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Methods in Molecular Biology

Won-Hee Song, Peter Sutovsky
A cell-free system using oocyte extracts is a valuable tool to study early events of animal fertilization and examine protein-protein interactions difficult to observe in whole cells. The process of postfertilization sperm mitophagy assures timely elimination of paternal, sperm-contributed mitochondria carrying potentially corrupted mitochondrial DNA (mtDNA). Cell-free systems would be especially advantageous for studying postfertilization sperm mitophagy as large amounts of oocyte extracts can be incubated with hundreds to thousands of spermatozoa in a single trial, while only one spermatozoon per zygote can be examined by whole-cell approaches...
May 11, 2018: Methods in Molecular Biology
Frédéric Auvré, Julien Coutier, Michèle T Martin, Nicolas O Fortunel
Genetic and epigenetic characterization of the large cellular diversity observed within tissues is essential to understanding the molecular networks that ensure the regulation of homeostasis, repair, and regeneration, but also pathophysiological processes. Skin is composed of multiple cell lineages and is therefore fully concerned by this complexity. Even within one particular lineage, such as epidermal keratinocytes, different immaturity statuses or differentiation stages are represented, which are still incompletely characterized...
May 8, 2018: Methods in Molecular Biology
Ehsan Taghiabadi, Bahareh Beiki, Nasser Aghdami, Amir Bajouri
Various natural and synthetic biomaterials have been applied as skin substitutes for regenerating damaged skin. Here, we describe a straightforward method for fabrication of a tissue-engineered skin substitute by seeding human fetal fibroblasts on acellular human amniotic membrane (HAM). Fetal fibroblasts are achieved from the skin of normal and non-macerated fetus of 11-14 weeks old after spontaneous pregnancy termination. Acellular HAM is obtained by separation of the outer membrane of the chorion and removing its epithelial cells...
May 8, 2018: Methods in Molecular Biology
Xiaoxiao Xiaoxiao, Shiyang Dong, Yaojiong Wu
Skin-derived precursors (SKPs) have been shown recently to be capable of inducing hair genesis and hair follicle regeneration. Here, we describe a protocol for SKP isolation and culture based on our experience and previous publications.
May 8, 2018: Methods in Molecular Biology
Meilang Xue, Suat Dervish, Christopher J Jackson
Skin epidermis is a continuous self-renewal tissue maintained by interfollicular epidermal stem cells (IESCs) that reside in the basal layer of epidermis. IESCs also contribute to the repair and regeneration of the epidermis during wound healing. The great plasticity and easy accessibility afforded by IESCs make them a promising source of stem cells for scientific research and clinical applications. Thus, simple methods to isolate and define pure and viable IESCs are a valuable resource. Here, we provide a method for isolating IESCs from human skin epidermis...
May 5, 2018: Methods in Molecular Biology
Ke Yang, Weiming Qiu, Pei-Rong Gu, Mingxing Lei
Coordinated regeneration of melanocyte stem cells (McSCs) and hair follicle stem cells (HSCs) contributes to generation of pigmented hairs. Synchronous regeneration of McSCs with activation of HSCs occurs not only during initiation of a new hair cycle in vivo but also during reconstitution of hair follicles in vitro. The duration of the quiescent state of these stem cells becomes longer and longer in lifespan of mammals, leading to a decreased regenerative ability to form hair follicles. Here, we describe methods to activate McSCs during hair follicle regeneration in vivo, and isolate melanocytes from neonatal mouse skin to generate an immortalized cell line of melanocyte progenitors in vitro, aiming to use them for studying melanogenesis and future clinical application...
April 28, 2018: Methods in Molecular Biology
Lei Chen, Daniel Radke, Shaohai Qi, Feng Zhao
Split thickness skin grafts (STSGs) are one of the standard treatments available for full thickness wound repair when full thickness grafts (FTGs) are not viable, such as in the case of wounds with large surface areas. The donor sites of STSGs may be harvested repeatedly, but STSG transplants are still limited by insufficient blood supply at the early stages of wound healing. Prevascularized human mesenchymal stem cell (hMSC) sheets may accelerate wound healing and improve regeneration by providing preformed vessel structures and angiogenic factors to overcome this limitation...
