Advanced modular self‐inactivating lentiviral expression vectors for multigene interventions in mammalian cells and in vivo transduction

In recent years, lentiviral expression systems have gained an unmatched reputation among the gene therapy community for their ability to deliver therapeutic transgenes into a wide variety of difficult‐to‐transfect/transduce target tissues (brain, hematopoietic system, liver, lung, retina) without eliciting significant humoral immune responses. We have cloned a construction kit‐like self‐inactivating lentiviral expression vector family which is compatible to state‐of‐the‐art packaging and pseudotyping technologies and contains, besides essential cis‐acting lentiviral sequences, (i) unparalleled polylinkers with up to 29 unique sites for restriction endonucleases, many of which recognize 8 bp motifs, (ii) strong promoters derived from the human cytomegalovirus immediate‐early promoter (PhCMV) or the human elongation factor 1α (PhEF1α), (iii) PhCMV- or PPGK- (phosphoglycerate kinase promoter) driven G418 resistance markers or fluorescent protein‐based expression tracers and (iv) tricistronic expression cassettes for coordinated expression of up to three transgenes. In addition, we have designed a size‐optimized series of highly modular lentiviral expression vectors (pLenti Module) which contain, besides the extensive central polylinker, unique restriction sites flanking any of the 5′U3, R‐U5‐ψ+‐SD, cPPT‐RRE‐SA and 3′LTRΔU3 modules or placed within the 5′U3 (-78 bp) and 3′LTRΔU3 (8666 bp). pLentiModule enables straightforward cassette‐type module swapping between lentiviral expression vector family members and facilitates the design of Tat‐independent (replacement of 5′LTR by heterologous promoter elements), regulated and self‐excisable proviruses (insertion of responsive operators or LoxP in the 3′LTRΔU3 element). We have validated our lentiviral expression vectors by transduction of a variety of insect, chicken, murine and human cell lines as well as adult rat cardiomyocytes, rat hippocampal slices and chicken embryos. The novel multi‐purpose construction kit‐like vector series described here is compatible with itself as well as many other (non‐viral) mammalian expression vectors for straightforward exchange of key components (e.g. promoters, LTRs, resistance genes) and will assist the gene therapy and tissue engineering communities in developing lentiviral expression vectors tailored for optimal treatment of prominent human disease

Prospects of micromass culture technology in tissue engineering

Tissue engineering of bone and cartilage tissue for subsequent implantation is of growing interest in cranio- and maxillofacial surgery. Commonly it is performed by using cells coaxed with scaffolds. Recently, there is a controversy concerning the use of artificial scaffolds compared to the use of a natural matrix. Therefore, new approaches called micromass technology have been invented to overcome these problems by avoiding the need for scaffolds. Technically, cells are dissociated and the dispersed cells are then reaggregated into cellular spheres. The micromass technology approach enables investigators to follow tissue formation from single cell sources to organised spheres in a controlled environment. Thus, the inherent fundamentals of tissue engineering are better revealed. Additionally, as the newly formed tissue is devoid of an artificial material, it resembles more closely the in vivo situation. The purpose of this review is to provide an insight into the fundamentals and the technique of micromass cell culture used to study bone tissue engineering

State-of-the-art of 3D cultures (organs-on-a-chip) in safety testing and pathophysiology.

Integrated approaches using different in vitro methods in combination with bioinformatics can (i) increase the success rate and speed of drug development; (ii) improve the accuracy of toxicological risk assessment; and (iii) increase our understanding of disease. Three-dimensional (3D) cell culture models are important building blocks of this strategy which has emerged during the last years. The majority of these models are organotypic, i.e., they aim to reproduce major functions of an organ or organ system. This implies in many cases that more than one cell type forms the 3D structure, and often matrix elements play an important role. This review summarizes the state of the art concerning commonalities of the different models. For instance, the theory of mass transport/metabolite exchange in 3D systems and the special analytical requirements for test endpoints in organotypic cultures are discussed in detail. In the next part, 3D model systems for selected organs--liver, lung, skin, brain--are presented and characterized in dedicated chapters. Also, 3D approaches to the modeling of tumors are presented and discussed. All chapters give a historical background, illustrate the large variety of approaches, and highlight up- and downsides as well as specific requirements. Moreover, they refer to the application in disease modeling, drug discovery and safety assessment. Finally, consensus recommendations indicate a roadmap for the successful implementation of 3D models in routine screening. It is expected that the use of such models will accelerate progress by reducing error rates and wrong predictions from compound testing

Determination of absolute H atom concentrations in low-pressure flames by two-photon laser-excited fluorescence

