Maternal fructose consumption affects the metabolic functions of offspring later in life. However, the molecular mechanism remains poorly understood. Differences of microRNA expression profile and DNA methylation status are a candidate mechanism to explain the developmental programming that contributes to the development of a metabolic disorder. This study examined the transgenerational effect of maternal fructose consumption from the perspective of epigenetic modification. To do this, we collected serum and liver tissues from male offspring rats that were exposed to maternal distilled water or 20% fructose water during gestation and lactation. A decreased serum high-density lipoprotein cholesterol (HDL-C) level was observed in the offspring of fructose-fed dams at postnatal day (PD) 160. Given research indicating a role of liver X receptor alpha (LXRA) in cholesterol metabolism, we analyzed Lxra expression. Real-time polymerase chain reaction analysis demonstrated that offspring that were delivered from fructose-fed dams exhibited decreased Lxra gene expression in their liver tissue. There is a well-established association between Lxra expression and the level of DNA methylation and miR-206 expression. Pyrosequencing assays revealed no differences in the level of DNA methylation in the Lxra promoter region, whereas miR-206 expression was increased in the liver at PD 60 and 160. Our data indicate that early-life exposure to maternal fructose results in changing of miR-206 expression level in the liver that suppresses the expression of Lxra. This phenomenon may be associated with the decreased serum HDL-C level in offspring.

Cells control organelle size with great precision and accuracy to maintain optimal physiology, but the mechanisms by which they do so are largely unknown. Cilia and flagella are simple organelles in which a single measurement, length, can represent size. Maintenance of flagellar length requires an active transport process known as intraflagellar transport, and previous measurements suggest that a length-dependent feedback regulates intraflagellar transport. But the question remains: how is a length-dependent signal produced to regulate intraflagellar transport appropriately? Several conceptual models have been suggested, but testing these models quantitatively requires that they be cast in mathematical form. Here, we derive a set of mathematical models that represent the main broad classes of hypothetical size-control mechanisms currently under consideration. We use these models to predict the relation between length and intraflagellar transport, and then compare the predicted relations for each model with experimental data. We find that three models-an initial bolus formation model, an ion current model, and a diffusion-based model-show particularly good agreement with available experimental data. The initial bolus and ion current models give mathematically equivalent predictions for length control, but fluorescence recovery after photobleaching experiments rule out the initial bolus model, suggesting that either the ion current model or a diffusion-based model is more likely correct. The general biophysical principles of the ion current and diffusion-based models presented here to measure cilia and flagellar length can be generalized to measure any membrane-bound organelle volume, such as the nucleus and endoplasmic reticulum.

Global fructose consumption is on the rise; however, maternal high-fructose intake may have adverse effects on offspring. We previously demonstrated that excessive fructose intake by rat dams altered steroidogenic gene transcription in the hippocampus of offspring. Herein, we examined how maternal high-fructose intake influences the regulation of adrenal glucocorticoid levels in offspring. Rat dams received 20% fructose solution during gestation and lactation. After weaning, the offspring were provided normal water. Maternal high-fructose intake did not alter mRNA expression levels of adrenal corticosterone-synthesizing and corticosterone-inactivating proteins or the circulating adrenocorticotropic hormone levels of offspring at postnatal day (PD) 21; however, it increased circulating corticosterone levels and decreased mRNA and protein levels of adrenal 5α-reductase type 1 and 11β-hydroxysteroid dehydrogenase type 2 in offspring at PD160. Furthermore, maternal high-fructose intake enhanced DNA methylation of the adrenal 5α-reductase 1 promoter region in PD160 offspring. Thus, maternal high-fructose intake was found to affect adrenal steroid hormone clearance in adult offspring - at least in part - through epigenetic mechanisms.

A recent study has reported that incidence of chronic kidney disease (CKD) is higher in evacuees, but the molecular mechanism still remains unclear. One plausible hypothesis is a change in vascular function following to psychological distress. In order to assess molecular mechanisms underlying this association, we examined whether cardiovascular disease (CVD)-associated miRNAs (miR-126, miR-197, and miR-223) were associated with CKD among Japanese elderly survivors after an earthquake.

