Recovery from spindle checkpoint-mediated arrest requires a novel Dnt1-dependent APC/C activation mechanism.

The activated spindle assembly checkpoint (SAC) potently inhibits the anaphase-promoting complex/cyclosome (APC/C) to ensure accurate chromosome segregation at anaphase. Early studies have recognized that the SAC should be silenced within minutes to enable rapid APC/C activation and synchronous segregation of chromosomes once all kinetochores are properly attached, but the underlying silencers are still being elucidated. Here, we report that the timely silencing of SAC in fission yeast requires dnt1+, which causes severe thiabendazole (TBZ) sensitivity and increased rate of lagging chromosomes when deleted. The absence of Dnt1 results in prolonged inhibitory binding of mitotic checkpoint complex (MCC) to APC/C and attenuated protein levels of Slp1Cdc20, consequently slows the degradation of cyclin B and securin, and eventually delays anaphase entry in cells released from SAC activation. Interestingly, Dnt1 physically associates with APC/C upon SAC activation. We propose that this association may fend off excessive and prolonged MCC binding to APC/C and help to maintain Slp1Cdc20 stability. This may allow a subset of APC/C to retain activity, which ensures rapid anaphase onset and mitotic exit once SAC is inactivated. Therefore, our study uncovered a new player in dictating the timing and efficacy of APC/C activation, which is actively required for maintaining cell viability upon recovery from the inhibition of APC/C by spindle checkpoint.

Analysis of the potential role of fission yeast PP2A in spindle assembly checkpoint inactivation.

As a surveillance mechanism, the activated spindle assembly checkpoint (SAC) potently inhibits the E3 ubiquitin ligase APC/C (anaphase-promoting complex/cyclosome) to ensure accurate chromosome segregation. Although the protein phosphatase 2A (PP2A) has been proposed to be both, directly and indirectly, involved in spindle assembly checkpoint inactivation in mammalian cells, whether it is similarly operating in the fission yeast Schizosaccharomycer pombe has never been demonstrated. Here, we investigated whether fission yeast PP2A is involved in SAC silencing by following the rate of cyclin B (Cdc13) destruction at SPBs during the recovery phase in nda3-KM311 cells released from the inhibition of APC/C by the activated spindle checkpoint. The timing of the SAC inactivation is only slightly delayed when two B56 regulatory subunits (Par1 and Par2) of fission yeast PP2A are absent. Overproduction of individual PP2A subunits either globally in the nda3-KM311 arrest-and-release system or locally in the synthetic spindle checkpoint activation system only slightly suppresses the SAC silencing defects in PP1 deletion (dis2Δ) cells. Our study thus demonstrates that the fission yeast PP2A is not a key regulator actively involved in SAC inactivation.

A Semi-Supervised Stacked Autoencoder Using the Pseudo Label for Classification Tasks.

The efficiency and cognitive limitations of manual sample labeling result in a large number of unlabeled training samples in practical applications. Making full use of both labeled and unlabeled samples is the key to solving the semi-supervised problem. However, as a supervised algorithm, the stacked autoencoder (SAE) only considers labeled samples and is difficult to apply to semi-supervised problems. Thus, by introducing the pseudo-labeling method into the SAE, a novel pseudo label-based semi-supervised stacked autoencoder (PL-SSAE) is proposed to address the semi-supervised classification tasks. The PL-SSAE first utilizes the unsupervised pre-training on all samples by the autoencoder (AE) to initialize the network parameters. Then, by the iterative fine-tuning of the network parameters based on the labeled samples, the unlabeled samples are identified, and their pseudo labels are generated. Finally, the pseudo-labeled samples are used to construct the regularization term and fine-tune the network parameters to complete the training of the PL-SSAE. Different from the traditional SAE, the PL-SSAE requires all samples in pre-training and the unlabeled samples with pseudo labels in fine-tuning to fully exploit the feature and category information of the unlabeled samples. Empirical evaluations on various benchmark datasets show that the semi-supervised performance of the PL-SSAE is more competitive than that of the SAE, sparse stacked autoencoder (SSAE), semi-supervised stacked autoencoder (Semi-SAE) and semi-supervised stacked autoencoder (Semi-SSAE).

Real-Time In Situ Volatile Organic Compound Sensing by a Dual-Emissive Polynuclear Ln-MOF with Pronounced LnIII Luminescence Response.

