Electrospun Aligned Coaxial Nanofibrous Scaffold for Cardiac Repair.
Abstract Cardiovascular diseases (CVDs) are one of the leading causes of mortality worldwide and a number one killer in the USA. Cell-based approaches to treat CVDs have only shown modest improvement due to poor survival, retention, and engraftment of the transplanted cells in the ischemic myocardium. Recently, tissue engineering and the use of 3D scaffolds for culturing and delivering stem cells for ischemic heart disease are gaining rapid potential. Here, we describe a protocol for the fabrication of aligned coaxial nanofibrous scaffold comprising of a polycaprolactone (PCL) core and gelatin shell. Furthermore, ...
Source: Mol Biol Cell - August 20, 2020 Category: Molecular Biology Authors: Sridharan D, Palaniappan A, Blackstone BN, Powell HM, Khan M Tags: Methods Mol Biol Source Type: research

Generation of Myocardial Ischemic Wounds and Healing with Stem Cells.
Abstract Myocardial ischemia is a common manifestation of cardiovascular diseases (CVD) that affects the health and lives of millions of people worldwide. While numerous treatment options exist that address cardiac damage after ischemic injury, none of these can repair damaged cardiac tissue. Stem cell-mediated therapy is an emerging approach for cardiac tissue regeneration that has shown promise in preclinical models and in clinical studies. However, much more research in this field must be carried out to bring effective stem cell therapies to clinical settings. This protocol discusses the methods for generation ...
Source: Mol Biol Cell - August 20, 2020 Category: Molecular Biology Authors: Rolph D, Das H Tags: Methods Mol Biol Source Type: research

Topical Adoptive Transfer of Plasmacytoid Dendritic Cells for Corneal Wound Healing.
Abstract Plasmacytoid dendritic cells (pDCs) are crucial for corneal homeostasis through secretion of various anti-angiogenic molecules and growth factors. Due to its avascular nature, only a limited number of adoptively transferred cells home to the cornea, when administered systemically. In addition, local adoptive transfer of cells poses several challenges and the clinical application of commonly used techniques is limited. Herein, we detail a novel approach for local adoptive transfer of pDCs to the cornea for the treatment of corneal wounds. This approach utilizes a commonly used fibrin sealant as a means of ...
Source: Mol Biol Cell - August 20, 2020 Category: Molecular Biology Authors: Jamali A, Kenyon BM, Ortiz G, Bayraktutar BN, Sendra VG, Hamrah P Tags: Methods Mol Biol Source Type: research

Principles Underlying Cryopreservation and Freeze-Drying of Cells and Tissues.
Abstract Cryopreservation and freeze-drying can be used to preserve cells or tissues for prolonged periods. Vitrification, or ice-free cryopreservation, is an alternative to cryopreservation that enables cooling cells to cryogenic temperatures in the absence of ice. The processing pathways involved in (ice-free) cryopreservation and freeze-drying of cells and tissues, however, can be very damaging. In this chapter, we describe the principles underlying preservation of cells for which freezing and drying are normally lethal processes as well as for cells that are able to survive in a reversible state of suspended a...
Source: Mol Biol Cell - August 18, 2020 Category: Molecular Biology Authors: Wolkers WF, Oldenhof H Tags: Methods Mol Biol Source Type: research

Principles of Ice-Free Cryopreservation by Vitrification.
Abstract Vitrification is an alternative to cryopreservation by freezing that enables hydrated living cells to be cooled to cryogenic temperatures in the absence of ice. Vitrification simplifies and frequently improves cryopreservation because it eliminates mechanical injury from ice, eliminates the need to find optimal cooling and warming rates, eliminates the importance of differing optimal cooling and warming rates for cells in mixed cell type populations, eliminates the need to find a frequently imperfect compromise between solution effects injury and intracellular ice formation, and can enable chilling injury...
Source: Mol Biol Cell - August 18, 2020 Category: Molecular Biology Authors: Fahy GM, Wowk B Tags: Methods Mol Biol Source Type: research

Mathematical Modeling and Optimization of Cryopreservation in Single Cells.
Abstract Cryobiology is a multiscale and interdisciplinary field. The scope and scale of interactions limit the gains that can be made by one theory or experiment alone. Because of this, modeling has played a critical role in both explaining cryobiological phenomena and predicting improved protocols. Modeling facilitates understanding of the biophysical and some of the biochemical mechanisms of damage during all phases of cryopreservation including CPA equilibration and cooling and warming. Moreover, as a tool for optimization of cryopreservation protocols, modeling has yielded many successes. Modern cryobiologica...
Source: Mol Biol Cell - August 18, 2020 Category: Molecular Biology Authors: Benson JD Tags: Methods Mol Biol Source Type: research

