Rationale: In experimental models, lung fibrosis is dependent on transforming growth factor (TGF)-b signaling. TGF-b is secreted in a latent complex with its propeptide, and TGF-b activators release TGF-b from this complex. Because the integrin avb6 is a major TGF-b activator in the lung, inhibition of avb6-mediated TGF-b activation is a logical strategy to treat lung fibrosis. Objectives: To determine, by genetic and pharmacologic approaches, whether murine radiation-induced lung fibrosis is dependent on avb6. Methods: Wild-type mice, avb6-deficient (Itgb6 2/2 ) mice, and mice heterozygous for a Tgfb1 mutation that eliminates integrin-mediated activation (Tgfb1 1/RGE ) were exposed to 14 Gy thoracic radiation. Some mice were treated with an anti-avb6 monoclonal antibody or a soluble TGF-b receptor fusion protein. avb6 expression was determined by immunohistochemistry. Fibrosis, inflammation, and gene expression patterns were assessed 20-32 weeks postirradiation. Measurements and Main Results: b6 Integrin expression increased within the alveolar epithelium 18 weeks postirradiation, just before onset of fibrosis. Itgb6 2/2 mice were completely protected from fibrosis, but not from late radiation-induced mortality. Anti-avb6 therapy (1-10 mg/kg/wk) prevented fibrosis, but only higher doses (6-10 mg/kg/wk) caused lung inflammation similar to that in Itgb6 2/2 mice. Tgfb1-haploinsufficient mice were also protected from fibrosis. Conclusions: avb6-Mediated TGF-b activation is required for radiationinduced lung fibrosis. Together with previous data, our results demonstrate a robust requirement for avb6 in distinct fibrosis models. Inhibition of avb6-mediated TGF-b activation is a promising new approach for antifibrosis therapy.

SummaryMice that lack activity of αvβ6-and αvβ8-integrins reproduce the abnormalities of Tgfb1-and Tgfb3-null mice

SUMMARYThe characterization of mesenchymal progenitors is central to understanding development, postnatal pathology and evolutionary adaptability. The precise identity of the mesenchymal precursors that generate the coronal suture, an important structural boundary in mammalian skull development, remains unclear. We show in mouse that coronal suture progenitors originate from hedgehog-responsive cephalic paraxial mesoderm (Mes) cells, which migrate rapidly to a supraorbital domain and establish a unidirectional lineage boundary with neural crest (NeuC) mesenchyme. Lineage tracing reveals clonal and stereotypical expansion of supraorbital mesenchymal cells to form the coronal suture between E11.0 and E13.5. We identify engrailed 1 (En1) as a necessary regulator of cell movement and NeuC/Mes lineage boundary positioning during coronal suture formation. In addition, we provide genetic evidence that En1 functions upstream of fibroblast growth factor receptor 2 (Fgfr2) in regulating early calvarial osteogenic differentiation, and postulate that it plays an additional role in precluding premature osteogenic conversion of the sutural mesenchyme.

Sonic Hedgehog (Shh) signaling is essential during embryonic lung development, but its role in postnatal lung development and adult lung are not known. Using Gli1(nlacZ) reporter mice to identify cells with active Hh signaling, we found that Gli1(nlacZ)-positive mesenchymal cells are densely and diffusely present up to 2 weeks after birth and decline in number thereafter. In adult mice, Gli1(nlacZ)-positive cells are present around large airways and vessels and are sparse in alveolar septa. Hh-stimulated cells are mostly fibroblasts; only 10% of Gli1(nlacZ)-positive cells are smooth muscle cells, and most smooth muscle cells do not have activation of Hh signaling. To assess its functional relevance, we influenced Hh signaling in the developing postnatal lung and adult injured lung. Inhibition of Hh signaling during early postnatal lung development causes airspace enlargement without diminished alveolar septation. After bleomycin injury in the adult lung, there are abundant Gli1(nlacZ)-positive mesenchymal cells in fibrotic lesions and increased numbers of Gli1(nlacZ)-positive cells in preserved alveolar septa. Inhibition of Hh signaling with an antibody against all Hedgehog isoforms does not reduce bleomycin-induced fibrosis, but adenovirus-mediated overexpression of Shh increases collagen production in this model. Our data provide strong evidence that Hh signaling can regulate lung stromal cell function in two critical scenarios: normal development in postnatal lung and lung fibrosis in adult lung.

Sonic Hedgehog (Shh) signaling regulates mesenchymal proliferation and differentiation during embryonic lung development. In the adult lung, Shh signaling maintains mesenchymal quiescence and is dysregulated in diseases such as idiopathic pulmonary fibrosis and chronic obstructive pulmonary disease. Our previous data implicated a role for Shh in postnatal lung development. Here, we report a detailed analysis of Shh signaling during murine postnatal lung development. We show that Shh pathway expression and activity during alveolarization (postnatal day [P] 0-P14) are distinct from those during maturation (P14-P24). This biphasic pattern is paralleled by the transient presence of Gli1;α-smooth muscle actin (α-SMA) myofibroblasts in the growing alveolar septal tips. Carefully timed inhibition of Hedgehog (Hh) signaling during alveolarization defined mechanisms by which Shh influences the mesenchymal compartment. First, interruption of Hh signaling at earlier time points results in increased lung compliance and wall structure defects of increasing severity, ranging from moderately enlarged alveolar airspaces to markedly enlarged airspaces and fewer secondary septa. Second, Shh signaling is required for myofibroblast differentiation: Hh inhibition during early alveolarization almost completely eliminates Gli1;α-SMA cells at the septal tips, and Gli1-lineage tracing revealed that Gli1 cells do not undergo apoptosis after Hh inhibition but remain in the alveolar septa and are unable to express α-SMA. Third, Shh signaling is vital to mesenchymal proliferation during alveolarization, as Hh inhibition decreased proliferation of Gli1 cells and their progeny. Our study establishes Shh as a new alveolarization-promoting factor that might be affected in perinatal lung diseases that are associated with impaired alveolarization.