Lanthanide metal-organic frameworks (Ln-MOFs) are promising for luminescence detection of volatile organic compound (VOC) vapors, but usually suffer from the silent or quenched Ln3+ emission. Herein, we report a new dual-emissive Eu-MOF composed of the coordinatively unsaturated Eu9 clusters that afford abundant open metal sites to form a confined "binding pocket" to facilitate the preconcentration and recognition of VOCs. Single-crystal structural analyses reveal that specific analytes can replace the OH oscillators in the first coordination sphere of Eu3+ and form a unique hydrogen-bonding second-sphere adduct tying adjacent Eu9 clusters together to minimize their nonradiative vibrational decay. With the promoted Eu3+ luminescence, the MOF realizes real-time in situ visual sensing of THF vapor (<1 s) and shows a quantitative ratiometric response to the vapor pressure with a limit of detection down to 17.33 Pa. Also, it represents a top-performing ratiometric luminescent thermometer.

Phosphoregulation of the cytokinetic protein Fic1 contributes to fission yeast growth polarity establishment.

Cellular polarization underlies many facets of cell behavior, including cell growth. The rod-shaped fission yeast Schizosaccharomyces pombe is a well-established, genetically tractable system for studying growth polarity regulation. S. pombe cells elongate at their two cell tips in a cell cycle-controlled manner, transitioning from monopolar to bipolar growth in interphase when new ends established by the most recent cell division begin to extend. We previously identified cytokinesis as a critical regulator of new end growth and demonstrated that Fic1, a cytokinetic factor, is required for normal polarized growth at new ends. Here, we report that Fic1 is phosphorylated on two C-terminal residues, which are each targeted by multiple protein kinases. Endogenously expressed Fic1 phosphomutants cannot support proper bipolar growth, and the resultant defects facilitate the switch into an invasive pseudohyphal state. Thus, phosphoregulation of Fic1 links the completion of cytokinesis to the re-establishment of polarized growth in the next cell cycle. These findings broaden the scope of signaling events that contribute to regulating S. pombe growth polarity, underscoring that cytokinetic factors constitute relevant targets of kinases affecting new end growth.This article has an associated First Person interview with Anthony M. Rossi, joint first author of the paper.

The molecular chaperone Hsp90 regulates heterochromatin assembly through stabilizing multiple complexes in fission yeast.

In the fission yeast Schizosaccharomyces pombe, both RNAi machinery and RNAi-independent factors mediate transcriptional and posttranscriptional silencing and heterochromatin formation. Here, we show that the silencing of reporter genes at major native heterochromatic loci (centromeres, telomeres, mating-type locus and rDNA regions) and an artificially induced heterochromatin locus is alleviated in a fission yeast hsp90 mutant, hsp90-G84C Also, H3K9me2 enrichment at heterochromatin regions, especially at the mating-type locus and subtelomeres, is compromised, suggesting heterochromatin assembly defects. We further discovered that Hsp90 is required for stabilization or assembly of the RNA-induced transcriptional silencing (RITS) and Argonaute siRNA chaperone (ARC) RNAi effector complexes, the RNAi-independent factor Fft3, the shelterin complex subunit Poz1 and the Snf2/HDAC-containing repressor complex (SHREC). Our ChIP data suggest that Hsp90 regulates the efficient recruitment of the methyltransferase/ubiquitin ligase complex CLRC by shelterin to chromosome ends and targeting of the SHREC and Fft3 to mating type locus and/or rDNA region. Finally, our genetic analyses demonstrated that increased heterochromatin spreading restores silencing at subtelomeres in the hsp90-G84C mutant. Thus, this work uncovers a conserved factor critical for promoting RNAi-dependent and -independent heterochromatin assembly and gene silencing through stabilizing multiple effectors and effector complexes.

Core-shell-structured magnetic covalent organic frameworks for effective extraction of parabens prior to their determination by HPLC.

Covalent organic framework (COF)-decorated magnetic nanoparticles (Fe3O4@DhaTab) with core-shell structure have been synthesized by one-pot method. The prepared Fe3O4@DhaTab was well characterized, and parameters of magnetic solid-phase extraction (MSPE) for parabens were also investigated in detail. Under optimized conditions, the adsorbent dosage was only 3 mg and extraction time was 10 min. The developed Fe3O4@DhaTab-based MSPE-HPLC analysis method offered good linearity (0.01-20 µg mL-1) with R2 (0.999) and low limits of detection (3.3-6.5 µg L-1) using UV detector at 254 nm. The proposed method was applied to determine four parabens in environmental water samples with recoveries in the range 64.0-105% and relative standard deviations of 0.16-7.8%. The adsorption mechanism was explored and indicated that porous DhaTab shell provided π-π, hydrophobic, and hydrogen bonding interactions in the MSPE process. The results revealed the potential of magnetic-functionalized COFs in determination of environmental contaminants.