Mathematical Modeling of Protectant Transport in Tissues.
Abstract Mass transfer of protectant chemicals is a fundamental aspect of cryopreservation and freeze-drying protocols. As such, mass transfer modeling is useful for design of preservation methods. Cell membrane transport modeling has been successfully used to guide design of preservation methods for isolated cells. For tissues, though, there are several mass transfer modeling challenges that arise from phenomena associated with cells being embedded in a tissue matrix. Both cells and the tissue matrix form a barrier to the free diffusion of water and protective chemicals. Notably, the extracellular space becomes i...
Source: Mol Biol Cell - August 18, 2020 Category: Molecular Biology Authors: Warner RM, Higgins AZ Tags: Methods Mol Biol Source Type: research

High-Speed Video Cryomicroscopy for Measurement of Intracellular Ice Formation Kinetics.
Abstract Quantitative information about the kinetics and cumulative probability of intracellular ice formation is necessary to develop minimally damaging freezing procedures for the cryopreservation of cells and tissues. Conventional cryomicroscopic assays, which rely on indirect evidence of intracellular freezing (e.g., opacity changes in the cell cytoplasm), can yield significant errors in the estimated kinetics. In contrast, the formation and growth of intracellular ice crystals can be accurately detected using temporally resolved imaging methods (i.e., video recording at sub-millisecond resolution). Here, deta...
Source: Mol Biol Cell - August 18, 2020 Category: Molecular Biology Authors: Karlsson JOM Tags: Methods Mol Biol Source Type: research

Use of In Situ Fourier Transform Infrared Spectroscopy in Cryobiological Research.
Abstract In this chapter, we describe how Fourier transform infrared spectroscopy (FTIR) can be applied in cryobiological research to study: structure and thermal properties of biomolecules in cells and tissues, physical properties of cryopreservation and freeze-drying formulations, and permeation of molecules into cells and tissues. An infrared spectrum gives information about characteristic molecular vibrations of specific groups in molecules, whereas the temperature dependence of specific infrared bands may reveal information about conformational and phase changes. Infrared spectroscopy is minimally invasive an...
Source: Mol Biol Cell - August 18, 2020 Category: Molecular Biology Authors: Wolkers WF, Oldenhof H Tags: Methods Mol Biol Source Type: research

Raman Cryomicroscopic Imaging and Sample Holder for Spectroscopic Subzero Temperature Measurements.
Abstract Raman spectroscopy has been gaining in popularity for noninvasive analysis of single cells. Raman spectra and images deliver meaningful information regarding the biochemical, biophysical, and structural properties of cells in various states. Low-temperature Raman spectroscopy has been applied to verify the presence of ice inside a frozen cell and to illustrate the distribution of both penetrating and non-penetrating cryoprotectants. This chapter delineates Raman cryomicroscopic imaging of single cells as well as sample handling for spectroscopic measurements at subzero temperature. The experimental s...
Source: Mol Biol Cell - August 18, 2020 Category: Molecular Biology Authors: Yu G, Li R, Hubel A Tags: Methods Mol Biol Source Type: research

Frozen Blood Reserves.
Abstract Frozen blood reserves are an important component in meeting blood needs. The idea behind a frozen blood reserve is twofold: to freeze units of rare blood types for later use by patients with special transfusion needs and for managing special transfusion circumstances. The permeating additive glycerol is used as a cryoprotectant to protect red blood cells (RBCs) from freezing damage. The use of thawed RBCs has been hampered by a 24-h outdating period due to the potential bacterial contamination when a functionally open system is used for addition and removal of the glycerol. The introduction of an automate...
Source: Mol Biol Cell - August 18, 2020 Category: Molecular Biology Authors: Lagerberg JW Tags: Methods Mol Biol Source Type: research

Chemically Defined, Clinical-Grade Cryopreservation of Human Adipose Stem Cells.
glu A Abstract Adipose-derived stem cells (ASCs) reside in the stromal compartment of adipose tissue and can be easily harvested in large quantities through a clinically safe liposuction procedure. ASCs do not induce immunogenic reactions and rather exert immunosuppressive effects. Therefore, they can be used for both autologous and allogeneic transplantations. They hold great promise for cell-based therapies and tissue engineering. A prerequisite to the realization of this promise is the development of successful cryopreservation methods for ASCs. In this chapter, we describe a xeno-free- and chemically defined c...
Source: Mol Biol Cell - August 18, 2020 Category: Molecular Biology Authors: López M, Eroglu A Tags: Methods Mol Biol Source Type: research

