Silicon-doped calcium phosphates; the critical effect of synthesis routes on the biological performance.
In this study, the effect of using different types of fuel and various amounts of Si4+ ions on the biological properties of silicon-doped calcium phosphates (CaPs), which were synthesized using solution combustion method were investigated. X-ray diffraction (XRD) patterns showed that hydroxyapatite/beta-tricalcium phosphate (HA/βTCP) was crystallized in all synthesized samples. The synthesized sample using glycine as fuel, which doped with 0.1 mol Si4+ ions exhibited the most desirable properties. Consecutively, the zeta potential and specific surface area were enhanced from -20 to -27 mV and 38 to 146 ...
Source: Mol Biol Cell - April 15, 2020 Category: Molecular Biology Authors: Kermani F, Gharavian A, Mollazadeh S, Kargozar S, Youssefi A, Vahdati Khaki J Tags: Mater Sci Eng C Mater Biol Appl Source Type: research

Touching the Surface: Diverse Roles for the Flagellar Membrane in Kinetoplastid Parasites.
Abstract SUMMARYWhile flagella have been studied extensively as motility organelles, with a focus on internal structures such as the axoneme, more recent research has illuminated the roles of the flagellar surface in a variety of biological processes. Parasitic protists of the order Kinetoplastida, which include trypanosomes and Leishmania species, provide a paradigm for probing the role of flagella in host-microbe interactions and illustrate that this interface between the flagellar surface and the host is of paramount importance. An increasing body of knowledge indicates that the flagellar membrane serves a mult...
Source: Mol Biol Cell - April 3, 2020 Category: Molecular Biology Authors: Kelly FD, Sanchez MA, Landfear SM Tags: Microbiol Mol Biol Rev Source Type: research

Microbiota-Propelled T Helper 17 Cells in Inflammatory Diseases and Cancer.
Abstract SUMMARYTechnologies allowing genetic sequencing of the human microbiome are opening new realms to discovery. The host microbiota substantially impacts immune responses both in immune-mediated inflammatory diseases (IMIDs) and in tumors affecting tissues beyond skin and mucosae. However, a mechanistic link between host microbiota and cancer or IMIDs has not been well established. Here, we propose T helper 17 (TH17) lymphocytes as the connecting factor between host microbiota and rheumatoid or psoriatic arthritides, multiple sclerosis, breast or ovarian cancer, and multiple myeloma. We theorize that similar...
Source: Mol Biol Cell - March 7, 2020 Category: Molecular Biology Authors: Bellone M, Brevi A, Huber S Tags: Microbiol Mol Biol Rev Source Type: research

Endoplasmic reticulum stress in the cellular release of damage-associated molecular patterns.
Abstract Several pathological and inflammatory disorders induce a cytoprotective endoplasmic reticulum (ER) stress that aims at reestablishing tissue homeostasis, yet can also ignite lethal signaling pathways leading to apoptotic cell death when ER stress endures. Cells that undergo episodes of ER stress in response to pathological malfunction or cytotoxic agents can expose and release immunomodulatory damaged-associated molecular patterns (DAMPs) on their surface and into the extracellular space, respectively. Immunosuppressive DAMPs inhibit the transfer of antigens from stressed cells to antigen-presenting cells...
Source: Mol Biol Cell - March 7, 2020 Category: Molecular Biology Authors: Wu Chuang A, Kepp O, Kroemer G, Bezu L Tags: Int Rev Cell Mol Biol Source Type: research

The role of mitochondria-associated membranes in cellular homeostasis and diseases.
Abstract Mitochondria and endoplasmic reticulum (ER) are fundamental in the control of cell physiology regulating several signal transduction pathways. They continuously communicate exchanging messages in their contact sites called MAMs (mitochondria-associated membranes). MAMs are specific microdomains acting as a platform for the sorting of vital and dangerous signals. In recent years increasing evidence reported that multiple scaffold proteins and regulatory factors localize to this subcellular fraction suggesting MAMs as hotspot signaling domains. In this review we describe the current knowledge about MAMs' dy...
Source: Mol Biol Cell - March 7, 2020 Category: Molecular Biology Authors: Perrone M, Caroccia N, Genovese I, Missiroli S, Modesti L, Pedriali G, Vezzani B, Vitto VAM, Antenori M, Lebiedzinska-Arciszewska M, Wieckowski MR, Giorgi C, Pinton P Tags: Int Rev Cell Mol Biol Source Type: research

Sarcoplasmic reticulum and calcium signaling in muscle cells: Homeostasis and disease.
ero S Abstract The sarco/endoplasmic reticulum is an extensive, dynamic and heterogeneous membranous network that fulfills multiple homeostatic functions. Among them, it compartmentalizes, stores and releases calcium within the intracellular space. In the case of muscle cells, calcium released from the sarco/endoplasmic reticulum in the vicinity of the contractile machinery induces cell contraction. Furthermore, sarco/endoplasmic reticulum-derived calcium also regulates gene transcription in the nucleus, energy metabolism in mitochondria and cytosolic signaling pathways. These diverse and overlapping processes req...
Source: Mol Biol Cell - March 7, 2020 Category: Molecular Biology Authors: Bravo-Sagua R, Parra V, Muñoz-Cordova F, Sanchez-Aguilera P, Garrido V, Contreras-Ferrat A, Chiong M, Lavandero S Tags: Int Rev Cell Mol Biol Source Type: research