Dynamic Full-Color Tuning of Organic Chromophore in a Multi-Stimuli-Responsive 2D Flexible MOF.

Developing universal stimuli-responsive materials capable of emitting a broad spectrum of colors is highly desirable. Herein, we deliberately grafted a conformation-adaptable organic chromophore into the established coordination space of a flexible metal-organic framework (MOF). In terms of the coupled structural transformations and the space confinement, the chromophore in the MOF matrix underwent well-regulated conformational changes under physical and chemical stimuli, simultaneously displaying thermo-, piezo-, and solvato-fluoro-chromism with color tunability over the visible range. Owing to the resilient nature and the reduced dimensionality of the selected coordination space, all three color modulations behaved in a sensitive and self-reversible manner, each following a linear correlation of the emission maximum with stimulus. Single-crystal X-ray diffraction of the variable-temperature structures and solvent-inclusion crystals elucidated the intricate color varying mechanisms.

Bone marrow cells are differentiated into MDSCs by BCC-Ex through down-regulating the expression of CXCR4 and activating STAT3 signalling pathway.

Studies showed that the increase of myeloid-derived suppressor cells (MDSCs) in tumour microenvironment is closely related to the resistant treatment and poor prognosis of metastatic breast cancer. However, the effect of tumour-derived exosomes on MDSCs and its mechanism are not clear. Here, we reported that breast cancer cells (4T1)-secreted exosomes (BCC-Ex) were able to differentiate bone marrow cells into MDSCs and significantly inhibited the proliferation of T lymphocytes to provide an immunosuppressive microenvironment for cancer cells in vivo and in vitro. The number of MDSCs in bone marrow and spleen of 4T1 tumour-bearing mice and BCC-Ex infused mice was significantly higher than that of normal mice, whereas the number of T lymphocytes in spleen was significantly decreased. In addition, BCC-Ex markedly promoted the differentiation of MDSCs from bone marrow cells or bone marrow cells derived macrophages, seen as the increased expressions of MDSCs-related functional proteins Arginase-1 (Arg-1) and inducible nitric oxide synthase (iNOS). Furthermore, BCC-Ex significantly down-regulated the expressions of chemokine receptor CXCR4 and markedly up-regulated the levels of inflammatory cytokines IL-6 and IL-10 in bone marrow cells and macrophages and remarkably inhibited the division and proliferation of T cells. Importantly, CXCR4 agonist, CXCL12, could reverse the function of BCC-Ex, indicating that BCC-Ex-induced MDSCs might be dependent on the down-regulation of CXCR4. Western blot showed that BCC-Ex significantly promoted the phosphorylation of STAT3 in bone marrow cells, resulting in the inhibitions of the proliferation and apoptosis of bone marrow cells, and the aggravation of the differentiation of bone marrow cells into MDSCs.

PODN is a prognostic biomarker and correlated with immune infiltrates in osteosarcoma.

BACKGROUND: Osteosarcoma was the most common primary bone malignancy in children and adolescents. It was imperative to identify effective prognostic biomarkers for this cancer. This study was aimed to identify potential crucial genes of osteosarcoma by integrated bioinformatics analysis. METHODS: Identification of differentially expressed genes from public data gene expression profiles (GSE42352), functional and pathway enrichment analysis, protein-protein interaction (PPI) network construction and module analysis, Cox regression and survival analysis was conducted. RESULTS: Totally 17 co-differential genes were found to be differentially expressed. These genes were enriched in biological processes, Kyoto Encyclopedia of Genes and Genomes (KEGG) and Gene Set Enrichment Analysis (GSEA) pathway of inflammatory immune response. PPI network was constructed with 63 differentially expressed genes that co-existed between the test set and the validation set. The area under the receiver operating characteristic curve (AUC value) was 0.855, which indicated that the expression of PODN had a good diagnostic value for osteosarcoma. Furthermore, Cox regression and survival analysis revealed 5 genes were statistically significant. CONCLUSIONS: PODN was regarded as a potential biomarker for the diagnosis and prognosis of osteosarcoma, ACTA2, COL6A1, FAP, OLFML2B and COL6A3, can be used as potential prognostic indicators for osteosarcoma.

Identification of a novel six autophagy-related genes signature for the prognostic and a miRNA-related autophagy predictor for anti-PD-1 therapy responses in prostate cancer.

