We thank Dr. Amy Skubitz and Dr. Kristin Boylan (University of Minnesota) for the pilot data sets and Dr. Paul Piehowski, Dr. Tao Liu, and Dr. Karin Rodland (Pacific Northwest National Laboratories (PNNL)) for their expertise in the sample collection, and processing of the PTF samples and generation of the TMT-labeled MS data used in this study. This project was funded in part by the Minnesota Ovarian Cancer Alliance (MOCA), the National Institutes of Health/National Cancer Institute Grant Number: 5R01CA262153 (A.P.N.S.), 1R21CA267707 (P.D.J and T.J.G.), and the National Institutes of Health/National Cancer Institute Grant Number: P30CA077598 (P.D.J. and T.J.G.).

Galaxy-Enabled Metaproteomics: Insights into Host-Microbiome Interaction

The authors declare no conflict of interest.

Clinical metaproteomics research offers potential breakthroughs for clinical studies, but challenges in its implementation persist. The lower abundance of microbial proteins relative to the host proteins in most samples hinders the detection and characterization of non-host proteins6,10. Dependence on large protein sequence databases for accurate peptide and protein identification and quantification, along with complexities of taxonomically and functionally annot...

Mass spectrometry (MS)-based metaproteomics identifies and quantifies microbial and human proteins from clinical samples. This approach provides a new understanding of microbiome responses to disease and uncovers potential mediators of host-microbiome interactions1,2. Although metaproteomic analysis of clinical samples can uncover the microbiome&#39;s interactions with its host environment, the field still faces many challenges. One main challenge is the relatively high abundance of host (human) proteins, which hampers the identification of lower abundant microbial proteins. Moreover, MS-based metaproteomics depends on the use of very large protein sequence databases. These databases comprise microbial proteomes that are present in the sample, which can result in a large database containing millions of sequences. Following the generation of tandem mass spectrometry (MS/MS) spectra from tryptically digested proteins, the MS/MS spectra are searched against large protein sequence databases, matching a peptide sequence to each spectrum (peptide-spectrum match, or PSM). However, sensitivity decreases, and the potential for false positives increases with large databases used for metaproteomics3. Additionally, conserved protein sequences across taxa and insufficient annotation of encoded proteins limit taxonomic and functional annotations for detected peptides and proteins4,5. We present a bioinformatics workflow for effective metaproteomic analysis of clinical samples that addresses many of these challenges and provides accessible software resources for researchers to investigate host-microbiome dynamics underlying human disease.
Clinical metaproteomics has been used to investigate diverse sample types, including feces and vaginal swabs, among others, to decipher pathogenic mechanisms in diseases and conditions6,7,8,9,10,11,12,13,14,15,16,17,18,19,20. Here, we use a metaproteomic bioinformatics workflow to analyze a subset of MS/MS data from Pap test fluid (PTF) samples from ovarian cancer (OVCA) and non-OVCA patients21. The software tools and workflow are accessible via the Galaxy platform, which streamlines the development and execution of complex clinical metaproteomic workflows22,23,24,25. Galaxy is an open-source platform that is designed for bioinformatics and computational biology. It provides a web-based environment for the use of open-source tools and workflows where academic researchers can perform and share complex data analyses. A thriving global community of software developers, data scientists, and end-users maintains the Galaxy ecosystem, including the Galaxy Training Network (GTN; https://training.galaxyproject.org/),&#160;which offers online and on-demand training resources22,23,24,25,26,27. Our workflow aims to reveal a new understanding of host-microbe dynamics in clinical samples as well as generate novel, well-characterized peptide targets of interest for developing targeted MS-based clinical assays for further study of clinical samples6,20,28. Furthermore, this manuscript intends to highlight the clinical metaproteomics workflow methodology. More detailed and beginner-friendly guides are provided in the GTN (https://training.galaxyproject.org/) as it is a valuable resource that can be used in parallel with this manuscript for users seeking additional explanations not covered. The Galaxy community has authored numerous manuscripts to aid beginner users of the Galaxy platform20,21,22,23,24,25,26,27.
