Localisation of apical and baso-lateral polarity proteins is not altered in BCs.

<p>Confocal microscope images of stage 15 wild-type embryonic hindguts (BCs: white arrowheads, PCs: blue arrowheads). Anterior is left. (<b>A–A″</b>) Hindgut stained for the polarity markers Crb (magenta), Baz (grey) and Dlg (green). Only Crb is upregulated in the BCs (A and A″), while Baz (A′ and A″) and Dlg (A″) show the same amount and localisation as in the PCs. (<b>B–B″</b>) Hindgut stained for the polarity markers Crb (magenta) and <i>D</i>Par6 (green). Crb is enriched in the BCs (B and B″) but <i>D</i>Par6 localises only to the SAR as in the PCs (B′ and B″).</p

Localisation of Crb in BCs is independent from its known protein binding motifs.

<p>(<b>A</b>) Schematic representation of the Crb protein and its variants used in this study. Green rectangles: EGF-like repeats, brown hexagons: laminin A G like domains, grey bar: transmembrane domain (TM). (<b>B</b>) Amino acid sequences of the cytoplasmic tails of wild-type and mutant Crb proteins used in this study. Blue: FERM domain-binding motif, brown: PDZ domain-binding motif. Red: point mutations. (<b>C–D″</b>) Confocal microscopy images of cross sections through the large intestine of stage 15 homozygous <i>crb^8F105</i> (C–C″) and <i>foscrb_Y10A,ΔERLI</i> (D–D″) embryos stained with anti-Crb (magenta), anti-α-Spectrin (grey) and anti-Sas (green). Crb is upregulated and apically localised in the BCs (white arrowheads), but is not detectable in the PCs.</p

Crb, but not the Crb complex members Sdt and <i>D</i>PATJ, is enriched in the BCs of the embryonic hindgut.

<p>(A–D″) Confocal microscope images of stage 15 wild-type embryos. (<b>A</b>) Dorsal view of a whole mount embryo stained with anti-Crb, showing apical localisation in the PCs (blue arrowhead) and strong enrichment in the BCs (white arrowhead) of the hindgut (hg). Anterior is left. (<b>B</b>) Cross section through the large intestine stained with anti-Crb to show strong apical accumulation of Crb in the BCs (white arrowheads) and SAR localisation in the PCs (blue arrowhead). (<b>C–D″</b>) Confocal microscope images of embryonic hindguts stained with anti-Crb (magenta in C, C″, D and D″), anti-Sdt (grey in C′ and C″) and anti-<i>D</i>PATJ (grey in D′ and D″) as well as anti-Dlg (green in C″ and D″). BCs (white arrowheads) accumulate Crb (C, C″, D, D″) but not Sdt (C′ and C″) or <i>D</i>PATJ (D′, and D″). PCs (C–D″, blue arrowheads) localise Crb (C, C″, D, D″), Sdt (C′ and C″) and <i>D</i>PATJ (D′ and D″) in the SAR. (<b>E</b>) Box Plot showing the fluorescence intensity of anti-Crb staining in PCs and BCs of stage 15 wild-type embryos. The line within the box represents the median value; the whiskers represent the maximum and minimum values; *** indicate p-value <0.001, assessed by two-sided Student's <i>t-</i>test.(<b>F, G</b>) Box Plot showing the Pearson's correlation coefficient of Crb and Sdt, <i>D</i>PATJ, <i>D</i>Lin-7, Baz, <i>D</i>Par6, aPKC and Dlg in PCs (F) and BCs (G) of stage 15 wild-type embryos. The line within the box represents the median value; the whiskers represent the maximum and minimum values. Note the difference in the scale of the Pearson's correlation coefficient in F and G.</p

Loss of Crb from the BCs alters the apical membrane structure.

<p>(<b>A–B″</b>) Electron micrographs of cross sections through the large intestines of stage 16 wild-type (A–A″) and homozygous mutant <i>crb^11A22</i> (B–B″) embryos. In A and B, BCs are outlined by red lines, the rectangles indicate areas enlarged in A′, A″, B′ and B″. White arrowheads in A′, A″, B′ and B″ point to the adherens junctions between the BCs and PCs. BCs form longer and more regular microvilli than the PCs in wild-type (A–A″) and homozygous <i>crb^11A22</i> mutant embryos (B–B″). (<b>C</b>) Graph showing the mean length of microvilli in the BCs of stage 16 wild-type and <i>crb^11A22</i> mutant embryos ± standard deviation. s refers to the number of embryos analysed; n refers to the number of microvilli analysed. ***indicate p-value <0.001, assessed by two-sided Student's <i>t-</i>test.</p

Crb overexpression in PCs leads to an expansion of the apical membrane domain.