BACKGROUND: Autophagy is a highly conserved homeostatic process in the human body that is responsible for the elimination of aggregated proteins and damaged organelles. Several autophagy-related genes (ARGs) contribute to the process of tumorigenesis and metastasis of prostate cancer (PCa). Also, miRNAs have been proven to modulate autophagy by targeting some ARGs. However, their potential role in PCa still remains unclear. METHODS: An univariate Cox proportional regression model was used to identify 17 ARGs associated with the overall survival (OS) of PCa. Then, a multivariate Cox proportional regression model was used to construct a 6 autophagy-related prognostic genes signature. Patients were divided into low-risk group and high-risk group using the median risk score as a cutoff value. High-risk patients had shorter OS than low-risk patients. Furthermore, the signature was validated by ROC curves. Regarding mRNA and miRNA, 12 differentially expressed miRNAs (DEMs) and 1073 differentially expressed genes (DEGs) were detected via the GEO database. We found that miR-205, one of the DEMs, was negatively regulated the expression of ARG (NKX2-3). Based on STRING analysis results, we found that the NKX2-3 was moderately related to the part of genes among the 6 autophagy-related genes prognostic signature. Further, NKX 2-3 was significantly correlated with OS and some clinical parameters of PCa by cBioProtal. By gene set enrichment analysis (GSEA). Lastly, we demonstrated that the association between NKX2-3 and tumor mutation burden (TMB) and PDCD1 (programmed cell death 1) of PCa. RESULTS: We identified that the six ARGs expression patterns are independent predictors of OS in PCa patients. Furthermore, our results suggest that ARGs and miRNAs are inter-related. MiR-205 was negatively regulated the expression of ARG (NKX2-3). Further analysis demonstrated that NKX2-3 may be a potential biomarker for predicting the efficacy of anti-PD-1 therapy in PCa. CONCLUSIONS: The current study may offer a novel autophagy-related prognostic signature and may identify a promising miRNA-ARG pathway for predicting the efficacy of anti-PD-1 therapy in PCa.

Exosomes derived from human umbilical cord mesenchymal stem cells alleviate acetaminophen-induced acute liver failure through activating ERK and IGF-1R/PI3K/AKT signaling pathway.

This study aimed to investigate the therapeutic potential of human umbilical cord mesenchymal stem cells derived exosomes (hUCMSC-Exo) in acute liver failure (ALF) in mice as well as its underlying mechanism. We found that a single tail vein administration of hucMSC-Exo effectively enhanced the survival rate, inhibited apoptosis in hepatocytes, and improved liver function in APAP-induced mouse model of ALF. Furthermore, the deletion of glutathione (GSH) and superoxide dismutase (SOD), generation of malondialdehyde (MDA), and the over production of cytochrome P450 E1 (CYP2E1) and 4-hydroxynonenal (4-HNE) caused by APAP were also inhibited by hucMSC-Exo, indicating that hucMSC-Exo inhibited APAP-induced apoptosis of hepatocytes by reducing oxidative stress. Moreover, hucMSC-Exo significantly down-regulated the levels of inflammatory cytokines IL-6, IL-1ß, and TNF-α in APAP-treated livers. Western blot showed that hucMSC-Exo significantly promoted the activation of ERK1/2 and IGF-1R/PI3K/AKT signaling pathways in APAP-injured LO2 cells, resulting in the inhibition of apoptosis of LO2 cells. Importantly, PI3K inhibitor LY294002 and ERK1/2 inhibitor PD98059 could reverse the function of hucMSC-Exo on APAP-injured LO2 cells in some extent. Our results suggest that hucMSC-Exo offer antioxidant hepatoprotection against APAP in vitro and in vivo by inhibitiing oxidative stress-induced apoptosis via upregulation of ERK1/2 and PI3K/AKT signaling pathways.

miR-143 is implicated in growth plate injury by targeting IHH in precartilaginous stem cells.

Precartilaginous stem cells (PCSCs) are able to initiate chondrocyte and bone development. The present study aimed to investigate the role of miR-143 and the underlying mechanisms involved in PCSC proliferation. In a rat growth plate injury model, tissue from the injury site was collected and the expression of miR-143 and its potential targets was determined. PCSCs were isolated from the rabbits' distal epiphyseal growth plate. Cell viability, DNA synthesis, and apoptosis were determined with MTT, BrdU, and flow cytometric analysis, respectively. Real time PCR and western blot were performed to detect the mRNA and protein expression of the indicated genes. Indian hedgehog (IHH) was identified as a target gene for miR-143 with luciferase reporter assay. Decreased expression of miR-143 and increased expression of IHH gene were observed in the growth plate after injury. miR-143 mimics decreased cell viability and DNA synthesis and promoted apoptosis of PCSCs. Conversely, siRNA-mediated inhibition of miR-143 led to increased growth and suppressed apoptosis of PCSCs. Transfection of miR-143 decreased luciferase activity of wild-type IHH but had no effect when the 3'-UTR of IHH was mutated. Furthermore, the effect of miR-143 overexpression was neutralized by overexpression of IHH. Our study showed that miR-143 is involved in growth plate behavior and regulates PCSC growth by targeting IHH, suggesting that miR-143 may serve as a novel target for PCSC-related diseases.