All supplementary tables (e.g., tool parameters) and figures (e.g., example plots) for this manuscript have been provided as separate files and are referenced accordingly. Current tool versions within Galaxy version 2.3.0 were used for this manuscript. Therefore, results may differ slightly depending on Galaxy and tool version updates. The Galaxy platform and its tools are open-source and can be used for academic research purposes.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Collapse Collection</td>
			<td>GalaxyP</td>
			<td>Galaxy Version 5.1.1</td>
			<td>Combines a dataset list collection into a single file (in the order of the list)</td>
		</tr>
		<tr>
			<td>Concatenate datasets</td>
			<td>GalaxyP</td>
			<td>Galaxy Version 0.1.1</td>
			<td>Concatenate files tail-to-head</td>
		</tr>
		<tr>
			<td>Cut</td>
			<td>GalaxyP</td>
			<td>Galaxy Version 1.0.2</td>
			<td>Cut (select) specified columns from a file</td>
		</tr>
		<tr>
			<td>FASTA Merge Files and Filter Unique Sequences</td>
			<td>GalaxyP</td>
			<td>Galaxy Version 1.2.0</td>
			<td>Concatenate FASTA database files together</td>
		</tr>
		<tr>
			<td>FastaCLI</td>
			<td>GalaxyP</td>
			<td>Galaxy Version 4.0.41+galaxy1</td>
			<td>Appends decoy sequences to FASTA files</td>
		</tr>
		<tr>
			<td>FASTA-to-Tablular</td>
			<td>GalaxyP</td>
			<td>Galaxy Version 1.1.0</td>
			<td>Convert FASTA-formatted sequences to TAB-delimited format</td>
		</tr>
		<tr>
			<td>Filter</td>
			<td>GalaxyP</td>
			<td>Galaxy Version 1.1.1</td>
			<td>Filter columns using simple expressions</td>
		</tr>
		<tr>
			<td>Filter Tabular</td>
			<td>GalaxyP</td>
			<td>Galaxy Version 3.3.0</td>
			<td>Filter a tabular file via line filters</td>
		</tr>
		<tr>
			<td>Galaxy Europe (EU) server</td>
			<td>GalaxyP</td>
			<td></td>
			<td>https://usegalaxy.eu/</td>
		</tr>
		<tr>
			<td>Group</td>
			<td>GalaxyP</td>
			<td>Galaxy Version 2.1.4</td>
			<td>Group a file by a particular column and perform aggregate functions</td>
		</tr>
		<tr>
			<td>Identification Parameters</td>
			<td>GalaxyP</td>
			<td>Galaxy Version 4.0.41+galaxy1</td>
			<td>Set identification parameters for SearchGUI/PeptideShaker</td>
		</tr>
		<tr>
			<td>Learning Pathway: Clinical metaproteomics workflows within Galaxy</td>
			<td>GalaxyP</td>
			<td></td>
			<td>https://training.galaxyproject.org/training-material/learning-pathways/clinical-metaproteomics.html</td>
		</tr>
		<tr>
			<td>MaxQuant</td>
			<td>GalaxyP</td>
			<td>Galaxy Version 2.0.3.0+galaxy0 (Discovery module); Galaxy Version 1.6.17.0+galaxy4 (Quantification module)</td>
			<td>Quantitative proteomics software package for analysis of large mass spectrometric data files</td>
		</tr>
		<tr>
			<td>MetaNovo</td>
			<td>GalaxyP</td>
			<td>Galaxy Version 1.9.4+galaxy4</td>
			<td>Search MS/MS data against a FASTA database (of known proteins) to produce a targeted database (of matched proteins) for mass spectrometry analysis</td>
		</tr>
		<tr>
			<td>msconvert</td>
			<td>GalaxyP</td>
			<td>Galaxy Version 3.0.20287.2</td>
			<td>Convert and/or filter mass spectrometry files</td>
		</tr>
		<tr>
			<td>MSstatsTMT</td>
			<td>GalaxyP</td>
			<td>Galaxy Version 2.0.0+galaxy1</td>
			<td>R-based package for detection of differentially abundant proteins in shotgun mass spectrometry-based proteomic experiments using tandem mass tag (TMT) labeling</td>
		</tr>
		<tr>
			<td>PepQuery2</td>
			<td>GalaxyP</td>
			<td>Galaxy Version 2.0.2+galaxy0</td>
			<td>Peptide-centric search engine for identification and/or validating known and novel peptides of interest</td>
		</tr>
		<tr>
			<td>PeptideShaker</td>
			<td>GalaxyP</td>
			<td>Galaxy Version 2.0.33+galaxy1</td>
			<td>Interpret results from SearchGUI for protein identification</td>
		</tr>
		<tr>
			<td>Protein Database Downloader</td>
			<td>GalaxyP</td>
			<td>Galaxy Version 0.3.4</td>
			<td>Download specified protein sequences as a FASTA file</td>
		</tr>
		<tr>
			<td>Query Tabular</td>
			<td>GalaxyP</td>
			<td>Galaxy Version 3.3.0</td>
			<td>Load tabular files intoa&#160; SQLite database</td>
		</tr>
		<tr>
			<td>Remove beginning</td>
			<td>GalaxyP</td>
			<td>Galaxy Version 1.0.0</td>
			<td>Remove the specified number of (header) lines from a file</td>
		</tr>
		<tr>
			<td>SearchGUI</td>
			<td>GalaxyP</td>
			<td>Galaxy Version 4.0.41+galaxy1</td>
			<td>Run search engines on MGF peak lists and prepare results for input to Peptide Shaker</td>
		</tr>
		<tr>
			<td>Select</td>
			<td>GalaxyP</td>
			<td>Galaxy Version 1.0.4</td>
			<td>Select lines that match an expression</td>
		</tr>
		<tr>
			<td>Unipept</td>
			<td>GalaxyP</td>
			<td>Galaxy Version 4.5.1</td>
			<td>Retrieve UniProt entries and taxonomic information for tryptic peptides</td>
		</tr>
		<tr>
			<td>UniProt</td>
			<td>GalaxyP</td>
			<td>Galaxy Version 2.3.0</td>
			<td>Download proteome as a XML (UniProtXML) or FASTA file from UniProtKB</td>
		</tr>
	</tbody>
</table>