Endoplasmic reticulum in viral infection.
Abstract Virus exploits host cellular machinery to replicate and form new viral progeny and endoplasmic reticulum (ER) plays central role in the interplay between virus and host cell. Here I will discuss how cellular functions of ER being utilized by viruses from different families during different stages of pathogenesis. Flow of knowledge related to this area of research based on interdisciplinary approach, using biochemical and cell biological assays coupled with advanced microscopy strategies, is pushing our understanding of the virus-ER interaction during infection to the next level. PMID: 32138901 [PubMe...
Source: Mol Biol Cell - March 7, 2020 Category: Molecular Biology Authors: Bagchi P Tags: Int Rev Cell Mol Biol Source Type: research

Targeting endoplasmic reticulum stress and autophagy as therapeutic approaches for neurological diseases.
Abstract Neurological diseases are multifactorial, devastating diseases that are causative for various neurodegenerative disorders. Emerging evidence points that accumulation of unfolded, misfolded, insoluble, and damaged proteins inside the CNS cells such as microglia, astrocytes, neurons, oligodendrocytes, pericytes, and endothelial cells, leads to endoplasmic reticulum (ER) stress and dysregulated autophagy, which, in turn, sets the stage for ensuing neuropathogenesis. Studies have also demonstrated that chronic ER stress/unfolded protein response (UPR) activates autophagy, and conversely, that blockade of auto...
Source: Mol Biol Cell - March 7, 2020 Category: Molecular Biology Authors: Thangaraj A, Sil S, Tripathi A, Chivero ET, Periyasamy P, Buch S Tags: Int Rev Cell Mol Biol Source Type: research

Emerging roles of the unfolded protein response (UPR) in the nervous system: A link with adaptive behavior to environmental stress?
z G, Hetz C Abstract Stressors elicit a neuroendocrine response leading to increased levels of glucocorticoids, allowing the organism to adapt to environmental changes and maintain homeostasis. Glucocorticoids have a broad effect in the body, modifying the activity of the immune system, metabolism, and behavior through the activation of receptors in the limbic system. Chronic exposition to stressors operates as a risk factor for psychiatric diseases such as depression and posttraumatic stress disorder. Among the cellular alterations observed as a consequence of environmental stress, alterations to organelle functi...
Source: Mol Biol Cell - March 7, 2020 Category: Molecular Biology Authors: Díaz-Hung ML, Martínez G, Hetz C Tags: Int Rev Cell Mol Biol Source Type: research

Type I interferons and endoplasmic reticulum stress in health and disease.
We describe how ER stress regulates type I IFNs production or sensing, or how type I IFNs may induce ER stress, in various circumstances like microbial infections, autoimmunity, diabetes, cancer and other ER stress-related contexts. PMID: 32138904 [PubMed - in process] (Source: Mol Biol Cell)
Source: Mol Biol Cell - March 7, 2020 Category: Molecular Biology Authors: Sprooten J, Garg AD Tags: Int Rev Cell Mol Biol Source Type: research

Preface: Endoplasmic reticulum in health and disease.
PMID: 32138905 [PubMed - in process] (Source: Mol Biol Cell)
Source: Mol Biol Cell - March 7, 2020 Category: Molecular Biology Authors: Kepp O, Galluzzi L Tags: Int Rev Cell Mol Biol Source Type: research

Combining canine mesenchymal stromal cells and hyaluronic acid for cartilage repair.
In this study, we investigated the chondrogenic potential of canine adipose-derived mesenchymal stromal cells (ASCs) combined to high molecular weight hyaluronic acid (HA) in vitro, and their therapeutic effect in dogs with chronic osteoarthritis (OA) associated with bilateral hip dysplasia. Canine ASCs were characterized after conventional 2D culture or 3D culture in HA, showing adequate immunophenotype, proliferation and trilineage differentiation, as well as chondrogenesis after cultivation in HA. ASC/HA constructs were used to treat 12 dogs with OA, sequentially assigned to control, ASC and ASC/HA groups. Animals were ...
Source: Mol Biol Cell - March 7, 2020 Category: Molecular Biology Authors: Wits MI, Tobin GC, Silveira MD, Baja KG, Braga LMM, Sesterheim P, Camassola M, Nardi NB Tags: Genet Mol Biol Source Type: research

