Insufficient phosphorylation of STAT5 in Tregs inhibits the expression of BLIMP-1 but not IRF4, reduction the proportion of Tregs in pediatric aplastic anemia.

Deficiency in regulatory T cells (Tregs) is an important mechanism underlying the pathogenesis of pediatric aplastic anemia, but its specific mechanism is unclear. In our study, we aimed to investigate whether IL-2/STAT5 can regulate the proliferation of Tregs in aplastic anemia (AA) by regulating their expression of B lymphocyte-induced mature protein-1 (BLIMP-1) or interferon regulatory factor 4 (IRF4). Through clinical research and animal experiments, we found that poor activation of the IL-2/STAT5 signaling pathway may leads to low expression of BLIMP-1 in Tregs of children with AA, which leads to defects in the differentiation and proliferation of Tregs in AA. In AA model mice, treatment with IL-2c reversed the decrease in Treg proportions and reduction in Blimp-1 expression in Tregs by increasing the phosphorylation of Stat5 in Tregs. In AA, deficiency of IRF4 expression in Tregs is closely related to the deficiency of Tregs, but is not regulated by the IL-2/STAT5 pathway.

Enhancement of PRMT6 binding to a novel germline GATA1 mutation associated with congenital anemia.

Mutations in the master hematopoietic transcription factor GATA1 are often associated with functional defects in erythropoiesis and megakaryopoiesis. In this study, we identified a novel GATA1 germline mutation (c.1162delGG, p.Leu387Leufs*62) in a patient with congenital anemia and occasional thrombocytopenia. The C-terminal GATA1, a rarely studied mutational region, undergoes frameshifting translation as a consequence of this double-base deletion mutation. To investigate the specific function and pathogenic mechanism of this mutant, in vitro mutant models of stable re-expression cells were generated. The mutation was subsequently validated to cause diminished transcriptional activity of GATA1 and defective differentiation of erythroid and megakaryocytes. Using proximity labeling and mass spectrometry, we identified selective alterations in the proximal protein networks of the mutant, revealing decreased binding to a set of normal GATA1-interaction proteins, including the essential co-factor FOG1. Notably, our findings further demonstrated enhanced recruitment of the protein arginine methyltransferase PRMT6, which mediates histone modification at H3R2me2a and represses transcription activity. We also found an enhanced binding of this mutant GATA1/PRMT6 complex to the transcriptional regulatory elements of GATA1's target genes. Moreover, treatment of the PRMT6 inhibitor MS023 could partially rescue the inhibited transcriptional and impaired erythroid differentiation caused by the GATA1 mutation. Taken together, our results provide molecular insights into erythropoiesis in which mutation leads to partial loss of GATA1 function and the broader role of PRMT6 and its inhibitor MS023 in congenital anemia, highlighting PRMT6 binding as a negative factor of GATA1 transcriptional activity in aberrant hematopoiesis.

Crosstalk between PML and p53 in response to TGF-ß1: A new mechanism of cardiac fibroblast activation.

Cardiac fibrosis is a common pathological cardiac remodeling in a variety of heart diseases, characterized by the activation of cardiac fibroblasts. Our previous study uncovered that promyelocytic leukemia protein (PML)-associated SUMO processes is a new regulator of cardiac hypertrophy and heart failure. The present study aimed to explore the role of PML in cardiac fibroblasts activation. Here we found that PML is significantly upregulated in cardiac fibrotic tissue and activated cardiac fibroblasts treated with transforming growth factor-ß1 (TGF-ß1). Gain- and loss-of-function experiments showed that PML impacted cardiac fibroblasts activation after TGF-ß1 treatment. Further study demonstrated that p53 acts as the transcriptional regulator of PML, and participated in TGF-ß1 induced the increase of PML expression and PML nuclear bodies (PML-NBs) formation. Knockdown or pharmacological inhibition of p53 produced inhibitory effects on the activation of cardiac fibroblasts. We further found that PML also may stabilize p53 through inhibiting its ubiquitin-mediated proteasomal degradation in cardiac fibroblasts. Collectively, this study suggests that PML crosstalk with p53 regulates cardiac fibroblasts activation, which provides a novel therapeutic strategy for cardiac fibrosis.