There are concerns about the negative effects of fructose intake during pregnancy on the next generation. We have previously reported that offspring from dams fed with fructose during gestation and lactation demonstrate abnormal lipid metabolism in the liver. In this study, we aimed to elucidate the molecular mechanism of the effects of maternal high-fructose corn syrup (HFCS) consumption on offspring. Pregnant Sprague-Dawley rats were fed with 20% HFCS water solution during gestation and lactation. Offspring were put on a normal diet after weaning, and the serum parameters and gene expression patterns were studied at predetermined intervals. Offsprings from pregnant rats fed with 20% HFCS (HFCS group) developed insulin resistance and hyperlipidemia at 60 d of age. RNA-seq analysis demonstrated that peroxisome proliferator-activated receptor α (PPARα) expression is downregulated by maternal HFCS intake. Hepatic Pparα expression in the HFCS group appeared to be suppressed by the enhanced DNA methylation of its promoter region. It is suggested that the development of insulin resistance and hyperlipidemia in the HFCS group may be attributable to aberrant Pparα methylation in the offspring liver. Pparα hypermethylation may be one of molecular mechanism underlying the toxicity of maternal fructose intake.

We examined whether orally administered octacosanol, a long-chain aliphatic saturated alcohol, improves the features of high fructose-induced metabolic syndrome in rats. Five-week-old rats were fed a high fructose diet containing 60% fructose for 3 weeks. Then, the high fructose fed rats received a daily single oral administration of octacosanol (10 or 100 mg/kg body weight) with high fructose feeding for one week. Three- or four-week high fructose feeding increased insulin resistance, serum insulin, triglyceride, total cholesterol, free fatty acids, uric acid, and lipid peroxide concentrations, and hepatic triglyceride and cholesterol contents significantly and decreased serum high-density lipoprotein cholesterol and adiponectin concentrations significantly but did not affect blood pressure and hepatic lipid peroxide and reduced glutathione contents. Four-week high fructose feeding decreased hepatic ascorbic acid content significantly. Oral administration of octacosanol (10 or 50 mg/kg body weight) to high fructose-fed rats for the last 1-week fructose diet feeding attenuated these changes except serum insulin level and insulin resistance significantly and increased hepatic reduced glutathione content significantly. The higher dose of Oct decreased hepatic lipid peroxide content significantly. These results indicate that orally administered octacosanol improves dyslipidemia, hyperuricemia, hypoadiponectinemia, and oxidative stress associated with the features of high fructose-induced metabolic syndrome rats.

Eukaryotic cilia and flagella are microtubule-based organelles whose relatively simple shape makes them ideal for investigating the fundamental question of organelle size regulation. Most of the flagellar materials are transported from the cell body via an active transport process called intraflagellar transport (IFT). The rate of IFT entry into flagella, known as IFT injection, has been shown to negatively correlate with flagellar length. However, it remains unknown how the cell measures the length of its flagella and controls IFT injection. One of the most-discussed theoretical models for length sensing to control IFT is the ion-current model, which posits that there is a uniform distribution of Ca2+ channels along the flagellum and that the Ca2+ current from the flagellum into the cell body increases linearly with flagellar length. In this model, the cell uses the Ca2+ current to negatively regulate IFT injection. The recent discovery that IFT entry into flagella is regulated by the phosphorylation of kinesin through a calcium-dependent protein kinase has provided further impetus for the ion-current model. To test this model, we measured and manipulated the levels of Ca2+ inside of Chlamydomonas flagella and quantified IFT injection. Although the concentration of Ca2+ inside of flagella was weakly correlated with the length of flagella, we found that IFT injection was reduced in calcium-deficient flagella, rather than increased as the model predicted, and that variation in IFT injection was uncorrelated with the occurrence of flagellar Ca2+ spikes. Thus, Ca2+ does not appear to function as a negative regulator of IFT injection, hence it cannot form the basis of a stable length control system.

This study aimed to analyze differences in sensitivity to hepatic lipid metabolism at different ages, through DNA methylation, using an experimental rat model of high-fructose corn syrup (HFCS) intake.

Upon cell cycle exit, centriole-to-basal body transition facilitates cilia formation. The centriolar protein Cp110 is a regulator of this process and cilia inhibitor, but its positive roles in ciliogenesis remain poorly understood. Using Xenopus we show that Cp110 inhibits cilia formation at high levels, while optimal levels promote ciliogenesis. Cp110 localizes to cilia-forming basal bodies and rootlets, and is required for ciliary adhesion complexes that facilitate Actin interactions. The opposing roles of Cp110 in ciliation are generated in part by coiled-coil domains that mediate preferential binding to centrioles over rootlets. Because of its dual role in ciliogenesis, Cp110 levels must be precisely controlled. In multiciliated cells, this is achieved by both transcriptional and post-transcriptional regulation through ciliary transcription factors and microRNAs, which activate and repress cp110 to produce optimal Cp110 levels during ciliogenesis. Our data provide novel insights into how Cp110 and its regulation contribute to development and cell function.