Chemically Defined and Xeno-Free Cryopreservation of Human-Induced Pluripotent Stem Cells.
Abstract Human-induced pluripotent stem cells (hiPSCs) can be derived from a variety of biopsy samples and have an unlimited capacity for self-renewal and differentiation into almost any cell type in the body. Therefore, hiPSCs offer unprecedented opportunities for patient-specific cell therapies, modeling of human diseases, biomarker discovery, and drug testing. However, clinical applications of hiPSCs require xeno-free and, ideally, chemically defined methods for their generation, expansion, and cryopreservation. In this chapter, we present a chemically defined and xeno-free slow freezing method for hiPSCs along...
Source: Mol Biol Cell - August 18, 2020 Category: Molecular Biology Authors: Seremak J, Eroglu A Tags: Methods Mol Biol Source Type: research

Protocol for Cryopreservation of Endothelial Monolayers.
Abstract One of the major challenges in the preservation of complex tissues is the cryosensitivity of the endothelium, the single layer of cells lining blood vessels, corneas, and other tissues. The increasing importance of endothelial monolayers in tissue-engineered constructs for transplantation and research warrants the need to develop protocols for the successful cryopreservation of cells in monolayers. In this chapter, we describe a recently published cryopreservation protocol that we developed based on examination of various factors that influence the post-thaw recovery of endothelial monolayers. To efficien...
Source: Mol Biol Cell - August 18, 2020 Category: Molecular Biology Authors: Marquez-Curtis LA, Eskandari N, McGann LE, Elliott JAW Tags: Methods Mol Biol Source Type: research

Vitrification of Heart Valve Tissues.
Abstract Application of the original vitrification protocol used for pieces of heart valves to intact heart valves has evolved over time. Ice-free cryopreservation by Protocol 1 using VS55 is limited to small samples (1-3 mL total volume) where relatively rapid cooling and warming rates are possible. VS55 cryopreservation typically provides extracellular matrix preservation with approximately 80% cell viability and tissue function compared with fresh untreated tissues. In contrast, ice-free cryopreservation using VS83, Protocols 2 and 3, permits preservation of large samples (80-100 mL total volume) with...
Source: Mol Biol Cell - August 18, 2020 Category: Molecular Biology Authors: Brockbank KGM, Chen Z, Greene ED, Campbell LH Tags: Methods Mol Biol Source Type: research

Cryopreservation of Plant Cell Lines Using Alginate Encapsulation.
Abstract Plant cell cultures consist of single cells or cell clusters growing as callus or suspension. Such cell cultures may be able to produce secondary metabolites and/or possess embryogenic potential. Therefore, they can be used for very different purposes in research, biotechnological applications, as well as for plant propagation. Cryopreservation is the only practical method to preserve such cultures until they are needed. Different cryopreservation approaches that have been developed for differentiated plant tissues including slow freezing, vitrification, and encapsulation/dehydration have also been applie...
Source: Mol Biol Cell - August 18, 2020 Category: Molecular Biology Authors: Schumacher HM, Westphal M, Heine-Dobbernack E Tags: Methods Mol Biol Source Type: research

Freeze-Drying of Lactic Acid Bacteria: A Stepwise Approach for Developing a Freeze-Drying Protocol Based on Physical Properties.
Abstract Freeze-drying or lyophilization has become a reference process for preserving lactic acid bacteria. The development of stable freeze-dried lactic acid bacteria (LAB) requires maintaining the biological activity of the cells and the macroscopic porous structure while increasing the efficiency of the manufacturing process. Physical properties of protective solutions, such as glass transition and collapse temperatures, are key elements not only for process optimization but also for the stability of freeze-dried LAB. This chapter provides a stepwise approach for developing a protective formulation for the&nbs...
Source: Mol Biol Cell - August 18, 2020 Category: Molecular Biology Authors: Fonseca F, Girardeau A, Passot S Tags: Methods Mol Biol Source Type: research

Strand-Specific RNA-Seq Applied to Malaria Samples.
Abstract Over the past few years only, next-generation sequencing technologies became accessible and many applications were rapidly derived, such as the development of RNA-seq, a technique that uses deep sequencing to profile whole transcriptomes. RNA-seq has the power to discover new transcripts and splicing variants, single nucleotide variations, fusion genes, and mRNA levels-based expression profiles. Preparing RNA-seq libraries can be delicate and usually obligates buying expensive kits that require large amounts of stating materials. The method presented here is flexible and cost-effective. Using this method,...
Source: Mol Biol Cell - August 18, 2020 Category: Molecular Biology Authors: Lu XM, Le Roch K Tags: Methods Mol Biol Source Type: research

Quantitative Analysis of Plant miRNA Primary Transcripts.
Abstract MicroRNAs control plant development and are key regulators of plant responses to biotic and abiotic stresses. Thus, their expression must be carefully controlled since both excess and deficiency of a given microRNA may be deleterious to plant cell. MicroRNA expression regulation can occur at several stages of their biogenesis pathway. One of the most important of these regulatory checkpoints is transcription efficiency. mirEX database is a tool for exploration and visualization of plant pri-miRNA expression profiles. It includes results obtained using high-throughput RT-qPCR platform designed to monitor p...
Source: Mol Biol Cell - August 18, 2020 Category: Molecular Biology Authors: Dolata J, Zielezinski A, Stepien A, Kruszka K, Bielewicz D, Pacak A, Jarmolowski A, Karlowski W, Szweykowska-Kulinska Z Tags: Methods Mol Biol Source Type: research

