Investigating cell uptake of guanidinium-rich RAFT polymers : impact of comonomer and monomer distribution

A range of well-defined guanidinium-rich linear polymers with demonstrable efficiency for cellular internalization were developed. A protected guanidinium-functional acrylamide monomer (di-Boc-guanidinium ethyl acrylamide, GEAdiBoc) was synthesized and then polymerized via RAFT polymerization to yield well-defined homopolymers, which were then deprotected and functionalized with a fluorescein dye to observe and quantify their cellular uptake. The cellular uptake of these homopolymers was first compared to analogous polyarginines, which are commonly used in modern drug delivery. Following this, a range of well-defined guanidinium-rich copolymers were prepared in which the monomer distribution was varied using a convenient one-pot sequential RAFT polymerization approach. Systematic quantification of the cell uptake of these compounds, supported by fluorescent confocal microscopy data, revealed that while the overall hydrophobicity of the resulting copolymers has a direct impact on the amount of copolymer taken up by cells, the distribution of monomers has an influence on both the extent of uptake and the relative extent to which each route of internalization (endocytosis vs direct translocation) is exploited

Tmem26 Is Dynamically Expressed during Palate and Limb Development but Is Not Required for Embryonic Survival

The Tmem26 gene encodes a novel protein that we have previously shown to be regulated by hedgehog signalling in the mouse limb. We now report that Tmem26 expression is spatially and temporally restricted in other regions of the mouse embryo, most notably the facial primordia. In particular, Tmem26 expression in the mesenchyme of the maxillary and nasal prominences is coincident with fusion of the primary palate. In the secondary palate, Tmem26 is expressed in the palatal shelves during their growth and fusion but is downregulated once fusion is complete. Expression was also detected at the midline of the expanding mandible and at the tips of the eyelids as they migrate across the cornea. Given the spatio-temporally restricted expression of Tmem26, we sought to uncover a functional role in embryonic development through targeted gene inactivation in the mouse. However, ubiquitous inactivation of Tmem26 led to no overt phenotype in the resulting embryos or adult mice, suggesting that TMEM26 function is dispensable for embryonic survival

Investigating Cell Uptake of Guanidinium-Rich RAFT Polymers: Impact of Comonomer and Monomer Distribution

A range of well-defined guanidinium-rich linear polymers with demonstrable efficiency for cellular internalization were developed. A protected guanidinium-functional acrylamide monomer (di-Boc-guanidinium ethyl acrylamide, GEA^diBoc) was synthesized and then polymerized via RAFT polymerization to yield well-defined homopolymers, which were then deprotected and functionalized with a fluorescein dye to observe and quantify their cellular uptake. The cellular uptake of these homopolymers was first compared to analogous polyarginines, which are commonly used in modern drug delivery. Following this, a range of well-defined guanidinium-rich copolymers were prepared in which the monomer distribution was varied using a convenient one-pot sequential RAFT polymerization approach. Systematic quantification of the cell uptake of these compounds, supported by fluorescent confocal microscopy data, revealed that while the overall hydrophobicity of the resulting copolymers has a direct impact on the amount of copolymer taken up by cells, the distribution of monomers has an influence on both the extent of uptake and the relative extent to which each route of internalization (endocytosis vs direct translocation) is exploited

The receptors CD96 and CD226 oppose each other in the regulation of natural killer cell functions

CD96, CD226 (DNAM-1) and TIGIT belong to an emerging family of receptors that interact with nectin and nectin-like proteins. CD226 activates natural killer (NK) cell-mediated cytotoxicity, whereas TIGIT reportedly counterbalances CD226. In contrast, the role of CD96, which shares the ligand CD155 with CD226 and TIGIT, has remained unclear. In this study we found that CD96 competed with CD226 for CD155 binding and limited NK cell function by direct inhibition. As a result, Cd96 -/- mice displayed hyperinflammatory responses to the bacterial product lipopolysaccharide (LPS) and resistance to carcinogenesis and experimental lung metastases. Our data provide the first description, to our knowledge, of the ability of CD96 to negatively control cytokine responses by NK cells. Blocking CD96 may have applications in pathologies in which NK cells are important

Expression of <i>Tmem26</i> in wild-type embryos by section in situ hybridisation.

<p>(<b>A–D, E–G,J</b>) radioisotopic; (<b>H,I</b>) DIG detection. (<b>A–D</b>) Transverse sections through the secondary palate at indicated stages. At 12.5 dpc and 13.5 dpc <i>Tmem26</i> is expressed in mesenchyme of the vertical palatal shelves. At 14.5 dpc the shelves have fused but the medial epithelial seam still remains and expression within the mesenchyme is apparent. By 15.5 dpc the epithelial seam is absent and <i>Tmem26</i> expression is virtually undetectable. (<b>A′–D′</b>) Corresponding serial sections stained with toluidine blue. (<b>E–G</b>) Sections through the facial prominences and primary palate region at indicated stages. <i>Tmem26</i> is not detected at 10.5 dpc but is upregulated at 11.5 dpc and 12.5 dpc. (<b>E′–G′</b>) Corresponding sections stained with toluidine blue. (<b>H</b>) Parasagittal section through the lateral hindbrain at 12.5 dpc, showing expression in the nucleus of the seventh facial nerve region within the pons. (<b>I</b>) Longitudinal section through the stomach at 11.5 dpc, showing expression in the anterior stomach mesenchyme. (<b>J</b>) Magnification of the left eyelid from <b>D</b>, (<b>J′</b>) same section counterstained with haematoxylin. 1p-primary palate, bt-mesencyme at the base of the tongue, c-cornea, el-eyelid, em-extraocular mesenchyme and developing extrinsic muscles of the eye, ep-epithelim, L-eye lens, lnp-lateral nasal prominence, n7-nucleus of the seventh facial nerve, mn-mandible, mnp-medial nasal prominence, mx-maxillary prominence, ns-nasal septum, p-palatal shelf, pd-eyelid periderm, st-stomach mesenchyme. Size bars indicate 1 mm.</p

Gene targeting strategy for the <i>Tmem26</i> locus.