[Expression and Significance of BLIMP-1 in Regulatory T Cells of Children with Aplastic Anemia].

OBJECTIVE: To study the expression of B lymphocyte-induced mature protein-1 (BLIMP-1) in regulatory T cells (Tregs) of children with aplastic anemia (AA), and analyze its correlation with the number of Tregs and the levels of inhibitory cytokines interleukin (IL)-10 and transforming growth factor (TGF)-ß in plasma. METHODS: The peripheral blood samples of 10 newly diagnosed AA children and 10 healthy children were collected for experiment. qPCR was used to detect FOXP3 and PRDM1 mRNA expression levels. Flow cytometry was used to detect the proportion of Tregs, the expression of BLIMP-1 in Tregs, and the levels of cytokines such as IL-2, IL-17A, IL-6, interferon (IFN)-γ, IL-10 and TGF-ß in plasma. Pearson correlation model was used to evaluate the relationship between the expression of BLIMP-1 in Treg and the number of Tregs, as well as the levels of IL-10 and TGF-ß in plasma. RESULTS: Compared with control group, the proportion of Tregs in peripheral blood of AA children was decreased significantly (P<0.001); The plasma levels of proinflammatory cytokines IL-2, IL-6 and IFN-γ in AA children were increased significantly (P=0.033, P=0.031, P=0.006), and IL-17A also was increased but the difference was not statistically significant (P=0.052), while anti-inflammatory cytokines IL-10 and TGF-ß were significantly reduced (P=0.048, P=0.002). The relative expressions level of FOXP3 and PRDM1 mRNA in AA children were significantly lower than those in control group (P=0.037, P=0.016). The expression of BLIMP-1 protein in Tregs of AA children was significantly lower than that in control group (P<0.001). The expression level of BLIMP-1 protein in Tregs was positively correlated with the percentage of Tregs in lymphocytes (r=0.671, P=0.001), and was also positively correlated with the levels of IL-10 and TGF-ß in plasma (r=0.500, P=0.029; r=0.486, P=0.030). CONCLUSION: The expression of BLIMP-1 in Tregs of AA children is impaired, and the low expression of BLIMP-1 is related to the decrease of the number in Tregs and IL-10 and TGF-ß expressions.

Targeting MEX3A attenuates metastasis of breast cancer via ß-catenin signaling pathway inhibition.

Metastasis is the major cause of mortality in patients with breast cancer. Understanding the metastatic mechanism to guide clinical diagnoses and the treatment of breast cancer remains a challenge. We found that the expression of Mex-3 RNA binding family member A (MEX3A) was upregulated significantly and related to tumor grade in breast cancer. The results of in vitro and in vivo studies showed that knockdown of MEX3A inhibited the metastasis and impaired the stemness of breast cancer cells. Furthermore, activation of the ß-catenin signaling pathway was discovered as a molecular intermediate of MEX3A-mediated regulation. We also found that ectopic expression of ß-catenin restored the migration ability, invasion ability, and CD44+/CD24- percentage of MDA-MB-231 and BT549 cells when MEX3A was depleted. In addition, we revealed that MEX3A positively regulated the expression of ß-catenin by downregulating Dickkopf WNT signaling pathway inhibitor 1 (DKK1) expression. Therefore, a previously undiscovered role of MEX3A comprising a critical contribution to promoting metastasis and maintaining the stemness of breast cancer via the Wnt/ß-catenin pathway was demonstrated in the present study.

Polymorphisms and haplotypes of IL2RA, IL10, IFNG, IRF5, and CCR2 are associated with Epstein-Barr virus-associated hemophagocytic lymphohistiocytosis in children.

