SUMMARY Although cerebrovascular impairment is a known common driver of both Vascular dementia (VaD) and Alzheimer’s disease (AD), the underlying mechanisms remain poorly defined. Substantial evidence has demonstrated that brain-derived estrogen is essential for cerebrovascular health and neuroprotection. Consequently, therapeutic strategies that can replicate the beneficial effects of estrogen replacement therapy (ERT) in the brain while avoiding its peripheral risks are being actively pursued. Here, we demonstrate that tripeptidyl peptidase II (TPP2) is essential for cerebrovascular homeostasis in adult female mice by orchestrating intracellular Ca²⁺ distribution and local estrogen biosynthesis. Specifically, TPP2 deficiency triggers a vicious cycle of Ca²⁺ imbalance and estrogen deficiency, thereby disrupting the anticipatory unfolded protein response (UPR). This disruption consequently drives aberrant pexophagic flux and ultimately depletes ether-linked phosphatidylcholine (PC-O)—the essential building block of endothelial cell (EC) membranes. In addition, we identified PC-O as a key facilitator of choline uptake through FLVCR2 and therefore its deficiency causes subsequent choline depletion as well as significantly decreased acetylcholine (ACh). Consistent with the fact that choline is a building block of PC and that ACh is a primary driver of vascular dilation, TPP2 depletion leads to significant cerebrovascular degeneration characterized by narrowed lumens, decreased EC number, and abnormal aggregation of ECs within the blood vessel. Additionally, AAV-mediated specific expression of Far1 in ECs to promote ether-PC biosynthesis significantly increases cerebrovascular volume and diameter in hippocampi of adult female TPP2 knockout (T2KO) mice. Importantly, ectopic expression of Far1 in ECs not only significantly ameliorates memory impairment in global and conditional TPP2-depleted female mice, but also significantly improves memory performance of naturally aged female mice. In total, our findings establish TPP2 as a key determinant of cerebrovascular homeostasis in adult female mice, positioning it as a novel therapeutic candidate for the treatment of VaD and AD.

This study reveals that GCN1 regulates chloroplast‐encoded protein synthesis by translationally controlling the nuclear gene RH39, which mediates post‐maturation processing of chloroplast 23S rRNA, thereby enabling Arabidopsis to adapt chloroplast translation to light‐dark cycles.

Nitrogen (N), phosphorus (P) or potassium (K) deficiency in plants can lead to a decrease in amino acid and protein synthesis. However, it is unknown how protein translation gets repressed during macronutrient deficiencies. Previous research has shown that general control non-depressible 1 (GCN1) cooperate with GCN2 to phosphorylate the alpha subunit of eukaryotic translation initiation factor (eIF2α). In this study, we observed phosphorylation of eIF2α under N, P, and K deficiencies, which was found to be lost in gcn1. Mutant gcn1 displayed higher sensitivity to macronutrient deficiencies compared to the wild-type (WT). The evidence of in situ reactive oxygen species (ROS) accumulation in leaves indicated that macronutrient starvation triggers ROS production. Treatment with Dimethylthiourea (DMTU), a ROS scavenger, eliminated ROS and reversed eIF2α phosphorylation induced by nutrient deficiency. Moreover, it was discovered that protein translation was reduced under N or K deficiency in the WT but not in gcn1, whereas under P deprivation, protein translation was reduced in both the WT and gcn1. We additionally found that DMTU can partially recover translation inhibition under N or K deprivation. Taken together, it is concluded that GCN1-GCN2-eIF2α pathway is regulated by ROS and is essential for plant survival under macronutrient starvation conditions.

The phytohormone abscisic acid (ABA) plays important roles in plant growth, development and adaptative responses to abiotic stresses. SNF1-related protein kinase 2s (SnRK2) are key components that activate the ABA core signaling pathway. NUCLEAR PORE ANCHOR (NUA) is a component of the nuclear pore complex (NPC) that involves in deSUMOylation through physically interacting with the EARLY IN SHORT DAYS 4 (ESD4) SUMO protease. However, it is not clear how NUA functions with SnRK2 and ESD4 to regulate ABA signaling. In our study, we found that nua loss-of-function mutants exhibited pleiotropic ABA-hypersensitive phenotype. We also found that ABA-responsive genes remarkably up-regulated in nua by exogenous ABA. The nua snrk2.2 snrk2.3 triple mutant and nua abi5 double mutant partially rescued the ABA-hypersensitive phenotype of nua, thereby suggesting that NUA is epistatic to SnRK2s. Additionally, we observed that esd4-3 mutant was also ABA-hypersensitive. NUA and ESD4 were further demonstrated to physically interact with SnRK2s and negatively regulate ABA signaling by reducing SnRK2s stability. Taken together, our findings uncover a new regulatory mechanism that can modulate ABA signaling.