Non-alcoholic fatty liver disease (NAFLD) is closely associated with obesity, metabolic syndrome, and type II diabetes mellitus. Recently, circulating microRNAs (miRNAs) have been proposed as useful disease biomarkers. We examined whether circulating miRNAs, such as miR-20a, miR-27a, and miR-126, were useful biomarkers for NAFLD. We conducted a cross-sectional analysis of 527 subjects aged 39 years or older who had undergone a health examination in the Yakumo Study. Of the residents, 92 were diagnosed with NAFLD using a registered medical sonographer. Serum miR-20a, miR-27a and miR-126 levels were measured by quantitative real-time PCR. We then calculated the odds ratios for serum miRNA level changes according to the severity of NAFLD using normal liver status as the reference group. Serum levels of miR-20a and 27a, but not miR-126, were significantly lower in NAFLD subjects than normal subjects. Serum miR-20a and miR-27a levels were significantly lower in both male and female severe NAFLD subjects. Logistic regression analysis showed a significant relationship between low circulating miR-20a and 27a levels and severe NAFLD. Down-regulated circulating miR-20a and 27a levels were significantly associated with severe NAFLD in the general population. Circulating miR-20a and miR-27a may be useful biomarkers for severe NAFLD.

Outer dense fibre 2 (Odf2 or ODF2) is a cytoskeletal protein required for flagella (tail)-beating and stability to transport sperm cells from testes to the eggs. There are infertile males, including human patients, who have a high percentage of decapitated and decaudated spermatozoa (DDS), whose semen contains abnormal spermatozoa with tailless heads and headless tails due to head-neck separation. DDS is untreatable in reproductive medicine. We report for the first time a new type of Odf2-DDS in heterozygous mutant Odf2+/- mice. Odf2+/- males were infertile due to haploinsufficiency caused by heterozygous deletion of the Odf2 gene, encoding the Odf2 proteins. Odf2 haploinsufficiency induced sperm neck-midpiece separation, a new type of head-tail separation, leading to the generation of headneck sperm cells or headnecks composed of heads with necks and neckless tails composed of only the main parts of tails. The headnecks were immotile but alive and capable of producing offspring by intracytoplasmic headneck sperm injection (ICSI). The neckless tails were motile and could induce capacitation but had no significant forward motility. Further studies are necessary to show that ICSI in humans, using headneck sperm cells, is viable and could be an alternative for infertile patients suffering from Odf2-DDS.

Length control of flagella represents a simple and tractable system to investigate the dynamics of organelle size. Models for flagellar length control in the model organism Chlamydomonas reinhardtii have focused on the length dependence of the intraflagellar transport (IFT) system, which manages the delivery and removal of axonemal subunits at the tip of the flagella. One of these cargoes, tubulin, is the major axonemal subunit, and its frequency of arrival at the tip plays a central role in size control models. However, the mechanisms determining tubulin dynamics at the tip are still poorly understood. We discovered a loss-of-function mutation that leads to shortened flagella and found that this was an allele of a previously described gene, SHF1, whose molecular identity had not been determined. We found that SHF1 encodes a Chlamydomonas orthologue of Crescerin, previously identified as a cilia-specific TOG-domain array protein that can bind tubulin via its TOG domains and increase tubulin polymerization rates. In this mutant, flagellar regeneration occurs with the same initial kinetics as in wild-type cells but plateaus at a shorter length. Using a computational model in which the flagellar microtubules are represented by a differential equation for flagellar length combined with a stochastic model for cytoplasmic microtubule dynamics, we found that our experimental results are best described by a model in which Crescerin/SHF1 binds tubulin dimers in the cytoplasm and transports them into the flagellum. We suggest that this TOG-domain protein is necessary to efficiently and preemptively increase intraflagella tubulin levels to offset decreasing IFT cargo at the tip as flagellar assembly progresses.