a clinical metaproteomics workflow implemented within galaxy bioinformatics platform to analyze host-microbiome interactions underlying human disease

Clinical metaproteomics reveals host-microbiome interactions underlying diseases. However, challenges to this approach exist. In particular, the characterization of microbial proteins present in low abundance relative to host proteins is difficult. Other significant challenges are attributed to using very large protein sequence databases, which impedes sensitivity and accuracy during peptide and protein identification from mass spectrometry data in addition to retrieving taxonomy and functional annotations and performing statistical analysis. To address these problems, we present an integrated bioinformatics workflow for mass spectrometry-based metaproteomics that combines custom protein sequence database generation, peptide-spectrum match generation and verification, quantification, taxonomic and functional annotations, and statistical analysis. This workflow also offers characterization of human proteins (while prioritizing microbial proteins), thus offering insights into host-microbe dynamics in disease. The tools and workflow are deployed in the Galaxy ecosystem, enabling the development, optimization, and dissemination of these computational resources. We have applied this workflow for metaproteomic analysis of numerous clinical sample types, such as nasopharyngeal swabs and bronchoalveolar lavage fluid. Here, we demonstrate its utility via the analysis of residual fluid from cervical swabs. The complete workflow and accompanying training resources are accessible on the Galaxy Training Network to equip non-experts and experienced researchers with the necessary knowledge and tools to analyze their data.

The general protocol described here was demonstrated on MS/MS files obtained from a subset of PTF samples21. Do et al.21 analyzed four MS/MS files from PTF samples that were collected following procedures described by Boylan et al.29and Afiuni-Zadel et al.30. This workflow prioritizes microbial proteins but offers the flexibility for the characterization of human proteins in parallel with microbial proteins21...

A Clinical Metaproteomics Workflow Implemented within Galaxy Bioinformatics Platform to Analyze Host-Microbiome Interactions Underlying Human Disease

Clinical metaproteomics offers insights into the human microbiome and its contributions to disease. We harnessed the computational power of the Galaxy platform to develop a modular bioinformatics workflow that facilitates complex mass spectrometry-based metaproteomic analysis and characterization of diverse clinical sample types relevant to studies of disease.

Clinical metaproteomics reveals host-microbiome interactions underlying diseases. However, challenges to this approach exist. In particular, the ...

a-clinical-metaproteomics-workflow-implemented-within-galaxy

Research

JoVE Journal

Immunology and Infection

414 Views.  University of Minnesota. Clinical metaproteomics offers insights into the human microbiome and its contributions to disease. We harnessed the computational power of the Galaxy platform to develop a modular bioinformatics workflow that facilitates complex mass spectrometry-based metaproteomic analysis and characterization of diverse clinical sample types relevant to studies of disease.