We have previously demonstrated that General Control Non-derepressible 1 (AtGCN1) is essential for translation inhibition under cold stress through interacting with GCN2 to phosphorylate eukaryotic translation initiation factor 2 (eIF2). Here, we report that the flower time of the atgcn1 mutant is later than that of the wild type (WT), and some siliques of atgcn1 cannot develop and produce seeds. Total and polysomal RNA of atgcn1-1 and wild type (WT) after cold treatments were sequenced. The sequencing results show that the mutation of atgcn1 selectively alters the expression of genes at both transcriptional and translational levels. The classification of AtGCN1 target genes reveals that AtGCN1 regulated gens are involved in flower development, seed dormancy and seed development, response to osmotic stress, amino acid biosynthesis, photosynthesis, cell wall organization, protein transport and localization, lipid biosynthesis, transcription, macroautophagy, proteolysis and cell death. Further analysis of AtGCN1 regulated genes at translational levels shows that the Kozak sequence and uORFs (upstream open reading frame) of transcripts affect translation selection. These results show that AtGCN1 is required for the expression of selective genes in Arabidopsis.

Tillering ability is a key agronomy trait for wheat ( Triticum aestivum L.) production. Studies on a dwarf monoculm wheat mutant ( dmc ) showed that ARF11 played an important role in tillering of wheat. In this study, a total of 67 ARF family members were identified and clustered to two main classes with four subgroups based on their protein structures. The promoter regions of T. aestivum ARF ( TaARF ) genes contain a large number of cis -acting elements closely related to plant growth and development, and hormone response. The segmental duplication events occurred commonly and played a major role in the expansion of TaARFs . The gene collinearity degrees of the ARFs between wheat and other grasses, rice and maize, were significantly high. The evolution distances among TaARFs determine their expression profiles, such as homoeologous genes have similar expression profiles, like TaARF4-3A-1 , TaARF4-3A-2 and their homoeologous genes. The expression profiles of TaARFs in various tissues or organs indicated TaARF3 , TaARF4 , TaARF9 and TaARF22 and their homoeologous genes played basic roles during wheat development. TaARF4 , TaARF9 , TaARF12 , TaARF15 , TaARF17 , TaARF21 , TaARF25 and their homoeologous genes probably played basic roles in tiller development. qRT-PCR analyses of 20 representative TaARF genes revealed that the abnormal expressions of TaARF11 and TaARF14 were major causes constraining the tillering of dmc . Indole-3-acetic acid (IAA) contents in dmc were significantly less than that in Guomai 301 at key tillering stages. Exogenous IAA application significantly promoted wheat tillering, and affected the transcriptions of TaARFs . These data suggested that TaARFs as well as IAA signaling were involved in controlling wheat tillering. This study provided valuable clues for functional characterization of ARFs in wheat.

利用CRISPR-Cas9(Clustered regularly interspaced short palindromic repeats-CRISPR-associated nuclease 9)系统对植物进行定点编辑的技术已经日渐成熟,但对于被编辑植株后代的鉴定依旧费时费力,以拟南芥SDP1(Sugar-dependent 1)基因为编辑对象,优化编辑植株后代的鉴定方法.对SDP1基因序列进行分析表明,SDP1基因外显子上含有PAM(Protospacer adjacent motif)序列,且其上游合适的酶切位点有4个,选择第二外显子上含有最常见的SacⅠ酶切位点的20 bp序列作为基因敲除的靶位点,构建了CRISPR-SDP1-Cas9载体,实现对SDP1基因的定点编辑.对经过潮霉素筛选的T1株系DNA进行酶切鉴定和测序峰图分析,选出SacⅠ酶切为3条带的杂合编辑植株,随机挑选的71株T1植株中有24株发生了杂合编辑,编辑效率为16.9％.在48株T2转基因植株中有5株发生了纯合编辑,经测序编辑位点均为SacⅠ酶切位点上碱基插入.综上,选用待编辑基因上PAM序列附近合适的酶切位点作为编辑靶点,通过酶切和测序峰图分析来鉴定后代植株,相比于传统方法对后代植株提取DNA、PCR扩增靶位点、连接T载体后进行多次测序,此方法方便、快捷且经济实用,为CRISPR-Cas9基因编辑后代鉴定提供了简单、快捷的方法.