OBJECTIVE: Cytokine storms are central to the development of Epstein-Barr virus-associated hemophagocytic lymphohistiocytosis (EBV-HLH). Previous studies have shown that single-nucleotide polymorphisms (SNPs) of cytokine genes may be associated with the development of EBV-HLH in children. As such, we investigated the association between susceptibility to EBV-HLH in children and SNPs and haplotypes of genes encoding interleukin-2 receptor subunit alpha (IL2RA), interleukin-10 (IL10), interferon gamma (IFNG), interferon regulatory factor 5 (IRF5), and C-C chemokine receptor 2 (CCR2). METHODS: Sixty-six children with EBV-HLH and 58 healthy EBV-seropositive controls were enrolled in this study. SNPs of IL2RA rs2104286, rs12722489, and rs11594656; IL10 rs1800896, rs1800871, and rs1800872; IFNG rs2430561, IRF5 rs2004640, and CCR2 rs1799864 were assayed and genotyped using the SNaPshot technique. RESULTS: Frequencies of the A allele of IL2RA rs2104286 and IL10 rs1800896, and C allele of IL-10 rs1800872 were significantly higher in the EBV-HLH group than in the control group. The AA genotype of IL2RA rs2104286 and IL10 rs1800896, and the CC genotype of IL10 rs1800872 might be associated with a significantly high risk of EBV-HLH. However, the frequencies of genotypes and alleles of IL2RA rs2104286, IL10 rs1800871, IFNG rs2430561, IRF5 rs2004640, and CCR2 rs1799864 were similar in both groups. Additionally, IL2RA AGT (rs2104286-rs12722489-rs11594656) and IL10 ACC (rs1800896-rs1800871-rs1800872) haplotypes were also associated with an increased risk of EBV-HLH. CONCLUSIONS: SNPs of IL2RA rs2104286, IL10 rs1800896 and rs1800872 and the haplotypes of IL2RA AGT and IL10 ACC were highly associated with susceptibility to EBV-HLH in children.

RANK promotes colorectal cancer migration and invasion by activating the Ca2+-calcineurin/NFATC1-ACP5 axis.

The tumor necrosis factor (TNF) receptor superfamily member 11a (TNFRSF11a, also known as RANK) was demonstrated to play an important role in tumor metastasis. However, the specific function of RANK in colorectal cancer (CRC) metastasis and the underlying mechanism are unknown. In this study, we found that RANK expression was markedly upregulated in CRC tissues compared with that in matched noncancerous tissues. Increased RANK expression correlated positively with metastasis, higher TNM stage, and worse prognosis in patients with CRC. Overexpression of RANK promoted CRC cell metastasis in vitro and in vivo, while knockdown of RANK decreased cell migration and invasion. Mechanistically, RANK overexpression significantly upregulated the expression of tartrate-resistant acid phosphatase 5 (TRAP/ACP5) in CRC cells. Silencing of ACP5 in RANK-overexpressing CRC cells attenuated RANK-induced migration and invasion, whereas overexpression of ACP5 increased the migration and invasion of RANK-silencing cells. The ACP5 expression was transcriptionally regulated by calcineurin/nuclear factor of activated T cells c1 (NFATC1) axis. The inhibition of calcineurin/NFATC1 significantly decreased ACP5 expression, and attenuated RANK-induced cell migration and invasion. Furthermore, RANK induced phospholipase C-gamma (PLCγ)-mediated inositol-1,4,5-trisphosphate receptor (IP3R) axis and stromal interaction molecule 1 (STIM1) to evoke calcium (Ca2+) oscillation. The RANK-mediated intracellular Ca2+ mobilization stimulated calcineurin to dephosphorylate NFATC1 and induce NFATC1 nuclear translocation. Both blockage of PLCγ-IP3R axis and STIM1 rescued RANK-induced NFATC1 nuclear translocation, ACP5 expression, and cell metastasis. Our study revealed the functional expression of RANK in human CRC cells and demonstrated that RANK induced the Ca2+-calcineurin/NFATC1-ACP5 axis in the regulation of CRC metastasis, that might be amenable to therapeutic targeting.