Any proposed mechanism for organelle size control should be able to account not only for average size but also for the variation in size. We analyzed cell-to-cell variation and within-cell variation of length for the two flagella in Chlamydomonas, finding that cell-to-cell variation is dominated by cell size, whereas within-cell variation results from dynamic fluctuations. Fluctuation analysis suggests tubulin assembly is not directly coupled with intraflagellar transport (IFT) and that the observed length fluctuations reflect tubulin assembly and disassembly events involving large numbers of tubulin dimers. Length variation is increased in long-flagella mutants, an effect consistent with theoretical models for flagellar length regulation. Cells with unequal flagellar lengths show impaired swimming but improved gliding, raising the possibility that cells have evolved mechanisms to tune biological noise in flagellar length. Analysis of noise at the level of organelle size provides a way to probe the mechanisms determining cell geometry.

We previously reported that maternal fructose consumption increases blood corticosterone levels in rat offspring. However, the underlying mechanism of action remains unclear. In the present study, we aimed to elucidate the molecular mechanism by which maternal high-fructose corn syrup (HFCS) intake increases circulating GC levels in rat offspring (GC; corticosterone in rodents and cortisol in humans). Female Sprague Dawley rats received HFCS solution during gestation and lactation. The male offspring were fed distilled water from weaning to 60 days of age. We investigated the activities of GC-metabolizing enzymes (11β-Hsd1 and 11β-Hsd2) in various tissues (i.e., liver, kidney, adrenal glands, muscle, and white adipose tissue) and epigenetic modification. 11β-Hsd2 activity decreased in the kidney of the HFCS-fed dams. Moreover, the epigenetic analysis suggested that miR-27a reduced Hsd11b2 mRNA expression in the kidney of offspring. Maternal HFCS-induced elevation of circulating GC levels in offspring may be explained by a decrease in 11β-Hsd2 activity via renal miR-27a expression. The present study may allow us to determine one of the mechanisms of GC elevation in rat offspring that is often observed in the developmental origins of the health and disease (DOHaD) phenomenon.

The maintenance of flagellar length is believed to require both anterograde and retrograde intraflagellar transport (IFT). However, it is difficult to uncouple the functions of retrograde transport from anterograde, as null mutants in dynein heavy chain 1b (DHC1b) have stumpy flagella, demonstrating solely that retrograde IFT is required for flagellar assembly. We isolated a Chlamydomonas reinhardtii mutant (dhc1b-3) with a temperature-sensitive defect in DHC1b, enabling inducible inhibition of retrograde IFT in full-length flagella. Although dhc1b-3 flagella at the nonpermissive temperature (34°C) showed a dramatic reduction of retrograde IFT, they remained nearly full-length for many hours. However, dhc1b-3 cells at 34°C had strong defects in flagellar assembly after cell division or pH shock. Furthermore, dhc1b-3 cells displayed altered phototaxis and flagellar beat. Thus, robust retrograde IFT is required for flagellar assembly and function but is dispensable for the maintenance of flagellar length. Proteomic analysis of dhc1b-3 flagella revealed distinct classes of proteins that change in abundance when retrograde IFT is inhibited.

In addition to the popular method of fluorescent protein fusion, live cell protein imaging has now seen more and more application of epitope tags. The small size of these tags may reduce functional perturbation and enable signal amplification. To address their background issue, we adapt self-complementing split fluorescent proteins as epitope tags for live cell protein labelling. The two tags, GFP11 and sfCherry11 are derived from the eleventh β-strand of super-folder GFP and sfCherry, respectively. The small size of FP11-tags enables a cost-effective and scalable way to insert them into endogenous genomic loci via CRISPR-mediated homology-directed repair. Tandem arrangement FP11-tags allows proportional enhancement of fluorescence signal in tracking intraflagellar transport particles, or reduction of photobleaching for live microtubule imaging. Finally, we show the utility of tandem GFP11-tag in scaffolding protein oligomerization. These experiments illustrate the versatility of FP11-tag as a labelling tool as well as a multimerization-control tool for both imaging and non-imaging applications.

Cilia/flagella are assembled and maintained by the process of intraflagellar transport (IFT), a highly conserved mechanism involving more than 20 IFT proteins. However, the functions of individual IFT proteins are mostly unclear. To help address this issue, we focused on a putative IFT protein TTC26/DYF13. Using live imaging and biochemical approaches we show that TTC26/DYF13 is an IFT complex B protein in mammalian cells and Chlamydomonas reinhardtii. Knockdown of TTC26/DYF13 in zebrafish embryos or mutation of TTC26/DYF13 in C. reinhardtii, produced short cilia with abnormal motility. Surprisingly, IFT particle assembly and speed were normal in dyf13 mutant flagella, unlike in other IFT complex B mutants. Proteomic and biochemical analyses indicated a particular set of proteins involved in motility was specifically depleted in the dyf13 mutant. These results support the concept that different IFT proteins are responsible for different cargo subsets, providing a possible explanation for the complexity of the IFT machinery. DOI: http://dx.doi.org/10.7554/eLife.01566.001.

