Full humanization of the glycolytic pathway in Saccharomyces cerevisiae.

Although transplantation of single genes in yeast plays a key role in elucidating gene functionality in metazoans, technical challenges hamper humanization of full pathways and processes. Empowered by advances in synthetic biology, this study demonstrates the feasibility and implementation of full humanization of glycolysis in yeast. Single gene and full pathway transplantation revealed the remarkable conservation of glycolytic and moonlighting functions and, combined with evolutionary strategies, brought to light context-dependent responses.

A toolkit for rapid CRISPR-SpCas9 assisted construction of hexose-transport-deficient Saccharomyces cerevisiae strains.

Hexose transporter-deficient yeast strains are valuable testbeds for the study of sugar transport by native and heterologous transporters. In the popular Saccharomyces cerevisiae strain EBY.VW4000, deletion of 21 transporters completely abolished hexose transport.

A protocol for introduction of multiple genetic modifications in Saccharomyces cerevisiae using CRISPR/Cas9.

Here, two methods are described for efficient genetic modification of Saccharomyces cerevisiae using CRISPR/Cas9. The first method enables the modification of a single genetic locus using in vivo assembly of a guide RNA (gRNA) expression plasmid without the need for prior cloning. A second method using in vitro assembled plasmids that could contain up to two gRNAs was used to simultaneously introduce up to six genetic modifications (e.

A CRISPR/Cas9-based exploration into the elusive mechanism for lactate export in Saccharomyces cerevisiae.

CRISPR/Cas9-based genome editing allows rapid, simultaneous modification of multiple genetic loci in Saccharomyces cerevisiae. Here, this technique was used in a functional analysis study aimed at identifying the hitherto unknown mechanism of lactate export in this yeast. First, an S.

FnCpf1: a novel and efficient genome editing tool for Saccharomyces cerevisiae.

Cpf1 is a new class II family of CRISPR-Cas RNA-programmable endonucleases with unique features that make it a very attractive alternative or complement to Cas9 for genome engineering. Using constitutively expressed Cpf1 from Francisella novicida, the present study demonstrates that FnCpf1 can mediate RNA-guided DNA cleavage at targeted genomic loci in the popular model and industrial yeast Saccharomyces cerevisiae. FnCpf1 very efficiently and precisely promoted repair DNA recombination with efficiencies up to 100%.

Nanopore sequencing enables near-complete de novo assembly of Saccharomyces cerevisiae reference strain CEN.PK113-7D.

The haploid Saccharomyces cerevisiae strain CEN.PK113-7D is a popular model system for metabolic engineering and systems biology research. Current genome assemblies are based on short-read sequencing data scaffolded based on homology to strain S288C.

CRISPR/Cas9: a molecular Swiss army knife for simultaneous introduction of multiple genetic modifications in Saccharomyces cerevisiae.

A variety of techniques for strain engineering in Saccharomyces cerevisiae have recently been developed. However, especially when multiple genetic manipulations are required, strain construction is still a time-consuming process. This study describes new CRISPR/Cas9-based approaches for easy, fast strain construction in yeast and explores their potential for simultaneous introduction of multiple genetic modifications.

HLA class II expression on human epidermal Langerhans cells in situ: upregulation during the elicitation of allergic contact dermatitis.

An immunoelectron-microscopic technique was applied to investigate the localization of molecules that are involved in the elicitation of allergic contact dermatitis in human epidermal cells in situ. Langerhans cells in the epidermis of lesions showed a strongly increased cell surface expression of HLA class II molecules as compared with normal skin. In addition, a high number of intracellularly located HLA class II molecules were present in Langerhans cells of lesional epidermis, suggesting increased biosynthesis of these molecules during the elicitation process.

Internalization of MHC class I molecules is a prerequisite for endocytosis of endorphin by lymphocytes.

The nature of the interaction between gamma-type endorphins and the HLA class I molecules was studied by immunoelectronmicroscopy. The HLA molecules were not involved in the actual binding of endorphin to the cell. In contrast, for the endocytosis of gamma-endorphin, co-internalization of the HLA class I molecules is essential.

Differences in low density lipoprotein receptor expression in the suprabasal layer of normal and psoriatic epidermis.

Previous morphological experiments on the distribution of binding sites for low density lipoprotein (LDL) on normal and psoriatic epidermis in situ, done with the LDL-gold technique [Mommaas-Kienhuis AM, et al. J Invest Dermatol 89: 513-517, 1987.] showed an unequivocal correlation between the ability to bind LDL-gold complexes and the state of keratinocyte differentiation.

Ultrastructural localization of HLA-DR and HLA-DQ molecules in Langerhans cells and B cells: an immunoelectronmicroscopic study.

Using the immunoelectronmicroscopic techniques of Lowicryl embedding and ultracryomicrotomy, the intracellular distribution of HLA class II molecules was investigated on a human B cell line and on human Langerhans cells. These techniques enabled us to localize the HLA class II molecules on fixed specimens in which mobilization or clustering induced by cross linkage with antibodies is ruled out. Compared with Lowicryl embedding, the ultracryomicrotomy clearly showed more labeling.

