Prolyl hydroxylase inhibition protects against murine MC903-induced skin inflammation by downregulating TSLP.

Previously, we reported an anti-inflammatory effect of mTORC1 in a mouse model of type 2 skin inflammation. TSLP, one of the epithelial cell-derived cytokines, was upregulated by Raptor deficiency or rapamycin treatment, which was inhibited by dimethyloxalylglycine (DMOG). However, it remains unclear how DMOG regulates TSLP expression and type 2 skin inflammation. In this study, we investigated the protective effect of DMOG on MC903 (calcipotriol)-induced type 2 skin inflammation. Morphological and immunological changes were assessed by H-E staining, flow cytometry and RT-qPCR. DMOG treatment attenuated MC903-induced skin inflammation in a T cell-independent manner. The anti-inflammatory effect of DMOG was accompanied by downregulation of TSLP and IL-33, and supplementation with recombinant TSLP and IL-33 abolished the effect of DMOG. MC903 increased ROS levels in skin tissue, which was prevented by DMOG. Furthermore, the ROS scavenger N-acetylcysteine (NAC) downregulated TSLP and ameliorated MC903-induced skin inflammation, as did DMOG. Finally, the effect of DMOG on ROS and TSLP was reduced by HIF knockdown. These results suggest that DMOG downregulates TSLP and ROS through the HIF pathway, which reduces MC903-induced skin inflammation.

Site-specific phosphorylation of ZYG-1 regulates ZYG-1 stability and centrosome number.

Spindle bipolarity is critical for genomic integrity. As centrosome number often dictates bipolarity, tight control of centrosome assembly is vital for faithful cell division. The master centrosome regulator ZYG-1/Plk4 plays a pivotal role in this process. In C. elegans, casein kinase II (CK2) negatively regulates centrosome duplication by controlling centrosome-associated ZYG-1 levels. Here, we investigated CK2 as a regulator of ZYG-1 and its impact on centrosome assembly. We show that CK2 phosphorylates ZYG-1 in vitro and physically interacts with ZYG-1 in vivo. Depleting CK2 or blocking ZYG-1 phosphorylation at CK2 target sites leads to centrosome amplification. Non-phosphorylatable ZYG-1 mutants exhibit elevated ZYG-1 levels, leading to increased ZYG-1 and downstream factors at centrosomes, thus driving centrosome amplification. Moreover, inhibiting the 26S proteasome prevents degradation of the phospho-mimetic ZYG-1. Our findings suggest that CK2-dependent phosphorylation of ZYG-1 controls ZYG-1 levels via proteasomal degradation to limit centrosome number.

Site-Specific Phosphorylation of ZYG-1 Regulates ZYG-1 Stability and Centrosome Number.

Spindle bipolarity is critical for genomic integrity. Given that centrosome number often dictates mitotic bipolarity, tight control of centrosome assembly is vital for the fidelity of cell division. The kinase ZYG-1/Plk4 is a master centrosome factor that is integral for controlling centrosome number and is modulated by protein phosphorylation. While autophosphorylation of Plk4 has been extensively studied in other systems, the mechanism of ZYG-1 phosphorylation in C. elegans remains largely unexplored. In C. elegans, Casein Kinase II (CK2) negatively regulates centrosome duplication by controlling centrosome-associated ZYG-1 levels. In this study, we investigated ZYG-1 as a potential substrate of CK2 and the functional impact of ZYG-1 phosphorylation on centrosome assembly. First, we show that CK2 directly phosphorylates ZYG-1 in vitro and physically interacts with ZYG-1 in vivo. Intriguingly, depleting CK2 or blocking ZYG-1 phosphorylation at putative CK2 target sites leads to centrosome amplification. In the non-phosphorylatable (NP)-ZYG-1 mutant embryo, the overall levels of ZYG-1 are elevated, leading to an increase in centrosomal ZYG-1 and downstream factors, providing a possible mechanism of the NP-ZYG-1 mutation to drive centrosome amplification. Moreover, inhibiting the 26S proteasome blocks degradation of the phospho-mimetic (PM)-ZYG-1, while the NP-ZYG-1 mutant shows partial resistance to proteasomal degradation. Our findings suggest that site-specific phosphorylation of ZYG-1, partly mediated by CK2, controls ZYG-1 levels via proteasomal degradation, limiting centrosome number. We provide a mechanism linking CK2 kinase activity to centrosome duplication through direct phosphorylation of ZYG-1, which is critical for the integrity of centrosome number.

kin-3 genetically suppresses sur-6 in centrosome assembly during Caenorhabditis elegans embryogenesis.