April 28, 2018: Methods in Molecular Biology
Barbora Dvořánková, Lukáš Lacina, Karel Smetana
Cancer-associated fibroblasts (CAFs) represent a crucial component of cancer microenvironment. CAFs significantly influence biological properties of various types of cancer in terms of local aggressiveness, recurrence, and metastatic behaviour. This chapter summarizes a simple protocol for isolation of normal fibroblasts and their cancer-associated counterparts from normal human skin and mucosa, respectively, as well as from samples of human tumours such as basal/squamous carcinoma, melanoma, and breast cancer, and employment of this procedure for other types of cancer is possible...
April 20, 2018: Methods in Molecular Biology
L M L Laurens, J L Olstad, D W Templeton
Accurately determining protein content is important in the valorization of algal biomass in food, feed, and fuel markets, where these values are used for component balance calculations. Conversion of elemental nitrogen to protein is a well-accepted and widely practiced method, but depends on developing an applicable nitrogen-to-protein conversion factor. The methodology reported here covers the quantitative assessment of the total nitrogen content of algal biomass and a description of the methodology that underpins the accurate de novo calculation of a dedicated nitrogen-to-protein conversion factor...
April 20, 2018: Methods in Molecular Biology
Huw Morgan, Carlotta Olivero, Girish K Patel
The cancer stem cell model states that a subset of tumor cells, called "cancer stem cells," can initiate and propagate tumor growth through self-renewal, high proliferative capacity, and their ability to recreate tumor heterogeneity. In basal cell carcinoma (BCC), we have shown that tumor cells that express the cell surface protein CD200 fulfill the cancer stem cell hypothesis. CD200+ CD45- BCC cells represent 0.05-3.96% of all BCC cells and reside in small clusters at the tumor periphery. Using a novel, reproducible in vivo xenograft growth assay, we determined that tumor-initiating cell (TIC) frequencies are approximately 1 per 1...
April 20, 2018: Methods in Molecular Biology
Tara K Sigdel, Carrie D Nicora, Wei-Jun Qian, Minnie M Sarwal
Analysis of native or endogenous peptides in biofluids can provide valuable insight into disease mechanisms. Furthermore, the detected peptides may also have utility as potential biomarkers for noninvasive monitoring of human diseases. The noninvasive nature of urine collection and the abundance of peptides in the urine make analysis by high-throughput "peptidomics" methods an attractive approach for investigating the pathogenesis of renal disease. However, urine peptidomics methodologies can be problematic with regard to difficulties associated with sample preparation...
April 6, 2018: Methods in Molecular Biology
Yaling Yu, Li Liu, Jianse Zhang, Zairong Wei, Jin Mei
The decellularized (DC) scaffolds retain three-dimensional structures for the stimulation of cell growth, with components of the extracellular matrix (ECM) relatively conserved between species. The strategy based on decellularized scaffolds provides a new way for organ regeneration, with a number of prominent advances having been reported in the past few years. While their lack of biomechanical strength and excessive degradation limit the clinical applications, therefore it is urgent to modify the DC scaffolds to improve the performance...
April 6, 2018: Methods in Molecular Biology
Lina Dagnino, Melissa Crawford
In this article, we provide a method to isolate primary epidermal melanocytes from reporter mice, which also allow targeted gene inactivation. The mice from which these cells are isolated are bred into a Rosa26mT/mG reporter background, which results in GFP expression in the targeted melanocytic cell population. These cells are isolated and cultured to >95% purity. The cells can be used for gene expression studies, clonogenic experiments, and biological assays, such as capacity for migration. Melanocytes are slow moving cells, and we also provide a method to measure motility using individual cell tracking and data analysis...
