Steroid-associated hip joint collapse in bipedal emus

In this study we established a bipedal animal model of steroid-associated hip joint collapse in emus for testing potential treatment protocols to be developed for prevention of steroid-associated joint collapse in preclinical settings. Five adult male emus were treated with a steroid-associated osteonecrosis (SAON) induction protocol using combination of pulsed lipopolysaccharide (LPS) and methylprednisolone (MPS). Additional three emus were used as normal control. Post-induction, emu gait was observed, magnetic resonance imaging (MRI) was performed, and blood was collected for routine examination, including testing blood coagulation and lipid metabolism. Emus were sacrificed at week 24 post-induction, bilateral femora were collected for micro-computed tomography (micro-CT) and histological analysis. Asymmetric limping gait and abnormal MRI signals were found in steroid-treated emus. SAON was found in all emus with a joint collapse incidence of 70%. The percentage of neutrophils (Neut %) and parameters on lipid metabolism significantly increased after induction. Micro-CT revealed structure deterioration of subchondral trabecular bone. Histomorphometry showed larger fat cell fraction and size, thinning of subchondral plate and cartilage layer, smaller osteoblast perimeter percentage and less blood vessels distributed at collapsed region in SAON group as compared with the normal controls. Scanning electron microscope (SEM) showed poor mineral matrix and more osteo-lacunae outline in the collapsed region in SAON group. The combination of pulsed LPS and MPS developed in the current study was safe and effective to induce SAON and deterioration of subchondral bone in bipedal emus with subsequent femoral head collapse, a typical clinical feature observed in patients under pulsed steroid treatment. In conclusion, bipedal emus could be used as an effective preclinical experimental model to evaluate potential treatment protocols to be developed for prevention of ON-induced hip joint collapse in patients

Ir(III)-Catalyzed Direct C–H Functionalization of Arylphosphine Oxides: A Strategy for MOP-Type Ligands Synthesis

Diazo compounds as coupling partners are efficiently applied to Ir­(III)-catalyzed direct C–H functionalization of arylphosphine oxides. Involving C–H activation, carbene insertion, and tautomerism, this reaction proceeds under mild conditions, thus proving an approach to the synthesis of MOP-type ligand precursor in a single step. The utility of this transformation has been further demonstrated in ligand synthesis as well as in the construction of phosphole framework

A canonical Hh signaling pathway is required to control NB proliferation but not neuronal differentiation.

<p>(A–I′) NB clones of different genotypes: <i>wt</i> (A–A′, D–D′, and G–G′), <i>ptc^S2</i> (B–B′, E–E′, and H–H′) and <i>smo^IA3</i> (C–C′, F–F′, and I–I′) were marked by CD8:GFP (green). Dpn was expressed in the nuclei of <i>wt</i> NBs (A–A′), and its expression remained unchanged in <i>smo^IA3</i> NB (C–C′). However <i>ptc^S2</i> NB had decreased levels of Dpn expression (B–B′) compared to <i>wt</i> NBs located outside of the clone (arrows). <i>wt</i> and <i>smo^IA3</i> NBs showed a Pros crescent during mitosis (D–D′ for <i>wt</i>; unpublished data for <i>smo^IA3</i>) and did not show visible nuclear Pros expression during interphase (F–F′ for <i>smo^IA3</i>; unpublished data for <i>wt</i>), but <i>ptc^S2</i> interphase NBs showed nuclear-localized Pros (E–E′). (G–G′) <i>wt</i> NB clone showing three undifferentiated GMCs (arrowheads) which lacked Elav expression. (H–H′) In a <i>ptc^S2</i> NB clone all of the cells surrounding the NB expressed Elav. (I–I′) In <i>smo^IA3</i> NB clones an enlarged cluster of cells surrounding the NB did not express Elav. (J–M′) Homozygous <i>ci⁹⁴</i> clones as marked by the absence of GFP. (J–L′) Three consecutive <i>z</i>-sections (6 µm apart from each other) of a <i>ci⁹⁴</i> clone exhibited areas that are both Dpn and Elav negative, occupied by GMC-like cells (arrows). The pink dotted line marked the outline of the NB (J–K). (M–M′) Pros was localized to the GMCs and neurons of <i>ci⁹⁴</i>. (N–N″) Over-expression of constitutively active form of <i>ci</i> using <i>act</i>-GAL4 flip-out system showed a single undifferentiated GMC (arrow) within the clone. (D–L, N) DNA was stained with ToPRO3. Scale bar = 10 µm. (O) Quantification of three different cell fates based on the absence or presence of Dpn and Elav expressions for both type I and type II NB clones in <i>wt</i>, <i>ptc^S2</i>, and <i>smo^IA3</i> backgrounds. Error bars showed standard error of the mean (SEM). Statistical significance was determined using Student's <i>t</i> test; *<i>p</i><0.05; **<i>p</i><0.003.</p

Hh signaling acts as a functional link between the temporal cascade and the asymmetric division machinery.

