Over-expression of Fih-1 inhibits the formation of angiogenic vessels during development.

<p>(<b>A</b>) Whole-mount <i>in situ</i> hybridization with <i>fli1</i>a and <i>kdrl</i> in control or <i>fih-1</i> mRNA-injected embryos. In fih-1 mRNA-injected embryos, the number of intersegmental vessels (black arrowheads) was substantially reduced. (<b>B</b>) Microangiography of <i>fih-1</i> mRNA-injected embryos shows a lack of circulation in intersegmental vessels. (<b>C</b>) Ectopic expression of <i>vegf-aa₁₆₅</i> restored impaired angiogenic vessel formation in <i>fih-1</i> mRNA-injected embryos at 52 hpf. Arrowheads point to intersegmental vessels which fail to anastomose at the dorsal-most part of the embryos. (<b>D</b>) Schematic diagram of two constructs that encode either N-terminus or C-terminus deleted Fih-1. Both N-terminus only Fih-1 (Fih-1ΔC) and C-terminus only Fih-1 (Fih-1ΔN) contain JmjC domain and metal binding sites which are essential for hydroxylation of HIF-1α. (<b>E</b>) C-terminus of Fih-1 is essential for its anti-angiogenic activity. While full length <i>fih-1</i> or <i>fih-1ΔN</i> mRNA-injected embryos inhibit angiogenic sprouts of intersegmental vessels, <i>fih-1ΔC</i> mRNA-injected embryos at 72 hpf do not show any obvious vascular defects. Arrowheads point to angiogenic sprouts which fail to extend to the dorsal-most side of the embryos.</p

Fih-1 negatively regulates Vegf-aa₁₆₅ in zebrafish embryos.

<p>(<b>A</b>) At 28 hpf, both <i>fih-1</i> and <i>vegf-aa₁₆₅</i> transcript are selectively expressed within the anterior somites and ventral mesoderm. (<b>B</b>) Attenuation of Fih-1 activity in zebrafish elevates the level of <i>vegf-aa₁₆₅</i> expression at 50 hpf. Arrowheads point to <i>vegf-aa₁₆₅</i> expression in somites caused by lack of Fih-1 activity. Similarly, the level of <i>vegfaa</i> transcript in 48 hpf control or <i>fih-1</i> MO-injected embryos was evaluated by quantitative RT-PCR. Error bars are standard deviation (right). (<b>C</b>) Ectopic expression of Fih-1 decreases the level of <i>vegf-aa₁₆₅</i> expression in a dose-dependent manner at 28 hpf. (<b>D</b>) Functional relationship between Fih-1 and Vegf-aa₁₆₅. Ectopic expression of Vegf-aa₁₆₅ under the regulation of <i>hsp70l</i> promoter caused a similar vascular phenotype as observed in <i>fih-1</i> MO-injected embryos. Moreover, inhibition of Vegf-A signaling can drastically reduce the exuberant angiogenic sprouts in <i>fih-1</i> MO-injected embryos at 80 hpf. Arrows point to ectopic sprouts.</p

Fih-1 negatively modulates angiogenesis during development.

<p>(<b>A</b>) Validation of the morpholino (MO) targeting <i>fih-1</i>. Injection of <i>fih-1</i> MO interferes with mRNA splicing, leading to the retention of an intron and premature translation termination. (<b>B</b>) Gross morphology of fih-1 MO-injected embryos in comparison with control MO-injected embryos. No obvious gross morphological defects were observed at 55 hpf. (<b>C</b>) At 50 hpf, injection of <i>fih-1</i> MO did not cause any obvious increase in angiogenic sprouts, however, exuberant angiogenic sprouts emerge from the intersegmental vessels (ISVs) in <i>fih-1</i> MO-injected embryos at 80 and 122 hpf. (<b>D</b>) Concomitant injection of <i>fih-1</i> mRNA can rescue exuberant angiogenesis caused by <i>fih-1</i> MO-injected embryos. On the right column, control (top, n = 12), <i>fih-1</i> MO (middle, n = 20), or <i>fih-1</i> MO and <i>fih-1</i> mRNA (bottom, n = 20) injected embryos. Arrows point to ectopic angiogenic sprouts. Quantification of ectopic angiogenic sprouts per embryo is shown on the right. Asterisk notes statistical significance in the number of ISVs between <i>fih-1</i> MO and <i>fih-1</i> MO and <i>fih-1</i> mRNA injected embryos. Error bars are standard deviation.</p

Expression pattern of <i>fih-1</i>, <i>vhl</i>, and <i>hif-1α</i>.

