Meshed Subvolumes for "Advantages of ionic conductors over electronic conductors as infiltrates in solid oxide fuel cell cathodes".

The meshed sub-volume input files (*.inp) and simulation output files (*.csv) are hosted at the linked folders, respectively MeshedFiles and csv_Outputs.   The link to the folders is :  https://drive.google.com/drive/folders/1cnusA0HayBuxmNUzfHsrAjNvAz_pbicG?usp=share_link If no Readme files exist in folders, contact [email protected] for details of each.  If Readme files exist,  they will explain filenaming, contents, and structure. </p

A Complex of BBS1 and NPHP7 Is Required for Cilia Motility in Zebrafish

<div><p>Bardet-Biedl syndrome (BBS) and nephronophthisis (NPH) are hereditary autosomal recessive disorders, encoded by two families of diverse genes. BBS and NPH display several overlapping phenotypes including cystic kidney disease, retinitis pigmentosa, liver fibrosis, <i>situs inversus</i> and cerebellar defects. Since most of the BBS and NPH proteins localize to cilia and/or their appendages, BBS and NPH are considered ciliopathies. In this study, we characterized the function of the transcription factor Nphp7 in zebrafish, and addressed the molecular connection between BBS and NPH. The knockdown of zebrafish <i>bbs1</i> and <i>nphp7.2</i> caused similar phenotypic changes including convergent extension defects, curvature of the body axis, hydrocephalus, abnormal heart looping and cystic pronephros, all consistent with an altered ciliary function. Immunoprecipitation assays revealed a physical interaction between BBS1 and NPHP7, and the simultaneous knockdown of z<i>bbs1</i> and z<i>nphp7.2</i> enhanced the cystic pronephros phenotype synergistically, suggesting a genetic interaction between z<i>bbs1</i> and z<i>nphp7.2 in vivo</i>. Deletion of zBbs1 or zNphp7.2 did not compromise cilia formation, but disrupted cilia motility. Although NPHP7 has been shown to act as transcriptional repressor, our studies suggest a crosstalk between BBS1 and NPHP7 in regulating normal function of the cilium.</p></div

Knockdown of <i>znphp7.2</i> showed normal development of cilia.

<p>(A) Images of live zebrafish embryos at the 8–10 somite stage. Kupffer's vesicle is located in the dashed box. (B) Measurement of relative KV area with setting control as ‘1.00’. The knockdown of z<i>nphp7.2</i> did not significantly affect on KV development and area. (C) Images of cilia in the KV of 8–10 somite stage were stained for acetylated tubulin. Scale bar = 10 µm. (D) zNphp7.2-deficient embryos showed shortened length of cilia in KV compared to control embryos. (E) Staining of acetylated tubulin in the anterior pronephric tubule of morphants at 48 hpf displayed that the overall distribution of cilia remained unchanged compared to control (Scale bar = 10 µm) even though (F) the knockdown either of z<i>bbs1</i> showed longer and the knockdown of z<i>nphp7.2</i> showed shorter cilia. (PT, Pronephric Tubule) (G and H) The morphology and length of cilia in posterior pronephric tubule appeared unchanged in combined knockdown of z<i>bbs1</i> and z<i>nphp7.2</i> compared to single knockdown of z<i>bbs1</i> or z<i>nphp7.2</i> or compared to Cont MO injected embryos. (PT, Pronephric Tubule) (I) The morphants of z<i>bbs1</i> and z<i>nphp7.2</i> displayed normal ultrastructure of motile cilia in pronephric tubule without recognizable deficiency in dynein arms (outer dynein arm marked by arrowhead). The numbers (n) in the graphs are the number of total cilia which were examined. 4–6 individual embryos per group were examined.</p

Expression of z<i>bbs1</i> and z<i>nphp7</i>.

<p>(A) Identification of 2 NPHP7 homologues in zebrafish: Zebrafish Nphp7.1 (zNphp7.1) and zebrafish Nphp7.2 (zNphp7.2) consist of 446 and 489 amino acids respectively. Amino acid sequence alignment showed that zNphp7.1 shares 43.9% identity and 50.8% similarity with the human NPHP7/GLIS2 (hNPHP7); zNphp7.2 was 51.4% identical and 60.2% similar to the human homologue. The ZF domains of zNphp7.1 and zNphp7.2 were 89.3% and 91.3% identical with those of the human homologue, respectively. (<b>*</b>, completely conserved; <b>.</b>, identical in 2 sequences or belonging to same type of amino acid group in 2 or 3 sequences) (B) Semi-quantitative RT-PCR reveals maternal transcript expression for z<i>nphp7.1</i> and z<i>nphp7.2</i> whereas z<i>bbs1</i> is not expressed maternally nor at 6 hpf. 2 maternal splice products were identified for z<i>nphp7.2</i> (open arrowhead: Transcript 1; filled arrowhead: Transcript 2). The transcript 2 of z<i>nphp7.2</i> is expressed only maternally. Sequencing of the lower splice product revealed an excision of 18 bp corresponding to amino acid (aa 101–118) (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0072549#pone.0072549.s001" target="_blank">Fig. S1</a>). (C) Semi-quantitative RT-PCR with organ specific cDNA from adult zebrafish indicated that z<i>bbs1</i> is expressed in kidney, eye and testis. z<i>nphp7.1</i> and z<i>nphp7.2</i> are expressed in other organs including kidney, eye, heart, testis, gut and muscle.</p

Genetic interaction between z<i>bbs1</i> and z<i>nphp7.2 in vivo</i>.

<p>Injected zebrafish embryos were assessed for the incidence of pronephric cysts. (A) Cont MO injected embryos with normal pronephros. While the majority of z<i>bbs1</i> or z<i>nphp7.2</i> morphants (suboptimal dose) showed pronephros of normal morphology, those of combined knockdown exhibited pronephros with cysts. Cysts are marked by asterisks. (Black scale bar = 500 µm, White scale bar = 100 µm) (B) Pronephric cysts were detectable after individual injections of <i>znphp7.2</i> MO at 0.1 mM and <i>zbbs1</i> MO at 0.2 mM with the level of 17% and 10% respectively whereas the combined knockdown caused pronephric cysts in 59% of microinjected embryos. The final MO concentration for injection was 0.3 mM in all conditions to keep total MO dose constant. Therefore suboptimal doses of z<i>bbs1</i> and z<i>nphp7.2</i> MO were combined with Cont MO to obtain this final concentration of 0.3 mM. The numbers in the brackets (n) are the numbers of total embryos which were examined.</p

zBbs1 and zNphp7.2 morphant embryos showed defects in convergent extension movements.

<p>(A) Representative side views of control embryos and morphants demonstrate the analysis of the body gap angle which is inversely related to length of the body. Embryos were randomly picked at 8–10 somite stage. Open arrowhead marks first somite, filled arrowhead ninth somite respectively. Scale bar = 200 µm. (B) The expression of <i>krx20</i>/<i>myod1</i> in morphant embryos showed shortened body axis with significantly broader somites compared to control. (C) The morphants of z<i>bbs1</i> and z<i>nphp7.2</i> showed wider body gap angle compared to control embryos. The numbers in the brackets (n) are the numbers of total embryos which were examined.</p