Protein phosphorylation plays a critical role in cell cycle progression. In Caenorhabditis elegans , Casein Kinase II (CK2) negatively regulates centrosome assembly, and Protein Phosphatase 2A (PP2A) SUR-6 /B55 acts as a positive regulator of centrosome duplication, suggesting CK2 and PP2A SUR-6 /B55 play opposing roles in centrosome assembly. Here, we examined the genetic interaction between kin-3 , encoding the catalytic subunit of CK2, and sur-6 , encoding the PP2Aregulatory subunit SUR-6 /B55, in Caenorhabditis elegans embryos. Our results show that kin-3 (RNAi) partially restores normal centrosome duplication and embryonic viability to hypomorphic sur-6 mutants, suggesting that kin-3 genetically suppresses sur-6 in centrosome assembly during Caenorhabditis elegans embryogenesis.

The C. elegans Casein Kinase II is associated with meiotic DNA in fertilized oocytes.

By using CRISPR/Cas9 genome-editing, we have generated epitope-tagged KIN-3 and KIN-10 expressing strains at the endogenous C-terminal loci in Caenorhabditis elegans . We observed that both the catalytic (KIN-3::V5) and regulatory (KIN-10::2xMyc) subunits of the Casein Kinase II (CK2) holoenzyme complex are associated with meiotic DNA, enriched in the midvalent rings during meiotic divisions in fertilized C. elegans oocytes.

CD4+ T cells play an essential role in chronic MC903-induced skin inflammation.

MC903 skin inflammation model is one of well-characterized murine models of atopic dermatitis and driven by TSLP-mediated type 2 inflammation. Since it can be prepared simply by repetitive applications of MC903 and shows consistent clinical results, this model has been widely used. However, in contrast to human atopic dermatitis which is chronic and closely related to TH2 cells, MC903 induces inflammations temporarily and even in the absence of T cells. Here, we modified the MC903 treatment schedule and developed a chronic MC903-induced skin inflammation model. Mice were sensitized with a high dose of MC903 and challenged with a low dose of MC903. Prior to challenge, mice were allowed to recover completely from the inflammation which occurred during the sensitization. The challenge of MC903 induced skin swelling and type 2 inflammations more rapidly, which was dependent on CD4+ T cells and IL-33. We expect that our mouse model will be beneficial for studying the late course of atopic dermatitis.

APC/CFZR-1 regulates centrosomal ZYG-1 to limit centrosome number.

Aberrant centrosome numbers are associated with human cancers. The levels of centrosome regulators positively correlate with centrosome number. Thus, tight control of centrosome protein levels is critical. In Caenorhabditis elegans, the anaphase-promoting complex/cyclosome and its co-activator FZR-1 (APC/CFZR-1), a ubiquitin ligase, negatively regulates centrosome assembly through SAS-5 degradation. In this study, we report the C. elegans ZYG-1 (Plk4 in humans) as a potential substrate of APC/CFZR-1. Inhibiting APC/CFZR-1 or mutating a ZYG-1 destruction (D)-box leads to elevated ZYG-1 levels at centrosomes, restoring bipolar spindles and embryonic viability to zyg-1 mutants, suggesting that APC/CFZR-1 influences centrosomal ZYG-1 via the D-box motif. We also show the Slimb/ßTrCP-binding (SB) motif is critical for ZYG-1 degradation, substantiating a conserved mechanism by which ZYG-1/Plk4 stability is regulated by the SKP1-CUL1-F-box (Slimb/ßTrCP)-protein complex (SCFSlimb/ßTrCP)-dependent proteolysis via the conserved SB motif in C. elegans. Furthermore, we show that co-mutating ZYG-1 SB and D-box motifs stabilizes ZYG-1 in an additive manner, suggesting that the APC/CFZR-1 and SCFSlimb/ßTrCP ubiquitin ligases function cooperatively for timely ZYG-1 destruction in C. elegans embryos where ZYG-1 activity remains at threshold level to ensure normal centrosome number.

