The role of Ephrins-B1 and - B2 during fetal rat lung development

Background/Aims: The knowledge of the molecular network that governs fetal lung branching is an essential step towards the discovery of novel therapeutic targets against pulmonary pathologies. Lung consists of two highly branched systems: airways and vasculature. Ephrins and its receptors, Eph, have been implicated in cardiovascular development, angiogenesis and vascular remodeling. This study aims to clarify the role of these factors during lung morphogenesis. Methods: Ephrins-B1, -B2 and receptor EphB4 expression pattern was assessed in fetal rat lungs between 15.5 and 21.5 days post-conception, by immunohistochemistry. Fetal rat lungs were harvested at 13.5 dpc, cultured during 4 days and treated with increasing doses of ephrins-B1 and -B2 and the activity of key signaling pathways was assessed. Results: Ephrin-B1 presents mesenchymal expression, whereas ephrin-B2 and its receptor EphB4 were expressed by the epithelium. Both ephrins stimulated pulmonary branching. Moreover, while ephrin-B1 did not affect the pathways studied, ephrin-B2 supplementation decreased activity of JNK, ERK and STAT. This study characterizes the expression pattern of ephrins-B1, -B2 and EphB4 receptor throughout rat lung development. Conclusion: Our data highlight a possible role of ephrins as molecular stimulators of lung morphogenesis. Moreover, it supports the idea that classical vascular factors might play a role as airway growth promoters.We would like to thank Luis Martins and Miguel Carneiro for histological technical support and help on animal euthanasia. This project was funded by Fundacao para a Ciencia e a Tecnologia (PTDC/SAU-OBD/108051/2008). PPT was supported by POPH/ FSE by Fundacao para a Ciencia e a Tecnologia (reference SFRH/BD/73660/2010) and FOP was supported by Fundacao para a Ciencia e a Tecnologia (reference UMINHO/BII/172/2009)

Leukemia Inhibitory Factor in Rat Fetal Lung Development: Expression and Functional Studies

Background: Leukemia inhibitory factor (LIF) and interleukin-6 (IL-6) are members of the family of the glycoprotein 130 (gp130)-type cytokines. These cytokines share gp130 as a common signal transducer, which explains why they show some functional redundancy. Recently, it was demonstrated that IL-6 promotes fetal lung branching. Additionally, LIF has been implicated in developmental processes of some branching organs. Thus, in this study LIF expression pattern and its effects on fetal rat lung morphogenesis were assessed. Methodology/Principal Findings: LIF and its subunit receptor LIFRa expression levels were evaluated by immunohistochemistry and western blot in fetal rat lungs of different gestational ages, ranging from 13.5 to 21.5 days post-conception. Throughout all gestational ages studied, LIF was constitutively expressed in pulmonary epithelium, whereas LIFRa was first mainly expressed in the mesenchyme, but after pseudoglandular stage it was also observed in epithelial cells. These results point to a LIF epithelium-mesenchyme cross-talk, which is known to be important for lung branching process. Regarding functional studies, fetal lung explants were cultured with increasing doses of LIF or LIF neutralizing antibodies during 4 days. MAPK, AKT, and STAT3 phosphorylation in the treated lung explants was analyzed. LIF supplementation significantly inhibited lung growth in spite of an increase in p44/42 phosphorylation. On the other hand, LIF inhibition significantly stimulated lung growth via p38 and Akt pathways

LIFRα expression pattern during fetal lung development (from 13.5 until 21.5 dpc).

<p>(A) IHC studies revealed that LIFRα was first mainly expressed in the mesenchyme, but after pseudoglandular stage it was also observed in epithelial cells throughout gestation. Original magnification: Upper panel – ×100; Bottom panel – ×200. (B) IHC negative controls: Left – omission of the primary antibody; Center – non-immune goat IgG isotype control; Right – simultaneous omission of the primary and secondary antibodies. In all negative controls immunoreactive LIFRα staining was not observed. (C) Western blot analysis of LIFRα throughout the gestation (190 kDa). Control loading was performed using β-tubulin (55 kDa). (D) Relative LIFRα protein levels expressed in arbitrary units normalized for β-tubulin. No significant difference was observed between gestational ages.</p

Analysis of intracellular signaling pathways that mediates anti-LIF actions on lung branching.