Maternal transmission of mitochondrial diseases.
Abstract Given the major role of the mitochondrion in cellular homeostasis, dysfunctions of this organelle may lead to several common diseases in humans. Among these, maternal diseases linked to mitochondrial DNA (mtDNA) mutations are of special interest due to the unclear pattern of mitochondrial inheritance. Multiple copies of mtDNA are present in a cell, each encoding for 37 genes essential for mitochondrial function. In cases of mtDNA mutations, mitochondrial malfunctioning relies on mutation load, as mutant and wild-type molecules may co-exist within the cell. Since the mutation load associated with disease m...
Source: Mol Biol Cell - March 7, 2020 Category: Molecular Biology Authors: Chiaratti MR, Macabelli CH, Augusto Neto JD, Grejo MP, Pandey AK, Perecin F, Collado MD Tags: Genet Mol Biol Source Type: research

Cooperation and interplay between base and nucleotide excision repair pathways: From DNA lesions to proteins.
Abstract Base and nucleotide excision repair (BER and NER) pathways are normally associated with removal of specific types of DNA damage: small base modifications (such as those induced by DNA oxidation) and bulky DNA lesions (such as those induced by ultraviolet or chemical carcinogens), respectively. However, growing evidence indicates that this scenario is much more complex and these pathways exchange proteins and cooperate with each other in the repair of specific lesions. In this review, we highlight studies discussing the involvement of NER in the repair of DNA damage induced by oxidative stress, and BER par...
Source: Mol Biol Cell - March 7, 2020 Category: Molecular Biology Authors: Kumar N, Moreno NC, Feltes BC, Menck CF, Houten BV Tags: Genet Mol Biol Source Type: research

Direct eNAMPT Involvement in Pulmonary Hypertension and Vascular Remodeling: Transcriptional Regulation by SOX and HIF2 α.
CONCLUSIONS: eNAMPT is a novel and attractive therapeutic target essential to PAH vascular remodeling. PMID: 32142369 [PubMed - as supplied by publisher] (Source: Mol Biol Cell)
Source: Mol Biol Cell - March 6, 2020 Category: Molecular Biology Authors: Sun X, Sun BL, Babicheva A, Vanderpool R, Oita RC, Casanova N, Tang H, Gupta A, Lynn H, Gupta G, Rischard F, Sammani S, Kempf CL, Moreno-Vinasco L, Ahmed M, Camp SM, Wang J, Desai AA, Yuan JX, Garcia JGN Tags: Am J Respir Cell Mol Biol Source Type: research

ZCCHC3 modulates TLR3-mediated signaling by promoting recruitment of TRIF to TLR3.
Abstract Toll-like receptor 3 (TLR3)-mediated signaling is important for host defense against RNA virus. Upon viral RNA stimulation, TIR domain-containing adaptor inducing IFN-β (TRIF) is recruited to TLR3 and then undergoes oligomerization, which is required for the recruitment of downstream molecules to transmit signals. Here we identified ZCCHC3 as a positive regulator of TLR3-mediated signaling. Overexpression of ZCCHC3 promoted transcription of downstream antiviral genes stimulated by the synthetic TLR3 ligand poly(I:C). ZCCHC3-deficiency markedly inhibited TLR3- but not TLR4-mediated induction of type I...
Source: Mol Biol Cell - March 5, 2020 Category: Molecular Biology Authors: Zang R, Lian H, Zhong X, Yang Q, Shu HB Tags: J Mol Cell Biol Source Type: research

Separation of mononuclear cells and identification of B lymphocytes: A comprehensive experiment for medical students.
Abstract In teaching of experiments for medical students, by using the isolation of mononuclear cells and identification of B lymphocytes, an experiment was developed that integrated biochemistry, cytology, and immunology techniques, from which the students performed a series of operations including spleen separation, isolating mononuclear cells, and identifying B lymphocytes. From the immunocytochemistry detection, we included advanced equipment-a flow cytometer-in the experiment, which detected the B lymphocytes more accurately. Moreover, we applied modern information technology to the teaching of experiments by...
Source: Mol Biol Cell - March 5, 2020 Category: Molecular Biology Authors: Li J, Gao F, Jin C, Shi X, Zhang C, Jia S, Xu J, Lv L, Yang W, Xu L Tags: Biochem Mol Biol Educ Source Type: research

GNAS promotes inflammation-related hepatocellular carcinoma progression by promoting STAT3 activation.
Conclusions: Our findings for the first time indicate a tumor-promoting role of GNAS in inflammation-related HCC progression and provide a novel potential target for HCC therapy. PMID: 32123532 [PubMed - in process] (Source: Mol Biol Cell)
Source: Mol Biol Cell - March 4, 2020 Category: Molecular Biology Authors: Ding H, Zhang X, Su Y, Jia C, Dai C Tags: Cell Mol Biol Lett Source Type: research