<p>(<b>A</b>) RT-PCR analysis showing expression of <i>fih-1</i>, <i>hif-1a</i>, <i>vhl</i>, and <i>β-actin</i> during development. (<b>B</b>) Whole-mount <i>in situ</i> hybridization of <i>fih-1</i> during development. As early as 8 hpf, <i>fih-1</i> expression can be detected. At 14 hpf, <i>fih-1</i> is strongly expressed at the midbrain-hindbrain boundary and eye. At 25 hpf, expression of <i>fih-1</i> is expanded to the optic vesicle and ventral mesoderm. The expression of <i>fih-1</i> in midbrain-hindbrain boundary, eye, optic vesicle and ventral mesoderm is maintained at later stages. (<b>C</b>) Expression of <i>hif1α</i>, a known target of Fih-1, at 25 hpf and 36 hpf. (<b>D</b>) Expression of <i>vhl</i>, which is known to interact and synergize with Fih-1, at the same developmental stage. Both <i>hif1α</i> and <i>vhl</i> express within the similar anatomical region as <i>fih-1</i>.</p

Table_3_Characterization of exosomal microRNAs in preterm infants fed with breast milk and infant formula.XLSX

Breastfeeding not only reduces infection-related morbidity, but also increases growth of preterm infants. Advantages of breast milk (BM) for preterm infants are significant. They continue to be studied. However, because not all preterm infants can receive breastfeeding, bovine-based infant formula (IF) is used as an alternative, which may increase the risk of several preterm complications. Exosomes isolated from biofluids are emerging as biomarkers in research of various diseases. Here, we characterized miRNA contents of exosomes in urine and serum samples of preterm infants who were BM and IF fed and performed transcriptomic analysis of small RNA libraries. We identified significantly up-regulated 6 miRNAs and 10 miRNAs, respectively. Gene Ontology (GO) analysis revealed that target genes of these miRNAs might participate in neuronal development, immunity modulation, detoxification of reactive oxygen species, and transmembrane exchange. Our data suggest that exosome-based systemic screening for preterm infants with breastfeeding might be a screening tool for identifying target molecules involved in therapy for preterm infants in neonatal intensive care unit (NICU) and for future application as nutraceutical formulations or pharmaceuticals.</p

Table_2_Characterization of exosomal microRNAs in preterm infants fed with breast milk and infant formula.XLSX

Breastfeeding not only reduces infection-related morbidity, but also increases growth of preterm infants. Advantages of breast milk (BM) for preterm infants are significant. They continue to be studied. However, because not all preterm infants can receive breastfeeding, bovine-based infant formula (IF) is used as an alternative, which may increase the risk of several preterm complications. Exosomes isolated from biofluids are emerging as biomarkers in research of various diseases. Here, we characterized miRNA contents of exosomes in urine and serum samples of preterm infants who were BM and IF fed and performed transcriptomic analysis of small RNA libraries. We identified significantly up-regulated 6 miRNAs and 10 miRNAs, respectively. Gene Ontology (GO) analysis revealed that target genes of these miRNAs might participate in neuronal development, immunity modulation, detoxification of reactive oxygen species, and transmembrane exchange. Our data suggest that exosome-based systemic screening for preterm infants with breastfeeding might be a screening tool for identifying target molecules involved in therapy for preterm infants in neonatal intensive care unit (NICU) and for future application as nutraceutical formulations or pharmaceuticals.</p

Table_4_Characterization of exosomal microRNAs in preterm infants fed with breast milk and infant formula.XLSX

