Establishing a platform cell factory through engineering of yeast acetyl-CoA metabolism

Production of fuels and chemicals by industrial biotechnology requires efficient, safe and flexible cell factory platforms that can be used for production of a wide range of compounds. Here we developed a platform yeast cell factory for efficient provision of acetyl-CoA that serves as precursor metabolite for a wide range of industrially interesting products. We demonstrate that the platform cell factory can be used to improve the production of α-santalene, a plant sesquiterpene that can be used as a perfume by four-fold. This strain would be a useful tool to produce a wide range of acetyl-CoA-derived products.     

DNA fingerprinting—a valuable new technique for the characterisation of cell lines

DNA fingerprinting is an important new development for the authentication of cell lines. Multilocus methods such as those developed by Alec Jeffreys provide information on a wide range of genetic loci throughout the human genome and thus give a useful genetic “snap-shot” of a cell culture. Our work has shown that Jeffreys multilocus fingerprinting method can be applied to cell lines from a wide range of animals including reptiles, birds, fish and diverse mammals. It can also differentiate very closely related cell lines including those from the same mouse strain. Routine fingerprint analysis has enabled an unprecedented level of confidence in the consistency of cell stocks. Our results demonstrate that this straightforward method represents a powerful and readily interpreted system for cell authentication and exclusion of cross-contamination.     

Mag fura staining of fungi

Mag fura is a fluorescent stain that can be used to identify those parts of filamentous fungi that have active cell membranes. As the stain is held within the chitin of the cell wall, where it responds to locally high concentrations of divalent ions being transported across the cell membrane. This has now been tested on a broad range of fungi, including four filamentous species and three yeasts. The characteristic response of the stain appeared in areas of the cell wall where chitin might be localised. This supports the hypothesis, as well as extending the range of use of this stain.     

Electroporation and ultrasound enhanced non-viral gene delivery in vitro and in vivo

Non-viral vectors are less efficient than the use of viral vectors for delivery of genetic material to cells in vitro and especially in vivo. However, viral vectors involve the use of foreign proteins that can stimulate both the innate and acquired immune response. In contrast, plasmid DNA can be delivered without carrier proteins and is non-immunogenic. Plasmid gene delivery can be enhanced by the use of physical methods that aid the passage of the plasmid through the cell membrane. Electroporation and microbubble-enhanced ultrasound are two of the most effective physical delivery methods and these can be applied to a range of different cell types in vitro and a broad range of tissues in vivo. Both techniques also have the advantage that, unlike viral vectors, they can be used to target specific tissues with systemic delivery. Although electroporation is often the more efficient of the two, microbubble-enhanced ultrasound causes less damage and is less invasive. This review provides an introduction to the methodology and summarises the range of cells and tissues that have been genetically modified using these techniques.     

Cell Surface Profiling Using High-Throughput Flow Cytometry: A Platform for Biomarker Discovery and Analysis of Cellular Heterogeneity

Cell surface proteins have a wide range of biological functions, and are often used as lineage-specific markers. Antibodies that recognize cell surface antigens are widely used as research tools, diagnostic markers, and even therapeutic agents. The ability to obtain broad cell surface protein profiles would thus be of great value in a wide range of fields. There are however currently few available methods for high-throughput analysis of large numbers of cell surface proteins. We describe here a high-throughput flow cytometry (HT-FC) platform for rapid analysis of 363 cell surface antigens. Here we demonstrate that HT-FC provides reproducible results, and use the platform to identify cell surface antigens that are influenced by common cell preparation methods. We show that multiple populations within complex samples such as primary tumors can be simultaneously analyzed by co-staining of cells with lineage-specific antibodies, allowing unprecedented depth of analysis of heterogeneous cell populations. Furthermore, standard informatics methods can be used to visualize, cluster and downsample HT-FC data to reveal novel signatures and biomarkers. We show that the cell surface profile provides sufficient molecular information to classify samples from different cancers and tissue types into biologically relevant clusters using unsupervised hierarchical clustering. Finally, we describe the identification of a candidate lineage marker and its subsequent validation. In summary, HT-FC combines the advantages of a high-throughput screen with a detection method that is sensitive, quantitative, highly reproducible, and allows in-depth analysis of heterogeneous samples. The use of commercially available antibodies means that high quality reagents are immediately available for follow-up studies. HT-FC has a wide range of applications, including biomarker discovery, molecular classification of cancers, or identification of novel lineage specific or stem cell markers.     

Skeletal myogenesis by human embryonic stem cells

We have examined the myogenic potential of human embryonic stem （hES） cells in a xeno-transplantation animal model. Here we show that precursors differentiated from hES cells can undergo myogenesis in an adult environment and give rise to a range of cell types in the myogenic lineage. This study provides direct evidences that hES cells can regenerate both muscle and satellite cells in vivo and are another promising cell type for treating muscle degenerative disorders in addition to other myogenic cell types.     