Bioinformatics for Cancer Immunotherapy.
öwer M Abstract Our immune system plays a key role in health and disease as it is capable of responding to foreign antigens as well as acquired antigens from cancer cells. Latter are caused by somatic mutations, the so-called neoepitopes, and might be recognized by T cells if they are presented by HLA molecules on the surface of cancer cells. Personalized mutanome vaccines are a class of customized immunotherapies, which is dependent on the detection of individual cancer-specific tumor mutations and neoepitope (i.e., prediction, followed by a rational vaccine design, before on-demand production. The developme...
Source: Mol Biol Cell - March 4, 2020 Category: Molecular Biology Authors: Holtsträter C, Schrörs B, Bukur T, Löwer M Tags: Methods Mol Biol Source Type: research

In Silico Prediction of Tumor Neoantigens with TIminer.
Abstract Tumor neoantigens are at the core of immunological tumor control and response to immunotherapy. In silico prediction of tumor neoantigens from next-generation sequencing (NGS) data is possible but requires the assembly of complex, multistep computational pipelines and extensive data preprocessing. Using public data from two cancer cell lines, here we show how TIminer, a framework to perform immunogenomics analyses, can be easily used to assemble and run customized pipelines to predict cancer neoantigens from multisample NGS data. PMID: 32124316 [PubMed - in process] (Source: Mol Biol Cell)
Source: Mol Biol Cell - March 4, 2020 Category: Molecular Biology Authors: Kirchmair A, Finotello F Tags: Methods Mol Biol Source Type: research

Improving MHC-I Ligand Identification by Incorporating Targeted Searches of Mass Spectrometry Data.
Abstract Effective immunotherapies rely on specific activation of immune cells. Class I major histocompatibility complex (MHC-I) bound peptide ligands play a major role in dictating the specificity and activation of CD8+ T cells and hence are important in developing T cell-based immunotherapies. Mass spectrometry-based approaches are most commonly used for identifying these MHC-bound peptides, wherein the MS/MS spectra are compared against a reference proteome database. Unfortunately, the effectiveness of matching the immunopeptide MS/MS spectra to a reference proteome database is hindered by inflated search space...
Source: Mol Biol Cell - March 4, 2020 Category: Molecular Biology Authors: Konda P, Murphy JP, Gujar S Tags: Methods Mol Biol Source Type: research

Identification of Epitope-Specific T Cells in T-Cell Receptor Repertoires.
Abstract Recognition of cancer epitopes by T cells is fundamental for the activation of targeted antitumor responses. As such, the identification and study of epitope-specific T cells has been instrumental in our understanding of cancer immunology and the development of personalized immunotherapies. To facilitate the study of T-cell epitope specificity, we developed a prediction tool, TCRex, that can identify epitope-specific T-cell receptors (TCRs) directly from TCR repertoire data and perform epitope-specificity enrichment analyses. This chapter details the use of the TCRex web tool. PMID: 32124320 [PubMed ...
Source: Mol Biol Cell - March 4, 2020 Category: Molecular Biology Authors: Gielis S, Moris P, Bittremieux W, De Neuter N, Ogunjimi B, Laukens K, Meysman P Tags: Methods Mol Biol Source Type: research

Modeling and Viewing T Cell Receptors Using TCRmodel and TCR3d.
Abstract The past decade has seen a rapid increase in T cell receptor (TCR) sequences from single cell cloning and repertoire-scale high throughput sequencing studies. Many of these TCRs are of interest as potential therapeutics or for their implications in autoimmune disease or effective targeting of pathogens. As it is impractical to characterize the structure or targeting of the vast majority of these TCRs experimentally, advanced computational methods have been developed to predict their 3D structures and gain mechanistic insights into their antigen binding and specificity. Here, we describe the use of a ...
Source: Mol Biol Cell - March 4, 2020 Category: Molecular Biology Authors: Gowthaman R, Pierce BG Tags: Methods Mol Biol Source Type: research

In Silico Cell-Type Deconvolution Methods in Cancer Immunotherapy.
Abstract Several computational methods have been proposed to infer the cellular composition from bulk RNA-seq data of a tumor biopsy sample. Elucidating interactions in the tumor microenvironment can yield unique insights into the status of the immune system. In immuno-oncology, this information can be crucial for deciding whether the immune system of a patient can be stimulated to target the tumor. Here, we shed a light on the working principles, capabilities, and limitations of the most commonly used methods for cell-type deconvolution in immuno-oncology and offer guidelines for method selection. PMID: 3212...
Source: Mol Biol Cell - March 4, 2020 Category: Molecular Biology Authors: Sturm G, Finotello F, List M Tags: Methods Mol Biol Source Type: research

Immunedeconv: An R Package for Unified Access to Computational Methods for Estimating Immune Cell Fractions from Bulk RNA-Sequencing Data.
Abstract Since the performance of in silico approaches for estimating immune-cell fractions from bulk RNA-seq data can vary, it is often advisable to compare results of several methods. Given numerous dependencies and differences in input and output format of the various computational methods, comparative analyses can become quite complex. This motivated us to develop immunedeconv, an R package providing uniform and user-friendly access to seven state-of-the-art computational methods for deconvolution of cell-type fractions from bulk RNA-seq data. Here, we show how immunedeconv can be installed and applied to a ty...
Source: Mol Biol Cell - March 4, 2020 Category: Molecular Biology Authors: Sturm G, Finotello F, List M Tags: Methods Mol Biol Source Type: research

