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Podocin is a key protein of the kidney podocyte slit diaphragm protein complex, an important part of the glomerular filtration barrier. Mutations in the human podocin gene NPHS2 cause familial or sporadic forms of renal disease owing to the disruption of filtration barrier integrity. The exclusive expression of NPHS2 in podocytes reflects its unique function and raises interesting questions about its transcriptional regulation. Here, we further define a 2.5-kb zebrafish nphs2 promoter fragment previously described and identify a 49-bp podocyte-specific transcriptional enhancer using Tol2-mediated G 0 transgenesis in zebrafish. Within this enhancer, we identified a cis-acting element composed of two adjacent DNA-binding sites (FLAT-E and forkhead) bound by transcription factors Lmx1b and FoxC. In zebrafish, double knockdown of Lmx1b and FoxC orthologs using morpholino doses that caused no or minimal phenotypic changes upon individual knockdown completely disrupted podocyte development in 40% of injected embryos. Cooverexpression of the two genes potently induced endogenous nphs2 expression in zebrafish podocytes. We found that the NPHS2 promoter also contains a cis-acting Lmx1b-FoxC motif that binds LMX1B and FoxC2. Furthermore, a genome-wide search identified several genes that carry the Lmx1b-FoxC motif in their promoter regions. Among these candidates, motif-driven podocyte enhancer activity of CCNC and MEIS2 was functionally analyzed in vivo. Our results show that podocyte expression of some genes is combinatorially regulated by two transcription factors interacting synergistically with a common enhancer. This finding provides insights into transcriptional mechanisms required for normal and pathologic podocyte functions. Normal glomerular filtration function depends on structural integrity of the filtration barrier. Glomerular podocytes play a key role in establishing and maintaining this unique filtration barrier structure. Mature podocytes are characterized by cell cycle arrest, foot process formation, and the presence of the slit diaphragm, 1 which bridges the gaps between the interdigitating foot processes of neighboring podocytes and functions as a size-selective filtration barrier. 2,3 For their differentiation, as well as for the maintenance of their complex architecture, podocytes require the expression of several specific genes in a correct spatial and temporal fashion. This notion is supported by the identification of many mutations in podocyte-expressed genes as the underlying cause of inherited renal diseases. 4 Moreover, recent studies from genetically modified mice

Multiple Osteochondromas (MO; previously known as multiple hereditary exostosis) is an autosomal dominant genetic condition that is characterized by the formation of cartilaginous bone tumours (osteochondromas) at multiple sites in the skeleton, secondary bursa formation and impingement of nerves, tendons and vessels, bone curving, and short stature. MO is also known to be associated with arthritis, general pain, scarring and occasional malignant transformation of osteochondroma into secondary peripheral chondrosarcoma. MO patients present additional complains but the relevance of those in relation to the syndromal background needs validation. Mutations in two enzymes that are required during heparan sulphate synthesis (EXT1 or EXT2) are known to cause MO. Previously, we have used zebrafish which harbour mutations in ext2 as a model for MO and shown that ext2−/− fish have skeletal defects that resemble those seen in osteochondromas. Here we analyse dental defects present in ext2−/− fish. Histological analysis reveals that ext2−/− fish have very severe defects associated with the formation and the morphology of teeth. At 5 days post fertilization 100% of ext2−/− fish have a single tooth at the end of the 5th pharyngeal arch, whereas wild-type fish develop three teeth, located in the middle of the pharyngeal arch. ext2−/− teeth have abnormal morphology (they were shorter and thicker than in the WT) and patchy ossification at the tooth base. Deformities such as split crowns and enamel lesions were found in 20% of ext2+/− adults. The tooth morphology in ext2−/− was partially rescued by FGF8 administered locally (bead implants). Our findings from zebrafish model were validated in a dental survey that was conducted with assistance of the MHE Research Foundation. The presence of the malformed and/or displaced teeth with abnormal enamel was declared by half of the respondents indicating that MO might indeed be also associated with dental problems.

Background Circular RNA circFADS2 plays protective roles in LPS-induced inflammation, which promotes sepsis, suggesting its involvement in sepsis. Methods Expression of circFADS2, mature miR-15a-5p, and miR-15a-5p precursor in plasma samples from sepsis patients and healthy controls was determined by RT-qPCR. The circFADS2 expression vector was transfected in lung cells, followed by the measurement of the expression levels of mature miR-15a-5p and miR-15a-5p precursor to study the role of circFADS2 in miR-15a-5p maturation. Cell apoptosis was analyzed by cell apoptosis assay. Results CircFADS2 was upregulated in sepsis and inversely correlated with mature miR-15a-5p, but not miR-15a-5p precursor. In lung cells, circFADS2 overexpression decreased the level of mature miR-15a-5p, but not miR-15a-5p precursor. LPS treatment decreased miR-15a-5p expression and increased circFADS2 level. Cell apoptosis analysis showed that circFADS2 overexpression reduced miR-15a-5p overexpression-induced apoptosis of LPS-treated lung cells. Conclusions CircFADS2 is upregulated in sepsis to suppress LPS-induced lung cell apoptosis by inhibiting miR-15a-5p maturation.

