Mutations in the Motile Cilia Gene DNAAF1 Are Associated with Neural Tube Defects in Humans

Neural tube defects (NTDs) are severe malformations of the central nervous system caused by complex genetic and environmental factors. Among genes involved in NTD, cilia-related genes have been well defined and found to be essential for the completion of neural tube closure (NTC). We have carried out next-generation sequencing on target genes in 373 NTDs and 222 healthy controls, and discovered eight disease-specific rare mutations in cilia-related gene DNAAF1. DNAAF1 plays a central role in cytoplasmic preassembly of distinct dynein-arm complexes, and is expressed in some key tissues involved in neural system development, such as neural tube, floor plate, embryonic node, and brain ependyma epithelial cells in zebrafish and mouse. Therefore, we evaluated the expression and functions of mutations in DNAAF1 in transfected cells to analyze the potential correlation of these mutants to NTDs in humans. One rare frameshift mutation (p.Gln341Argfs*10) resulted in significantly diminished DNAAF1 protein expression, compared to the wild type. Another mutation, p.Lys231Gln, disrupted cytoplasmic preassembly of the dynein-arm complexes in cellular assay. Furthermore, results from NanoString assay on mRNA from NTD samples indicated that DNAAF1 mutants altered the expression level of NTC-related genes. Altogether, these findings suggest that the rare mutations in DNAAF1 may contribute to the susceptibility for NTDs in humans

Different epigenetic alterations are associated with abnormal IGF2/Igf2 upregulation in neural tube defects.

The methylation status of DNA methylation regions (DMRs) of the imprinted gene IGF2/Igf2 is associated with neural tube defects (NTDs), which are caused by a failure of the neural tube to fold and close and are the second-most common birth defect; however, the characterization of the expression level of IGF2/Igf2 in neural tissue from human fetuses affected with NTDs remains elusive. More importantly, whether abnormal chromatin structure also influences IGF2/Igf2 expression in NTDs is unclear. Here, we investigated the transcriptional activity of IGF2/Igf2 in normal and NTD spinal cord tissues, the methylation status of different DMRs, and the chromatin structure of the promoter. Our data indicated that in NTD samples from both human fetuses and retinoic acid (RA)-treated mouse fetuses, the expression level of IGF2/Igf2 was upregulated 6.41-fold and 1.84-fold, respectively, compared to controls. H19 DMR1, but not IGF2 DMR0, was hypermethylated in human NTD samples. In NTD mice, h19 DMR1 was stable, whereas the chromatin structure around the promoter of Igf2 might be loosened, which was displayed by higher H3K4 acetylation and lower H3K27 trimethylation. Therefore, the data revealed that IGF2/Igf2 expression can be ectopically up-regulated by dual epigenetic factors in NTDs. In detail, the upregulation of IGF2/Igf2 is likely controlled by hypermethylation of H19 DMR1 in human NTDs, however, in acute external RA-induced NTD mice it is potentially determined by more open chromatin structure

Replacement of soybean meal by urea or starea in beef cattle diet: I. igestibility, nitrogen balance, ruminal and blood parameters; II. performance and III. evaluation of digestibility markers.

Com o objetivo de avaliar a substituição de uma fonte de proteína verdadeira (farelo de soja; dieta deficiente em PDR), por uréia ou amiréia (A-150S - fonte de nitrogênio não protéico de suposta liberação gradativa de nitrogênio; dietas adequadas em PDR), foram realizados três experimentos. Experimento I: Seis machos da raça Nelore, não castrados, com peso médio inicial de 420 kg, foram utilizados em quadrado latino 3x3 duplicado, avaliando-se: a digestibilidade, o balanço de nitrogênio, parâmetros ruminais e sanguíneos (capítulo 3); a estimativa da digestibilidade no trato gastrintestinal utilizando indicadores externo e internos comparados com colheita total de fezes (capítulo 6). O volumoso utilizado foi o BIN (20% da MS). A digestibilidade da MS, MO, CNF, EE, PB e o NDT não diferiram (P>0,05) entre os tratamentos. A digestibilidade da FDA e FDN foram superiores (P0,05) nos valores de pH, AGV total, acetato, propionato, butirato e relação acetato:propionato do fluido ruminal. A concentração de nitrogênio amoniacal no fluido ruminal foi superior (P0,05) entre os tratamentos. A estimativa da digestibilidade utilizando o óxido de cromo apresentou resultados similares (P>0,05) à colheita total de fezes. O mesmo foi observado com a lignina no tratamento deficiente em PDR (FS), mas nos de uréia e amiréia, os coeficientes de digestibilidade foram subestimados (P0,05) entre si. Experimento III: realizou-se outro experimento de desempenho similar ao anterior, utilizando-se apenas outro volumoso (45% de BTPV e 5% de BIN) e os animais estavam na fase de terminação (capitulo 5). O tratamento FS apresentou maior (P0,05) entre si. A amiréia promoveu resultados similares a uréia convencional no consumo dos nutrientes, digestibilidade, parâmetros ruminais e sanguíneos e no desempenho de bovinos de corte confinados.Experiment I: Six Nellore bulls, with 420 kg of body weight, were used to evaluate the replacement of a true protein source (soybean meal-SBM), in an inadequate RDP diet, by urea or starea (non protein nitrogen source supposedly of slow N release), being the last two N sources in an adequate RDP diet. Sugar cane bagasse in natura (BIN) was the only source of diet forage (20% of DM). This trial evaluated: digestibility, ruminal parameters, ruminal ammonia, blood parameters and N balance (chapter 3); total tract digestibility estimated by using internal and external markers compared to total feces collection (chapter 6). DM, OM, NFC, EE, CP and TDN digestibilities (%) did not differ (P>0.05) among treatments. ADF and NDF digestibilities (%) were higher (P0.05) on pH, total VFA, acetate, propionate, butirate and acetate:propionate ratio. Ruminal ammonia N concentration was greater (P0.05) urinary N loss. N retention (g/d and % of ingested) and protein biological value (N retention, % of N digestible) were higher (P0.05) among treatment (chapter 3). The digestibility estimated by using cromium oxide was similar (P>0.05) to that of using total feces collection. Lignin used as an internal marker resulted in similar pattern as feces collection when the diet contained soybean meal (RDP deficient diet), however, when the diet N was urea or starea, the digestibility coefficients were underestimated (P0.05). Experiment III: Another performance trial was done (chapter 5), similar to Exp. II, differing only by the forage portion of the diet (45% hidrolized sugar cane bagasse-BTPV and 5% bagasse in natura-BIN) for finishing cattle. DM intake, ADG and feed conversion were 8.99, 7.43 and 7.69 kg/day; 0.983, 0.368 and 0.404 kg/day and 9.56, 20.14 and 19.54 kg DM/kg gain for SBM, urea and starea treatments, respectively. SBM had the higher (P0.05). Starea showed similar results to urea