Thioredoxin-interacting protein (TXNIP) inhibits the activity of thioredoxin (TXN), leading to increased oxidative stress. Expression of the TXNIP gene is regulated by DNA methylation. However, no study has reported the influence of lifestyle factors on TXNIP DNA methylation. Our goal was to determine the association between smoking habits and TXNIP DNA methylation levels in a Japanese population. We conducted a cross-sectional study of 417 subjects (180 males and 237 females) participating in a health examination. We used a pyrosequencing assay to determine TXNIP DNA methylation levels in leukocytes. The mean TXNIP DNA methylation level in current smokers (75.3%) was significantly lower than that in never and ex-smokers (never: 78.1%, p < 0.001; ex: 76.9%, p = 0.013). Multivariable logistic regression analyses showed that the OR for TXNIP DNA hypomethylation was significantly higher in current smokers than that in never smokers, and significantly higher in current smokers with years of smoking ≥ 35 and Brinkman Index ≥ 600 compared to that in non-smokers. In conclusion, we found that current smokers had TXNIP DNA hypomethylation compared to never and ex-smokers. Moreover, long-term smoking and high smoking exposure also were associated with TXNIP DNA hypomethylation.

Organoids, which are self-organizing three-dimensional cultures, provide models that replicate specific cellular components of native tissues or facets of organ complexity. We describe a simple method to generate organoid cultures using isolated human tracheobronchial epithelial cells grown in mixed matrix components and supplemented at day 14 with the Wnt pathway agonist R-spondin 2 (RSPO2) and the bone morphogenic protein antagonist Noggin. In contrast to previous reports, our method produces differentiated tracheobronchospheres with externally orientated apical membranes without pretreatments, providing an epithelial model to study cilia formation and function, disease pathogenesis, and interaction of pathogens with the respiratory mucosa. Starting from 3 × 105 cells, organoid yield at day 28 was 1,720 ± 302. Immunocytochemistry confirmed the cellular localization of airway epithelial markers, including CFTR, Na+/K+ ATPase, acetylated-α-tubulin, E-cadherin, and ZO-1. Compared to native tissues, expression of genes related to bronchial differentiation and ion transport were similar in organoid and air-liquid interface (ALI) cultures. In matched primary cultures, mean organoid cilia length was 6.1 ± 0.2 µm, similar to that of 5.7 ± 0.1 µm in ALI cultures, and ciliary beating was vigorous and coordinated with frequencies of 7.7 ± 0.3 Hz in organoid cultures and 5.3 ± 0.8 Hz in ALI cultures. Functional measurement of osmotically induced volume changes in organoids showed low water permeability. The generation of numerous single testable units from minimal starting material complements prior techniques. This culture system may be useful for studying airway biology and pathophysiology, aiding diagnosis of ciliopathies, and potentially for high-throughput drug screening.

Epithelial responses to the cytokine interleukin-13 (IL-13) cause airway obstruction in asthma. Here we utilized multiple genomic techniques to identify IL-13-responsive regulatory elements in bronchial epithelial cells and used these data to develop a CRISPR interference (CRISPRi)-based therapeutic approach to downregulate airway obstruction-inducing genes in a cell type- and IL-13-specific manner. Using single-cell RNA sequencing (scRNA-seq) and acetylated lysine 27 on histone 3 (H3K27ac) chromatin immunoprecipitation sequencing (ChIP-seq) in primary human bronchial epithelial cells, we identified IL-13-responsive genes and regulatory elements. These sequences were functionally validated and optimized via massively parallel reporter assays (MPRAs) for IL-13-inducible activity. The top secretory cell-selective sequence from the MPRA, a novel, distal enhancer of the sterile alpha motif pointed domain containing E-26 transformation-specific transcription factor (SPDEF) gene, was utilized to drive CRISPRi and knock down SPDEF or mucin 5AC (MUC5AC), both involved in pathologic mucus production in asthma. Our work provides a catalog of cell type-specific genes and regulatory elements involved in IL-13 bronchial epithelial response and showcases their use for therapeutic purposes.