Video: A Clinical Metaproteomics Workflow Implemented within Galaxy Bioinformatics Platform to Analyze Host-Microbiome Interactions Underlying Human Disease

An In vitro Co-infection Model to Study Plasmodium falciparum-HIV-1 Interactions in Human Primary Monocyte-derived Immune Cells

Plasmodium falciparum, the causative agent of the deadliest form of malaria, and human immunodeficiency virus type-1 (HIV-1) are among the most important health problems worldwide, being responsible for a total of 4 million deaths annually1. Due to their extensive overlap in developing regions, especially Sub-Saharan Africa, co-infections with malaria and HIV-1 are common, but the interplay between the two diseases is poorly understood. Epidemiological reports have suggested that malarial infection transiently enhances HIV-1 replication and increases HIV-1 viral load in co-infected individuals2,3. Because this viremia stays high for several weeks after treatment with antimalarials, this phenomenon could have an impact on disease progression and transmission.
The cellular immunological mechanisms behind these observations have been studied only scarcely. The few in vitro studies investigating the impact of malaria on HIV-1 have demonstrated that exposure to soluble malarial antigens can increase HIV-1 infection and reactivation in immune cells. However, these studies used whole cell extracts of P. falciparum schizont stage parasites and peripheral blood mononuclear cells (PBMC), making it hard to decipher which malarial component(s) was responsible for the observed effects and what the target host cells were4,5. Recent work has demonstrated that exposure of immature monocyte-derived dendritic cells to the malarial pigment hemozoin increased their ability to transfer HIV-1 to CD4+ T cells6,7, but that it decreased HIV-1 infection of macrophages8. To shed light on this complex process, a systematic analysis of the interactions between the malaria parasite and HIV-1 in different relevant human primary cell populations is critically needed.
Several techniques for investigating the impact of HIV-1 on the phagocytosis of micro-organisms and the effect of such pathogens on HIV-1 replication have been described. We here present a method to investigate the effects of P. falciparum-infected erythrocytes on the replication of HIV-1 in human primary monocyte-derived macrophages. The impact of parasite exposure on HIV-1 transcriptional/translational events is monitored by using single cycle pseudotyped viruses in which a luciferase reporter gene has replaced the Env gene while the effect on the quantity of virus released by the infected macrophages is determined by measuring the HIV-1 capsid protein p24 by ELISA in cell supernatants.

An In vitro Co-infection Model to Study Plasmodium falciparum-HIV-1 Interactions in Human Primary Monocyte-derived Immune Cells

We have developed an in vitro malaria-HIV-1 co-infection model to study the impact of Plasmodium falciparum on the HIV-1 replicative cycle in human primary monocyte-derived macrophages. This versatile system can easily be adapted to other primary cell types susceptible to HIV-1 infection.

Plasmodium falciparum, the causative agent  of the deadliest form of malaria, and human immunodeficiency virus type-1 (HIV-1) are among the most important ...

Enumeration of Major Peripheral Blood Leukocyte Populations for Multicenter Clinical Trials Using a Whole Blood Phenotyping Assay

Cryopreservation of peripheral blood leukocytes is widely used to preserve cells for immune response evaluations in clinical trials and offers many advantages for ease and standardization of immunological assessments, but detrimental effects of this process have been observed on some cell subsets, such as granulocytes, B cells, and dendritic cells 1-3. Assaying fresh leukocytes gives a more accurate picture of the in vivo state of the cells, but is often difficult to perform in the context of large clinical trials. Fresh cell assays are dependent upon volunteer commitments and timeframes and, if time-consuming, their application can be impractical due to the working hours required of laboratory personnel. In addition, when trials are conducted at multiple centers, laboratories with the resources and training necessary to perform the assays may not be located in sufficient proximity to clinical sites. To address these issues, we have developed an 11-color antibody staining panel that can be used with Trucount tubes (Becton Dickinson; San Jose, CA) to phenotype and enumerate the major leukocyte populations within the peripheral blood, yielding more robust cell-type specific information than assays such as a complete blood count (CBC) or assays with commercially-available panels designed for Trucount tubes that stain for only a few cell types. The staining procedure is simple, requires only 100 &mu;l of fresh whole blood, and takes approximately 45 minutes, making it feasible for standard blood-processing labs to perform. It is adapted from the BD Trucount tube technical data sheet (<a href="http://www.bdbiosciences.com/external_files/is/doc/tds/Package_Inserts_CE/live/web_enabled/23-3483-06_PI_Trucount tubes_CE_EN.pdf" target="_blank">version 8/2010</a>). The staining antibody cocktail can be prepared in advance in bulk at a central assay laboratory and shipped to the site processing labs. Stained tubes can be fixed and frozen for shipment to the central assay laboratory for multicolor flow cytometry analysis. The data generated from this staining panel can be used to track changes in leukocyte concentrations over time in relation to intervention and could easily be further developed to assess activation states of specific cell types of interest. In this report, we demonstrate the procedure used by blood-processing lab technicians to perform staining on fresh whole blood and the steps to analyze these stained samples at a central assay laboratory supporting a multicenter clinical trial. The video details the procedure as it is performed in the context of a clinical trial blood draw in the HIV Vaccine Trials Network (HVTN).

