function and is usually used in combination with biophysical techniques. It is ...

Utilizing Time-Resolved Protein-Induced Fluorescence Enhancement to Identify Stable Local Conformations One &#945;-Synuclein Monomer at a Time

Using spectroscopic rulers to track multiple conformations of single biomolecules and their dynamics have revolutionized the understanding of structural dynamics and its contributions to biology. While the FRET-based ruler reports on inter-dye distances in the 3-10 nm range, other spectroscopic techniques, such as protein-induced fluorescence enhancement (PIFE), report on the proximity between a dye and a protein surface in the shorter 0-3 nm range. Regardless of the method of choice, its use in measuring freely-diffusing biomolecules one at a time retrieves histograms of the experimental parameter yielding separate centrally-distributed sub-populations of biomolecules, where each sub-population represents either a single conformation that stayed unchanged within milliseconds, or multiple conformations that interconvert much faster than milliseconds, and hence an averaged-out sub-population. In single-molecule FRET, where the reported parameter in histograms is the inter-dye FRET efficiency, an intrinsically disordered protein, such as the &#945;-Synuclein monomer in buffer, was previously reported as exhibiting a single averaged-out sub-population of multiple conformations interconverting rapidly. While these past findings depend on the 3-10 nm range of the FRET-based ruler, we sought to put this protein to the test using single-molecule PIFE, where we track the fluorescence lifetime of site-specific sCy3-labeled &#945;-Synuclein proteins one at a time. Interestingly, using this shorter range spectroscopic proximity sensor, sCy3-labeled &#945;-Synuclein exhibits several lifetime sub-populations with distinctly different mean lifetimes that interconvert in 10-100 ms. These results show that while &#945;-Synuclein might be disordered globally, it nonetheless attains stable local structures. In summary, in this work we highlight the advantage of using different spectroscopic proximity sensors that track local or global structural changes one biomolecule at a time.

Time-resolved single-molecule protein-induced fluorescence enhancement is a useful fluorescence spectroscopic proximity sensor sensitive to local structural changes in proteins. Here we show it can be used to uncover stable local conformations in &#945;-Synuclein, which is otherwise known as globularly unstructured and unstable when measured using the longer range FRET ruler.

Using spectroscopic rulers to track multiple conformations of single biomolecules and their dynamics have revolutionized the understanding of structural ...