EPIC: A Tool to Estimate the Proportions of Different Cell Types from Bulk Gene Expression Data.
Abstract Gene expression profiling is nowadays routinely performed on clinically relevant samples (e.g., from tumor specimens). Such measurements are often obtained from bulk samples containing a mixture of cell types. Knowledge of the proportions of these cell types is crucial as they are key determinants of the disease evolution and response to treatment. Moreover, heterogeneity in cell type proportions across samples is an important confounding factor in downstream analyses.Many tools have been developed to estimate the proportion of the different cell types from bulk gene expression data. Here, we provide guid...
Source: Mol Biol Cell - March 4, 2020 Category: Molecular Biology Authors: Racle J, Gfeller D Tags: Methods Mol Biol Source Type: research

Computational Deconvolution of Tumor-Infiltrating Immune Components with Bulk Tumor Gene Expression Data.
Abstract Tumor-infiltrating immune cells play critical roles in immune-mediated tumor rejection and/or progression, and are key targets of immunotherapies. Estimation of different immune subsets becomes increasingly important with the decreased cost of high-throughput molecular profiling and the rapidly growing amount of cancer genomics data. Here, we present Tumor IMmune Estimation Resource (TIMER), an in silico deconvolution method for inference of tumor-infiltrating immune components. TIMER takes bulk tissue gene expression profiles measured with RNA-seq or microarray to evaluate the abundance of six immune cel...
Source: Mol Biol Cell - March 4, 2020 Category: Molecular Biology Authors: Li B, Li T, Liu JS, Liu XS Tags: Methods Mol Biol Source Type: research

Cell-Type Enrichment Analysis of Bulk Transcriptomes Using xCell.
Abstract Tissues are a complex milieu of cell types of different lineages and subtypes, each with its own unique transcriptomic profile. Bulk transcriptome profiling is therefore the sum of the cell-type-specific gene expression weighted by cell-type proportion in the given sample. Deconvolution of gene expression profiles allows to reconstruct the cellular composition of tissues. xCell is a robust computational method that converts gene expression profiles to enrichment scores of 64 immune and stroma cell types across samples. Here, we described the method, discuss correct usage, and demonstrate an analysis of a ...
Source: Mol Biol Cell - March 4, 2020 Category: Molecular Biology Authors: Aran D Tags: Methods Mol Biol Source Type: research

Chemotaxis Assay for Marsupenaeus japonicas Hemocytes and Application for the Development of an Oral Immunostimulant Against White Spot Syndrome Virus.
In this study, we aimed to optimize the conditions for a Boyden chamber chemotaxis assay using hemocytes (granulocytes) from cultured kuruma shrimp, Marsupenaeus japonicas (Mj) and the optimal conditions were: 5 μm-pore-size Polyvinylpyrrolidone membrane; culture buffer at pH 7.0; and chemotactic factor N-formyl-methionyl-leucyl-phenylalanine (fMLP) 10-8 mol/L; 4 h incubation time. We then applied the chemotaxis assay to develop an oral immunostimulant against white spot syndrome virus (WSSV), which results in high mortality rates in several shrimp species worldwide. We focused on the kelp Laminaria japonica, as this sp...
Source: Mol Biol Cell - March 3, 2020 Category: Molecular Biology Authors: Imai T, Takahashi Y Tags: Front Cell Dev Biol Source Type: research

Membrane Extracts from Plant Tissues.
We describe this method for the isolation of different organelles from one plant tissue for the example of Arabidopsis thaliana. All compartments are retrieved by utilizing differential centrifugation with organelle-specific parameters. PMID: 32112316 [PubMed - as supplied by publisher] (Source: Mol Biol Cell)
Source: Mol Biol Cell - March 2, 2020 Category: Molecular Biology Authors: Bodensohn U, Ruland C, Ladig R, Schleiff E Tags: Methods Mol Biol Source Type: research

Reconstitution of Membrane Proteins into Platforms Suitable for Biophysical and Structural Analyses.
Abstract Integral membrane proteins have historically been challenging targets for biophysical research due to their low solubility in aqueous solution. Their importance for chemical and electrical signaling between cells, however, makes them fascinating targets for investigators interested in the regulation of cellular and physiological processes. Since membrane proteins shunt the barrier imposed by the cell membrane, they also serve as entry points for drugs, adding pharmaceutical research and development to the interests. In recent years, detailed understanding of membrane protein function has significantl...
Source: Mol Biol Cell - March 2, 2020 Category: Molecular Biology Authors: Schmidpeter PAM, Sukomon N, Nimigean CM Tags: Methods Mol Biol Source Type: research