Bittner J, Kohse-Höinghaus K, Meier U, Kelm S, Just T. Determination of absolute H atom concentrations in low-pressure flames by two-photon laser-excited fluorescence. Combustion and Flame. 1988;71(1):41-50.A calibration technique is demonstrated which allows the determination of absolute atom concentrations in flames: two-photon laser-excited fluorescence signals from known atom concentrations generated in a discharge flow reactor are related to the fluorescence signals in a flame under identical excitation and detection conditions. With this method, absolute H atom profiles in several low-pressure hydrogen-oxygen flames have been obtained. For the same flame conditions, local temperature and absolute OH concentration profiles have been determined using laser-induced fluorescence (LIF). The experimental results are compared to the predictions of a one-dimensional flame model. The application of the new calibration technique to the atom detection in hydrocarbon flames is discussed

Study of the chemotactic response of multicellular spheroids in a microfluidic device

YesWe report the first application of a microfluidic device to observe chemotactic migration in multicellular spheroids. A microfluidic device was designed comprising a central microchamber and two lateral channels through which reagents can be introduced. Multicellular spheroids were embedded in collagen and introduced to the microchamber. A gradient of fetal bovine serum (FBS) was established across the central chamber by addition of growth media containing serum into one of the lateral channels. We observe that spheroids of oral squamous carcinoma cells OSC–19 invade collectively in the direction of the gradient of FBS. This invasion is more directional and aggressive than that observed for individual cells in the same experimental setup. In contrast to spheroids of OSC–19, U87-MG multicellular spheroids migrate as individual cells. A study of the exposure of spheroids to the chemoattractant shows that the rate of diffusion into the spheroid is slow and thus, the chemoattractant wave engulfs the spheroid before diffusing through it.This work has been supported by National Research Program of Spain (DPI2011-28262-c04-01) and by the project "MICROANGIOTHECAN" (CIBERBBN, IMIBIC and SEOM). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript

Nck2 promotes human melanoma cell proliferation, migration and invasion in vitro and primary melanoma-derived tumor growth in vivo

<p>Abstract</p> <p>Background</p> <p>Nck1 and Nck2 adaptor proteins are involved in signaling pathways mediating proliferation, cytoskeleton organization and integrated stress response. Overexpression of Nck1 in fibroblasts has been shown to be oncogenic. Through the years this concept has been challenged and the consensus is now that overexpression of either Nck cooperates with strong oncogenes to transform cells. Therefore, variations in Nck expression levels in transformed cells could endorse cancer progression.</p> <p>Methods</p> <p>Expression of Nck1 and Nck2 proteins in various cancer cell lines at different stages of progression were analyzed by western blots. We created human primary melanoma cell lines overexpressing GFP-Nck2 and investigated their ability to proliferate along with metastatic characteristics such as migration and invasion. By western blot analysis, we compared levels of proteins phosphorylated on tyrosine as well as cadherins and integrins in human melanoma cells overexpressing or not Nck2. Finally, in mice we assessed tumor growth rate of human melanoma cells expressing increasing levels of Nck2.</p> <p>Results</p> <p>We found that expression of Nck2 is consistently increased in various metastatic cancer cell lines compared with primary counterparts. Particularly, we observed significant higher levels of Nck2 protein and mRNA, as opposed to no change in Nck1, in human metastatic melanoma cell lines compared with non-metastatic melanoma and normal melanocytes. We demonstrated the involvement of Nck2 in proliferation, migration and invasion in human melanoma cells. Moreover, we discovered that Nck2 overexpression in human primary melanoma cells correlates with higher levels of proteins phosphorylated on tyrosine residues, assembly of Nck2-dependent pY-proteins-containing molecular complexes and downregulation of cadherins and integrins. Importantly, we uncovered that injection of Nck2-overexpressing human primary melanoma cells into mice increases melanoma-derived tumor growth rate.</p> <p>Conclusions</p> <p>Collectively, our data indicate that Nck2 effectively influences human melanoma phenotype progression. At the molecular level, we propose that Nck2 in human primary melanoma promotes the formation of molecular complexes regulating proliferation and actin cytoskeleton dynamics by modulating kinases or phosphatases activities that results in increased levels of proteins phosphorylated on tyrosine residues. This study provides new insights regarding cancer progression that could impact on the therapeutic strategies targeting cancer.</p

Genome-wide association study identifies multiple risk loci for renal cell carcinoma

Previous genome-wide association studies (GWAS) have identified six risk loci for renal cell carcinoma (RCC). We conducted a meta-analysis of two new scans of 5,198 cases and 7,331 controls together with four existing scans, totalling 10,784 cases and 20,406 controls of European ancestry. Twenty-four loci were tested in an additional 3,182 cases and 6,301 controls. We confirm the six known RCC risk loci and identify seven new loci at 1p32.3 (rs4381241, P=3.1 × 10−10), 3p22.1 (rs67311347, P=2.5 × 10−8), 3q26.2 (rs10936602, P=8.8 × 10−9), 8p21.3 (rs2241261, P=5.8 × 10−9), 10q24.33-q25.1 (rs11813268, P=3.9 × 10−8), 11q22.3 (rs74911261, P=2.1 × 10−10) and 14q24.2 (rs4903064, P=2.2 × 10−24). Expression quantitative trait analyses suggest plausible candidate genes at these regions that may contribute to RCC susceptibility