Objective This study aimed to identify the predictors and threshold of failure in neonatal acute respiratory distress syndrome. Methods Newborns with severe acute respiratory distress syndrome aged 0–28 days and gestational age ≥36 weeks were included in the study if their cases were managed with non-extra corporal membrane oxygenation treatments. Patients were divided into two groups according to whether they died before discharge. Predictors of non-extra corporal membrane oxygenation treatment failure were sought, and the threshold of predictors was calculated. Results A total of 103 patients were included in the study. A total of 77 (74.8%) survived hospitalization and were discharged, whereas 26 (25.2%) died. Receiver operating characteristic analysis of oxygen index, pH, base excess, and combinations of these indicators demonstrated the advantage of the combination of oxygen index and base excess over the others variables regarding their predictive ability. The area under the curve for the combination of oxygen index and base excess was 0.865. When the cut-off values of oxygen index and base excess were 30.0 and −7.4, respectively, the sensitivity and specificity for predicting death were 77.0% and 84.0%, respectively. The model with base excess added a net reclassification improvement of 0.090 to the model without base excess. Conclusion The combination of oxygen index and base excess can be used as a predictor of outcomes in neonates receiving non-extra corporal membrane oxygenation treatment for acute respiratory distress syndrome. In neonates with acute respiratory distress syndrome, if oxygen index >30 and base excess <−7.4, non-extra corporal membrane oxygenation therapy is likely to lead to death.

BackgroundMutations in the EXT genes disrupt polymerisation of heparan sulphates (HS) and lead to the development of osteochondroma, an isolated/sporadic- or a multifocal/hereditary cartilaginous bone tumour. Zebrafish (Danio rerio) is a very powerful animal model which has shown to present the same cartilage phenotype that is commonly seen in mice model and patients with the rare hereditary syndrome, Multiple Osteochondroma (MO).MethodsZebrafish dackel (dak) mutant that carries a nonsense mutation in the ext2 gene was used in this study. A panel of molecular, morphological and biochemical analyses was used to assess at what step bone formation is affected and what mechanisms underlie changes in the bone formation in the ext2 mutant.ResultsDuring bone development in the ext2-/- zebrafish, chondrocytes fail to undergo terminal differentiation; and pre-osteoblasts do not differentiate toward osteoblasts. This inadequate osteogenesis coincides with increased deposition of lipids/fats along/in the vessels and premature adipocyte differentiation as shown by biochemical and molecular markers. Also, the ext2-null fish have a muscle phenotype, i.e. muscles are shorter and thicker. These changes coexist with misshapen bones. Normal expression of runx2 together with impaired expression of osterix and its master regulator - xbp1 suggest that unfolded protein responses might play a role in MO pathogenesis.ConclusionsHeparan sulphates are required for terminal differentiation of the cartilaginous template and consecutive formation of a scaffold that is needed for further bone development. HS are also needed for mesenchymal cell differentiation. At least one copy of ext2 is needed to maintain the balance between bone and fat lineages, but homozygous loss of the ext2 function leads to an imbalance between cartilage, bone and fat lineages. Normal expression of runx2 and impaired expression of osterix in the ext2-/- fish indicate that HS are required by osteoblast precursors for their further differentiation towards osteoblastic lineage. Lower expression of xbp1, a master regulator of osterix, suggests that HS affect the ‘unfolded protein response’, a pathway that is known to control bone formation and lipid metabolism. Our observations in the ext2-null fish might explain the musculoskeletal defects that are often observed in MO patients.

Pulmonary artery sling (PA sling) often presents as a life-threatening condition requiring urgent surgical correction. We reported 32 cases of PA sling in children who were followed up postoperatively in the past 6 years. All patients with PA slings who were admitted to the hospital from January 2012 to December 2017 and underwent surgery were retrospectively analyzed. The mean age of the 32 patients at repair was 16.97 months (range, 15 days to 128 months). Six patients required ventilator assistance for respiratory failure. All children underwent left pulmonary artery (LPA) reimplantation (n = 32), and 3 patients needed reimplantation slide tracheoplasty (n = 3) due to ventilation weaning failure. Four patients died, 27 patients survived until discharge, and 18 patients were followed up. Pulmonary computed tomography imaging and echocardiography were performed in 18 patients who were followed up. After LPA reimplantation, the tracheal carina area was significantly enlarged compared to that preoperation (p = 0.0002). In this follow-up cohort study, 75% of the patients who underwent LPA reimplantation survived until discharge. The survivors had subsequently well-developed pulmonary arteries and tracheas.

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