Flowering time is an important agronomic trait that determines the distribution and adaptation of plants. The accurate prediction of flowering time in elite germplasm is very critical for maize breeding. However, the molecular mechanisms underlying photoperiod response remain elusive in maize. Here we cloned the flowering time controlling gene, ZmDPS10-2, by map-based cloning and confirmed that ZmDPS10-2 is a positive regulator of flowering time in maize under long-day conditions using ChIP-Seq and CRISPER-Cas9 mutagenesis system. More specifically ZmDPS10-2 promotes ZmNF-YA3 transcription through binding to the promoter region of ZmNF-YA3. Then ZmNF-YA3 negatively regulates the transcription of ZmAP2 by binding ZmAP2 promoter. Finally, ZmAP2 suppresses the expression of ZMM4 to delay flowering time. The cascading regulation of flowering time by ZmDPS10-2 in maize that has not been reported in other species such as rice or Arabidopsis thaliana. Then we created an integrated gene regulatory network of flowering time in maize using ZmDPS10-2, ZmNF-YA3, ZmAP2, ZMM4, and CONZ1 as the backbone plus 12 previous reported key maize flowering genes, ten enhancers via ChIA-PET, 41 genes identified by ChIP-Seq, and 49 through Hi-C technology. The PIF3 being identified by both ChIP-Seq and Hi-C experiments is a hub linking DPS10-2 and other known flowering genes. This network provides in-depth understanding of genetic mechanism of maize flowering time and serves as invaluable resource for the plant community to tackle this very important agronomic trait.

Two zinc(II) complexes, [Zn4(HOQ)6Ac2] (I) (HOQ = 8-hydroxylquinoline) and [Zn4(MeQ)6Ac2] (II) (MeQ = 2-methyl-8-hydroxylquinoline), were synthesized and characterized by IR spectroscopy, ESI-MS spectrometry, elemental analysis and single crystal X-ray diffraction analysis (CIF files CCDC nos. 1433544 (I) and 1433546 (II)). The in vitro cytotoxicity of the two complexes, which was first reported, was evaluated by MTT assay against a series of tumor cell lines as well as HL-7702 normal liver cell line. The results indicated that they showed significantly higher cytotoxicity than cispltain on BEL-7404 cells with IC50 values of 11.85 ± 0.06 μM (I) and 8.40 ± 0.07 μM (II), respectively. Further apoptosis mechanism studies on BEL-7404 cells suggested that their antitumor activities were achieved through cell apoptosis and arrest at G1 or S phase. The decline of mitochondrial membrane potential, the elevation of reactive oxygen species and cytoplasmic calcium concentration ([Ca2+]c), the raise of caspase-3/9 activity indicated that complexes I and II induced apoptosis of BEL-7404 by a mitochondrial dysfunction pathway. Investigations on the binding properties of complexes I and II to ct-DNA by UV-Vis, circular dichroism spectra and agarose gel electrophoresis indicated that the two complexes could bind with ct-DNA via an intercalative mode.

MicroRNAs (miRNAs) regulate gene expression and play critical roles in growth and development as well as stress responses in eukaryotes. miRNA biogenesis in plants requires a processing complex that consists of the core components DICER-LIKE 1 (DCL1), SERRATE (SE) and HYPONASTIC LEAVES (HYL1). Here we show that inactivation of functionally redundant members of the SnRK2 kinases, which are the core components of abscisic acid (ABA) and osmotic stress signaling pathways, leads to reduction in miRNA accumulation under stress conditions. Further analysis revealed that the steady state level of HYL1 protein in plants under osmotic stress is dependent on the SnRK2 kinases. Additionally, our results suggest that the SnRK2 kinases physically associate with the miRNA processing components SE and HYL1 and can phosphorylate these proteins in vitro. These findings reveal an important role for the SnRK2 kinases in the regulation of miRNA accumulation and establish a mechanism by which ABA and osmotic stress signaling is linked to miRNA biogenesis.