March 27, 2018: Methods in Molecular Biology
Carlotta Olivero, Huw Morgan, Girish K Patel
Epithelia are under constant threat from environmental carcinogens and none more so than squamous epithelia, which form the outermost linings of our bodies. Hence malignancies of squamous epithelia are collectively the most common cancer type and with the highest mortality, despite a constant cell turnover and only relatively rare long-lived adult tissue stem cells. Genetic analysis from SCC whole genome sequencing reveals commonality in mutated genes, despite various etiological factors. Most SCC types have been shown to exhibit hierarchical growth, in which a high frequency of cancer stem cells is associated with poor prognosis...
March 27, 2018: Methods in Molecular Biology
Xusheng Wang, Shiyang Dong, Yaojiong Wu
Recent studies have shown that epidermal stem cells derived from the epidermis of are able to form hair follicles in the presence of hair follicle-inducing cells. Here we describe the method that we have used to isolate and cultivate epidermal stem cells.
March 27, 2018: Methods in Molecular Biology
Stefano Sol, Dario Antonini, Caterina Missero
The interfollicular epidermis regenerates from a heterogeneous population of basal cells undergoing either self-renewal or terminal differentiation, thereby balancing cell loss in tissue turnover or in wound repair. In this chapter, we describe a reliable and simple method for isolating interfollicular epithelial stem cells from the skin of newborn mice or from tail and ear skin of adult mice using fluorescence-activated cell sorting (FACS). We also provide a detailed protocol for culturing interfollicular epidermal stem cells and to assess their proliferative potential and self-renewing ability...
March 27, 2018: Methods in Molecular Biology
Jaana Koistinen, Mervi Sjöblom, Kristian Spilling
Most algae do not use silicon in any form with one notable exception, diatoms. Silicon is a major constituent of diatoms. Diatoms are characterized by high growth rates and are often one of the key groups in forming algal blooms in natural waters, and as such it is an interesting group for cultivation. In this chapter we present methods for determining dissolved silica (DSi) and biogenic silica (BSi), oxide forms of silicon, based on colorimetric methods. BSi is determined after filtration and alkaline digestion...
March 27, 2018: Methods in Molecular Biology
Jaana Koistinen, Mervi Sjöblom, Kristian Spilling
Nitrogen (N) is one of the key nutrients for algal growth and is an integral part of many cellular components, for example in proteins. Being able to determine the inorganic and organic pools of N is consequently critical for algal cultivation. In this chapter we present the methods we use for determining dissolved inorganic nitrogen (DIN), dissolved organic nitrogen (DON), and particulate organic nitrogen (PON). The methods presented here for DIN rely on colorimetric methods and those of DON and PON on filtration and high temperature catalytic oxidation...
March 27, 2018: Methods in Molecular Biology
Wen Li, Shupeng Li, Yifang Li, Xiaoying Lin, Yongquan Hu, Tian Meng, Baojin Wu, Rongrong He, Du Feng
Immunofluorescence is an invaluable technique widely used in cell biology. This technique allows visualization of the subcellular distribution of different target proteins or organelles, by specific recognition of the antibody to the endogenous protein itself or to its antigen via the epitope. This technique can be used on tissue sections, cultured cells, or individual cells. Meanwhile, immunofluorescence can also be used in combination with non-antibody fluorescent staining, such as DAPI or fluorescent fusion proteins, e...
March 27, 2018: Methods in Molecular Biology
Emily M Mackay, Oliver F Bathe
In recent years, hundreds of candidate protein biomarkers have been identified using discovery-based proteomics. Despite the large number of candidate biomarkers, few proteins advance to clinical validation. Here, we describe a hypothesis driven approach to identify candidate biomarkers, previously characterized in the literature, with the highest probability of clinical applicability. A ranking method, the hypothesis directed biomarker ranking (HDBR) system, was developed to score candidate biomarkers based on seven criteria deemed important in the selection of clinically useful biomarkers...
March 23, 2018: Methods in Molecular Biology
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