Lysophosphatidylcholine aggravates contact hypersensitivity by promoting neutrophil infiltration and IL17 expression.

Lysophosphatidylcholine (LPC) is a bioactive lysolipid known to contribute to the development of lung allergic diseases. However, it remains unknown whether LPC possesses proinflammatory properties in the skin as well. Here, we investigated this issue by injection of LPC into the murine contact hypersensitivity (CHS) model induced by 2,4-dinitrofluorobenzene (DNFB). LPC increased the expression of IL17, recruited more neutrophils, and eventually aggravated the CHS in the skins. Moreover, the effects of LPC diminished after neutralizing IL17 or depleting neutrophils. Mechanistically, LPC upregulated not only IL17 but also CXCL1 and CXCL2 in a G2A-dependent manner. Taken together, our study demonstrated that the upregulation of LPC could contribute to allergic skin inflammation by increasing IL17 expression and neutrophil recruitment via G2A receptor. [BMB Reports 2021; 54(4): 203-208].

APC/CFZR-1 Controls SAS-5 Levels To Regulate Centrosome Duplication in Caenorhabditis elegans.

As the primary microtubule-organizing center, centrosomes play a key role in establishing mitotic bipolar spindles that secure correct transmission of genomic content. For the fidelity of cell division, centrosome number must be strictly controlled by duplicating only once per cell cycle. Proper levels of centrosome proteins are shown to be critical for normal centrosome number and function. Overexpressing core centrosome factors leads to extra centrosomes, while depleting these factors results in centrosome duplication failure. In this regard, protein turnover by the ubiquitin-proteasome system provides a vital mechanism for the regulation of centrosome protein levels. Here, we report that FZR-1, the Caenorhabditis elegans homolog of Cdh1/Hct1/Fzr, a coactivator of the anaphase promoting complex/cyclosome (APC/C), an E3 ubiquitin ligase, functions as a negative regulator of centrosome duplication in the C. elegans embryo. During mitotic cell division in the early embryo, FZR-1 is associated with centrosomes and enriched at nuclei. Loss of fzr-1 function restores centrosome duplication and embryonic viability to the hypomorphic zyg-1(it25) mutant, in part, through elevated levels of SAS-5 at centrosomes. Our data suggest that the APC/CFZR-1 regulates SAS-5 levels by directly recognizing the conserved KEN-box motif, contributing to proper centrosome duplication. Together, our work shows that FZR-1 plays a conserved role in regulating centrosome duplication in C. elegans.

The Zebrafish curly fry Is Required for Proper Centrosome and Mitotic Spindle Assembly.

The zebrafish curly fry (cfy) mutation leads to a dramatic increase in mitotic index and cell death starting during neural tube formation. The mutant phenotype is cell autonomous and does not result from defects in apical/basal polarity within the neuroepithelium. The increase in mitotic index could be due to increased proliferation or cell cycle arrest in mitosis. cfy embryos were analyzed to examine these two possibilities. By labeling embryos with a pulse of BrdU and anti-phospho-histone 3 and examining the DNA content by fluorescence activated cell sorting, we show that cfy mutants exhibit no increase in proliferation, but a significant increase in the number of cells arrested in mitosis. Furthermore, time-lapse microscopy in vivo confirmed that a great majority of dividing cells arrest during mitosis and that these mitotically arrested cells die in cfy embryos. Finally, immunostaining and confocal microscopy in cfy mutant embryos revealed that mitotic cells in mutants contain aberrant centrosomes and often exhibit monopolar spindles, thereby leading to mitotic cell cycle arrest. Our results suggest that the cfy gene is required for proper centrosome assembly and mitotic spindle formation, therefore critical for normal cell division.

Vitamin C enhances the expression of IL17 in a Jmjd2-dependent manner.

