Antibody-free magnetic cell sorting of genetically modified primary human CD4+ T cells by one-step streptavidin affinity purification.

Existing methods for phenotypic selection of genetically modified mammalian cells suffer disadvantages of time, cost and scalability and, where antibodies are used to bind exogenous cell surface markers for magnetic selection, typically yield cells coated with antibody-antigen complexes and beads. To overcome these limitations we have developed a method termed Antibody-Free Magnetic Cell Sorting in which the 38 amino acid Streptavidin Binding Peptide (SBP) is displayed at the cell surface by the truncated Low Affinity Nerve Growth Receptor (LNGFRF) and used as an affinity tag for one-step selection with streptavidin-conjugated magnetic beads. Cells are released through competition with the naturally occurring vitamin biotin, free of either beads or antibody-antigen complexes and ready for culture or use in downstream applications. Antibody-Free Magnetic Cell Sorting is a rapid, cost-effective, scalable method of magnetic selection applicable to either viral transduction or transient transfection of cell lines or primary cells. We have optimised the system for enrichment of primary human CD4+ T cells expressing shRNAs and exogenous genes of interest to purities of >99%, and used it to isolate cells following Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/Cas9 genome editing

The disruption of proteostasis in neurodegenerative diseases

Cells count on surveillance systems to monitor and protect the cellular proteome which, besides being highly heterogeneous, is constantly being challenged by intrinsic and environmental factors. In this context, the proteostasis network (PN) is essential to achieve a stable and functional proteome. Disruption of the PN is associated with aging and can lead to and/or potentiate the occurrence of many neurodegenerative diseases (ND). This not only emphasizes the importance of the PN in health span and aging but also how its modulation can be a potential target for intervention and treatment of human diseases.info:eu-repo/semantics/publishedVersio

Yki/YAP, Sd/TEAD and Hth/MEIS Control Tissue Specification in the Drosophila Eye Disc Epithelium

During animal development, accurate control of tissue specification and growth are critical to generate organisms of reproducible shape and size. The eye-antennal disc epithelium of Drosophila is a powerful model system to identify the signaling pathway and transcription factors that mediate and coordinate these processes. We show here that the Yorkie (Yki) pathway plays a major role in tissue specification within the developing fly eye disc epithelium at a time when organ primordia and regional identity domains are specified. RNAi-mediated inactivation of Yki, or its partner Scalloped (Sd), or increased activity of the upstream negative regulators of Yki cause a dramatic reorganization of the eye disc fate map leading to specification of the entire disc epithelium into retina. On the contrary, constitutive expression of Yki suppresses eye formation in a Sd-dependent fashion. We also show that knockdown of the transcription factor Homothorax (Hth), known to partner Yki in some developmental contexts, also induces an ectopic retina domain, that Yki and Scalloped regulate Hth expression, and that the gain-of-function activity of Yki is partially dependent on Hth. Our results support a critical role for Yki- and its partners Sd and Hth - in shaping the fate map of the eye epithelium independently of its universal role as a regulator of proliferation and survival

Engineering T cells for cancer therapy

It is generally accepted that the immune system plays an important role in controlling tumour development. However, the interplay between tumour and immune system is complex, as demonstrated by the fact that tumours can successfully establish and develop despite the presence of T cells in tumour. An improved understanding of how tumours evade T-cell surveillance, coupled with technical developments allowing the culture and manipulation of T cells, has driven the exploration of therapeutic strategies based on the adoptive transfer of tumour-specific T cells. The isolation, expansion and re-infusion of large numbers of tumour-specific T cells generated from tumour biopsies has been shown to be feasible. Indeed, impressive clinical responses have been documented in melanoma patients treated with these T cells. These studies and others demonstrate the potential of T cells for the adoptive therapy of cancer. However, the significant technical issues relating to the production of natural tumour-specific T cells suggest that the application of this approach is likely to be limited at the moment. With the advent of retroviral gene transfer technology, it has become possible to efficiently endow T cells with antigen-specific receptors. Using this strategy, it is potentially possible to generate large numbers of tumour reactive T cells rapidly. This review summarises the current gene therapy approaches in relation to the development of adoptive T-cell-based cancer treatments, as these methods now head towards testing in the clinical trial setting

Identification of Retinal Transformation Hot Spots in Developing Drosophila Epithelia