A Revised Adaptation of the Smart-Seq2 Protocol for Single-Nematode RNA-Seq.
Abstract The advancement of transcriptomic studies in plant parasitic nematodes will greatly benefit from the development of single-nematode RNA-seq methods. Since many plant parasitic nematodes are obligate parasites, it is often difficult to efficiently obtain sufficient amounts of nematodes for transcriptomic studies. Here we have adapted SMART-Seq2 for single-nematode RNA-seq requiring only an individual nematode for a sample replicate. This protocol provides a detailed step-by-step procedure of the RNA-seq workflow starting from lysis of the nematode to quantification of transcripts using a user-friendly onli...
Source: Mol Biol Cell - August 18, 2020 Category: Molecular Biology Authors: Chang D, Serra L, Lu D, Mortazavi A, Dillman A Tags: Methods Mol Biol Source Type: research

Analysis of RBP Regulation and Co-regulation of mRNA 3' UTR Regions in a Luciferase Reporter System.
Abstract The luciferase reporter assay is a widely used tool to study the cis and trans factors controlling regulation of gene expression. In this assay, regulatory elements can be fused to the luciferase gene, and as a result effect protein output by changing rates of transcription, rates of translation, or mRNA stability. This protocol focuses on probing the function of RNA-binding proteins (RBPs) through their interactions with the 3' untranslated region (UTR), thus examining gene expression regulation on the mRNA level. Assessment of 3' UTR sequence requirements, as well as single and co-regulatory roles of RB...
Source: Mol Biol Cell - August 18, 2020 Category: Molecular Biology Authors: Sternburg EL, Karginov FV Tags: Methods Mol Biol Source Type: research

Extraction of Small RNAs by Titanium Dioxide Nanofibers.
Abstract MicroRNAs (miRNAs) are small RNAs, that bind to mRNA targets and regulate their translation. Functional study of miRNAs and exploration of their utility as disease markers require miRNA extraction from biological samples, which contain large amounts of interfering compounds for downstream RNA identification and quantification. The most common extraction methods employ either silica columns or TRIzol reagent, but these approaches afford low recovery for small RNAs, possibly due to their short strand lengths. Here, we describe the fabrication of titanium dioxide nanofibers and the optimal extraction conditi...
Source: Mol Biol Cell - August 18, 2020 Category: Molecular Biology Authors: Jimenez LA, Zhong W Tags: Methods Mol Biol Source Type: research

Purification and Analysis of Chloroplast RNAs in Arabidopsis.
Abstract Chloroplasts are essential semiautonomous plant organelles responsible for photosynthesis, which generates sugars and oxygen vital for the entire biosphere. Additionally, chloroplasts regulate energy production, metabolite synthesis, and stress responses in plants and algae. Chloroplasts possess a notably complex RNA metabolism that includes RNA processing, editing, splicing, and regulation by various RNA-binding proteins. Highly purified chloroplasts, free of nuclear/cytoplasmic contaminants are desirable when studying chloroplast RNA metabolism. Here, we describe an efficient protocol to obtain highly p...
Source: Mol Biol Cell - August 18, 2020 Category: Molecular Biology Authors: Wang H, Jin H Tags: Methods Mol Biol Source Type: research

Small RNA Isolation and Library Construction for Expression Profiling of Small RNAs from Neurospora crassa and Fusarium oxysporum and Analysis of Small RNAs in Fusarium oxysporum-Infected Plant Root Tissue.
Abstract Due to crucial roles in gene regulation, noncoding small RNAs (sRNAs) of 20-30 nucleotides (nt) have been intensively studied in mammals and plants and are implicated in significant diseases and metabolic disorders. Elucidation of biogenesis mechanisms and functional characterization of sRNAs is often achieved using tools such as separation of small-sized RNA and deep sequencing. Although RNA interference pathways, such as quelling and meiotic silencing, have been well-described in Neurospora crassa, knowledge of sRNAs in other filamentous fungi is still limited compared to other eukaryotes. As a prerequi...
Source: Mol Biol Cell - August 18, 2020 Category: Molecular Biology Authors: Ouyang SQ, Park G, Ji HM, Borkovich KA Tags: Methods Mol Biol Source Type: research