<p>(A) Western blot analysis of p38, p44/42, JNK1/2, Akt and STAT3, and to diphosphorylated forms of p38 (dp-p38), p44/42 (dp-p44/42), SAPK/JNK (dp-JNK1/2), Akt (dp-Akt) and STAT3 (dp-STAT3) in control IgG lung explants (IgG) and treated with anti-LIF IgG (Anti-LIF). Control loading was performed using β-tubulin (55 kDa). (B) Semi-quantitative analysis of expression of phosphorylated forms of these intracellular signaling pathways. Results are presented as arbitrary units normalized for β-tubulin. p<0.05: ^*<i>vs.</i> IgG.</p

LIF expression pattern during fetal lung development (from 13.5 until 21.5 dpc).

<p>(A) IHC studies revealed that LIF expression was localized to airway epithelium. Original magnification: Upper panel – ×100; Bottom panel – ×200. (B) IHC negative controls: Left – omission of the primary antibody; Center – non-immune goat IgG isotype control; Right – simultaneous omission of the primary and secondary antibodies. In all negative controls immunoreactive LIF staining was not observed. (C) Western blot analysis of LIF throughout gestation (45 kDa). Control loading was performed using β-tubulin (55 kDa). (D) Relative LIF protein levels expressed in arbitrary units normalized for β-tubulin. p<0.05: ^*<i>vs.</i> 13.5 dpc, ^§<i>vs.</i> 15.5 dpc, ^‡<i>vs.</i> 17.5 dpc, ^¥<i>vs.</i> 19.5 dpc.</p

LIF neutralizing studies in a fetal lung explant culture system.

<p>(A) Representative examples of lung explants treated daily with normal IgG (control; 1 µg/mL), anti-LIF IgG (1 µg/mL) or FGF-10 (500 ng/mL), after 4 days in culture. Original magnification ×25. (B) Number of total airway buds; (C) Epithelial perimeter; (D) Area; (E) External perimeter of treated lung explants. Inhibition of LIF action significantly stimulated lung branching in a similar way than FGF-10. Results are expressed as D₄/D₀ ratio. p<0.05: ^*<i>vs.</i> control IgG.</p

Analysis of intracellular signaling pathways that mediates LIF actions on lung growth.

<p>(A) Western blot analysis of p38, p44/42, JNK1/2, Akt and STAT3, and to diphosphorylated forms of p38 (dp-p38), p44/42 (dp-p44/42), SAPK/JNK (dp-JNK1/2), Akt (dp-Akt) and STAT3 (dp-STAT3) in control lung explants (C) and treated with LIF at 40 ng/mL (LIF). Control loading was performed using β-tubulin (55 kDa). p38 corresponds to 38 kDa. p44/42 correspond to 44 and 42 kDa, respectively. JNK1 and 2 correspond to 46 and 54 kDa, respectively. Akt corresponds to 60 kDa. STAT3 corresponds to two bands, 79 and 86 kDa. (B) Semi-quantitative analysis of expression of phosphorylated forms of these intracellular signaling pathways. Results are presented as arbitrary units normalized for β-tubulin. p<0.05: ^*<i>vs.</i> control.</p

Probiotics reshape the coral microbiome in situ without detectable off-target effects in the surrounding environment

Beneficial microorganisms for corals (BMCs), or probiotics, can enhance coral resilience against stressors in laboratory trials. However, the ability of probiotics to restructure the coral microbiome in situ is yet to be determined. As a first step to elucidate this, we inoculated putative probiotic bacteria (pBMCs) on healthy colonies of Pocillopora verrucosa in situ in the Red Sea, three times per week, during 3 months. pBMCs significantly influenced the coral microbiome, while bacteria of the surrounding seawater and sediment remained unchanged. The inoculated genera Halomonas , Pseudoalteromonas, and Bacillus were significantly enriched in probiotic-treated corals. Furthermore, the probiotic treatment also correlated with an increase in other beneficial groups (e.g., Ruegeria and Limosilactobacillus ), and a decrease in potential coral pathogens, such as Vibrio . As all corals (treated and non-treated) remained healthy throughout the experiment, we could not track health improvements or protection against stress. Our data indicate that healthy, and therefore stable, coral microbiomes can be restructured in situ, although repeated and continuous inoculations may be required in these cases. Further, our study provides supporting evidence that, at the studied scale, pBMCs have no detectable off-target effects on the surrounding microbiomes of seawater and sediment near inoculated corals.publishe

Desenvolvimento e desigualdades na América Latina : dilemas de longo curso

No início da terceira década do século XXI, o leitor é agraciado com uma obra coletiva de fôlego, inovadora, e certamente paradigmática. Estamos diante de um livro util para compreender e agir em favor do Desenvolvimento e, inversamente, para continuar lutando de forma mais eficiente contra o excesso de Desigualdade. Resta agradecer ao organizador do livro e aos autores de cada um dos capítulos pelo brilhante trabalho realizado. Del Prefácio de Carlos Frederico Dominguez Ávil