Name: a double humanized blt-mice model featuring a stable human-like gut microbiome and human immune system
Uploaded: 2019-08-30T13:30:00
Description: 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

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 studi...

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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;">Animal Feeding Needles 18G</td>
			<td style="width:225px;">Cadence Science</td>
			<td style="width:103px;">9928B</td>
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			<td height="42" style="height:42px;width:337px;">Clidox-s Activator</td>
			<td style="width:225px;">Pharmacal Research Laboratories</td>
			<td style="width:103px;">95120F</td>
			<td></td>
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		<tr height="42">
			<td height="42" style="height:42px;width:337px;">Clidox-s Base</td>
			<td style="width:225px;">Pharmacal Research Laboratories</td>
			<td style="width:103px;">96125F</td>
			<td></td>
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			<td height="21" style="height:21px;width:337px;">DGM 108 cage rack</td>
			<td style="width:225px;">Techniplast</td>
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			<td height="42" style="height:42px;width:337px;">Flat Brown Grocery Bag 3-5/8&#34;D x 6&#34;W x 11-1/16&#34;L&#160;</td>
			<td style="width:225px;">Grainger</td>
			<td style="width:103px;">12R063</td>
			<td></td>
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			<td height="21" style="height:21px;">FMT Upper Delivery Microbiota Preparations&#160;</td>
			<td style="width:225px;">OpenBiome</td>
			<td style="width:103px;">FMP30</td>
			<td></td>
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			<td height="21" style="height:21px;width:337px;">Grape Kool-Aid</td>
			<td style="width:225px;">Kraft Foods Inc.</td>
			<td style="width:103px;"></td>
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			<td height="21" style="height:21px;width:337px;">hCD19-PE/Cy5</td>
			<td style="width:225px;">Biolegend</td>
			<td style="width:103px;">302209</td>
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			<td height="21" style="height:21px;width:337px;">hCD3-PE</td>
			<td style="width:225px;">Biolegend</td>
			<td style="width:103px;">300408</td>
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			<td height="21" style="height:21px;width:337px;">hCD4-Alexa 700</td>
			<td style="width:225px;">Biolegend</td>
			<td style="width:103px;">300526</td>
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			<td height="21" style="height:21px;width:337px;">hCD45-FITC</td>
			<td style="width:225px;">Biolegend</td>
			<td style="width:103px;">304006</td>
			<td></td>
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			<td height="21" style="height:21px;width:337px;">hCD8-APC/Cy7</td>
			<td style="width:225px;">Biolegend</td>
			<td style="width:103px;">301016</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:337px;">Lactate Buffered Ringer&#39;s Solution</td>
			<td style="width:225px;">Boston BioProducts Inc&#160;</td>
			<td style="width:103px;">PY-906-500&#160;</td>
			<td></td>
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			<td height="21" style="height:21px;width:337px;">mCD45-APC</td>
			<td style="width:225px;">Biolegend</td>
			<td style="width:103px;">103111</td>
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			<td height="21" style="height:21px;width:337px;">Microvette 100 K3E</td>
			<td style="width:225px;">Microvette</td>
			<td style="width:103px;">20.1278.100</td>
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			<td height="42" style="height:42px;width:337px;">Neosporin First Aid Antibiotic/Pain Relieving Ointment</td>
			<td style="width:225px;">Neosporin</td>
			<td style="width:103px;"></td>
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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>
			<td></td>
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			<td height="21" style="height:21px;width:337px;">PrecisionGlide 25 G Needle</td>
			<td style="width:225px;">BD</td>
			<td style="width:103px;">305127</td>
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			<td height="21" style="height:21px;width:337px;">RS200 X-ray irradiator</td>
			<td style="width:225px;">RAD Source Technologies</td>
			<td style="width:103px;"></td>
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			<td height="21" style="height:21px;width:337px;">Sealsafe Plus GM500 microisolator cages</td>
			<td style="width:225px;">Techniplast</td>
			<td style="width:103px;"></td>
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			<td height="21" style="height:21px;width:337px;">Sterile Non-woven Gauze</td>
			<td style="width:225px;">Fisherbrand</td>
			<td style="width:103px;">22-028-558</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:337px;">Teklad global 16% protein irradiated mouse chow</td>
			<td style="width:225px;">Teklad</td>
			<td style="width:103px;">2916</td>
			<td></td>
		</tr>
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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 ...