Background: The retinal determination (RD) network is an evolutionarily conserved regulatory circuit that governs early events in the development of eyes throughout the animal kingdom. Ectopic expression of many members of this network leads to the transformation of non-retinal epithelia into eye tissue. An often-overlooked observation is that only particular cell-populations within a handful of tissues are capable of having their primary developmental instructions superseded and overruled. Methodology/Preliminary Findings: Here we confirm that indeed, only a discrete number of cell populations within the imaginal discs that give rise to the head, antenna, legs, wings and halteres have the cellular plasticity to have their developmental fates altered. In contrast to previous reports, we find that all transformable cell populations do not lie within the TGFb or Hedgehog signaling domains. Additionally neither signaling cascade alone is sufficient for non-retinal cell types to be converted into retinal tissue. The transformation ‘‘hot spots’ ’ that we have identified appear to coincide with several previously defined transdetermination ‘‘weak spots’’, suggesting that ectopic eye formation is less the result of one network overriding the orders of another, as previously thought, but rather is the physical manifestation of redirecting cell populations of enormous cellular plasticity. We also demonstrate that the initiation of eye formation in non-retinal tissues occurs asynchronously compared to that of the normal eye suggesting that retinal development is not under the control o

Yki/YAP, Sd/TEAD and Hth/MEIS Control Tissue Specification in the Drosophila Eye Disc Epithelium

During animal development, accurate control of tissue specification and growth are critical to generate organisms of reproducible shape and size. The eye-antennal disc epithelium of Drosophila is a powerful model system to identify the signaling pathway and transcription factors that mediate and coordinate these processes. We show here that the Yorkie (Yki) pathway plays a major role in tissue specification within the developing fly eye disc epithelium at a time when organ primordia and regional identity domains are specified. RNAi-mediated inactivation of Yki, or its partner Scalloped (Sd), or increased activity of the upstream negative regulators of Yki cause a dramatic reorganization of the eye disc fate map leading to specification of the entire disc epithelium into retina. On the contrary, constitutive expression of Yki suppresses eye formation in a Sd-dependent fashion. We also show that knockdown of the transcription factor Homothorax (Hth), known to partner Yki in some developmental contexts, also induces an ectopic retina domain, that Yki and Scalloped regulate Hth expression, and that the gain-of-function activity of Yki is partially dependent on Hth. Our results support a critical role for Yki- and its partners Sd and Hth - in shaping the fate map of the eye epithelium independently of its universal role as a regulator of proliferation and survival

Hypoxia-Induced Mitogenic Factor (HIMF/FIZZ1/RELMα) Recruits Bone Marrow-Derived Cells to the Murine Pulmonary Vasculature

. and localized to the media layer of the vessels. This finding suggests that these cells are of mesenchymal origin and differentiate toward myofibroblast and vascular smooth muscle. Structural location in the media of small vessels suggests a functional role in the lung vasculature. To examine a potential mechanism for HIMF-dependent recruitment of mesenchymal stem cells to the pulmonary vasculature, we performed a cell migration assay using cultured human mesenchymal stem cells (HMSCs). The addition of recombinant HIMF induced migration of HMSCs in a phosphoinosotide-3-kinase-dependent manner.These results demonstrate HIMF-dependent recruitment of BMD mesenchymal-like cells to the remodeling pulmonary vasculature

Tyrosine dephosphorylation of H2AX modulates apoptosis and survival decisions

Life and death fate decisions allow cells to avoid massive apoptotic death in response to genotoxic stress. Although the regulatory mechanisms and signalling pathways controlling DNA repair and apoptosis are well characterized, the precise molecular strategies that determine the ultimate choice of DNA repair and survival or apoptotic cell death remain incompletely understood. Here we report that a protein tyrosine phosphatase, EYA, is involved in promoting efficient DNA repair rather than apoptosis in response to genotoxic stress in mammalian embryonic kidney cells by executing a damage-signal-dependent dephosphorylation of an H2AX carboxy-terminal tyrosine phosphate (Y142). This post-translational modification determines the relative recruitment of either DNA repair or pro-apoptotic factors to the tail of serine phosphorylated histone H2AX (gamma-H2AX) and allows it to function as an active determinant of repair/survival versus apoptotic responses to DNA damage, revealing an additional phosphorylation-dependent mechanism that modulates survival/apoptotic decisions during mammalian organogenesis