Paper Microfluidics for Cell Analysis

Paper microfluidics has attracted much attention since its first introduction around one decade ago due to the merits such as low cost, ease of fabrication and operation, portability, and facile integration with other devices. The dominant application for paper microfluidics still lies in point-of-care testing (POCT), which holds great promise to provide diagnostic tools to meet the ASSURED criteria. With micro/nanostructures inside, paper substrates provide a natural 3D scaffold to mimic native cellular microenvironments and create excellent biointerfaces for cell analysis applications, such as long-term 3D cell culture, cell capture/phenotyping, and cell-related biochemical analysis (small molecules, protein DNA, etc.). This review summarizes cell-related applications based on various engineered paper microdevices and provides some perspectives for paper microfluidics-based cell analysis

Chromatin status in the promoter region of the <i>IGF2</i> gene is negatively associated with gene expression.

<p>(A) and (D): The genomic profile of genes displayed in UCSC (hg19), the genomic locus of target regions in ChIP assay (black block on the gene schematic) and the CpG island (green block underneath the gene schematic) assayed by the MASSARRAY platform are shown. The capital letter beside the block indicates the corresponding panel below. The number beside the block indicates the genomic location that was targeted. In the ChIP assay results (B and E), the enrichment in the y-axis represents the relative enrichment fold in which the higher enrichment in case or in control is designated as 1. C) and F) indicate the DNA methylation level. Notably, in F, the DNA methylation status of <i>KvDMR1</i> is shown.*: <i>P</i><0.05; **: <i>P</i><0.01 (Student's <i>t</i>-test). TSS: transcription start site.</p

Schematic for the imprinting mechanism of <i>IGF2</i> and <i>KCNQ1</i>.

<p>In (A), the rectangle represents each gene, and the black circle is methylated DNA. In (B), the green arrow in the lower panel represents lncRNAs <i>KCNQ1OT1</i>, and the hexagon represents <i>KvDMR1</i> that is methylated or not.</p

Hypermethylated <i>H19</i> DMR1 and stably methylated <i>IGF2</i> DMR0 in human fetuses with NTDs.

<p>The DNA methylation levels of the <i>H19</i> DMR1 (A) and <i>IGF2</i> DMR0 (B) were examined through matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry. In the upper panel in (A) and right panel in (B), the methylation status of all detected CpG sites was visualized. Each solid circle represents a “CG” site. (A)The number in upper panel refers to the CG site(s) in the lower histogram. (B) The number in the left panel refers to the site in the right histogram. ***: <i>P</i><0.0001 (Student's <i>t</i>-test).</p

More open chromatin structure in mice fetuses with RA-induced spina bifida.

<p>(A) The methylation level of <i>H19</i> DMR1 is stable in RA-treated mice. For details, see the legend in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0113308#pone-0113308-g003" target="_blank">Fig. 3</a>. (B) and (F): The genomic profile of genes displayed in UCSC (mm9), the genomic locus of target regions in ChIP assay (black block in the gene schematic) (corresponding results shown in C and D or G and H), and the CpG island (green block underneath the gene schematic) (corresponding results shown in E and I) detected by MASSARRAY assay are shown. The number beside the block indicates the genomic location that was targeted. In the ChIP assay results (C and D or G and H), the enrichment in the y-axis represents the relative fold enrichment in which the higher enrichment in case or control is designated as 1. *: <i>P</i><0.05; **: <i>P</i><0.01; ***: <i>P</i><0.0001 (Student's <i>t</i>-test). TSS: transcription start site.</p

Wide ectopic mRNA levels of imprinted genes in mouse fetuses with RA-induced spina bifida.

<p>(<b>A and B</b>) The real-time qPCR assay results indicate the fold change in imprinted genes on each chromosome in the spinal cord tissue of E18 RA-induced mice with NTDs compared with control. The x-axis represents the chromosome and the relative genomic position of each gene. The y-axis represents the relative mRNA abundance in NTDs compared with the control. The red column indicates a maternally expressed gene, while the blue column indicates a paternally expressed gene. *: <i>P</i><0.05; **: <i>P</i><0.01; ***: <i>P</i><0.0001 (Student's <i>t</i>-test).</p

Enhanced mRNA levels of <i>IGF2</i> in the spinal cord and brains of human fetuses with NTDs.

<p>(A) In the spinal cord and (B) brains of human fetuses with NTDs, <i>IGF2</i> mRNA levels were dramatically upregulated. *: <i>P</i><0.05; **: <i>P</i><0.01; ***: <i>P</i><0.0001 (Student's <i>t</i>-test).</p