Previously, we reported that vitamin C facilitates the CpG demethylation of Foxp3 enhancer in CD4＋Foxp3＋ regulatory T cells (Tregs) by enhancing the activity of a DNA demethylase ten-eleven-translocation (Tet). However, it is not clear whether vitamin C affects other helper T cell lineages like T helper type 17 (Th17) cells which are related with Tregs. Here, we show that the expression of interleukin-17A (IL17) increases with the treatment of vitamin C but not with other antioxidants. Interestingly, the upregulation of IL17 was not accompanied by DNA demethylation in Il17 promoter and was independent of Tet enzymes. Rather, vitamin C reduced the trimethylation of histone H3 lysine 9 (H3K9me3) in the regulatory elements of the Il17 locus, and the effects of vitamin C were abrogated by knockdown of jumonji-C domain-containing protein 2 (jmjd2). These results suggest that vitamin C can affect the expression of IL17 by modulating the histone demethylase activity. [BMB Reports 2017; 50(1): 49-54].

Casein kinase II is required for proper cell division and acts as a negative regulator of centrosome duplication in Caenorhabditis elegans embryos.

Centrosomes are the primary microtubule-organizing centers that orchestrate microtubule dynamics during the cell cycle. The correct number of centrosomes is pivotal for establishing bipolar mitotic spindles that ensure accurate segregation of chromosomes. Thus, centrioles must duplicate once per cell cycle, one daughter per mother centriole, the process of which requires highly coordinated actions among core factors and modulators. Protein phosphorylation is shown to regulate the stability, localization and activity of centrosome proteins. Here, we report the function of Casein kinase II (CK2) in early Caenorhabditis elegans embryos. The catalytic subunit (KIN-3/CK2α) of CK2 localizes to nuclei, centrosomes and midbodies. Inactivating CK2 leads to cell division defects, including chromosome missegregation, cytokinesis failure and aberrant centrosome behavior. Furthermore, depletion or inhibiting kinase activity of CK2 results in elevated ZYG-1 levels at centrosomes, restoring centrosome duplication and embryonic viability to zyg-1 mutants. Our data suggest that CK2 functions in cell division and negatively regulates centrosome duplication in a kinase-dependent manner.

Correction: ATX-2, The C. elegans Ortholog of Human Ataxin-2, Regulates Centrosome Size and Microtubule Dynamics.

[This corrects the article DOI: 10.1371/journal.pgen.1006370.].

DNA Demethylation of the Foxp3 Enhancer Is Maintained through Modulation of Ten-Eleven-Translocation and DNA Methyltransferases.

Stable expression of Foxp3 is ensured by demethylation of CpG motifs in the Foxp3 intronic element, the conserved non-coding sequence 2 (CNS2), which persists throughout the lifespan of regulatory T cells (Tregs). However, little is known about the mechanisms on how CNS2 demethylation is sustained. In this study, we found that Ten-Eleven-Translocation (Tet) DNA dioxygenase protects the CpG motifs of CNS2 from re-methylation by DNA methyltransferases (Dnmts) and prevents Tregs from losing Foxp3 expression under inflammatory conditions. Upon stimulation of Tregs by interleukin-6 (IL6), Dnmt1 was recruited to CNS2 and induced methylation, which was inhibited by Tet2 recruited by IL2. Tet2 prevented CNS2 re-methylation by not only the occupancy of the CNS2 locus but also by its enzymatic activity. These results show that the CNS2 methylation status is dynamically regulated by a balance between Tets and Dnmts which influences the expression of Foxp3 in Tregs.

ATX-2, the C. elegans Ortholog of Human Ataxin-2, Regulates Centrosome Size and Microtubule Dynamics.

Centrosomes are critical sites for orchestrating microtubule dynamics, and exhibit dynamic changes in size during the cell cycle. As cells progress to mitosis, centrosomes recruit more microtubules (MT) to form mitotic bipolar spindles that ensure proper chromosome segregation. We report a new role for ATX-2, a C. elegans ortholog of Human Ataxin-2, in regulating centrosome size and MT dynamics. ATX-2, an RNA-binding protein, forms a complex with SZY-20 in an RNA-independent fashion. Depleting ATX-2 results in embryonic lethality and cytokinesis failure, and restores centrosome duplication to zyg-1 mutants. In this pathway, SZY-20 promotes ATX-2 abundance, which inversely correlates with centrosome size. Centrosomes depleted of ATX-2 exhibit elevated levels of centrosome factors (ZYG-1, SPD-5, γ-Tubulin), increasing MT nucleating activity but impeding MT growth. We show that ATX-2 influences MT behavior through γ-Tubulin at the centrosome. Our data suggest that RNA-binding proteins play an active role in controlling MT dynamics and provide insight into the control of proper centrosome size and MT dynamics.