Low density lipoprotein receptor expression on keratinocytes in normal and psoriatic epidermis.

Biochemical and morphologic studies on the interaction of low density lipoprotein (LDL) with cultured normal keratinocytes and squamous carcinoma cells have shown a negative correlation between LDL receptor activity and terminal differentiation of the epidermal cells [Ponec M et al, J Invest Dermatol 83:436-440, 1984 and Vermeer, BJ et al, J Invest Dermatol 86:195-200, 1986]. Whether such in vitro studies pertain to the epidermis in vivo is not known. To obtain information on the distribution of LDL receptors in the epidermis in situ, morphologic studies were performed using LDL-gold as an ultrastructural marker.

Culture and characterization of rat middle-ear epithelium.

This study was performed to design a method for the culture of rat middle-ear epithelium and to apply the method to investigate the characteristics of this epithelium. Culture of explants of middle-ear epithelium in the presence of the epidermal growth factor was successful, whereas serial cultivation required 3T3 feeder cells in addition to the epidermal growth factor. Cultured middle-ear epithelium was studied by phase-contrast microscopy, transmission and scanning electron microscopy, and combined light and scanning electron microscopy (LM/SEM).

Binding and internalization of low-density lipoproteins in SCC25 cells and SV40 transformed keratinocytes. A morphologic study.

Binding of low-density lipoproteins (LDL) to the plasma membrane and internalization of low-density lipoprotein receptor complexes were investigated in an epithelial tumor cell derived from the tongue (SCC25) and in SV40-transformed keratinocytes (SVK14 cells). For light microscopic studies an immunofluorescence technique with antiapoprotein B as well as conjugation procedure by which a fluorochrome 1,1'-dioctadecyl-3,3,3',3'-tetramethyl-indocarbocyanide (DIL) was conjugated with LDL (LDL-DIL) was used. Binding of LDL to the plasma membrane at 4 degrees C was observed in most SCC25 cells but not in SVK14 cells.

Morphological studies on cellular detachment induced by antibody reactions directed against membrane associated antigens. An ultrastructural study.

The skin explant model was used to determine the effect of antibody reactions against membrane associated antigens on normal human keratinocytes. Addition of specific allo-antibodies against HLA class I antigens induced characteristic changes in the cells on the outermost region of the explant-outgrowth. A disorganization of the filopodia of these cells occurred and the edges of the cellular border were lifted from the substratum.

Calcium-mediated regulation of the low density lipoprotein receptor and intracellular cholesterol synthesis in human epidermal keratinocytes.

Unlike cells cultured under physiological Ca2+ concentrations (1-2 mM), keratinocytes cultured in media containing Ca2+ in low concentrations (less than 0.1 mM) do not stratify. The latter cells also differ with respect to several features of the regulation of cholesterol synthesis.

Modulation of low density lipoprotein receptor activity in squamous carcinoma cells by variation in cell density.

Morphological and biochemical studies on low density lipoprotein (LDL) receptor metabolism were performed in squamous carcinoma cells (SCC-15 and SCC-12F2). Modulation of terminal differentiation was achieved by culturing these cells at different cell densities. Studies on these cells cultured at low density (hardly any terminal differentiation) showed the following results: High affinity binding of LDL was excessive; LDL binding to SCC-15 cells was twice as high as that in SCC-12F2 cells and in fibroblasts.

Conjugates of colloidal gold with native and acetylated low density lipoproteins for ultrastructural investigations on receptor-mediated endocytosis by cultured human monocyte-derived macrophages.

The morphological aspects of the binding and internalization of low density lipoproteins (LDL) and acetylated low density lipoproteins (AcLDL) by cultured human monocyte-derived macrophages were investigated. For this purpose, LDL and AcLDL were conjugated to 20 nm colloidal gold particles. After incubation of the cells with the conjugated lipoproteins at 4 degrees C some LDL- or AcLDL-gold complexes were found to be attached to the cell surface, but without characteristic localization.

Inhibition of fermentation and growth in batch cultures of the yeast Brettanomyces intermedius upon a shift from aerobic to anaerobic conditions (Custers effect).

Aerobic growth of the yeast Brettanomyces intermedius CBS 1943 in batch culture on a medium containing glucose and yeast extract proceeded via a characteristic pattern. In the first phase of growth glucose was fermented to nearly equal amounts of ethanol and acetic acid. After glucose depletion, growth continued while the ethanol produced in the first phase was almost quantitatively converted to acetic acid.

Morphological studies on the binding of low-density lipoproteins and acetylated low-density lipoproteins to the plasma membrane of cultured monocytes.

Binding of low density lipoproteins (LDL) and acetyl-LDL to the plasma membrane of cultured swine monocytes was investigated by immunofluorescent and immunoelectron microscopy. Binding sites for native LDL, visualized on both the light microscopical and the ultrastructural level, were found to be comparable to those of cultured human fibroblasts. These techniques, however, failed to reveal binding of acetyl-LDL to the cell surface.