In this report, we demonstrate the staining and analysis steps of a phenotyping assay performed on fresh whole blood to enumerate major innate and adaptive leukocyte populations. We emphasize considerations for performing these procedures in the context of a multicenter clinical trial.

Cryopreservation of peripheral blood leukocytes is widely used to preserve cells for immune response evaluations in clinical trials and offers many ...

Flow Virometry to Analyze Antigenic Spectra of Virions and Extracellular Vesicles

Cells release small extracellular vesicles (EVs) into the surrounding media. Upon virus infection cells also release virions that have the same size of some of the EVs. Both virions and EVs carry proteins of the cells that generated them and are antigenically heterogeneous. In spite of their diversity, both viruses and EVs were characterized predominantly by bulk analysis. Here, we describe an original nanotechnology-based high throughput method that allows the characterization of antigens on individual small particles using regular flow cytometers. Viruses or extracellular vesicles were immunocaptured with 15 nm magnetic nanoparticles (MNPs) coupled to antibodies recognizing one of the surface antigens. The captured virions or vesicles were incubated with fluorescent antibodies against other surface antigens. The resultant complexes were separated on magnetic columns from unbound antibodies and analyzed with conventional flow cytometers triggered on fluorescence. This method has wide applications and can be used to characterize the antigenic composition of any viral- and non-viral small particles generated by cells in vivo and in vitro. Here, we provide examples of the usage of this method to evaluate the distribution of host cell markers on individual HIV-1 particles, to study the maturation of individual Dengue virions (DENV), and to investigate extracellular vesicles released into the bloodstream.

Viruses or extracellular vesicles were immunocaptured with 15 nm magnetic nanoparticles coupled to antibodies recognizing surface antigens. The captured virions or vesicles were labeled with fluorescent antibodies against other surface antigens. The resultant complexes were separated in high magnetic field and analyzed with conventional flow cytometers triggered on fluorescence.

Cells release small extracellular vesicles (EVs) into the surrounding media. Upon virus infection cells also release virions that have the same size of ...

Intracellular Staining and Flow Cytometry to Identify Lymphocyte Subsets within Murine Aorta, Kidney and Lymph Nodes in a Model of Hypertension

It is now well known that T lymphocytes play a critical role in the development of several cardiovascular diseases1,2,3,4,5. For example, studies from our group have shown that hypertension is associated with an excessive accumulation of T cells in the vessels and kidney during the development of experimental hypertension6. Once in these tissues, T cells produce several cytokines that affect both vascular and renal function leading to vasoconstriction and sodium and water retention1,2. To fully understand how T cells cause cardiovascular and renal diseases, it is important to be able to identify and quantify the specific T cell subsets present in these tissues. T cell subsets are defined by a combination of surface markers, the cytokines they secrete, and the transcription factors they express. The complexity of the T cell population makes flow cytometry and intracellular staining an invaluable technique to dissect the phenotypes of the lymphocytes present in tissues. Here, we provide a detailed protocol to identify the surface and intracellular markers (cytokines and transcription factors) in T cells isolated from murine kidney, aorta and aortic draining lymph nodes in a model of angiotensin II induced hypertension. The following steps are described in detail: isolation of the tissues, generation of the single cell suspensions, ex vivo stimulation, fixation, permeabilization and staining. In addition, several fundamental principles of flow cytometric analyses including choosing the proper controls and appropriate gating strategies are discussed.

