Interface-Induced Ag Monolayer Film for Surface-Enhanced Raman Scattering Detection of Water-Insoluble Enrofloxacin

Water-insoluble molecules usually show poor surface-enhanced Raman scattering (SERS) signals, because they are hardly adsorbed on the surface of most commonly used SERS substrates, such as aqueous Ag or Au colloids. In this work, a highly sensitive and reproducible Ag monolayer film (Ag MLF) SERS substrate prepared by self-assembly of Ag nanoparticles (Ag NPs) on water/oil interface can realize the trace SERS detection of water-insoluble enrofloxacin. The positively charged phase transfer catalyst can transfer the negatively charged Ag nanoparticles in aqueous solution to the water/oil interface. At the same time, the water-insoluble enrofloxacin can also be attracted to the interface because of its lipophilic group. The type/volume of the oil phase and phase transfer catalyst and the vortex mixing time were all optimized to maximize the SERS effect of Ag MLF. Results showed that trace water-insoluble enrofloxacin can be identified by Ag MLF and its detection sensitivity was significantly improved. The proposed novel Ag MLF can be further applied to detect other water-insoluble molecules in SERS.

     

Phosphorylation of Ago2 is required for its role in DNA double-strand break repair

Repair of DNA double-strand break(DSB) is critical for the maintenance of genome integrity. A class of DSB-induced small RNAs(di RNAs) has been shown to play an important role in DSB repair. In humans,di RNAs are associated with Ago2 and guide the recruitment of Rad51 to DSB sites to facilitate repair by homologous recombination(HR). Ago2 activity has been reported to be regulated by phosphorylation under normal and hypoxic conditions. However, the role of Ago2 phosphorylation in DNA damage repair is unexplored. Here, we show that S672, S828, T830, and S831 of human Ago2 are phosphorylated in response to ionizing radiation(IR). S672 A mutation of Ago2 leads to significant reduction in Rad51 foci formation and HR efficiency. We further show that defective association of Ago2 S672 A variant with DSB sites, instead of defects in di RNA and Rad51 binding, may account for decreased Rad51 foci formation and HR efficiency.Our study reveals a novel regulatory mechanism for the function of Ago2 in DNA repair.     

薇甘菊化感物质对土壤氮素矿化的影响及其化感利己作用

【目的】研究入侵植物薇甘菊提取物对土壤氮素矿化的影响及化感利己作用,为薇甘菊通过释放化感物质促进自身生长并排斥本地植物生长提供实验证据,为揭示薇甘菊的入侵机制提供理论依据。【方法】采用经典的化感生测实验、化感物质添加实验以及盆栽控制实验,对比研究薇甘菊及其本地伴生植物水提液和粗提物的化感生测效应、水提液和粗提物添加对土壤氮素的影响以及水提液添加对薇甘菊生长的影响。【结果】与本地伴生种相比,薇甘菊水提液的化感抑制作用较强且促进了土壤硝态氮的生成;而薇甘菊粗提物的化感作用较弱且抑制了土壤铵态氮和硝态氮的生成。水提液盆栽控制实验结果表明,与本地伴生种火炭母相比,薇甘菊水提液具有显著的化感利己作用,这与薇甘菊水提液处理后薇甘菊生长土壤中的蛋白酶活性增强和有效氮含量增加相一致。【结论】薇甘菊可通过水溶性化感物质促进有效氮的生成来实现化感利己作用。     

Histone H2A Ubiquitination Inhibits the Enzymatic Activity of H3 Lysine 36 Methyltransferases

Histone H3 lysine 27 (H3K27) methylation and H2A monoubiquitination (ubH2A) are two closely related histone modifications that regulate Polycomb silencing. Previous studies reported that H3K27 trimethylation (H3K27me3) rarely coexists with H3K36 di- or tri-methylation (H3K36me2/3) on the same histone H3 tails, which is partially controlled by the direct inhibition of the enzymatic activity of H3K27-specific methyltransferase PRC2. By contrast, H3K27 methylation does not affect the catalytic activity of H3K36-specific methyltransferases, suggesting other Polycomb mechanism(s) may negatively regulate the H3K36-specific methyltransferase(s). In this study, we established a simple protocol to purify milligram quantities of ubH2A from mammalian cells, which were used to reconstitute nucleosome substrates with fully ubiquitinated H2A. A number of histone methyltransferases were then tested on these nucleosome substrates. Notably, all of the H3K36-specific methyltransferases, including ASH1L, HYPB, NSD1, and NSD2 were inhibited by ubH2A, whereas the other histone methyltransferases, including PRC2, G9a, and Pr-Set7 were not affected by ubH2A. Together with previous reports, these findings collectively explain the mutual repulsion of H3K36me2/3 and Polycomb modifications.     