Studying RNA-Protein Interaction Using Riboproteomics.
Abstract Protein-protein interactions (PPI) are vital in regulating the biological and physiological functions in a given cell or organism. Proteomics, in conjunction with bioinformatic tools, represents the study involving the characterization of the protein content of the genome of a given biological system. Like PPI, an interaction between either coding or noncoding RNA and a complex set of host proteins protein plays an essential role in gene expression at translational, posttranscriptional, and epigenetic level. Although a wide range of techniques such as shotgun proteomics, MuDPIT, etc. are available for cha...
Source: Mol Biol Cell - August 18, 2020 Category: Molecular Biology Authors: Chaturvedi S, Rao ALN Tags: Methods Mol Biol Source Type: research

Small RNA Extraction and Quantification of Isolated Fungal Cells from Plant Tissue by the Sequential Protoplastation.
Abstract Small RNA (sRNA)-mediated RNA interference (RNAi) is critical for regulating both host immunity and pathogen virulence. Recent studies have revealed that RNA-silencing signals travel between different organisms and trigger gene silencing in trans, termed cross-kingdom RNAi. To investigate cross-kingdom RNAi, it is necessary to purify the fungal cells of interest from infected plants. Here, we present a method for small RNA extraction and quantification of isolated Botrytis cinerea cells from infected Arabidopsis leaves, by utilizing the differences between plant and fungal cell wall components (sequential...
Source: Mol Biol Cell - August 18, 2020 Category: Molecular Biology Authors: Cai Q, Jin H Tags: Methods Mol Biol Source Type: research

Predicting Hot Spots Using a Deep Neural Network Approach.
We present all the available resources, including code snippets, the main dataset, and the free and open-source modules/packages necessary for full replication of the protocol. The users should be able to develop an HS prediction model with accuracy, precision, recall, and AUROC of 0.96, 0.93, 0.91, and 0.86, respectively. PMID: 32804371 [PubMed - as supplied by publisher] (Source: Mol Biol Cell)
Source: Mol Biol Cell - August 18, 2020 Category: Molecular Biology Authors: Preto AJ, Matos-Filipe P, de Almeida JG, Mourão J, Moreira IS Tags: Methods Mol Biol Source Type: research

C-to-U RNA Editing: From Computational Detection to Experimental Validation.
Abstract The AID/APOBEC family of enzymes are cytidine deaminases that act upon DNA and RNA. Among APOBECs, the best characterized family member to act on RNA is the enzyme APOBEC1. APOBEC1-mediated RNA editing plays a key role in lipid metabolism and in maintenance of brain homeostasis. Editing can be easily detected in RNA-seq data as a cytosine to thymine (C-to-T) change with regard to the reference. However, there are many other sources of base conversions relative to reference, such as PCR errors, SNPs, and even DNA editing by mutator APOBECs. Furthermore, APOBEC1 exhibits disparate activity in different cell...
Source: Mol Biol Cell - August 1, 2020 Category: Molecular Biology Authors: Lerner T, Kluesner M, Tasakis RN, Moriarity BS, Papavasiliou FN, Pecori R Tags: Methods Mol Biol Source Type: research

Live-Cell Quantification of APOBEC1-Mediated RNA Editing: A Comparison of RNA Editing Assays.
Abstract APOBEC1 is a member of the AID/APOBECs, a group of deaminases responsible for the editing of C>U in both DNA and RNA. APOBEC1 is physiologically involved in C>U RNA editing: while hundreds of targets have been discovered in mice, in humans the only well-characterized target of APOBEC1 is the apolipoprotein B (ApoB) transcript. APOBEC1 edits a CAA codon into a stop codon, which causes the translation of a truncated form of ApoB. A number of assays have been developed to investigate this process. Early assays, poisoned primer extension and Sanger sequencing, have focused on accuracy and sensitivity bu...
Source: Mol Biol Cell - August 1, 2020 Category: Molecular Biology Authors: Chieca M, Torrini S, Conticello SG Tags: Methods Mol Biol Source Type: research

MicroRNA Editing Detection and Function: A Combined In Silico and Experimental Approach for the Identification and Validation of Putative Oncogenic Targets.
Abstract MicroRNAs (miRNAs) are a class of ~22 nt noncoding RNAs playing essential roles in the post-transcriptional regulation of gene expression, cell proliferation, and cell differentiation and are often found deregulated in several diseases including cancer.The A-to-I RNA editing, mediated by ADAR enzymes, is a diffuse post-transcriptional mechanism that converts the genetically coded adenosine (A) into inosine (I) at the RNA level. Among different RNA targets, the ADAR enzymes can also edit miRNA precursors. Specifically, a single nucleotide change (A/I) lying within the mature miRNA can alter ...
Source: Mol Biol Cell - August 1, 2020 Category: Molecular Biology Authors: Tassinari V, Cesarini V, Silvestris DA, Scafidi A, Cucina L, Gallo A Tags: Methods Mol Biol Source Type: research