Reconstitution of Proteoliposomes for Phospholipid Scrambling and Nonselective Channel Assays.
Abstract Phospholipid scramblases catalyze the rapid trans-bilayer movement of lipids down their concentration gradients. This process is essential for numerous cellular signaling functions including cell fusion, blood coagulation, and apoptosis. The importance of scramblases is highlighted by the number of human diseases caused by mutations in these proteins. Because of their indispensable function, it is essential to understand and characterize the molecular function of phospholipid scramblases. Powerful tools to measure lipid transport in cells are available. However, these approaches provide limited mechanisti...
Source: Mol Biol Cell - March 2, 2020 Category: Molecular Biology Authors: Falzone ME, Accardi A Tags: Methods Mol Biol Source Type: research

Stabilization and Crystallization of a Membrane Protein Involved in Lipid Transport.
Abstract Lipoteichoic acids (LTA) are ubiquitous cell wall components of Gram-positive bacteria. In Staphylococcus aureus LTA are composed of a polymer with 1,3-linked glycerol phosphate repeating units anchored to the plasma membrane. The anchor molecule is a lipid-linked disaccharide (anchor-LLD) synthesized at the cytoplasmic leaflet of the membrane. The anchor lipid becomes accessible at the outer leaflet of the membrane after the flippase LtaA catalyzes translocation. Recently we have elucidated the structure of LtaA using vapor diffusion X-ray crystallography and in situ annealing. We were able to obtain Lta...
Source: Mol Biol Cell - March 2, 2020 Category: Molecular Biology Authors: Zhang B, Perez C Tags: Methods Mol Biol Source Type: research

Membrane Protein Preparation for Serial Crystallography Using High-Viscosity Injectors: Rhodopsin as an Example.
Abstract Membrane proteins are highly interesting targets due to their pivotal role in cell function and disease. They are inserted in cell membranes, are often intrinsically flexible, and can adopt several conformational states to carry out their function. Although most overall folds of membrane proteins are known, many questions remain about specific functionally relevant intramolecular rearrangements that require experimental structure determination. Here, using the example of rhodopsin, we describe how to prepare and analyze membrane protein crystals for serial crystallography at room temperature, a new techni...
Source: Mol Biol Cell - March 2, 2020 Category: Molecular Biology Authors: Weinert T, Panneels V Tags: Methods Mol Biol Source Type: research

Using Bioengineered Bioluminescence to Track Stem Cell Transplantation In Vivo.
Abstract Bioluminescence imaging enables the real-time detection and tracking of engrafted cells in vivo noninvasively and dynamically. By detecting and quantifying the photons released from the oxidation of luciferin catalyzed by luciferase enzymes, this approach has proven effective in tracking engrafted stem cell survival and retention, making it a powerful tool to monitor cell fate after transplantation without animal sacrifice. Here we describe a protocol that allows luciferase-labeled stem cells to be imaged and tracked in vivo by bioluminescent imaging via an IVIS spectrum imaging system. PMID: 3211237...
Source: Mol Biol Cell - March 2, 2020 Category: Molecular Biology Authors: Han D, Wu JC Tags: Methods Mol Biol Source Type: research

Using Naturally Occurring Bioluminescent Enzymes to Track Specific Cell Populations.
Abstract Luminol-based bioluminescence imaging allows noninvasive tracking of oxidatively active cells such as neutrophils. Luminol is given intravenously or intraperitoneally, followed by bioluminescence imaging at 425 nm. Here we describe a method for tracking neutrophil extravasation into an inflammatory site, especially focusing on mammary carcinoma. PMID: 32112375 [PubMed - as supplied by publisher] (Source: Mol Biol Cell)
Source: Mol Biol Cell - March 2, 2020 Category: Molecular Biology Authors: Shrestha TB, Basel MT Tags: Methods Mol Biol Source Type: research

In Vivo Epithelial Metabolic Imaging Using a Topical Fluorescent Glucose Analog.
Abstract The demanding metabolic needs of cancer cells are met by aerobic glycolysis. While whole-body PET imaging methods exist for evaluating this metabolic response, these are not ideal for local, more detailed regions such as mucosal surfaces. Fluorescence imaging of glucose analogs with similarities to radiolabeled deoxyglucose used in PET, namely, fluorescent 2-deoxy-2-[(7-nitro-2,1,3-benzoxadiazol-4-yl)amino]-D-glucose (2-NBDG), offers such an alternative, particularly as this glucose analog may be delivered by local topical delivery. In this chapter, methods for in vivo epithelial imaging in a preclinical ...
Source: Mol Biol Cell - March 2, 2020 Category: Molecular Biology Authors: Villarreal P, Pal R, Vargas G Tags: Methods Mol Biol Source Type: research

Lipophilic Near-Infrared Dyes for In Vivo Fluorescent Cell Tracking.
Abstract Cells can be easily and noninvasively tracked in the body by labeling them with a lipophilic, near-infrared dye and using a live fluorescence imaging system to image the position of the dye in the body. Near-infrared dyes provide several advantages, primarily that tissue is mostly highly transparent to near-infrared light, resulting in clearer and more accurate images. Briefly, cells are labeled with a near-infrared dye such as DiR and injected into a disease model. The model is then imaged using the live fluorescence imaging system on an hourly and/or daily basis to track cell migration and final locatio...
Source: Mol Biol Cell - March 2, 2020 Category: Molecular Biology Authors: Basel MT Tags: Methods Mol Biol Source Type: research