<p>(<b>A</b>) Targeting construct for <i>Tmem26</i> conditional inactivation compared to the wild type allele and the floxed <i>Tmem26</i> allele after homologous recombination. In the presence of Cre recombinase, the region between <i>LoxP</i> sites will be excised, including exon 2, exon 2a (red) and the neomycin selection cassette (green). The neomycin selection cassette can be independently excised by <i>Flp</i> recombinase (<i>frt</i> sites). A Southern probe (open box) and PCR primers (small arrows) external to the targeting construct were used to screen stem cells for successful homologous recombination. An <i>Eco</i>RV site (E*) was introduced with the targeting construct and was diagnostic during Southern and PCR assays for 5′ <i>LoxP</i> integration. (<b>B</b>) Two transcripts were detected, one lacking exons 2 and 2a as predicted, and another variant in which exon 3 was also absent. In both cases exon 1 is retained, and the exons after the deleted exons are out of frame. A premature stop codon is introduced in both cases (<b>C</b>) Southern blot showing the wild type and mutant band arising from Cre-mediated excision (<i>Tmem26^Ex2−</i>).</p

Expression of <i>Tmem26</i> in wild-type embryos by whole mount in situ hybridisation analysis.

<p>(<b>A–E</b>) Whole embryos aged 11.0 dpc-14.5 dpc. No expression was detected before 11.0 dpc. (<b>F–J</b>) Forelimbs of corresponding embryos shown in <b>A–E</b>, dorsal view, limb anterior is to the top. (<b>K,L</b>) At 11.0 and 11.5 dpc <i>Tmem26</i> expression in the facial prominences is restricted primarily to mesenchyme underlying areas of facial prominence fusion and merging. (<b>M</b>) At 12.5 dpc striking expression is observed in the primary palate, and expression remains at the midline of the mandible. (<b>N</b>) After fusion is complete at 13.5 dpc, expression is restricted to maxillary tissue at the distal tip of the snout. (<b>O,P</b>) The developing secondary palate at 14.5 dpc, taken from below with the mandible removed, one showing the palatal shelves wide apart, and one as the shelves are coming together to fuse. <i>Tmem26</i> is expressed in the palatal shelves prior to fusion but downregulated upon palate fusion, most obvious at 15.5 dpc (<b>Q</b>). (<b>R</b>) Ventral and (<b>S</b>) lateral views of a 13.5 dpc genital tubercle. Dorsal-ventral and anterior-posterior axes are indicated. 1p-primary palate, 2p-secondary palatal shelves, e-external ear, em-extraoccular musculature, fl-forelimb, lnp-lateral nasal prominence, mes-medial epithelial seam, md-mandible, mnp-medial nasal prominence, mx-maxillary prominence, n-nasal opening, vcm-ventral cephalic mesenchyme.</p

Predicted gene and protein structure.

<p>(<b>A</b>) Predicted exon structure of <i>Tmem26</i>. (<b>B</b>) RT-PCR across exon2, showing the main <i>Tmem26</i> transcript incorporating exon2 only, and a minor variant generated by alternative splicing in some tissues (arrow), which incorporates an extra exon (2a). (<b>C</b>) The topographic prediction for TMEM26 generated by the program SVTtm. Other predictions vary dependent on the presence or absence of a leader sequence and the number of transmembrane domains. 1,2,3 – non-membrane loops; C-terminus, N-terminus.</p

Expression of <i>Tmem26</i> as determined by qRT-PCR.

<p>Graph shows the mean 2^−ΔCT and the standard error of the mean (SEM).</p

<i>Tmem26^{Ex2−/Ex2−}</i> mice appear phenotypically normal.

<p>(<b>A</b>) Dorsal and (<b>B</b>) ventral views of adult WT and <i>Tmem26^{Ex2−/Ex2−}</i> littermate mice. (<b>C–J</b>) Alcian blue/alizarin red staining of WT and <i>Tmem26^{Ex2−/Ex2−}</i> adult littermate skeletal preparations. (<b>C</b>) and (<b>D</b>) are at the same magnification. (<b>E,H</b>) Dorsal, (<b>F,I</b>) ventral and (<b>G,J</b>) lateral views of skull. In (<b>F,I</b>) the mandible has been removed to allow visualisation of the palate. (<b>K,L</b>) Transverse sections through the secondary palate of 15.5 dpc WT and <i>Tmem26^{Ex2−/Ex2−}</i> embryos. (<b>M</b>) Mean distance between the inner canthi of the eyes, and (<b>N</b>) mean length of the snout were compared between WT and <i>Tmem26^{Ex2−/Ex2−}</i> adult mice (n = 18 female mice, 20 male mice per genotype). (<b>O</b>) diagram showing the measurements taken. L-snout length; w-distance between the eyes. Error bars represent standard error of the mean (SEM), statistical analysis (Student's t-test) revealed no significant difference.</p