Knockdown of endogenous RNF4 exacerbates ischaemia-induced cardiomyocyte apoptosis in mice.

RNF4, a poly-SUMO-specific E3 ubiquitin ligase, is associated with protein degradation, DNA damage repair and tumour progression. However, the effect of RNF4 in cardiomyocytes remains to be explored. Here, we identified the alteration of RNF4 from ischaemic hearts and oxidative stress-induced apoptotic cardiomyocytes. Upon myocardial infarction (MI) or H2 O2 /ATO treatment, RNF4 increased rapidly and then decreased gradually. PML SUMOylation and PML nuclear body (PML-NB) formation first enhanced and then degraded upon oxidative stress. Reactive oxygen species (ROS) inhibitor was able to attenuate the elevation of RNF4 expression and PML SUMOylation. PML overexpression and RNF4 knockdown by small interfering RNA (siRNA) enhanced PML SUMOylation, promoted p53 recruitment and activation and exacerbated H2 O2 /ATO-induced cardiomyocyte apoptosis which could be partially reversed by knockdown of p53. In vivo, knockdown of endogenous RNF4 via in vivo adeno-associated virus infection deteriorated post-MI structure remodelling including more extensive interstitial fibrosis and severely fractured and disordered structure. Furthermore, knockdown of RNF4 worsened ischaemia-induced cardiac dysfunction of MI models. Our results reveal a novel myocardial apoptosis regulation model that is composed of RNF4, PML and p53. The modulation of these proteins may provide a new approach to tackling cardiac ischaemia.

Ascorbic Acid Sensitizes Colorectal Carcinoma to the Cytotoxicity of Arsenic Trioxide via Promoting Reactive Oxygen Species-Dependent Apoptosis and Pyroptosis.

Arsenic trioxide (ATO) is an effective therapeutic agent against acute promyelocytic leukemia (APL); however, its anti-tumor effect on solid tumors such as colorectal cancer (CRC) is still in debate. Ascorbic acid (AA) also produces a selective cytotoxic activity against tumor cells. Here, we exploit the potential benefit of ATO/AA combination in generating cytotoxicity to CRC cells, which may lay the groundwork for the potential combinational chemotherapy of CRCs. According to the results, we found that ATO and AA effectively inhibited the viability of human CRC cells in a synergistic manner. AA and ATO corporately activated caspase-3 to trigger apoptosis and upregulated the expression of caspase-1 and promoted formation of inflammasomes to induce pyroptosis. Furthermore, the stimulation of reactive oxygen species (ROS) overproduction was demonstrated as a subcellular mechanism for apoptosis and pyroptosis induced by ATO/AA combination treatment. Our findings suggest that ATO combination with a conventional dosage of AA offers an advantage for killing CRC cells. The synergistic action of ATO/AA combination might be considered a plausible strategy for the treatment of CRC and perhaps other solid tumors as well.

ICPMS-Based Specific Quantification of Phosphotyrosine: A Gallium-Tagging and Tyrosine-Phosphatase Mediated Strategy.

Low-abundance tyrosine phosphorylation is crucial to not only normal but also aberrant life processes. We designed and synthesized a photocleavable magnetic nanoparticle-based gallium tag for tagging and enrichment as well as UV-release of the phosphate-bearing molecules/ions in cells. HPLC/71Ga species-unspecific isotope dilution (71Ga-SUID) ICPMS was subsequently developed for specific and absolute quantification of phosphotyrosine (pY) under the assistance of a protein tyrosine phosphatase-1B (PTP-1B). pY quantification was thus achieved via determination of Ga in the Ga-phosphate complexes that come exclusively from the Ga-tagged pY. In this way, the method detection limit of pY reached down to 30 amol with the RSD lower than 5.70% (n = 5 at pmol level). Feasibility of this proposed method was validated using VNQIGTLSEpYIK, VNQIGTLpSEpYIK, and extracellular regulated protein kinase 1 peptide (-pTEpY-) standards with the recovery of more than 96% (n = 5). It was applied to the absolute quantification of pY in human breast cancer MCF-7 cells, indicating that pY increased by 1.60 nmol (61.1%) in 3.0 × 106 MCF-7 cells after 100 nM insulin stimulation. We believe that, not limited to pY quantification, this element-tagging and protease-specific reaction mediated ICPMS methodology will pave a simple path for ever more applications of ICPMS to the studies of quantitative protein post-translational modifications (PTMs) when suitable element-tags are designed and specific proteases are available toward targeted PTMs.