Energy Transfer Systems for In Vivo Tracking.
Abstract With recent advances, fluorescent imaging has gained momentum as an important tool for in vivo imaging. FRET systems consist of molecules that absorb in the near-infrared region which are efficient candidates for in vivo imaging, basic research, and clinical applications. Nontoxic, photostable fluorophores, such as fluorescent proteins and dyes, can successfully be used to visualize spatial and temporal dynamics of living cells. Selected cells to be injected are first tagged with the FRET-based biosensor and then injected to the living animal. Then, these foreign cells in the host body can be visualized u...
Source: Mol Biol Cell - March 2, 2020 Category: Molecular Biology Authors: Neri R, Yapa AS, Bossmann SH Tags: Methods Mol Biol Source Type: research

Using Bioengineered Fluorescence for Selective In Vivo and Ex Vivo Tracking of Intestinal Organoids Derived from Human Pluripotent Stem Cells.
Abstract For current and future applications of human intestinal organoids (hIOs) to various aspects of in vivo research and their potential clinical use, an efficient noninvasive system is needed to directly visualize the stage of intestinal differentiation and graft-host interactions and for further safety monitoring and efficacy. Here, we describe a detailed method for monitoring and histologically identifying implanted hIO-expressing eGFP and mCherry fluorescence under the kidney capsule of immunodeficient mice with fluorescence imaging (FLI). We then describe the orthotropic transplantation method of hIOs and...
Source: Mol Biol Cell - March 2, 2020 Category: Molecular Biology Authors: Lee H, Son MY Tags: Methods Mol Biol Source Type: research

Using iRFP Genetic Labeling Technology to Track Tumorogenesis of Transplanted CRISPR/Cas9-Edited iPSC in Skeletal Muscle.
Abstract Tumorigenesis and attendant safety risks are significant concerns of induced pluripotent stem cell (iPSC)-based therapies. Thus, it is crucial to evaluate iPSC proliferation, differentiation, and tumor formation after transplantation. Several approaches have been employed for tracking the donor cells, including fluorescent protein and luciferase, but both have limitations. Here, we introduce a protocol using iRFP genetic labeling technology to track tumor formation of iPSCs in skeletal muscle after CRISPR/Cas9 gene editing. PMID: 32112380 [PubMed - as supplied by publisher] (Source: Mol Biol Cell)
Source: Mol Biol Cell - March 2, 2020 Category: Molecular Biology Authors: Jin Y, Shen Y, Weintraub NL, Tang Y Tags: Methods Mol Biol Source Type: research

Upconversion Fluorescent Nanoprobe for Highly Sensitive In Vivo Cell Tracking.
Abstract Near-infrared (NIR)-to-visible upconversion nanomaterials (UCNPs) used as biomedical nanoprobes have considerable advantages over the traditional used "downconversion" fluorescent dyes. Functionalized upconversion nanoparticles (UCNPs) represent high sensitivity and great biocompatibility. Cells labeled with these UCNPs can be tracked for long term in vivo. Here we describe UCNP-PEG-ARG for highly sensitive in vivo cell tracking. PMID: 32112381 [PubMed - as supplied by publisher] (Source: Mol Biol Cell)
Source: Mol Biol Cell - March 2, 2020 Category: Molecular Biology Authors: Shen S, Wang C Tags: Methods Mol Biol Source Type: research

Magnetic Resonance Imaging of Single Cells.
Abstract This chapter discusses a methodology for simultaneously imaging stem cells and endothelial cells within polysaccharide-based scaffolds for tissue engineering. These scaffolds were then implanted into nude mice. Human mesenchymal stem cells (HMSCs) were labeled with the T1-marker Gd(III)-DOTAGA-functionalized polysiloxane nanoparticles (GdNPs), whereas endothelial umbilical vein cells (HUVECs) were labeled with citrate-stabilized maghemite nanoparticles (IONPs), which predominantly shorten the T2-relaxation times of the water molecules in scaffolds and tissue. Dual cell detection was achieved by performing...
Source: Mol Biol Cell - March 2, 2020 Category: Molecular Biology Authors: Kalubowilage M, Bossmann SH Tags: Methods Mol Biol Source Type: research

MRI Tracking of Dendritic Cells Loaded with Superparamagnetic Iron Oxide Nanoparticles.
Abstract Cell tracking via MRI has drawn much attention recently for its sensitive, deep, and real-time properties and high spatial resolution. In a previous chapter, the labeling and tracking of superparamagnetic iron oxide (SPIO)-nanoparticle-loaded stem cells have been well summarized (Sykova et al., Methods Mol Biol 750:79-90, 2011). Thus, in this chapter, we will mainly focus on the tracking of SPIO-nanoparticle-labeled mouse dendritic cells by MRI and provide a detailed protocol for cell labeling and in vivo tracking by a clinical 3.0T MRI scanner. Of note, this protocol is also suitable to be applied on oth...
Source: Mol Biol Cell - March 2, 2020 Category: Molecular Biology Authors: Zhu W, Xu Y, Jin R, Wu C, Ai H Tags: Methods Mol Biol Source Type: research