Our protocol is significant, because it can be use to reproducibly create mice with both a human gut microbiome and a human immune system. We call these mice, double humanized mice. The double humanized mouse model can be used for both basic and translational biomedical research, including the study of in vivo relationships between the human gut microbiome and human immune system.Demonstrating the procedure will be Jianshui Zhang, a postdoc, and Yilun Chang, a PhD grad student from our laboratory. To implant the liver and thymus tissues into the left kidney capsule, first, place the mouse in a hood. On the day of surgery, treat the mice with whole body sublethal irradiation at a 12 centigrays per gram of body weight dose.Shave each mouse around the left lateral and medial side after confirming an appropriate level of sedation. Apply ophthalmic ointment to the animal's eyes, and apply an ear tag as needed. Disinfect the skin with three sequential iodine and 70%isopropanol scrubs.When the skin has been prepped, use forceps to load one to one-to-six millimeter cubed human fetal liver tissue fragment into a trocar, followed by one one-to-1.6 millimeter cubed human fetal thymus tissue fragment. Use the forceps to lift the skin, and use a scalpel to make a small longitudinal cut into the skin. Extend the cut to 1.5 to two centimeters on the left side of the mouse.And use forceps to lift the muscle layer. Use scissors to open the muscle layer longitudinally, extending the cut as necessary to expose the kidney, and gently grasp the fatty tissue surrounding the kidney. Use a scalpel to make a one-to-two millimeter incision at the posterior end of the kidney capsule and slowly insert the preloaded trocar into the incision parallel to the long axis of the kidney.Release the tissues between the kidney capsule and kidney, and return the kidney and bowel to their normal positions. Then, use sutures to close the muscle layer and surgical staples to close the skin. Place the mouse into an autoclaved microisolator cage with monitoring until full recumbancy.Six hours after the surgery, place the mice under a heat lamp and disinfect the tails with 70%isopropanol. Inject 1.5 to five times 10-to-the-five CD34 positive human fetal liver tissue isolated hemopoietic stem cells in 200 microliters of PBS into one dilated tail vein per animal. Then stop any bleeding with gentle pressure and return the mice to their home cage.Nine to 12 weeks after the surgery, use an appropriately sized plastic comb restraint to isolate one leg of one humanized mouse, and spray the medial side of the isolated leg with 70%isopropanol. Spread antibiotic and pain-relieving ointment onto the collection site. Using a 25-gauge needle held at a 90 degree angle, puncture the saphenous vein to collect 50 to 100 microliters of blood into an EDTA-coated blood collection tube.When a suitable volume of blood has been obtained, apply pressure to the site with a sterile piece of gauze to stop the bleeding before returning the mouse to its cage. Then use the collected peripheral blood sample to assess the level of human immune cell reconstitution by flow cytometry using antibodies against human CD45, CD3, CD4, CD8, CD19 and mouse CD45. For fresh fecal sample collection, place individual autoclaved paper bags into a fume hood and place one mouse into each bag until each animal defecates.Then return the mice to their cage, and use sterile forceps to transfer each fecal sample into one 1.5-milliliter tube per mouse for minus 80 degrees Celsius storage. At the end of the 14-day antibiotic treatment, change the drinking water to autoclaved sterilized water, and transfer the mice into a new autoclaved cage. 24 and 48 hours after the cessation of antibiotics thaw properly prepared sources of fecal microbiota transplant material, and aliquot the samples in an anaerobic chamber under anaerobic conditions.To perform a fecal transplant, deliver 200 microliters of fecal microbiota transplant material via oral gavage to each experimental animal, and spread any remaining thawed material on the fur of humanized mice or onto the cage bedding. Here, an example of flow cytometry analysis of peripheral blood from a humanized BLT mouse 10 weeks post surgery is shown. T and B cell populations can both be identified, as well as CD4 and CD8 positive T cells subsets.In this graph the relative abundance of the human fecal donor samples used to transfer a gut microbiome to create double humanized mice can be observed. The phenotypic changes to the spleen and cecum induced by antibiotic treatment is similar to that observed in germ-free animals. This principal component analysis plot of representative 16S ribosomal RNA sequencing data reveals that double humanized mice have human-like gut microbiomes that are unique to the human donor sample.The most critical aspect of this protocol is to limit the introduction of unwanted bacteria to ensure the mice remain healthy and maintain the human gut microbiome. This model has the potential to advance personalized medicine and to allow us to look at how human disease impacts the gut microbiome while controlling for other mitigating factors.

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Research

JoVE Journal

Biochemistry

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 ...

Manipulating Living Cells to Construct Stable 3D Cellular Assembly Without Artificial Scaffold

Regenerative medicine and tissue engineering offer several advantages for the treatment of intractable diseases, and several studies have demonstrated the importance of 3-dimensional (3D) cellular assemblies in these fields. Artificial scaffolds have often been used to construct 3D cellular assemblies. However, the scaffolds used to construct cellular assemblies are sometimes toxic and may change the properties of the cells. Thus, it would be beneficial to establish a non-toxic method for facilitating cell-cell contact. In this paper, we introduce a novel method for constructing stable cellular assemblies by using optical tweezers with dextran. One of the advantages of this method is that it establishes stable cell-to-cell contact within a few minutes. This new method allows the construction of 3D cellular assemblies in a natural hydrophilic polymer and is expected to be useful for constructing next-generation 3D single-cell assemblies in the fields of regenerative medicine and tissue engineering.

We demonstrate a novel method for constructing a single-cell-based 3-dimensional (3D) assembly without an artificial scaffold.

Regenerative medicine and tissue engineering offer several advantages for the treatment of intractable diseases, and several studies have demonstrated 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 ...