Recruitment of TNF ligands to lipid rafts is mediated by their physical association with caveolin-1.

Activities of the tumour necrosis factor (TNF) family members are associated with their targeting to lipid rafts, specialised regions of the plasma membrane. Herein, we investigated the physical association of TNF and its family members cluster of differentiation 40 ligand (CD40L) and tumour necrosis factor-related apoptosis-inducing ligand with caveolin-1, a lipid raft resident protein. We discovered that the intracellular domains of TNF and CD40L interact with caveolin-1, and the membrane proximal region of TNF is required for the binding of caveolin-1 domains. Full-length TNF can form a complex with caveolin-1 in membrane rafts of HeLa cells, and caveolin-1 knockdown leads to impaired TNF transport to rafts. These findings provide the first evidence of a direct interaction between TNF, CD40L and caveolin-1 and suggest that caveolin-1 may be responsible for recruiting TNF to lipid rafts.

CRISPR editing of the GLI1 first intron abrogates GLI1 expression and differentially alters lineage commitment.

GLI1 is one of three GLI family transcription factors that mediate Sonic Hedgehog signaling, which plays a role in development and cell differentiation. GLI1 forms a positive feedback loop with GLI2 and likely with itself. To determine the impact of GLI1 and its intronic regulatory locus on this transcriptional loop and human stem cell differentiation, we deleted the region containing six GLI binding sites in the human GLI1 intron using CRISPR/Cas9 editing to produce H1 human embryonic stem cell (hESC) GLI1-edited clones. Editing out this intronic region, without removing the entire GLI1 gene, allowed us to study the effects of this highly complex region, which binds transcription factors in a variety of cells. The roles of GLI1 in human ESC differentiation were investigated by comparing RNA sequencing, quantitative-real time PCR (q-rtPCR), and functional assays. Editing this region resulted in GLI1 transcriptional knockdown, delayed neural commitment, and inhibition of endodermal and mesodermal differentiation during spontaneous and directed differentiation experiments. We found a delay in the onset of early osteogenic markers, a reduction in the hematopoietic potential to form granulocyte units, and a decrease in cancer-related gene expression. Furthermore, inhibition of GLI1 via antagonist GANT-61 had similar in vitro effects. These results indicate that the GLI1 intronic region is critical for the feedback loop and that GLI1 has lineage-specific effects on hESC differentiation. Our work is the first study to document the extent of GLI1 abrogation on early stages of human development and to show that GLI1 transcription can be altered in a therapeutically useful way.

Dephosphorylation of Fas-ligand and caveolin-1 is a prerequisite step in Fas-ligand - caveolin-1 complex formation and cell death stimulation.

Fas-ligand/CD178 belongs to the TNF family proteins and is the well-characterized inducer of cell death. We showed previously that the interaction of Fas-ligand and caveolin-1 is necessary for Fas-ligand translocation to rafts, and the subsequent induction of Fas-ligand-dependent cell death. Both molecules can undergo phosphorylation, however the role of the phosphorylation state of Fas-ligand and caveolin-1 in their physical association, and consequently in of Fas - mediated cell death induction is currently unknown. In this study, we show that in control cells Fas-ligand interaction with caveolin-1 is not observed, and both molecules are phosphorylated. The intracellular part of Fas-ligand was shown to form a complex with p59Fyn-kinase. Upon cell death activation, the expression and activity of p59Fyn-kinase decreases substantially, leading to the disruption of Fas-ligand - p59Fyn-kinase association, dephosphorylation of Fas-ligand and caveolin-1, and formation of a complex between them (Fas-ligand - caveolin-1). The analysis of the effects of kinase and phosphatase inhibitors revealed that phosphorylation of Fas-ligand and caveolin-1 at tyrosine residues suppressed Fas-mediated cell death. Thus, dephosphorylation of Fas-ligand and caveolin-1 is critical for triggering Fas-ligand-mediated apoptotic pathway and cell death execution.

Impairment of Fas-ligand-caveolin-1 interaction inhibits Fas-ligand translocation to rafts and Fas-ligand-induced cell death.

Fas-ligand/CD178 belongs to the TNF family proteins and can induce apoptosis through death receptor Fas/CD95. The important requirement for Fas-ligand-dependent cell death induction is its localization to rafts, cholesterol- and sphingolipid-enriched micro-domains of membrane, involved in regulation of different signaling complexes. Here, we demonstrate that Fas-ligand physically associates with caveolin-1, the main protein component of rafts. Experiments with cells overexpressing Fas-ligand revealed a FasL N-terminal pre-prolin-rich region, which is essential for the association with caveolin-1. We found that the N-terminal domain of Fas-ligand bears two caveolin-binding sites. The first caveolin-binding site binds the N-terminal domain of caveolin-1, whereas the second one appears to interact with the C-terminal domain of caveolin-1. The deletion of both caveolin-binding sites in Fas-ligand impairs its distribution between cellular membranes, and attenuates a Fas-ligand-induced cytotoxicity. These results demonstrate that the interaction of Fas-ligand and caveolin-1 represents a molecular basis for Fas-ligand translocation to rafts, and the subsequent induction of Fas-ligand-dependent cell death. A possibility of a similar association between other TNF family members and caveolin-1 is discussed.

A simple and sensitive two-tube multiplex PCR assay for simultaneous detection of ten meat species.

A sensitive and reliable technique for meat species identification is required to prevent food adulteration, particularly in meat production. This work developed an optimized multiplex PCR assay for simultaneous identification of five commonly consumed and five commonly banned meat species in meat products. We designed primers that specifically amplified mitochondrial ATPase subunit 8 gene regions of different lengths of bovine, ovine, swine, chicken, turkey, cat, dog, mouse and human DNAs. The developed multiplex PCR assay proved to be specific, sensitive down to 30pg DNA per reaction, reproducible and economical. It could be used with a variety of raw meats and processed food samples and is easily applicable in a routine laboratory analysis without specific sophisticated equipment.

Updates on the Production of Therapeutic Antibodies Using Human Hybridoma Technique.

Therapeutic antibodies are implicated into the very promising and fast growing area of pharmaceutics. Human hybridoma technology, allowing generation of natural human antibodies in a native form, seems to be the most direct way that require no additional modifications for production of therapeutic antibodies. However, technical difficulties in human hybridoma creation discovered in the 80s of the last century have switched the mainstream therapeutic antibody development into new directions like display and transgenic mice techniques. These approaches have provided remarkable achievements in antibody engineering within last 15 years, but also revealed other limitations. Thus, it is time to turn back to forgotten human hybridoma technology. In this review, we describe new advances in all components of human hybridoma technology and discuss challenges in generating novel therapeutic mABs based on hybridoma technologies.