<p>(A–B′) The excessive cytoplasmic Mira and weak Mira crescent seen in <i>ptc^S2</i> NB during mitosis (A–A′) can be rescued by removing one copy of <i>pros</i> (B–B′). (C–D) Similarly, the excessive cytoplasmic Mira and weak Mira crescent seen in <i>flfl⁷⁹⁵/flfl^N42</i> transheterozygous NB (C) can be rescued by removing a copy of <i>pros</i> (D). (E–F′) A <i>flfl⁷⁹⁵</i> NB showing weak Mira crescent and cytoplasmic Mira (E–E′). Such Mira localization defects can be rescued via the introduction of <i>ci^RNAi</i>. (G) Quantification of Mira localization phenotypes in various mutant backgrounds.</p

Hh signaling interacts genetically with <i>grh</i> to orchestrate NB cell cycle exit.

<p>(A–C′) <i>smo^IA3</i> clones (A–A′) marked with CD8:GFP (green) often contained a single NB that expressed Mira (blue) but was devoid of nuclear Pros (red) at 24 h APF. However, no Mira expressing cell could be found in <i>smo^IA3</i> clone that had Grh level reduced by RNA interference (B–B′). As a control, clones expressing <i>grh^RNAi</i> transgene (C–C′) alone did not contain any Mira expressing cell either. RNA mediated knock-down of <i>grh</i> in <i>smo^IA3</i> background at 96 h ALH (D–D′) significantly rescued the ectopic GMC-like cells phenotype as the number of cells negative for Dpn (red) and Elav (blue) plummeted to <i>wt</i> level (arrowheads). (E–E′) showed a control <i>grh^RNAi</i> clone with its GMCs pointed out by the arrowheads, at 96 h ALH. (F–I″) Over-expression of <i>grh</i> in <i>ptc^S2</i> clones substantially rescued the Mira delocalization and Dpn-expression defects in the interphase NBs at late L3 stage. More than 60% of the NB within <i>ptc^S2</i> clones (marked by CD8:GFP in green) displayed weak cortical Mira (red) and low intensity of nuclear Dpn (blue) as compared to the surrounding <i>wt</i> NBs (F–F″), while the rest of the interphase <i>ptc^S2</i> NBs had largely normal Mira localization and nuclear Dpn intensity (G–G″). <i>ptc^S2</i> clones that over-expressed <i>grh</i> had a higher frequency NBs with normal Mira localization and nuclear Dpn intensity (H–H″), whereas control NB that over-expressed <i>grh</i> was indistinguishable from <i>wt</i> NB in terms of Mira localization and nuclear Dpn intensity (I–I″). Scale bar = 10 µm.</p

The developmentally regulated cell cycle length of NB is affected by Hh signaling.

<p>(A–C) Live-imaging video stills of a 72-h ALH <i>wt</i> NB (A) marked by histone-red fluorescent protein (RFP) (red) and G147 (green), as well as those of 72 h ALH <i>ptc^S2</i> (B) and <i>smo^IA3</i> (C) NBs marked by histone-RFP (red) and CD8:GFP (green). Cell cycle lengths were determined by counting the time taken from the appearance of cleavage furrow from one division to the next, on the basis of the appearance of GMC bud (A and B) or nuclear membrane elongation when GMC budded off (C). (D) Quantification of cell cycle length in <i>wt</i>, <i>ptc^S2</i>, and <i>smo^IA3</i> NBs, at 48 h, 72 h, and 96 h ALH. <i>ptc^S2</i> NBs failed to divide at 96 h ALH under our live imaging conditions. Error bars represent standard deviation (SD). Statistical significance was determined using Student's <i>t</i> test; *<i>p</i><0.003; **<i>p</i><0.05.</p

An early transient pulse of Cas is required for later <i>hh</i> expression.