Practical range of effective dose for Cre recombinase-expressing recombinant adenovirus without cell toxicity in mammalian cells

The site-specific recombinase Cre is valuable for regulation of gene expression not only in vitro but also in vivo. We previously reported that replication-deficient recombinant adenovirus (rAd) expressing Cre can mediate efficient and strict regulation in 100% of cultured cells. Recently, the constitutive-expression of Cre using retrovirus or lentivirus vector reportedly inhibited cell-growth, but the effect of transient Cre expression have not yet been examined. Here we showed that an excess amount of Cre produced from Cre-expressing rAd caused a deleterious effect in cells even when Cre was transiently expressed. We used three rAds carrying promoters with different activities: the SV40 early promoter (AxSVENCre), the SR alpha promoter (AxSRCre) and the CAG promoter (AxCANCre). Cell toxicity was clearly caused by Cre itself and was distinguishable from that caused by rAd virions when the cytopathic effects of these rAds were compared with that of a control virus lacking the Cre expression unit. Cre toxicity was strongly correlated with the expression level of Cre. Importantly, AxSRCre and AxCANCre gave a 60-fold range of effective MOIs ("effective range") sufficient for gene activation without causing cell toxicity from either the rAd particles or Cre itself, while AxSVENCre failed to give such a range because the expression level of Cre was too low. When Cre was tagged with a nuclear localization signal (NLS), not only its activity but also Cre toxicity was increased fourfold, and the effective range was unchanged. Therefore, AxSRNCre might be more useful to control cell toxicity from the rAd virions than AxSRCre. Cre-induced cell toxicity can be avoided by pre-examining the "effective range" using the purpose cell lines before starting experiments utilizing the experiment of Cre-expressing rAd.     

Programmed cell death in cell cultures

In plants most instances of programmed cell death (PCD) occur in a number of related, or neighbouring, cells in specific tissues. However, recent research with plant cell cultures has demonstrated that PCD can be induced in single cells. The uniformity, accessibility and reduced complexity of cell cultures make them ideal research tools to investigate the regulation of PCD in plants. PCD has now been induced in cell cultures from a wide range of species including many of the so-called model species. We will discuss the establishment of cell cultures, the fractionation of single cells and isolation of protoplasts, and consider the characteristic features of PCD in cultured cells. We will review the wide range of methods to induce cell death in cell cultures ranging from abiotic stress, absence of survival signals, manipulation of signal pathway intermediates, through the induction of defence-related PCD and developmentally induced cell death.     

Geometry-induced inhomogeneity of distribution of cell adhesion molecules along branching processes

There is evidence that inhomogeneity of lateral distribution of cell adhesion molecules (CAM) in the plasma membrane is crucial for cell motility and growth. It is known that the cells move when the cell-substratum adhesiveness is within a certain critical range. We have carried out simulation studies of the factors governing inhomogeneous CAM distribution along uniform and branching cylinder-shaped cell processes on a flat substrate evenly covered with a ligand. Our model included the following mechanisms: intracellular transportation of CAM to an active (growing) part of the cell, installation in the plasma membrane, lateral diffusive redistribution of mobile CAM, formation and dissociation of CAM/ligand complexes, and CAM internalization by endocytosis. Since the rate of growth is two and one order of magnitude slower than the rate of trafficking and lateral diffusion, respectively, we analyzed steady distributions of CAM. We showed that interplay of the mechanisms included in the model may lead to the occurrence of CAM distributions with inhomogeneity, whose range is critical for translocation of an active part of the cell. It was shown that a difference in the diameters of asymmetric sister branches can cause different inhomogeneous distributions of CAM along these branches and thus can define conditions of their growth. Depending on the branching geometry, one or both sister branches may appear within this critical range, and a difference in the diameters may define a difference in the rate of growth and, correspondingly, in the length. This may constitute the basis for self-control of neurite outgrowth.     

Cell-biological applications of transfected-cell microarrays

Cell microarrays are a recent addition to the set of tools available for functional genomic studies. Each cell microarray is a slide with thousands of cell clusters that are each transfected with a defined DNA, which directs either the overproduction or the inhibition of a particular gene product. By using a range of detection assays, the phenotypic consequences of perturbing each gene in mammalian cells can be probed in a systematic, high-throughput fashion. Combining well-established methods for cellular investigation with the miniaturization and multiplexing capabilities of microarrays, cell arrays are a versatile tool that can be useful in many cell-biological applications.     

Control of Retinal Sensitivity : II. Lateral Interactions at the Outer Plexiform Layer

Test stimuli, presented at the center of the bipolar cell receptive field, spanning less than 2 log units of intensity, elicit the full range of graded response. The intensity range of test stimuli that elicits the graded response depends upon the background conditions. A higher range of log test intensities is required to elicit the graded bipolar response in the presence of surround backgrounds. But surround backgrounds can also serve to unsaturate the bipolar response and thereby increase sensitivity under certain conditions. The results suggest that a second stage of sensitivity-control is mediated by the horizontal cell system at the outer plexiform layer, concatenated with the effects of adaptation in the photoreceptors.     