This article provides detailed methodology to identify and quantify functional T lymphocyte subsets present within murine kidney, aorta and lymph nodes by intracellular staining and flow cytometry. The model of angiotensin II induced hypertension was chosen to explain, step-by-step, the procedures and fundamental principles of flow cytometry and intracellular staining.

It is now well known that T lymphocytes play a critical role in the development of several cardiovascular diseases1,2,3,4,5. For example, studies from our ...

A Suction Blister Protocol to Study Human T-cell Recall Responses In Vivo

Cutaneous antigen-recall models allow for studies of human memory responses in vivo. When combined with skin suction blister (SB) induction, this model offers accessibility to rare populations of antigen-specific T-cells representative of the cellular memory response as well as the cytokine microenvironment in situ.
This report describes the practical procedure of a cutaneous recall, an SB induction, and a harvest of antigen-specific T-cells. To exemplify the method, the tuberculin skin test is used for antigenic recall in individuals who, prior to this study, underwent a Bacillus Calmette-Gu&#233;rin vaccination against an infection with Mycobacterium tuberculosis. Finally, examples of multiplex and flow cytometric analyses of SB specimens are provided, illustrating high fractions of antigen-specific polyfunctional CD4+ T-cells available by this sampling method compared with cells isolated from the blood.
The method described here is safe and minimally invasive, provides a unique opportunity to study both innate and adaptive immune responses in vivo, and may be beneficial to a broad community of researchers working with cell-mediated immunity and human memory responses, in the context of vaccine development.

A Suction Blister Protocol to Study Human T-cell Recall Responses In Vivo

Here, we provide a demonstration of the suction blister cutaneous recall model. The model allows a simple access to study human in vivo adaptive immune responses, for instance in the context of vaccine development.

Cutaneous antigen-recall models allow for studies of human memory responses in vivo. When combined with skin suction blister (SB) induction, this model ...

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 ...

Generation of Knock-out Primary and Expanded Human NK Cells Using Cas9 Ribonucleoproteins

CRISPR/Cas9 technology is accelerating genome engineering in many cell types, but so far, gene delivery and stable gene modification have been challenging in primary NK cells. For example, transgene delivery using lentiviral or retroviral transduction resulted in a limited yield of genetically-engineered NK cells due to substantial procedure-associated NK cell apoptosis. We describe here a DNA-free method for genome editing of human primary and expanded NK cells using Cas9 ribonucleoprotein complexes (Cas9/RNPs). This method allowed efficient knockout of the TGFBR2 and HPRT1 genes in NK cells. RT-PCR data showed a significant decrease in gene expression level, and a cytotoxicity assay of a representative cell product suggested that the RNP-modified NK cells became less sensitive to TGF&#946;. Genetically modified cells could be expanded post-electroporation by stimulation with irradiated mbIL21-expressing feeder cells.

Here, we present a protocol to genetically modify primary or expanded human natural killer (NK) cells using Cas9 Ribonucleoproteins (Cas9/RNPs). By using this protocol, we generated human NK cells deficient for transforming growth factor&#8211;b receptor 2 (TGFBR2) and hypoxanthine phosphoribosyltransferase 1 (HPRT1).

CRISPR/Cas9 technology is accelerating genome engineering in many cell types, but so far, gene delivery and stable gene modification have been challenging ...

Bone Marrow Transplantation Platform to Investigate the Role of Dendritic Cells in Graft-versus-Host Disease

Allogeneic bone marrow transplantation (BMT) is an effective therapy for hematological malignancies due to the graft-versus-leukemia (GVL) effect to eradicate tumors. However, its application is limited by the development of graft-versus-host disease (GVHD), a major complication of BMT. GVHD is evoked when T-cells in the donor grafts recognizealloantigen expressed by recipient cells and mount unwanted immunological attacks against recipient healthy tissues. Thus, traditional therapies are designed to suppress donor T-cell alloreactivity. However, these approaches substantially impair the GVL effect so that the recipient&#39;s survival is not improved. Understanding the effects of therapeutic approaches on BMT, GVL, and GVHD, is thus essential. Due to the antigen-presenting and cytokine-secreting capacities to stimulate donor T-cells, recipient dendritic cells (DCs) play a significant role in the induction of GVHD. Therefore, targeting recipient DCs becomes a potential approach for controlling GVHD. This work provides a description of a novel BMT platform to investigate how host DCs regulate GVH and GVL responses after transplantation. Also presented is an effective BMT model to study the biology of GVHD and GVL after transplantation.

