SRSF3 and HNRNPH1 Regulate Radiation-Induced Alternative Splicing of Protein Arginine Methyltransferase 5 in Hepatocellular Carcinoma

Protein arginine methyltransferase 5 (PRMT5) is an epigenetic regulator which has been proven to be a potential target for cancer therapy. We observed that PRMT5 underwent alternative splicing (AS) and generated a spliced isoform PRMT5-ISO5 in hepatocellular carcinoma (HCC) patients after radiotherapy. However, the regulatory mechanism and the clinical implications of IR-induced PRMT5 AS are unclear. This work revealed that serine and arginine rich splicing factor 3 (SRSF3) silencing increased PRMT5-ISO5 level, whereas heterogeneous nuclear ribonucleoprotein H 1 (HNRNPH1) silencing reduced it. Then, we found that SRSF3 and HNRNPH1 competitively combined with PRMT5 pre-mRNA located at the region around the 3′- splicing site on intron 2 and the alternative 3′- splicing site on exon 4. IR-induced SRSF3 downregulation led to an elevated level of PRMT5-ISO5, and exogenous expression of PRMT5-ISO5 enhanced cell radiosensitivity. Finally, we confirmed in vivo that IR induced the increased level of PRMT5-ISO5 which in turn enhanced tumor killing and regression, and liver-specific Prmt5 depletion reduced hepatic steatosis and delayed tumor progression of spontaneous HCC. In conclusion, our data uncover the competitive antagonistic interaction of SRSF3 and HNRNPH1 in regulating PRMT5 splicing induced by IR, providing potentially effective radiotherapy by modulating PRMT5 splicing against HCC.     

Rice β-Glucosidase 4 (Os1βGlu4) Regulates the Hull Pigmentation via Accumulation of Salicylic Acid

Salicylic acid (SA) is a stress hormone synthesized in phenylalanine ammonia-lyase (PAL) and the branching acid pathway. SA has two interconvertible forms in plants: SAG (SA O-β-glucoside) and SA (free form). The molecular mechanism of conversion of SA to SAG had been reported previously. However, which genes regulate SAG to SA remained unknown. Here, we report a cytoplasmic β-glucosidase (β-Glu) which participates in the SA pathway and is involved in the brown hull pigmentation in rice grain. In the current study, an EMS-generated mutant brown hull 1 (bh1) displayed decreased contents of SA in hulls, a lower photosynthesis rate, and high-temperature sensitivity compared to the wild type (WT). A plaque-like phenotype (brown pigmentation) was present on the hulls of bh1, which causes a significant decrease in the seed setting rate. Genetic analysis revealed a mutation in LOC_Os01g67220, which encodes a cytoplasmic Os1βGlu4. The knock-out lines displayed the phenotype of brown pigmentation on hulls and decreased seed setting rate comparable with bh1. Overexpression and complementation lines of Os1βGlu4 restored the phenotype of hulls and normal seed setting rate comparable with WT. Subcellular localization revealed that the protein of Os1βGlu4 was localized in the cytoplasm. In contrast to WT, bh1 could not hydrolyze SAG into SA in vivo. Together, our results revealed the novel role of Os1βGlu4 in the accumulation of flavonoids in hulls by regulating the level of free SA in the cellular pool.     

Discovery of Flavonoids as Novel Inhibitors of ATP Citrate Lyase: Structure–Activity Relationship and Inhibition Profiles

ATP citrate lyase (ACLY) is a key enzyme in glucolipid metabolism and its aberrantly high expression is closely associated with various cancers, hyperlipemia and atherosclerotic cardiovascular diseases. Prospects of ACLY inhibitors as treatments of these diseases are excellent. To date, flavonoids have not been extensively reported as ACLY inhibitors. In our study, 138 flavonoids were screened and 21 of them were subjected to concentration–response curves. A remarkable structure–activity relationship (SAR) trend was found: ortho-dihydroxyphenyl and a conjugated system maintained by a pyrone ring were critical for inhibitory activity. Among these flavonoids, herbacetin had a typical structure and showed a non–aggregated state in solution and a high inhibition potency (IC₅₀ = 0.50 ± 0.08 μM), and therefore was selected as a representative for the ligand–protein interaction study. In thermal shift assays, herbacetin improved the thermal stability of ACLY, suggesting a direct interaction with ACLY. Kinetic studies determined that herbacetin was a noncompetitive inhibitor of ACLY, as illustrated by molecular docking and dynamics simulation. Together, this work demonstrated flavonoids as novel and potent ACLY inhibitors with a remarkable SAR trend, which may help design high–potency ACLY inhibitors. In–depth studies of herbacetin deepened our understanding of the interactions between flavonoids and ACLY.     

Extraction,Characterization, and Platelet Inhibitory Effects of Two Polysaccharides from the Cs-4 Fungus

Cardiovascular diseases are associated with platelet hyperactivity, and downregulating platelet activation is one of the promising antithrombotic strategies. This study newly extracted two polysaccharides (purified exopolysaccharides, EPSp and purified intercellular exopolysaccharides, IPSp) from Cordyceps sinensis Cs-4 mycelial fermentation powder, and investigated the effects of the two polysaccharides and their gut bacterial metabolites on platelet functions and thrombus formation. EPSp and IPSp are majorly composed of galactose, mannose, glucose, and arabinose. Both EPSp and IPSp mainly contain 4-Galp and 4-Glcp glycosidic linkages. EPSp and IPSp significantly inhibited human platelet activation and aggregation with a dose-dependent manner, and attenuated thrombus formation in mice without increasing bleeding risk. Furthermore, the EPSp and IPSp after fecal fermentation showed enhanced platelet inhibitory effects. The results have demonstrated the potential value of Cs-4 polysaccharides as novel protective ingredients for cardiovascular diseases.     