Collagen, stiffness, and adhesion: the evolutionary basis of vertebrate mechanobiology.
Abstract The emergence of collagen I in vertebrates resulted in a dramatic increase in the stiffness of the extracellular environment, supporting long-range force propagation and the development of low-compliant tissues necessary for the development of vertebrate traits including pressurized circulation and renal filtration. Vertebrates have also evolved integrins that can bind to collagens, resulting in the generation of higher tension and more efficient force transmission in the extracellular matrix. The stiffer environment provides an opportunity for the vertebrates to create new structures such as the stress f...
Source: Mol Biol Cell - August 1, 2020 Category: Molecular Biology Authors: Tang VW Tags: Mol Biol Cell Source Type: research

Established Biomarkers of Chronic Obstructive Pulmonary Disease Reflect Skeletal Muscle Integrity's Response to Exercise in an Animal Model of Pulmonary Emphysema.
PMID: 32735164 [PubMed - as supplied by publisher] (Source: Mol Biol Cell)
Source: Mol Biol Cell - August 1, 2020 Category: Molecular Biology Authors: Balnis J, Vincent CE, Jones AJ, Drake LA, Coon JJ, Lee CG, Elias JA, Singer HA, Jaitovich A Tags: Am J Respir Cell Mol Biol Source Type: research

The Ill Effects of IQOS on Airway Cells: Let's Not Get Burned All Over Again.
PMID: 32735165 [PubMed - as supplied by publisher] (Source: Mol Biol Cell)
Source: Mol Biol Cell - August 1, 2020 Category: Molecular Biology Authors: McAlinden KD, Eapen MS, Lu W, Sharma P, Sohal SS Tags: Am J Respir Cell Mol Biol Source Type: research

August Highlights/Papers by Junior Investigators/NIH News.
Authors: PMID: 32735166 [PubMed - as supplied by publisher] (Source: Mol Biol Cell)
Source: Mol Biol Cell - August 1, 2020 Category: Molecular Biology Tags: Am J Respir Cell Mol Biol Source Type: research

Progestogen addition with low-dose levonorgestrel intrauterine system in menopausal hormone treatment gives less normal breast tissue proliferation than oral norethisterone acetate or medroxyprogesterone acetate.
Conclusion E2V in combination with very low serum concentrations of LNG in the IUS gives no increase in proliferation in the normal breast. PMID: 32735552 [PubMed - as supplied by publisher] (Source: Mol Biol Cell)
Source: Mol Biol Cell - July 31, 2020 Category: Molecular Biology Authors: Lundström E, Virijevic I, Söderqvist G Tags: Horm Mol Biol Clin Investig Source Type: research

Characterizing Intracellular Proteomes for Microbes: An Experimental Approach Using Label-Free Protein Quantitation.
Abstract Genomic and transcriptomic studies generate a rich source of molecular information; however, neither a static genome nor the presence of a messenger RNA provides the most direct insight into the functional state of a microbial cell at any given time. Rather, it is the ensemble of proteins (i.e., proteome) that is the primary workhorse for most biological processes that are concurrent and coordinately active in a living cell. Currently, mass-spectrometry-based (MS) technologies provide unprecedented information into the composition of the proteome, shedding light on the numerous complex biological processe...
Source: Mol Biol Cell - July 30, 2020 Category: Molecular Biology Authors: Abraham PE, Hettich RL Tags: Methods Mol Biol Source Type: research

Dynamic Flux Analysis: An Experimental Approach of Fluxomics.
Abstract Metabolic flux analysis represents an essential perspective to understand cellular physiology and offers quantitative information to guide pathway engineering. A valuable approach for experimental elucidation of metabolic flux is dynamic flux analysis, which estimates the relative or absolute flow rates through a series of metabolic intermediates in a given pathway. It is based on kinetic isotope labeling experiments, liquid chromatography-mass spectrometry (LC-MS), and computational analysis that relate kinetic isotope trajectories of metabolites to pathway activity. Herein, we illustrate the mathematic ...
Source: Mol Biol Cell - July 30, 2020 Category: Molecular Biology Authors: Xiong W, Jiang H, Maness P Tags: Methods Mol Biol Source Type: research

Connecting Microbial Genotype with Phenotype in the Omics Era.
Abstract Although rational design-based metabolic engineering has been applied widely to obtain promising microbial biocatalysts, conventional strategies such as adaptive laboratory evolution (ALE) and mutagenesis are still efficient approaches to improve microorganisms for exceptional features such as a broad spectrum of substrate utilization, robustness of cell growth, as well as high titer, yield, and productivity of the target products. In this chapter, we describe the procedure to generate mutant strains with desired phenotypes using ALE and a new mutagenesis approach of Atmosphere and Room Temperature Plasma...
Source: Mol Biol Cell - July 30, 2020 Category: Molecular Biology Authors: Yang Y, Qiu M, Yang Q, Wang Y, Wei H, Yang S Tags: Methods Mol Biol Source Type: research