<p>(A–B) <i>hh</i> transcript (red) was detected mainly in the GMCs of 72 h ALH brain lobe (B), but not in that of 48 h ALH brain lobe (A). CD8:GFP (green) was driven with <i>elav</i>-Gal4 to mark the outlines of the cells. (C–C″) In situ hybridization of <i>hh</i> (red) in a <i>wt</i> clone showed that the transcript was expressed in the GMC adjacent to the Dpn-expressing NB. (D–D′) <i>In situ</i> hybridization using an intronic probe that detects <i>hh</i> pre-mRNA (red) in a <i>wt</i> clone showing <i>hh</i> expression in the mitotic NB (note the nuclear morphology, arrow), as well as in the GMC next to it (arrowhead). (E–E′) The mature form of <i>hh</i> mRNA (red) was detected in the cytoplasm of the NB (arrow) and to a lesser extent, the adjacent GMC (arrowhead). (F–H) Immuno-staining against Cas (red) showed its expression in cells (NBs and intermediate neural precursors [INPs]) that were also co-expressing Dpn (blue, arrowhead), as well as in some other neurons at different developmental time points. The outlines of the cells were marked with membrane GFP (green). The brain lobes were position such that the dorsal side is facing up and the dotted line indicated the midline of the brain. (I–J′) In situ hybridization of <i>hh</i> mRNA showed that embryonic Cas was required for normal <i>hh</i> expression. <i>cas²⁴</i> clones induced at 24 h ALH did not affect <i>hh</i> expression (I–I′) while <i>cas²⁴</i> clones induced during late embryonic stage could reduce or abolish <i>hh</i> expression (J–J′). Pon (blue) showed the outline of the newly born GMCs that typically expressed <i>hh</i> mRNA. (A–F, I–J) Scale bar = 10 µm for all panels. (K) ChIP for Flag-tagged Cas transfected into S2 cells showed that Cas was heavily enriched within the 6-kb region at the 5′ UTR of <i>hh</i> gene (orange box). There are 19 putative Cas binding sites within that region. The enrichment of any region of the chromatin was counted as the multiple of specific binding (anti-Flag) against non-specific binding (anti-IgG), and normalized to the enrichment at the actin promoter site (negative control). A known target of Cas, pdm-1 was used as a positive control (grey box) for comparison purposes. The value of the blue bars was the average enrichment (times) from three independent transfections and five independent ChIPs. Error bar correspond to standard error of the mean (SEM).</p

Hedgehog signaling affects the proliferation of NBs.

<p>(A–C) Third instar larval brains harbouring <i>wt</i> (A), <i>ptc^S2</i> (B), and <i>smo^IA3</i> (C) MARCM clones were immunostained to show the clone size (green, GFP channel), and Mira (red) localization. In <i>wt</i> clones (D–D′), and <i>smo^IA3</i> (F–F′) clones, the mitotic NBs showed a strong Mira crescent; but Mira was highly cytoplasmic in <i>ptc^S2</i> NBs (E–E′). In interphase <i>ptc^S2</i> NBs (G–G′, GFP clones), the cortical Mira was weakened or absent (yellow arrowheads) compared to their <i>wt</i> counterparts (yellow arrows). DNA was stained with either PH3 (D and F), or ToPRO3 (A–C, E, and G). (H) Quantification of NB mitotic index in <i>wt</i>, <i>ptc^S2</i>, <i>smo^IA3</i> and <i>hh^AC</i> clones based on the percentage of the NBs that expressed PH3 at 96 h ALH. (I–K) BrdU (red) incorporation in <i>wt</i> (I), <i>ptc^S2</i> (J), and <i>smo^IA3</i> (K) clones labeled by CD8:GFP (green) after 4 h of continuous feeding with BrdU. Scale bar = 10 µm.</p

Hh signaling affects the maintenance of <i>grh</i> expression.

<p>(A–D′) Grh (red) was expressed in <i>wt</i> NBs (marked by Dpn, blue) and some GMCs at 72 h ALH (A–A′) and 96 h ALH (B–B′). Grh expression was down regulated in the NBs and abolished in the GMCs by 12 h APF (C–C′) and its expression was barely visible at 24 h APF (D–D′). (E–G′) <i>ptc^S2</i> NB showed normal expression of Grh at 72 h ALH (E–E′) but its level quickly decreased by 96 h ALH (F–F′) and was completely abolished at 12 h APF (G–G′). (H–L′) Grh expression was detected in <i>smo^IA3</i> NB and GMCs at 72 h ALH (H–H′), and 96 h ALH (I–I′) but persisted until 12 h APF (J–J′) and failed to be down-regulated at 24 h APF (K–L′). (L) A <i>smo^IA3</i> NB at 24 h APF with persistent Grh expression amidst the wt background (non-green) in which all the NBs had down-regulated their Grh expression. Scale bar = 10 µm.</p

The model.

<p>(A) An early pulse of Cas at early larval stage primes the expression of <i>hh</i> in both NB and GMCs. The Hh ligand (unpublished data) acts in an autocrine and/or paracrine fashion to activate Hh signalling transduction in the NB. Among the outcomes of Hh signaling pathway activation in the context of postembryonic NB, <i>grh</i> expression is down-regulated and Pros moves into the nucleus, eventually leading to NB cell cycle exit at early pupal stage. The strength of Hh signaling activation is likely to be regulated PP4, which dampen Hh pathway activity by dephosphorylating Smo. <i>svp</i> probably constitutes a parallel pathway, which may or may not converge with <i>cas</i>→<i>hh</i> pathway at a point prior to the eventual NB cell cycle exit. Dotted lines and double question marks denote uncertainties while solid lines represent availability of experimental evidences. Triangular and diamond-shaped arrowheads imply positive and negative regulations respectively. This illustration is not drawn to scale. (B) Type I NB lineage trees at three different developmental stages, during which the expression of Grh (red), Cas (blue), Svp (yellow), Hh (green), and Elav (dark blue) are shown. The NB, GMC, and neuron are represented by circles with black, tan, and brown outlines, respectively. The green arrows show the autocrine/paracrine mode of Hh signaling during lineage progression.</p