Assessment of the Genetic Diversity of Different Job's Tears (Coix lacryma-jobi L.) Accessions and the Active Composition and Anticancer Effect of Its Seed Oil.

Job's tears (Coix lachryma-jobi L.) is an important crop used as food and herbal medicine in Asian countries. A drug made of Job's tears seed oil has been clinically applied to treat multiple cancers. In this study, the genetic diversity of Job's tears accessions and the fatty acid composition, triglyceride composition, and anti-proliferative effect of Job's tears seed oil were analyzed using morphological characteristics and ISSR markers, GC-MS, HPLC-ELSD, and the MTT method. ISSR analysis demonstrated low genetic diversity of Job's tears at the species level (h = 0.21, I = 0.33) and the accession level (h = 0.07, I = 0.10), and strong genetic differentiation (GST = 0.6702) among all accessions. It also clustered the 11 accessions into three cultivated clades corresponding with geographical locations and two evidently divergent wild clades. The grouping patterns based on morphological characteristics and chemical profiles were in accordance with those clustered by ISSR analysis. Significant differences in morphological characteristics, fatty acid composition, triglyceride composition, and inhibition rates of seed oil were detected among different accessions, which showed a highly significant positive correlation with genetic variation. These results suggest that the seed morphological characteristics, fatty acid composition, and triglyceride composition may be mainly attributed to genetic factors. The proportion of palmitic acid and linoleic acid to oleic acid displayed a highly significant positive correlation with the inhibition rates of Job's tears seed oil for T24 cells, and thus can be an important indicator for quality control for Job's tears.

GASZ and mitofusin-mediated mitochondrial functions are crucial for spermatogenesis.

Nuage is an electron-dense cytoplasmic structure in germ cells that contains ribonucleoproteins and participates in piRNA biosynthesis. Despite the observation that clustered mitochondria are associated with a specific type of nuage called intermitochondrial cement (pi-body), the importance of mitochondrial functions in nuage formation and spermatogenesis is yet to be determined. We show that a germ cell-specific protein GASZ contains a functional mitochondrial targeting signal and is largely localized at mitochondria both endogenously in germ cells and in somatic cells when ectopically expressed. In addition, GASZ interacts with itself at the outer membrane of mitochondria and promotes mitofusion in a mitofusin/MFN-dependent manner. In mice, deletion of the mitochondrial targeting signal reveals that mitochondrial localization of GASZ is essential for nuage formation, mitochondrial clustering, transposon repression, and spermatogenesis. MFN1 deficiency also leads to defects in mitochondrial activity and male infertility. Our data thus reveal a requirement for GASZ and MFN-mediated mitofusion during spermatogenesis.

Quantification and visualization of glutathione S-transferase omega 1 in cells using inductively coupled plasma mass spectrometry (ICP-MS) and fluorescence microscopy.