Successful control of viruses by NK cells--a balance of opposing forces?

Natural killer (NK) cells play a crucial role in limiting the severity of diseases caused by a range of viruses. Recent data have shown that the effector functions of NK cells can be specifically stimulated when NK cell activation receptors engage cellular major histocompatibility complex (MHC) class I-like ligands induced after infection or by specific viral gene products. However, to counter this NK cell response viruses have evolved an array of strategies to subvert efficient NK cell activation. These data indicate that the balance of host NK cell responses and viral NK cell escape mechanisms can be strategically poised as each strives for survival.     

Tracheotomy: a method for transplantation of stem cells to the lung

Lung disease is a leading cause of death and likely to become an epidemic given increases in pollution and smoking worldwide. Advances in stem cell therapy may alleviate many of the symptoms associated with lung disease and induce alveolar repair in adults. Concurrent with the ongoing search for stem cells applicable for human treatment, precise delivery and homing (to the site of disease) must be reassured for successful therapy. Here, I report that stem cells can safely be instilled via the trachea opening a non-stop route to the lung. This method involves a skin incision, caudal insertion of a cannula into and along the tracheal lumen, and injection of a stem cell vehicle mixture into airways of the lung. A broad range of media solutions and stabilizers can be instilled via tracheotomy, resulting in the ability to deliver a wider range of cell types. With alveolar epithelium confining these cells to the lumen, lung expansion and negative pressure during inhalation may also assist in stem cell integration. Tracheal delivery of stem cells, with a quick uptake and the ability to handle a large range of treatments, could accelerate the development of cell-based therapies, opening new avenues for treatment of lung disease.     

LKB1 loss of function studied in vivo

Recent developments have placed the serine/threonine kinase LKB1 on the crossroads linking energy metabolism, cell structure and cancer progression and that its deletion can affect tumorigenesis, metastasis, cell adhesion and polarity. LKB1 can regulate a host of different functions which all have potential to impact upon the initiation and progression of neoplastic disease. To understand the phenotypic consequences of LKB1 loss in a range of different settings, a number of animal models of loss of function have been generated and analyzed. In this review we summarize recent data generated from a range of these models, which reveal clear tissue specific differences in LKB1 function in vivo and in the consequences of its loss.     

Adhesion and spreading of cells on charged surfaces

Rival theories of cell adhesion are divided into long-range or contact, respectively. An experimental observation capable of deciding between them is the increasing attachment of negatively charged cells to surfaces of increasing negative charge density. These results would appear to refute hypotheses based solely on the balance between long-range electrostatic and dispersive forces; but they are not incompatible with bridging through polymer adsorption. The crucial difference is that negative groups on a surface are necessarily repulsive to the cell at long range, but at contact range they can strongly bind polarizable or amphoteric links of the polymer chains in the cell coat.     

Monoclonal antibodies block cell-cell adhesion in Dictyostelium discoideum

Of 39 monoclonal antibodies that bind the cell surface of aggregating Dictyostelium discoideum, 4 block 76-98% of cell-cell adhesion measured in an in vitro assay. The active antibodies all bind in the range of 10(6) antigenic sites/cell surface and react with more than one material on nitrocellulose blots prepared after polyacrylamide gel electrophoresis of whole aggregating cells in sodium dodecyl sulfate. Active antibodies can by grouped into two classes, each with two very similar members. Class I binds several molecules that are prominent in aggregating cells but scarce or undetectable in vegetative cells, blocks cell adhesion only in the presence of EDTA, and has no detectable effect on cell morphology. Class II binds a wide range of molecules present in both vegetative and aggregating cells, inhibits adhesion as well in the absence as in the presence of EDTA, and reversibly alters cell shape.     

A finite element model of cell deformation during magnetic bead twisting.

Magnetic twisting cytometry probes mechanical properties of an adherent cell by applying a torque to a magnetic bead that is tightly bound to the cell surface. Here we have used a three-dimensional finite element model of cell deformation to compute the relationships between the applied torque and resulting bead rotation and lateral bead translation. From the analysis, we computed two coefficients that allow the cell elastic modulus to be estimated from measurements of either bead rotation or lateral bead translation, respectively, if the degree of bead embedding and the cell height are known. Although computed strains in proximity of the bead can be large, the relationships between applied torque and bead rotation or translation remain virtually linear up to bead rotations of 15 degrees, above which geometrical nonlinearities become significant. This appreciable linear range stands in contrast to the intrinsically nonlinear force-displacement relationship that is observed when cells are indented during atomic force microscopy. Finally, these computations support the idea that adhesive forces are sufficient to keep the bead firmly attached to the cell surface throughout the range of working torques.     

Cell spreading: the power to simplify

A eukaryotic cell spreads over a substrate in distinct stages, with the earliest events characterized by passive adhesion and cell deformation. Recent work suggests a common physical mechanism can explain the early stages of cell spreading for a wide range of cell types and substrates.