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.     

Targeting PSEN1 by lnc-CYP3A43-2/miR-29b-2-5p to Reduce β Amyloid Plaque Formation and Improve Cognition Function

Presenilin-1 (PSEN1) is a crucial subunit within the γ-secretase complex and regulates β-amyloid (Aβ) production. Accumulated evidence indicates that n-butylidenephthalide (BP) acts effectively to reduce Aβ levels in neuronal cells that are derived from trisomy 21 (Ts21) induced pluripotent stem cells (iPSCs). However, the mechanism underlying this effect remains unclear. This article aims to investigate the possible mechanisms through which BP ameliorates the development of Alzheimer’s disease (AD) and verify the effectiveness of BP through animal experiments. Results from RNA microarray analysis showed that BP treatment in Ts21 iPSC-derived neuronal cells reduced long noncoding RNA (lncRNA) CYP3A43-2 levels and increased microRNA (miR)-29b-2-5p levels. Bioinformatics tool prediction analysis, biotin-labeled miR-29b-2-5p pull-down assay, and dual-luciferase reporter assay confirmed a direct negative regulatory effect for miRNA29b-2-5p on lnc-RNA-CYP3A43-2 and PSEN1. Moreover, BP administration improved short-term memory and significantly reduced Aβ accumulation in the hippocampus and cortex of 3xTg-AD mice but failed in miR-29b-2-5p mutant mice generated by CRISP/Cas9 technology. In addition, analysis of brain samples from patients with AD showed a decrease in microRNA-29b-2-5p expression in the frontal cortex region. Our results provide evidence that the LncCYP3A43-2/miR29-2-5p/PSEN1 network might be involved in the molecular mechanisms underlying BP-induced Aβ reduction.     

Rapid Detection of Fusarium oxysporum Using Insulated Isothermal PCR and a Rapid,Simple DNA Preparation Protocol

We developed an insulated isothermal PCR (iiPCR) method for the efficient and rapid detection of Fusarium oxysporum (Fo), which is a fungus that infects various hosts and causes severe crop losses. The Fo iiPCR method was sensitive enough to detect up to 100 copies of standard DNA template and 10 fg of Fo genomic DNA. In addition, it could directly detect 1 pg of mycelium and 10 spores of Fo without DNA extraction. Our study compared the performance of Fo iiPCR to that of three published in planta molecular detection methods—conventional PCR, SYBR green-based real-time PCR, and hydrolysis probe-based real-time PCR—in field detection of Fo. All diseased field samples yielded positive detection results with high reproducibility when subjected to an Fo iiPCR test combined with a rapid DNA extraction protocol compared to Fo iiPCR with an automated magnetic bead-based DNA extraction protocol. Intraday and interday assays were performed to ensure the stability of this new rapid detection method. The results of detection of Fo in diseased banana pseudostem samples demonstrated that this new rapid detection method was suitable for field diagnosis of Fusarium wilt and had high F1 scores for detection (the harmonic mean of precision and recall of detection) for all asymptomatic and symptomatic Fo-infected banana samples. In addition, banana samples at four growth stages (seedling, vegetative, flowering and fruiting, and harvesting) with mild symptoms also showed positive detection results. These results indicate that this new rapid detection method is a potentially efficient procedure for on-site detection of Fo.     

2cChIP-seq and 2cMeDIP-seq: The Carrier-Assisted Methods for Epigenomic Profiling of Small Cell Numbers or Single Cells

Chromatin immunoprecipitation coupled with high-throughput sequencing (ChIP-seq) can profile genome-wide epigenetic marks associated with regulatory genomic elements. However, conventional ChIP-seq is challenging when examining limited numbers of cells. Here, we developed a new technique by supplementing carrier materials of both chemically modified mimics with epigenetic marks and dUTP-containing DNA fragments during conventional ChIP procedures (hereafter referred to as 2cChIP-seq), thus dramatically improving immunoprecipitation efficiency and reducing DNA loss of low-input ChIP-seq samples. Using this strategy, we generated high-quality epigenomic profiles of histone modifications or DNA methylation in 10–1000 cells. By introducing Tn5 transposase-assisted fragmentation, 2cChIP-seq reliably captured genomic regions with histone modification at the single-cell level in about 100 cells. Moreover, we characterized the methylome of 100 differentiated female germline stem cells (FGSCs) and observed a particular DNA methylation signature potentially involved in the differentiation of mouse germline stem cells. Hence, we provided a reliable and robust epigenomic profiling approach for small cell numbers and single cells.     

A Novel Antithrombocytopenia Agent,Rhizoma cibotii,Promotes Megakaryopoiesis and Thrombopoiesis through the PI3K/AKT,MEK/ERK,and JAK2/STAT3 Signaling Pathways

Background: Cibotii rhizoma (CR) is a famous traditional Chinese medicine (TCM) used to treat bleeding, rheumatism, lumbago, etc. However, its therapeutic effects and mechanism against thrombocytopenia are still unknown so far. In the study, we investigated the effects of aqueous extracts of Cibotii rhizoma (AECRs) against thrombocytopenia and its molecular mechanism.Methods: Giemsa staining, phalloidin staining, and flow cytometry were performed to measure the effect of AECRs on the megakaryocyte differentiation in K562 and Meg-01 cells. A radiation-induced thrombocytopenia mouse model was constructed to assess the therapeutic actions of AECRs on thrombocytopenia. Network pharmacology and experimental verification were carried out to clarify its mechanism against thrombocytopenia. Results: AECRs promoted megakaryocyte differentiation in K562 and Meg-01 cells and accelerated platelet recovery and megakaryopoiesis with no systemic toxicity in radiation-induced thrombocytopenia mice. The PI3K/AKT, MEK/ERK, and JAK2/STAT3 signaling pathways contributed to AECR-induced megakaryocyte differentiation. The suppression of the above signaling pathways by their inhibitors blocked AERC-induced megakaryocyte differentiation. Conclusions: AECRs can promote megakaryopoiesis and thrombopoiesis through activating PI3K/AKT, MEK/ERK, and JAK2/STAT3 signaling pathways, which has the potential to treat radiation-induced thrombocytopenia in the clinic.