Vitamin C Facilitates Demethylation of the Foxp3 Enhancer in a Tet-Dependent Manner.

Demethylation of CpG motifs in the Foxp3 intronic element, conserved noncoding sequence 2 (CNS2), is indispensable for the stable expression of Foxp3 in regulatory T cells (Tregs). In this study, we found that vitamin C induces CNS2 demethylation in Tregs in a ten-eleven-translocation 2 (Tet2)-dependent manner. The CpG motifs of CNS2 in Tregs generated in vitro by TGF-ß (iTregs), which were methylated originally, became demethylated after vitamin C treatment. The conversion of 5-methylcytosin into 5-hydroxymethylcytosin was more efficient, and the methyl group from the CpG motifs of Foxp3 CNS2 was erased rapidly in iTregs treated with vitamin C. The effect of vitamin C disappeared in Tet2(-/-) iTregs. Furthermore, CNS2 in peripheral Tregs in vivo, which were demethylated originally, became methylated after treatment with a sodium-dependent vitamin C transporter inhibitor, sulfinpyrazone. Finally, CNS2 demethylation in thymic Tregs was also impaired in Tet2(-/-) mice, but not in wild type mice, when they were treated with sulfinpyrazone. Collectively, vitamin C was required for the CNS2 demethylation mediated by Tet proteins, which was essential for Foxp3 expression. Our findings indicate that environmental factors, such as nutrients, could bring about changes in immune homeostasis through epigenetic mechanisms.

New real-time heartbeat detection method using the angle of a single-lead electrocardiogram.

This study presents a new real-time heartbeat detection algorithm using the geometric angle between two consecutive samples of single-lead electrocardiogram (ECG) signals. The angle was adopted as a new index representing the slope of ECG signal. The method consists of three steps: elimination of high-frequency noise, calculation of the angle of ECG signal, and detection of R-waves using a simple adaptive thresholding technique. The MIT-BIH arrhythmia database, QT database, European ST-T database, T-wave alternans database and synthesized ECG signals were used to evaluate the performance of the proposed algorithm and compare with the results of other methods suggested in literature. The proposed method shows a high detection rate-99.95% of the sensitivity, 99.95% of the positive predictivity, and 0.10% of the fail detection rate on the four databases. The result shows that the proposed method can yield better or comparable performance than other literature despite the relatively simple process. The proposed algorithm needs only a single-lead ECG, and involves a simple and quick calculation. Moreover, it does not require post-processing to enhance the detection. Thus, it can be effectively applied to various real-time healthcare and medical devices.

Event synchronous adaptive filter based atrial activity estimation in single-lead atrial fibrillation electrocardiograms.

In this paper, an event synchronous adaptive filter (ESAF) is proposed to estimate atrial activity (AA) from a single-lead AF ECG in real time. The proposed ESAF is a kind of adaptive filter designed to have the reference fed with the impulse train synchronized with the R peak in a raw atrial fibrillation (AF) ECG and to input the timely delayed AF ECG into the primary input. To assess the performance, for ten simulated AF ECGs, the cross-correlation coefficient (ρ) and the normalized mean square error (NMSE) between estimated AAs and ten original simulated AAs were calculated and, for ten real AF ECGs, the ventricular residue (VR) in QRS interval and similarity (S) in non-QRS interval were computed. As a result, these four parameters were revealed as ρ = 0.938 ± 0.016 and NMSE = 0.243 ± 0.051 for simulated AF ECGs and VR = 1.190 ± 0.476 and S = 0.967 ± 0.041 for real AF ECGs. These results were found to be better than those of the averaged beat subtraction (ABS) method, which had been previously considered the only way to estimate AA automatically in real time. In conclusion, even with single-lead AF ECGs, the proposed method estimated AAs accurately and calculated the atrial fibrillatory frequencies, the most valuable index in AF maintenance and therapy evaluation, with a remarkably low computational cost.