The N‐terminal region of the Neurospora NDR kinase COT1 regulates morphology via its interactions with MOB2A/B

Nuclear Dbf2p‐related (NDR) protein kinases are important for cell differentiation and polar morphogenesis in various organisms, yet some of their functions are still elusive. Dysfunction of the Neurospora crassa NDR kinase COT1 leads to cessation of tip extension and hyperbranching. NDR kinases require the physical interaction between the kinase's N‐terminal region (NTR) and the MPS1‐binding (MOB) proteins for their activity and functions. To study the interactions between COT1 and MOB2 proteins, we mutated several conserved residues and a novel phosphorylation site within the COT1 NTR. The phenotypes of these mutants suggest that the NTR is required for COT1 functions in regulating hyphal elongation and branching, asexual conidiation and germination. Interestingly, while both MOB2A and MOB2B promote proper hyphal growth, they have distinct COT1‐dependent roles in regulation of macroconidiation. Immunoprecipitation experiments indicate physical association of COT1 with both MOB2A and MOB2B, simultaneously. Furthermore, the binding of the two MOB2 proteins to COT1 is mediated by different residues at the COT1 NTR, suggesting a hetero‐trimer is formed. Thus, although MOB2A/B may have some overlapping functions in regulating hyphal tip extension, their function is not redundant and they are both required for proper fungal development.     

Mutational analysis of MukE reveals its role in focal subcellular localization of MukBEF

Bacterial condensin MukBEF is essential for global folding of the Escherichia coli chromosome. MukB, a SMC (structural maintenance of chromosome) protein, comprises the core of this complex and is responsible for its ATP‐modulated DNA binding and reshaping activities. MukF serves as a kleisin that modulates MukB–DNA interactions and links MukBs into macromolecular assemblies. Little is known about the function of MukE. Using random mutagenesis, we generated six loss‐of‐function point mutations in MukE. The surface mutations clustered in two places. One of them was at or close to the interface with MukF while the other was away from the known interactions of the protein. All loss‐of‐function mutations affected focal localization of MukBEF in live cells. In vitro, however, only some of them interfered with the assembly of MukBEF into a complex or the ability of MukEF to disrupt MukB–DNA interactions. Moreover, some MukE mutants were able to join intracellular foci formed by endogenous MukBEF and most of the mutants were efficiently incorporated into MukBEF even in the presence of endogenous MukE. These data reveal that focal localization of MukBEF involves other activities besides DNA binding and that MukE plays a central role in them.     

A robust CRISPR–Cas9-based fluorescent reporter assay for the detection and quantification of DNA double-strand break repair

DNA double-strand breaks (DSBs) are highly cytotoxic lesions that can lead to chromosome rearrangements, genomic instability and cell death. Consequently, cells have evolved multiple mechanisms to efficiently repair DSBs to preserve genomic integrity. We have developed a DSB repair assay system, designated CDDR (CRISPR–Cas9-based Dual-fluorescent DSB Repair), that enables the detection and quantification of DSB repair outcomes in mammalian cells with high precision. CDDR is based on the introduction and subsequent resolution of one or two DSB(s) in an intrachromosomal fluorescent reporter following the expression of Cas9 and sgRNAs targeting the reporter. CDDR can discriminate between high-fidelity (HF) and error-prone non-homologous end-joining (NHEJ), as well as between proximal and distal NHEJ repair. Furthermore, CDDR can detect homology-directed repair (HDR) with high sensitivity. Using CDDR, we found HF-NHEJ to be strictly dependent on DNA Ligase IV, XRCC4 and XLF, members of the canonical branch of NHEJ pathway (c-NHEJ). Loss of these genes also stimulated HDR, and promoted error-prone distal end-joining. Deletion of the DNA repair kinase ATM, on the other hand, stimulated HF-NHEJ and suppressed HDR. These findings demonstrate the utility of CDDR in characterizing the effect of repair factors and in elucidating the balance between competing DSB repair pathways.     

CUDC‐907, a novel dual PI3K and HDAC inhibitor,in prostate cancer: Antitumour activity and molecular mechanism of action

Targeting the androgen receptor (AR) signalling pathway remains the main therapeutic option for advanced prostate cancer. However, resistance to AR‐targeting inhibitors represents a great challenge, highlighting the need for new therapies. Activation of the PI3K/AKT pathway and increased expression of histone deacetylases (HDACs) are common aberrations in prostate cancer, suggesting that inhibition of such targets may be a viable therapeutic strategy for this patient population. Previous reports demonstrated that combination of PI3K inhibitors (PI3KIs) with histone deacetylase inhibitors (HDACIs) resulted in synergistic antitumour activities against preclinical models of prostate cancer. In this study, we demonstrate that the novel dual PI3K and HDAC inhibitor CUDC‐907 has promising antitumour activity against prostate cancer cell lines in vitro and castration‐resistant LuCaP 35CR patient‐derived xenograft (PDX) mouse model in vivo. CUDC‐907‐induced apoptosis was partially dependent on Mcl‐1, Bcl‐xL, Bim and c‐Myc. Further, down‐regulation of Wee1, CHK1, RRM1 and RRM2 contributed to CUDC‐907‐induced DNA damage and apoptosis. In the LuCaP 35CR PDX model, treatment with CUDC‐907 resulted in significant inhibition of tumour growth. These findings support the clinical development of CUDC‐907 for the treatment of prostate cancer.