Breastfeeding not only reduces infection-related morbidity, but also increases growth of preterm infants. Advantages of breast milk (BM) for preterm infants are significant. They continue to be studied. However, because not all preterm infants can receive breastfeeding, bovine-based infant formula (IF) is used as an alternative, which may increase the risk of several preterm complications. Exosomes isolated from biofluids are emerging as biomarkers in research of various diseases. Here, we characterized miRNA contents of exosomes in urine and serum samples of preterm infants who were BM and IF fed and performed transcriptomic analysis of small RNA libraries. We identified significantly up-regulated 6 miRNAs and 10 miRNAs, respectively. Gene Ontology (GO) analysis revealed that target genes of these miRNAs might participate in neuronal development, immunity modulation, detoxification of reactive oxygen species, and transmembrane exchange. Our data suggest that exosome-based systemic screening for preterm infants with breastfeeding might be a screening tool for identifying target molecules involved in therapy for preterm infants in neonatal intensive care unit (NICU) and for future application as nutraceutical formulations or pharmaceuticals.</p

Table_1_Characterization of exosomal microRNAs in preterm infants fed with breast milk and infant formula.XLSX

Breastfeeding not only reduces infection-related morbidity, but also increases growth of preterm infants. Advantages of breast milk (BM) for preterm infants are significant. They continue to be studied. However, because not all preterm infants can receive breastfeeding, bovine-based infant formula (IF) is used as an alternative, which may increase the risk of several preterm complications. Exosomes isolated from biofluids are emerging as biomarkers in research of various diseases. Here, we characterized miRNA contents of exosomes in urine and serum samples of preterm infants who were BM and IF fed and performed transcriptomic analysis of small RNA libraries. We identified significantly up-regulated 6 miRNAs and 10 miRNAs, respectively. Gene Ontology (GO) analysis revealed that target genes of these miRNAs might participate in neuronal development, immunity modulation, detoxification of reactive oxygen species, and transmembrane exchange. Our data suggest that exosome-based systemic screening for preterm infants with breastfeeding might be a screening tool for identifying target molecules involved in therapy for preterm infants in neonatal intensive care unit (NICU) and for future application as nutraceutical formulations or pharmaceuticals.</p

Image_1_Characterization of exosomal microRNAs in preterm infants fed with breast milk and infant formula.PNG

Breastfeeding not only reduces infection-related morbidity, but also increases growth of preterm infants. Advantages of breast milk (BM) for preterm infants are significant. They continue to be studied. However, because not all preterm infants can receive breastfeeding, bovine-based infant formula (IF) is used as an alternative, which may increase the risk of several preterm complications. Exosomes isolated from biofluids are emerging as biomarkers in research of various diseases. Here, we characterized miRNA contents of exosomes in urine and serum samples of preterm infants who were BM and IF fed and performed transcriptomic analysis of small RNA libraries. We identified significantly up-regulated 6 miRNAs and 10 miRNAs, respectively. Gene Ontology (GO) analysis revealed that target genes of these miRNAs might participate in neuronal development, immunity modulation, detoxification of reactive oxygen species, and transmembrane exchange. Our data suggest that exosome-based systemic screening for preterm infants with breastfeeding might be a screening tool for identifying target molecules involved in therapy for preterm infants in neonatal intensive care unit (NICU) and for future application as nutraceutical formulations or pharmaceuticals.</p

Association of Factor Inhibiting HIF-1α (FIH-1) with Mindbomb.

<p>(<b>A</b>) Amino acid sequence comparison between human and zebrafish FIH-1/Fih-1. JmjC domain is marked as a red box. Region of FIH-1 which mediates its interaction with HIF-1α is marked as blue underline. Red asterisks indicate metal coordination sites. (<b>B</b>) Zebrafish Fih-1 co-localizes with Mib1 in transiently transduced Cos7 cells. (<b>C</b>) Zebrafish Fih-1 co-localizes with Hif1α in transiently transduced Cos7 cells. Expression vectors encoding HA-Mib1 and Fih-1-GFP (For B) or HA-Hif-1α and Fih-1-GFP (For C) were used.</p