CS12192, a Novel JAK3/JAK1/TBK1 Inhibitor,Synergistically Enhances the Anti-Inflammation Effect of Methotrexate in a Rat Model of Rheumatoid Arthritis

Rheumatoid arthritis (RA) is a common disease worldwide and is treated commonly with methotrexate (MTX). CS12192 is a novel JAK3 inhibitor discovered by Chipscreen Biosciences for the treatment of autoimmune diseases. In the present study, we examined the therapeutic effect of CS12192 against RA and explored if the combinational therapy of CS12192 and MTX produced a synergistic effect against RA in rat collagen-induced arthritis (CIA). Arthritis was induced in male Sprague-Dawley rats by two intradermal injections of bovine type II collagen (CII) and treated with MTX, CS12192, or the combination of CS12192 and MTX daily for two weeks. Effects of different treatments on arthritis score, X-ray score, pathology, and expression of inflammatory cytokines and biomarkers were examined. We found that treatment with either CS12192 or MTX produced a comparable therapeutic effect on CIA including: (1) significantly lowering the arthritis score, X-ray score, serum levels of rheumatic factor (RF), C-reactive protein (CRP), and anti-nuclear antibodies (ANA); (2) largely alleviating histopathological damage, reducing infiltration of Th17 cells while promoting Treg cells; (3) inhibiting the expression of inflammatory cytokines and chemokines such as IL-1β, TNF-α, IL-6, CCL2, and CXCL1. All these inhibitory effects were further improved by the combinational therapy with MTX and CS12192. Of importance, the combinational treatment also resulted in a marked switching of the Th17 to Treg and the M1 to M2 immune responses in synovial tissues of CIA. Thus, when compared to the monotherapy, the combination treatment with CS12192 and MTX produces a better therapeutic effect against CIA with a greater suppressive effect on T cells and macrophage-mediated joint inflammation.     

The Relationships among “STAY-GREEN” Trait,Post-Anthesis Assimilate Remobilization,and Grain Yield in Rice (Oryza sativa L.)

The mobilization and translocation of carbohydrates and mineral nutrients from vegetative plant parts to grains are pivotal for grain filling, often involving a whole plant senescence process. Loss of greenness is a hallmark of leaf senescence. However, the relationship between crop yield and senescence has been controversial for many years. Here, in this study, the overexpression and RNA interference lines of gene of OsNYC3 (Non-Yellow Coloring 3), a chlorophyll catabolism gene, were investigated. Furthermore, exogenous phytohormones were applied, and a treatment of alternate wetting and moderate drying (AWMD) was introduced to regulate the processes of leaf senescence. The results indicated that the delayed senescence of the “STAY-GREEN” trait of rice is undesirable for the process of grain filling, and it would cause a lower ratio of grain filling and lower grain weight of inferior grains, because of unused assimilates in the stems and leaves. Through the overexpression of OsNYC3, application of exogenous chemicals of abscisic acid (ABA), and water management of AWMD, leaf photosynthesis was less influenced, a high ratio of carbohydrate assimilates was partitioned to grains other than leaves and stems as labeled by ¹³C, grain filling was improved, especially for inferior spikelets, and activities of starch-synthesizing enzymes were enhanced. However, application of ethephon not only accelerated leaf senescence, but also caused seed abortion and grain weight reduction. Thus, plant senescence needs to be finely adjusted in order to make a contribution to crop productivity.     

Treatment of Periodontal Inflammation in Diabetic Rats with IL-1ra Thermosensitive Hydrogel

Periodontitis is a chronic inflammatory disease that is considered to be the main cause of adult tooth loss. Diabetes mellitus (DM) has a bidirectional relationship with periodontitis. Interleukin-1β (IL-1β) is an important pre-inflammatory factor, which participates in the pathophysiological process of periodontitis and diabetes. The interleukin-1 receptor antagonist (IL-1ra) is a natural inhibitor of IL-1, and the balance between IL-1ra and IL-1β is one of the main factors affecting chronic periodontitis (CP) and diabetes. The purpose of this study is to develop a drug carrier that is safe and nontoxic and can effectively release IL-1ra, which can effectively slow down the inflammation of periodontal tissues with diabetes, and explore the possibility of lowering the blood sugar of this drug carrier. Therefore, in this experiment, a temperature-sensitive hydrogel loaded with IL-1ra was prepared and characterized, and its anti-inflammatory effect in high-sugar environments in vivo and in vitro was evaluated. The results showed that the hydrogel could gel after 5 min at 37 °C, the pore size was 5–70 μm, and the cumulative release of IL-1ra reached 83.23% on the 21st day. Real-time polymerase chain reaction (qRT-PCR) showed that the expression of IL-1β, Interleukin 6 (IL-6), and tumor necrosis factor α (TNF-α) inflammatory factors decreased after the treatment with IL-1ra-loaded thermosensitive hydrogel. Histological evaluation and micro-computed tomography (Micro-CT) showed that IL-1ra-loaded thermosensitive hydrogel could effectively inhibit periodontal inflammation and reduce alveolar bone absorption in rats with diabetic periodontitis. It is worth mentioning that this hydrogel also plays a role in relieving hyperglycemia. Therefore, the temperature-sensitive hydrogel loaded with IL-1ra may be an effective method to treat periodontitis with diabetes.