Protease-Activated Sensors for In Vivo Imaging of Cell Populations.
Abstract Biosensors are important devices that can be used to obtain information from within a living organism. They can be implanted within living tissues in order to continuously monitor for changes. This allows for personalized, noninvasive medicine, since a baseline can be more accurately established and any deviations, even slight, can be detected. These devices have applications in the treatment of diseases such as diabetes and cancer, as well as the study of pathways of interest and tailored drug dosing. Proteases within the tumor microenvironment can be studied in vivo in order to indicate the effectivenes...
Source: Mol Biol Cell - March 2, 2020 Category: Molecular Biology Authors: Eschliman K, Bossmann SH Tags: Methods Mol Biol Source Type: research

Antibody-Targeted Magnetic Nanoparticles to Track Immune Cells In Vivo.
Abstract Brain tumors can prove difficult to diagnose and successfully treat. Gliomas, and in particular glioblastomas, are the most common type of primary brain tumor. The most difficult part about treating these tumors is the fact that they are able to migrate through the extracellular space inside the brain. Recurrence is also highly possible due to their invasive nature, leading to the destruction of nearby tissues. The migratory nature of these tumors makes imaging difficult. To combat this, antibodies can be conjugated to the surface of nanoparticles such as superparamagnetic iron oxide (SPIO) nanoparticles ...
Source: Mol Biol Cell - March 2, 2020 Category: Molecular Biology Authors: Eschliman K, Bossmann SH Tags: Methods Mol Biol Source Type: research

Stem Cell Tracking with Nanoparticle-Based Ultrasound Contrast Agents.
Abstract Cell therapy is revolutionizing modern medicine. To promote this emerging therapy, the ability to image and track therapeutic cells is critical to monitor the progress of the treatment. Ultrasound imaging is promising in tracking therapeutic cells but suffers from poor contrast against local tissues. Therefore, it is critical to increase the ultrasound contrast of therapeutic cells over local tissue at the injection site. Here, we describe a method to increase the ultrasound intensity of therapeutic cells with nanoparticles to make the injected therapeutic cells more visible. PMID: 32112386 [PubMed -...
Source: Mol Biol Cell - March 2, 2020 Category: Molecular Biology Authors: Chen F, Jokerst JV Tags: Methods Mol Biol Source Type: research

Gold Nanoparticles as a Computed Tomography Marker for Stem Cell Tracking.
Abstract Stem cell tracking is an essential prerequisite for effective stem cell therapy. Computed tomography (CT) imaging technique is an emerging quantitative tool to detect real time distribution of transplanted cells. Most of CT labels based on the high atomic number (Z) materials have concern over biocompatibility. The present book chapter describes a protocol for the use of biocompatible gold nanoparticles as a CT marker for efficient labeling of mesenchymal stem cells (MSCs) and subsequent cell tracking in rodent models. PMID: 32112387 [PubMed - as supplied by publisher] (Source: Mol Biol Cell)
Source: Mol Biol Cell - March 2, 2020 Category: Molecular Biology Authors: Nafiujjaman M, Kim T Tags: Methods Mol Biol Source Type: research

Comparison of the Efficacy and Sensitivity of Alternative PET Reporter Gene/PET Reporter Probe Systems That Minimize Biological Variables.
Abstract Positron emission tomography (PET) reporter genes (PRGs), when coupled with positron-emitting PET reporter probes (PRPs), are useful for tracking specific cell populations in cell-based therapies, in transgenic animal models, and in xenograft tumor progression experiments. The activities of incorporated PRGs in targeted cells can be monitored noninvasively by PET imaging in preclinical in vivo studies and clinical applications following systemic administration of the appropriate PRG. Here we describe a method that minimizes both design and variability of vector delivery vehicles for alternative PRGs and b...
Source: Mol Biol Cell - March 2, 2020 Category: Molecular Biology Authors: Xu S, Herschman HR Tags: Methods Mol Biol Source Type: research

Uncovering cell biology in the third dimension.
Abstract Developmental biology has long benefited from studies of classic model organisms. These model systems have provided the fundamental understanding of general principles of development, as well as insight into genes and signaling pathways that control unique aspects of cell fate specification and tissue morphogenesis. Because human brain development cannot be studied in vivo, scientists have relied on these model systems to study basic principles underlying the development of this complex organ as many of these genes and signaling pathways play conserved roles in human development. However, recent studies h...
Source: Mol Biol Cell - February 29, 2020 Category: Molecular Biology Authors: Robertson GL, Romero-Morales AI, Lippmann ES, Gama V Tags: Mol Biol Cell Source Type: research