<p>(<b>A</b>) Schematic representation of the large intestine with the dorsal domain (dd) in orange, the ventral domain (vd) in blue and the BCs in green. (<b>B–B″</b>) Confocal microscope images of a stage 15 embryonic hindgut expressing <i>UAS-crb^full</i> under the control of <i>en-</i>GAL4 in the PCs of the dorsal domain. The hindgut is stained with anti-Crb (magenta), anti-<i>D</i>Par6 (grey) and anti-Dlg (green). The insets show higher magnifications of the vd (outlined by the grey dotted line), which serves as control tissue and the dd (outlined by the blue dotted line) where the altered apico-basal polarity in cells overexpressing Crb is highlighted (due to the very strong overexpression of Crb in the dd, the gain of the microscope was strongly reduced).</p

Apical localisation of Crb in BCs is independent of its known interaction partner Sdt.

<p>Confocal microscopy images of a cross section through the large intestine of a stage 15 homozygous <i>sdt^K85</i> mutant embryo stained with anti-Crb (magenta), anti-α-Spectrin (grey) and anti-Sas (green). Crb is upregulated and apically localised in the BCs (white arrowheads), but is not detectable in the SAR of the PCs (A, A″). Sas, an apical marker of the monolayered epithelial tube, is reduced in the BCs (A′, A″).</p

The Crumbs_C isoform of Drosophila

Drosophila Crumbs (Crb) is a key regulator of epithelial polarity and fulfils a plethora of other functions, such as growth regulation, morphogenesis of photoreceptor cells and prevention of retinal degeneration. This raises the question how a single gene regulates such diverse functions, which in mammals are controlled by three different paralogs. Here, we show that in Drosophila different Crb protein isoforms are differentially expressed as a result of alternative splicing. All isoforms are transmembrane proteins that differ by just one EGF-like repeat in their extracellular portion. Unlike Crb_A, which is expressed in most embryonic epithelia from early stages onward, Crb_C is expressed later and only in a subset of embryonic epithelia. Flies specifically lacking Crb_C are homozygous viable and fertile. Strikingly, these flies undergo light-dependent photoreceptor degeneration despite the fact that the other isoforms are expressed and properly localised at the stalk membrane. This allele now provides an ideal possibility to further unravel the molecular mechanisms by which Drosophila crb protects photoreceptor cells from the detrimental consequences of light-induced cell stress

Outcomes of Antifungal Prophylaxis in High-Risk Haematological Patients (AML under Intensive Chemotherapy): The SAPHIR Prospective Multicentre Study

International audienceAntifungal prophylaxis (AFP) is recommended by international guidelines for patients with acute myeloid leukaemia (AML) undergoing induction chemotherapy and allogeneic hematopoietic cell transplantation. Nonetheless, treatment of breakthrough fungal infections remains challenging. This observational, prospective, multicentre, non-comparative study of patients undergoing myelosuppressive and intensive chemotherapy for AML who are at high-risk of invasive fungal diseases (IFDs), describes AFP management and outcomes for 404 patients (65.6% newly diagnosed and 73.3% chemotherapy naïve). Ongoing chemotherapy started 1.0 ± 4.5 days before inclusion and represented induction therapy for 79% of participants. In 92.3% of patients, posaconazole was initially prescribed, and 8.2% of all patients underwent at least one treatment change after 17 ± 24 days, mainly due to medical conditions influencing AFP absorption (65%). The mean AFP period was 24 ± 32 days, 66.8% stopped their prophylaxis after the high-risk period and 31.2% switched to a non-prophylactic treatment (2/3 empirical, 1/3 pre-emptive/curative). Overall, 9/404 patients (2.2%) were diagnosed with probable or proven IFDs. During the follow-up, 94.3% showed no signs of infection. Altogether, 20 patients (5%) died, and three deaths (0.7%) were IFD-related. In conclusion, AFP was frequently prescribed and well tolerated by these AML patients, breakthrough infections incidence and IFD mortality were low and very few treatment changes were required