A new cobalt(II) complex ( 1 ) of 5‐chloro‐8‐hydroxyquinoline was prepared and structurally characterized using infrared spectroscopy, electrospray ionization mass spectrometry, elemental analysis, single‐crystal X‐ray diffraction as well as powder X‐ray diffraction. Its biological activities including DNA binding and anticancer activity were investigated. The DNA binding study of complex 1 suggested that it interacted with calf thymus DNA mainly via an intercalative binding mode. The in vitro anticancer activity of complex 1 was screened against a series of tumor cell lines as well as the normal liver cell line HL‐7702 using MTT assay. complex 1 showed much higher cytotoxicity than corresponding metal salt and ligand towards the five tested tumor cell lines, in which T‐24 was the most sensitive tumor cell line towards 1, with IC 50 value of 7.04 ± 0.06 μM. complex 1 was found to greatly induce cell cycle arrest in T‐24 cells at S phase, and consequently to induce cell apoptosis in a dose‐dependent mode suggested by cell apoptosis analysis via Hoechst 33258 and acridine orange/ethidium bromide staining assays. The cell apoptosis mechanism of 1 was studied targeting the mitochondrion‐mediated pathway, since the apoptotic mechanism in the T‐24 cells treated by 1 was investigated by reactive oxygen species (ROS) detection, intracellular calcium concentration measurement and caspase‐9/3 activity assay. The results suggested that the cell apoptosis induced by 1 was closely related to the loss of mitochondrial membrane potential, ROS production and enhancement of intracellular [Ca 2 + ], which would trigger the caspase‐9/3 activation via a mitochondrial dysfunction pathway. Copyright © 2016 John Wiley & Sons, Ltd.

Two rhodium(III) complexes (Rh(OQ)3 (1) and Rh(BrQ)2(CH3OH)Cl (2), HOQ = 8-hydroxyquinoline, HBrQ = 5-bromo-8-hydroxyquinoline) of 8-hydroxylquinoline were synthesized and characterized. By MTT assay, the in vitro cytotoxicity of complexes 1 and 2, compared with HOQ, HBrQ and cisplatin, was evaluated towards a series of tumor cell lines as well as the normal liver cell line HL-7702. Complexes 1 and 2 showed higher cytotoxicity against the tested tumor cell lines than the corresponding ligands, among which T-24 was the most sensitive cell line for complexes 1 and 2 (IC50 = 13.42 μM for 1, 18.91 μM for 2). Compared with cisplatin, complex 1 exhibited higher cytotoxicity against T-24 cells but lower cytotoxicity against HL-7702(IC50 = 15.93 μM). Considering the better cytotoxicity of complex 1 than complex 2 against T-24 cells, the underlying anticancer molecular mechanisms were also investigated. DNA interaction studies revealed that complex 1 interacted with ct-DNA mainly via an intercalative binding mode. Further investigation of intracellular mechanisms revealed that complex 1 caused G2 phase cell cycle arrest and induced T-24 cell apoptosis in a dose-dependent mode. Targeting the mitochondrial pathway, the apoptotic mechanism in T-24 cells treated with 1 was studied by ROS detection, intracellular Ca2+ concentration measurements and caspase-9/3 activity assay, which suggested that complex 1 induced T-24 cell apoptosis by the disruption of mitochondrial-related mechanisms.

Three cobalt(ii) complexes with 8-hydroxyquinoline derivatives as ligands were synthesized. They exhibited strong proliferation inhibition activity against T-24 cancer cells, which induced cancer cell apoptosis via intrinsic caspase-mitochondria pathways.

In yeast, the interaction of General Control Non-derepressible 1 (GCN1) with GCN2 enables GCN2 to phosphorylate eIF2α (the alpha subunit of eukaryotic translation initiation factor 2) under a variety of stresses. Here, we cloned AtGCN1, an Arabidopsis homologue of GCN1. We show that AtGCN1 directly interacts with GCN2 and is essential for the phosphorylation of eIF2α under salicylic acid (SA), ultraviolet (UV), cold stress and amino acid deprivation conditions. Two mutant alleles, atgcn1-1 and atgcn1-2, which are defective in the phosphorylation of eIF2α, showed increased sensitivity to cold stress, compared with the wild type. Ribosome-bound RNA profiles showed that the translational state of mRNA was higher in atgcn1-1 than in the wild type. Our result also showed that cold treatment reduced the tendency of the tor mutant seedlings to produce purple hypocotyls. In addition, the kinase activity of TOR was transiently inhibited when plants were exposed to cold stress, suggesting that the inhibition of TOR is another pathway important for plants to respond to cold stress. In conclusion, our results indicate that the AtGCN1-mediated phosphorylation of eIF2α, which is required for inhibiting the initiation of protein translation, is essential for cold tolerance in Arabidopsis.