We report a novel activity-based and Cu-free click chemistry (CC) mediated methodology for glutathione S-transferase omega 1 (GSTO1) quantification using species-unspecific isotope dilution inductively coupled plasma mass spectrometry (SUID ICP-MS), in which dibenzylcyclooctyne-modified 2-chloroacetamide (DBCO-ChAcA) was designed and synthesized, meanwhile, as a navigator towards GSTO1 for subsequent N3-DOTA-Eu-tagging via Cu-free CC. Using (153)Eu-SUID ICP-MS coupled with size exclusion chromatography (SEC), the LOD (3σ) of GSTO1 reached 6.9 fmol with an RSD of 2.4% at the 0.1 µM level (n = 5) considering the recovery of GSTO1 on the SEC was 96.5 ± 2.4%. The GSTO1 contents in the cells of human hepatocellular carcinoma C7721 and breast carcinoma MCF-7 as well as normal hepatic C7701 without or with cis-platin administration were quantified to be from 1.2 µg/10,000 cells (n = 3, RSD = 4.5%) corresponding to 1.2 × 10(-2) ng per cell to 4.76 µg/10,000 cells (n = 3, RSD = 2.9%) corresponding to 4.76 × 10(-2) ng per cell. For a comparative study, DBCO-ChAcA-fluor 488-based fluorescence microscopy could not alone visualize GSTO1 in the cells but could together with those from the small SH-containing molecules such as GSH and that from extra N3-fluor 488 in the cells. This activity-based CC-mediated tagging/labeling strategy provided an opportunity for ICP-MS-based targeted protein quantification, and is very much expected to find its applications in biological mechanism study and the subsequent drug design.

A novel role of CDX1 in embryonic epicardial development.

The molecular mechanism that regulates epicardial development has yet to be understood. In this study, we explored the function of CDX1, a Caudal-related family member, in epicardial epithelial-to-mesenchymal transition (EMT) and in the migration and the differentiation of epicardium-derived progenitors into vascular smooth muscle cells. We detected a transient expression of CDX1 in murine embryonic hearts at 11.5 days post coitum (dpc). Using a doxycycline-inducible CDX1 mouse model, primary epicardium, and ex vivo heart culture, we further demonstrated that ectopic expression of CDX1 promoted epicardial EMT. In addition, a low-dose CDX1 induction led to enhanced migration and differentiation of epicardium-derived cells into α-SMA+ vascular smooth muscles. In contrast, either continued high-level induction of CDX1 or CDX1 deficiency attenuated the ability of epicardium-derived cells to migrate and to mature into smooth muscles induced by TGF-ß1. Further RNA-seq analyses showed that CDX1 induction altered the transcript levels of genes involved in neuronal development, angiogenesis, and cell adhesions required for EMT. Our data have revealed a previously undefined role of CDX1 during epicardial development, and suggest that transient expression of CDX1 promotes epicardial EMT, whereas subsequent down-regulation of CDX1 after 11.5 dpc in mice is necessary for further subepicardial invasion of EPDCs and contribution to coronary vascular endothelium or smooth muscle cells.

GASZ promotes germ cell derivation from embryonic stem cells.

Primordial germ cells (PGCs) are the first germ-line population that forms from the proximal epiblast of the developing embryo. Despite their biological importance, the regulatory networks whereby PGCs arise, migrate, and differentiate into gametes during embryonic development remains elusive, largely due to the limited number of germ cells in the early embryo. To elucidate the molecular mechanisms that govern early germ cell development, we utilized an in vitro differentiation model of embryonic stem cells (ESCs) and screened a series of candidate genes with specific expression in the adult reproductive organs. We discovered that gain of function of Gasz, a gene previously reported to participate in meiosis of postnatal spermatocytes, led to the most robust upregulation of PGC formation from both human and murine ESCs. In contrast, Gasz deficiency resulted in pronounced reduction of germ cells during ESC differentiation and decreased expression of MVH and DAZL in genital ridges during early embryonic development. Further analyses demonstrated that GASZ interacted with DAZL, a key germ cell regulator, to synergistically promote germ cell derivation from ESCs. Thus, our data reveal a potential role of GASZ during embryonic germ cell development and provide a powerful in vitro system for dissecting the molecular pathways in early germ cell formation during embryogenesis.

Directed graphs of DNA sequences and their numerical characterization.

In this paper we (1) introduce a directed graphical representation of DNA primary sequences; (2) describe a scheme that transforms the directed graph of a DNA sequence into an upper triangular matrix; (3) investigate whether or not the existing matrix-based invariants of DNA sequences are compatible for the upper triangular matrix representation. The utility of our method is illustrated by an examination of the similarity between human and other seven species.