cells r CRISPR/Cas r dCas9 r HoxB8 r gene regulation Additional supporting information may be found online in the Supporting Information section at the end of the article. Dendritic cells (DC) are regulated by intricate genetic programs, which determine DC development, subset specification, and DC functions [1,2]. Here, we provide a workflow for CRISPR/Cas9 targeting noncoding cis-regulatory elements of transcription in DC in conditionally immortalized HoxB8 multipotent hematopoietic progenitors (HoxB8 MPP). This includes (i) induced CRISPR/Cas9 editing of an IRF8 enhancer element in single-cell HoxB8 MPP and (ii) CRISPR/dCas9-mediated IRF8 promoter silencing.DC development from hematopoietic stem cells (HSC) and MPP entails sequential steps of lineage commitment and differentiation. First, HSC and MPP are committed to DC restricted common DC progenitors (CDP), which second further differentiate into specific DC subsets: antigen-presenting classical DC (cDC) type 1 and type 2 (cDC1 and cDC2, respectively) and interferon-producing plasmacytoid DC (pDC) [1][2][3]. DC differentiation involves several transcription factors and cis-acting regulatory elements that control DC fate and subset specification [1,2].
Outer membrane vesicles (OMVs), released from Gram-negative bacteria, have been attributed to intra-and interspecies communication and pathogenicity in diverse bacteria. OMVs carry various components including genetic material, toxins, signaling molecules, or proteins. Although the molecular mechanism(s) of cargo delivery is not fully understood, recent studies showed that transfer of the OMV content to surrounding cells is mediated by selective interactions. Here, we show that the phytopathogen Agrobacterium tumefaciens, the causative agent of crown gall disease, releases OMVs, which attach to the cell surface of various Gram-negative bacteria. The OMVs contain the conserved small lipoprotein Atu8019. An atu8019-deletion mutant produced wildtype-like amounts of OMVs with a subtle but reproducible reduction in cell-attachment. Otherwise, loss of atu8019 did not alter growth, susceptibility against cations or antibiotics, attachment to plant cells, virulence, motility, or biofilm formation. In contrast, overproduction of Atu8019 in A. tumefaciens triggered cell aggregation and biofilm formation. Localization studies revealed that Atu8019 is surface exposed in Agrobacterium cells and in OMVs supporting a role in cell adhesion. Purified Atu8019 protein reconstituted into liposomes interacted with model membranes and with the surface of several Gram-negative bacteria. Collectively, our data suggest that the small lipoprotein Atu8019 is involved in OMV docking to specific bacteria.
Thalidomide, a drug used for the treatment of multiple myeloma and inflammatory diseases, is also a teratogen that causes birth defects, such as limb truncations and microphthalmia, in humans. Thalidomideinduced limb truncations result from increased cell death during embryonic limb development and consequential disturbance of limb outgrowth. Here we demonstrate in primary human embryonic cells and in the chicken embryo that thalidomide-induced signaling through bone morphogenetic proteins (Bmps) protects active PTEN from proteasomal degradation, resulting in suppression of Akt signaling. As a consequence, caspase-dependent cell death is stimulated by the intrinsic and Fas death receptor apoptotic pathway. Most importantly, thalidomide-induced limb deformities and microphthalmia in chicken embryos could be rescued by a pharmacological PTEN inhibitor as well as by insulin, a stimulant of Akt signaling. We therefore conclude that perturbation of PTEN/Akt signaling and stimulation of caspase activity is central to the teratogenic effects of thalidomide.
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