A new iron(III) complex (1) of 5-nitro-8-hydroxylquinoline (HNOQ) was synthesized and structurally characterized in its solid state and solution state by IR, UV-Vis, electrospray ionization (ESI)-MS, elemental analysis, conductivity and X-ray single crystal diffraction analysis. The DNA binding study suggested that complex 1 interacted with calf thymus (ct)-DNA mainly via an intercalative binding mode. By 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, the in vitro cytotoxicity of complex 1, comparing with HNOQ and cisplatin, was screened towards a series of tumor cell lines as well as the normal liver cell line HL-7702. Complex 1 showed higher cytotoxicity towards the tested tumor cell lines but lower cytotoxicity towards HL-7702 than HNOQ, in which the T-24 was the most sensitive cell line for 1. Complex 1 caused G2 phase cell cycle arrest and induced cell apoptosis in T-24 cells in a dose-dependent mode, evidenced by changes in cell morphology. Targeting the mitochondrial pathway due to the redox potential of Fe(III)/Fe(II), the apoptotic mechanism in T-24 cells treated by 1 was investigated by reactive oxygen species (ROS) detection, intracellular [Ca(2+)] measurement and caspase-9 and caspase-3 activity assay. It suggested that complex 1 induced cell apoptosis by triggering the caspase-9 and caspase-3 activation via a mitochondrion-mediated pathway.

A new zinc(II) complex (1) of 5-chloro-8-hydroxylquinoline (HClQ) has been synthesized and characterized by IR spectroscopy, ESI-MS spectrometry, elemental analysis and single crystal X-ray diffraction analysis. The DNA binding property of complex (1) has been investigated by UV-vis spectroscopy, circular dichroism spectroscopy and agarose gel electrophoresis. The results indicate that complex (1) binds with ct-DNA via an intercalative mode, and also exhibits DNA cleavage activity. The in vitro cytotoxicity of complex (1) has been screened by MIT assay against a series of tumor cell lines as well as HL-7702 normal liver cell line and, compared with that of cisplatin. Complex (1) shows lower cytotoxicity than cisplatin towards both the tumor cell lines and the normal liver cell line, except the BEL-7404 liver tumor cell line. The IC50 value of (1) towards BEL-7404 is 7.04 +/- 0.06 mu g which is lower than that of cisplatin (25.08 +/- 0.12 mu M). This suggests that BEL-7404 is the most sensitive tumor cell line for (1). Complex (1) is found to induce cell apoptosis in the BEL-7404 cells by arresting the cell cycle at the S phase. The antitumor mechanism of (1) involves targeting the mitochondria-mediated pathway, since the ROS release and the cytoplasmic [Call increase in the tested tumor cells after incubation with (1). Flow cytometry assay on the cellular level confirms that the dysfunction of the mitochondria (indicated by the loss of mitochondrial membrane potential) and the release of ROS and Ca2+, directly cause the activation of caspase cascade, including caspase-9 as the initiator and caspase-3 as the executor. These results strongly suggest that the central Zn(II) as the coordinating centre plays the key role in enhancing the antitumor activity and actuating the potential apoptotic pathway for this kind of halogenated quinoline derivatives.

A zinc(ii) complex of HBrQ showed higher in vitro antitumor activity. It induced cell apoptosis in BEL-7404 cells via G2 phase arrest, led to mitochondria dysfunction and activation of caspase cascade. The central zinc(ii) should play a key role to enhance the antitumor effect

Nanowires from deposition of pyromellitic diimide (PMDI) from the gas phase and their unique excitation-wavelength-dependent photoluminescence were demonstrated. The luminescence peaks of the PMDI nanowires red-shifted as the excitation wavelength increased. The relationship between the luminescence peak and the excitation wavelength is nearly linear in a broad range of excitation.

The biological functions of WRKY transcription factors in plants have been widely studied, but their roles in abiotic stress are still not well understood. We isolated an ABA overly sensitive mutant, abo3, which is disrupted by a T-DNA insertion in At1g66600 encoding a WRKY transcription factor AtWRKY63. The mutant was hypersensitive to ABA in both seedling establishment and seedling growth. However, stomatal closure was less sensitive to ABA, and the abo3 mutant was less drought tolerant than the wild type. Northern blot analysis indicated that the expression of the ABA-responsive transcription factor ABF2/AREB1 was markedly lower in the abo3 mutant than in the wild type. The abo3 mutation also reduced the expression of stress-inducible genes RD29A and COR47, especially early during ABA treatment. ABO3 is able to bind the W-box in the promoter of ABF2in vitro. These results uncover an important role for a WRKY transcription factor in plant responses to ABA and drought stress.