Graft-versus-host disease is a major complication after allogeneic bone marrow transplantation. Dendritic cells play a critical role in the pathogenesis of graft-versus-host disease. The current article describes a novel bone marrow transplantation platform to investigate the role of dendritic cells in the development of graft-versus-host disease and the graft-versus-leukemia effect.

Allogeneic bone marrow transplantation (BMT) is an effective therapy for hematological malignancies due to the graft-versus-leukemia (GVL) effect to ...

Antibiotic Dereplication Using the Antibiotic Resistance Platform

One of the main challenges in the search for new antibiotics from natural product extracts is the re-discovery of common compounds. To address this challenge, dereplication, which is the process of identifying known compounds, is performed on samples of interest. Methods for dereplication such as analytical separation followed by mass spectrometry are time-consuming and resource-intensive. To improve the dereplication process, we have developed the antibiotic resistance platform (ARP). The ARP is a library of approximately 100 antibiotic resistance genes that have been individually cloned into Escherichia coli. This strain collection has many applications, including a cost-effective and facile method for antibiotic dereplication. The process involves the fermentation of antibiotic-producing microbes on the surface of rectangular Petri dishes containing solid medium, thereby allowing for the secretion and diffusion of secondary metabolites through the medium. After a 6 day fermentation period, the microbial biomass is removed, and a thin agar-overlay is added to the Petri dish to create a smooth surface and enable the growth of the E. coli indicator strains. Our collection of ARP strains is then pinned onto the surface of the antibiotic-containing Petri dish. The plate is next incubated overnight to allow for E. coli growth on the surface of the overlay. Only strains containing resistance to a specific antibiotic (or class) grow on this surface enabling rapid identification of the produced compound. This method has been successfully used for the identification of producers of known antibiotics and as a means to identify those producing novel compounds.

We describe a platform that utilizes a library of isogenic antibiotic resistant Escherichia coli for the dereplication of antibiotics. The identity of an antibiotic produced by bacteria or fungi can be deduced by the growth of E. coli expressing its respective resistance gene. This platform is economically effective and time-efficient.

One of the main challenges in the search for new antibiotics from natural product extracts is the re-discovery of common compounds. To address this ...

In Vitro Microfluidic Disease Model to Study Whole Blood-Endothelial Interactions and Blood Clot Dynamics in Real-Time

The formation of blood clots involves complex interactions between endothelial cells, their underlying matrix, various blood cells, and proteins. The endothelium is the primary source of many of the major hemostatic molecules that control platelet aggregation, coagulation, and fibrinolysis. Although the mechanism of thrombosis has been investigated for decades, in vitro studies mainly focus on situations of vascular damage where the subendothelial matrix gets exposed, or on interactions between cells with single blood components. Our method allows studying interactions between whole blood and an intact, confluent vascular cell network.
By utilizing primary human endothelial cells, this protocol provides the unique opportunity to study the influence of endothelial cells on thrombus dynamics and gives&#160;valuable insights into the pathophysiology of thrombotic disease. The use of custom-made microfluidic flow channels allows application of disease-specific vascular geometries and model specific morphological vascular changes. The development of a thrombus is recorded in real-time and quantitatively characterized by platelet adhesion and fibrin deposition. The effect of endothelial function in altered thrombus dynamics is determined by postanalysis through immunofluorescence staining of specific molecules.
The representative results describe the experimental setup, data collection, and data analysis. Depending on the research question, parameters for every section can be adjusted including cell type, shear rates, channel geometry, drug therapy, and postanalysis procedures. The protocol is validated by quantifying thrombus formation on the pulmonary artery endothelium of patients with chronic thromboembolic disease.

We present an in vitro vascular disease model to investigate whole blood interactions with patient-derived endothelium. This system allows the study of thrombogenic properties of primary endothelial cells under various circumstances. The method is especially suited to evaluate in situ thrombogenicity and anticoagulation therapy during different phases of coagulation.

The formation of blood clots involves complex interactions between endothelial cells, their underlying matrix, various blood cells, and proteins. The ...