To examine the characteristics of the interaction of the Fc⑀RI␥ ITAM with the SH2 domains of p72 syk , the binding of an 125 I-labeled dual phosphorylated Fc⑀RI␥ ITAM-based peptide to the p72 syk SH2 domains was monitored utilizing a novel scintillation proximity based assay. The K d for this interaction, determined from the saturation binding isotherm, was 1.4 nM. This high affinity binding was reflected in the rapid rate of association for the peptide binding to the SH2 domains. Competition studies utilizing a soluble C-terminal SH2 domain knockout and N-terminal SH2 domain knockouts revealed that both domains contribute cooperatively to the high affinity binding. Unlabeled dual phosphorylated peptide competed with the 125 I-labeled peptide for binding to the dual p72 syk SH2 domains with an IC 50 value of 4.8 nM. Monophosphorylated 24-mer Fc⑀RI␥ ITAM peptides, and phosphotyrosine also competed for binding, but with substantially higher IC 50 values. This, and other data discussed, suggest that high affinity binding requires both tyrosine residues to be phosphorylated and that the preferred binding orientation of the ITAM is such that the N-terminal phosphotyrosine occupies the C-terminal SH2 domain and the C-terminal phosphotyrosine occupies the N-terminal SH2 domain. Src homology 2 (SH2)1 domains are regions of approximately 100 -120 amino acid residues present in a variety of proteins including tyrosine kinases, tyrosine phosphatases, phospholipases, and other signal transducing proteins (1-6). These domains bind with high affinity to tyrosine containing motifs in associating proteins, such as specific cytokine and immunoglobulin receptor subunits, adapter proteins, tyrosine kinases, and other signaling molecules such as STATs, when these motifs are phosphorylated by the action of tyrosine kinases (1-5, 7, 8). This allows recruitment of SH2 domain-containing signaling molecules and phosphotyrosine-containing signaling molecules into receptor-linked signal transduction assemblies (9 -13). The tyrosine-containing motifs are composed of phosphorylated tyrosine residues followed by 3-4 amino acids (e.g. pYXX(L/I)) which carry the sequence-specific information for SH2 recognition (14 -23). These motifs can occur in isolation or in tandem, thus can bind single SH2 domains (e.g. of src related tyrosine kinases) (4, 24 -26) or dual SH2 domains (e.g. of p70 zap and p72 syk ) (27-34). Certain antigen receptor subunits, such as the subunit of the T cell receptor (TCR), the Ig␣ and Ig␤ subunits of the B cell receptor and the ␤ and ␥ subunits of the high affinity IgE receptor (Fc⑀RI), contain tyrosine motifs in tandem and these have been termed immunoglobulin receptor tyrosine activation motifs (ITAMs) (24, 27, 32, 34 -36). In hematopoietic cell signaling, tyrosine-phosphorylated ITAMs have been shown to be critical for signaling interactions via their association with the SH2 domains of tyrosine kinases. For example, p70zap and the src-related kinases p59 fyn and p56 lck , appear to play a role in TCR-mediated T cell a...

Gene deletion experiments have shown that the three TGFbeta isoforms regulate distinct developmental processes. Recent work by our group and others showed that the integrins alphavbeta6 and alphavbeta8 activate latent forms of TGFbeta1 and TGFbeta3. This raises the possibility that TGFbeta1 and TGFbeta3 act redundantly in developmental processes where both isoforms are expressed and activation is by integrins. To investigate this issue, we generated mice with defective integrin-mediated TGFbeta1 activation (Tgfb1(RGE/RGE)) that were also homozygous for a null mutation in the TGFbeta3 gene. Tgfb1(RGE/RGE); Tgfb3(-/-) mice have severely perturbed development of the brain vasculature that is highly similar to that in mice lacking alphavbeta8. Some Tgfb1(RGE/RGE); Tgfb3(+/-) and Tgfb1(RGE/RGE); Tgfb3(+/+) mice have milder, background-dependent versions of the phenotype. In addition, we found that Tgfb3 gene status influences embryonic lethality due to TGFbeta1 deficiency after limited backcrossing to the BALB/c background. Conversely, Tgfb1 gene status modifies the extent of palate fusion in Tgfb3(-/-) mice after limited backcrossing to the ICR background. Our results are consistent with a functional connection between TGFbeta1 and TGFbeta3 during development based on a shared mechanism of activation.

Summary Bio‐oil is a promising alternative energy source to crude oil for broad application prospects. Bio‐oil can help us avoid over‐reliance on petroleum resources and significantly reduce pollutants and greenhouse gas emissions, improve environment conditions, and protect ecological systems. However, bio‐oil applications have been impeded because of limited technologies, and poor bio‐oil quality has posed a great challenge. As such, considerable research efforts have been made for realizing its potential application value. Scientific and technical advancements on methods of bio‐oil quality improvement to date are reviewed, with an emphasis on advantages and disadvantages of each method. It also points out barriers and gives some recommendations to enhance bio‐oil combustion performance in the future. Copyright © 2017 John Wiley & Sons, Ltd.