Identification and Isolation of Human LGR5+ Cells Using an Antibody-Based Strategy.
Abstract Leucine-rich repeat-containing G protein-coupled receptor 5 (Lgr5) has been identified as a marker of stem cells across multiple tissues. Lgr5-expressing cells are also regulators of tissue homeostasis and wound repair, and drivers of carcinogenic progression. The majority of information about Lgr5-expressing cells derives from genetically engineered mouse models. Human studies have been limited by a lack of specific reagents and experimental procedures for the purification of these cells. We recently demonstrated that antibody-based purification can be used to obtain viable LGR5-expressing cells from hum...
Source: Mol Biol Cell - July 28, 2020 Category: Molecular Biology Authors: Dame MK, Huang S, Attili D, Spence JR, Colacino JA Tags: Methods Mol Biol Source Type: research

Immune-Mediated Specific Depletion of Intestinal Stem Cells.
Abstract Functional studies of specific stem cell populations often require depletion of tissue-specific stem cells in an in vivo model to allow for the interrogation of their contribution to the maintenance and/or regeneration of their home tissue. Depletion methods need an exquisite specificity to uniquely eliminate the target cell type. To achieve such specificity, a commonly used approach has been murine models with expression of the Diphtheria Toxin Receptor (DTR) in the cell of interest. The major caveat of using these DTR-expressing transgenic mice is the need to generate new DTR models for every new cell p...
Source: Mol Biol Cell - July 28, 2020 Category: Molecular Biology Authors: Sherman SE, Agudo J Tags: Methods Mol Biol Source Type: research

Analysis of Aged Dysfunctional Intestinal Stem Cells.
Abstract Aging is a multifactorial process. Organ maintenance and tissue regeneration are impaired upon aging mainly due to loss of stem cell function in organs that depend on stem cell in the adult. Intestine is such an organ, and upon aging intestinal regeneration is impaired due to decline of intestinal stem cell function. To determine the aging status of intestine and intestinal stem cells, histological analyses; analyses of the level of proliferation markers in tissue by immunofluorescence and/or quantitative RT-PCR; and gene expression analysis for stemness related genes in isolated crypts, intestinal stem c...
Source: Mol Biol Cell - July 28, 2020 Category: Molecular Biology Authors: Nalapareddy K, Geiger H Tags: Methods Mol Biol Source Type: research

Strategies for Measuring Induction of Fatty Acid Oxidation in Intestinal Stem and Progenitor Cells.
Abstract This protocol describes a multipronged approach that we have created to determine the transcriptional induction of fatty acid oxidation (FAO) genes in Lgr5high intestinal stem cells and a subsequent metabolomics-based approach for assessing fatty acid utilization in the mammalian intestinal crypt. More specifically, we describe methods for crypt isolation followed by a FACS-based purification of stem and progenitor populations and RNA-sequencing analysis. Using this workflow, we can determine both basal gene expression profiles of key metabolic genes as well as corresponding changes in response to altered...
Source: Mol Biol Cell - July 28, 2020 Category: Molecular Biology Authors: Cheng CW, Yilmaz OH, Mihaylova MM Tags: Methods Mol Biol Source Type: research

Visualization of Stem Cell Niche by Fluorescence Lifetime Imaging Microscopy.
Abstract Fluorescence lifetime imaging microscopy (FLIM), enabling live quantitative multiparametric analyses, is an emerging bioimaging approach in tissue engineering and regenerative medicine. When combined with stem cell-derived intestinal organoid models, FLIM allows for tracing stem cells and monitoring of their proliferation, metabolic fluxes, and oxygenation. It is compatible with the use of live Matrigel-grown intestinal organoids produced from primary adult stem cells, crypts, and transgenic Lgr5-GFP mice. In this chapter we summarize available experimental protocols, imaging platforms (one- and two-photo...
Source: Mol Biol Cell - July 28, 2020 Category: Molecular Biology Authors: Okkelman IA, Puschhof J, Papkovsky DB, Dmitriev RI Tags: Methods Mol Biol Source Type: research

Generation and Quantitative Imaging of Enteroid Monolayers.
Abstract The intestinal epithelium is a single layer of cells that plays a critical role in digestion, absorbs nutrients from food, and coordinates the delicate interplay between microbes in the gut lumen and the immune system. Epithelial homeostasis is crucial for maintaining health; disruption of homeostasis results in disorders including inflammatory bowel disease and cancer. The advent of 3D intestinal epithelial organoids has greatly advanced our understanding of the molecular underpinnings of epithelial homeostasis and disease. Recently, we developed an enteroid monolayer (2D) culture system that recapitulat...
Source: Mol Biol Cell - July 28, 2020 Category: Molecular Biology Authors: Sanman LE, Chen IW, Bieber JM, Thorne CA, Wu LF, Altschuler SJ Tags: Methods Mol Biol Source Type: research

Single-Cell Transcriptional Profiling of the Intestinal Epithelium.
Abstract Emerging single-cell technologies, like single-cell RNA sequencing (scRNA-seq), enable the study of heterogeneous biological systems at cellular resolution. By profiling the set of expressed transcripts in each cell, single-cell transcriptomics has allowed for the cataloging of the cellular constituents of multiple organs and tissues, both in health and disease. In addition, these technologies have provided mechanistic insights into cellular function, cell state transitions, developmental trajectories and lineage relationships, as well as helped to dissect complex, population-level responses to environmen...
Source: Mol Biol Cell - July 28, 2020 Category: Molecular Biology Authors: Capdevila C, Calderon RI, Bush EC, Sheldon-Collins K, Sims PA, Yan KS Tags: Methods Mol Biol Source Type: research

Single-Cell Studies of Intestinal Stem Cell Heterogeneity During Homeostasis and Regeneration.
acute;n P, Yan KS Abstract Single-cell RNA-sequencing (scRNA-seq) provides a unique opportunity to study heterogeneous cell populations within tissues, including the intestinal epithelium, to gain detailed molecular insights into their biology. Many new putative markers of intestinal stem cells and their progeny have been described using single-cell transcriptomics, which has contributed to the identification of novel subpopulations of mature cell types and insight into their developmental trajectories. This approach has revealed tremendous cellular heterogeneity within the intestinal epithelium that is concordant...
Source: Mol Biol Cell - July 28, 2020 Category: Molecular Biology Authors: Norkin M, Capdevila C, Calderon RI, Su T, Trifas M, Ordóñez-Morán P, Yan KS Tags: Methods Mol Biol Source Type: research

Primary Intestinal Epithelial Organoid Culture.
Abstract The intestinal epithelium is known as one of the most regenerative tissues in our body. The lining of the intestine is composed of a single layer of epithelial cells generated by rapidly renewing stem cells residing at the crypt bottoms, resulting in a flow of cells to the villus tips. The stereotypical crypt-villus architecture makes the intestine an ideal model for stem cell research. Based on recent advances in research of stem cell niche signals in vivo, we have established an intestinal epithelial stem cell culture method. Under this culture condition, single Lgr5+ intestinal stem cells (ISCs) or iso...
Source: Mol Biol Cell - July 28, 2020 Category: Molecular Biology Authors: Mizutani T, Clevers H Tags: Methods Mol Biol Source Type: research

In Vivo Human PSC-Derived Intestinal Organoids to Study Stem Cell Maintenance.
Abstract Human intestinal organoids (HIOs), derived from pluripotent stem cells, are a new tool to gain insights in gastrointestinal development, physiology, and associated diseases. Herein, we present a method for renal transplantation of HIOs in immunocompromised mice and subsequent analysis to study intestinal epithelial cell proliferation. In addition, we describe how to generate enteroids from transplanted HIOs. The method highlights the specific steps to successful engraftment and provides insight into the study of human intestinal stem cells. PMID: 32705643 [PubMed - in process] (Source: Mol Biol Cell)
Source: Mol Biol Cell - July 28, 2020 Category: Molecular Biology Authors: Vales S, Poling HM, Sundaram N, Helmrath MA, Mahe MM Tags: Methods Mol Biol Source Type: research

Generation of Knockout Gene-Edited Human Intestinal Organoids.
We describe the generation of knockout human embryonic stem cell lines that we then differentiate into mature human intestinal organoid tissue in Matrigel using several growth factors. We also discuss a pair of assays that can be used to study the integrity of the intestinal epithelial barrier of the human intestinal organoids under inflammatory stress conditions. PMID: 32705644 [PubMed - in process] (Source: Mol Biol Cell)
Source: Mol Biol Cell - July 28, 2020 Category: Molecular Biology Authors: Rajendra C, Wald T, Barber K, Spence JR, Fattahi F, Klein OD Tags: Methods Mol Biol Source Type: research

Direct Lineage Reprogramming of Mouse Fibroblasts to Acquire the Identity of Fetal Intestine-Derived Progenitor Cells.
Abstract Intestinal organoids are useful models for studying the characteristics of intestinal diseases and their treatment. However, a major limiting factor in their usability is the need for donor tissue fragments or pluripotent stem cells to generate the organoids. Here, we describe an approach to generate intestinal organoids from fibroblasts, a new source. We used direct reprogramming technology to generate cells with the properties of fetal intestine-derived progenitor cells (FIPCs) from mouse embryonic fibroblasts (MEFs). These induced FIPCs (iFIPCs) can give rise to cells resembling intestinal stem cells (...
Source: Mol Biol Cell - July 28, 2020 Category: Molecular Biology Authors: Miura S, Suzuki A Tags: Methods Mol Biol Source Type: research