Nicaraven inhibits TNFα-induced endothelial activation and inflammation through suppression of NF-κB signaling pathway.

Inflammation-induced activation and dysfunction of endothelial cells play an important role in the pathology of multiple vascular diseases. Nicaraven, a potent hydroxyl radical scavenger, has recently been found to have anti-inflammatory roles; however, the mechanism of its action is not fully understood. Here we investigated the effects of Nicaraven on tumor necrosis factor α (TNFα) - induced inflammatory response in human umbilical vein endothelial cells and we explore the underlying mechanisms related to the nuclear factor-κB (NF-κB) signaling pathway. Our results showed that Nicaraven significantly reduced the reactive oxygen species production after TNFα stimulation. Nicaraven suppressed TNFα-induced mRNA expression of multiple adhesion molecules and pro-inflammatory cytokines, including vascular cell adhesion molecule 1 (VCAM-1), intercellular adhesion molecule 1 (ICAM-1), E-selectin, MCP-1, TNFα, interleukin-1ß (IL-1ß), IL-6, and IL-8. In addition, Nicaraven inhibited monocyte adhesion and reduced the protein levels of VCAM-1 and ICAM-1. Mechanistically, Nicaraven prevented TNFα-induced activation of NF-κB signaling pathway by suppressing the phosphorylation of NF-κB p65, IκBα, and IκB kinase (IKK)α/ß, stabilizing IκBα, and inhibiting the translocation of p65 from cytosol to nucleus. Finally, we showed that Nicaraven improved the functions of endothelial cells, seen as the upregulation of endothelial nitric oxide synthase and increased nitric oxide levels. Our findings indicated that Nicaraven effectively inhibits TNFα-induced endothelial activation and inflammatory response at least partly through inhibiting NF-κB signaling pathway.

Characteristics and Therapeutic Potential of Human Amnion-Derived Stem Cells.

Stem cells including embryonic stem cells (ESCs), induced pluripotent stem cells (iPSCs) and adult stem cells (ASCs) are able to repair/replace damaged or degenerative tissues and improve functional recovery in experimental model and clinical trials. However, there are still many limitations and unresolved problems regarding stem cell therapy in terms of ethical barriers, immune rejection, tumorigenicity, and cell sources. By reviewing recent literatures and our related works, human amnion-derived stem cells (hADSCs) including human amniotic mesenchymal stem cells (hAMSCs) and human amniotic epithelial stem cells (hAESCs) have shown considerable advantages over other stem cells. In this review, we first described the biological characteristics and advantages of hADSCs, especially for their high pluripotency and immunomodulatory effects. Then, we summarized the therapeutic applications and recent progresses of hADSCs in treating various diseases for preclinical research and clinical trials. In addition, the possible mechanisms and the challenges of hADSCs applications have been also discussed. Finally, we highlighted the properties of hADSCs as a promising source of stem cells for cell therapy and regenerative medicine and pointed out the perspectives for the directions of hADSCs applications clinically.

Opening Magnetic Hysteresis by Axial Ferromagnetic Coupling: From Mono-Decker to Double-Decker Metallacrown.

Combining Ising-type magnetic anisotropy with collinear magnetic interactions in single-molecule magnets (SMMs) is a significant synthetic challenge. Herein we report a Dy[15-MCCu -5] (1-Dy) SMM, where a DyIII ion is held in a central pseudo-D5h pocket of a rigid and planar Cu5 metallacrown (MC). Linking two Dy[15-MCCu -5] units with a single hydroxide bridge yields the double-decker {Dy[15-MCCu -5]}2 (2-Dy) SMM where the anisotropy axes of the two DyIII ions are nearly collinear, resulting in magnetic relaxation times for 2-Dy that are approximately 200 000 times slower at 2 K than for 1-Dy in zero external field. Whereas 1-Dy and the YIII -diluted Dy@2-Y analogue do not show remanence in magnetic hysteresis experiments, the hysteresis data for 2-Dy remain open up to 6 K without a sudden drop at zero field. In conjunction with theoretical calculations, these results demonstrate that the axial ferromagnetic Dy-Dy coupling suppresses fast quantum tunneling of magnetization (QTM). The relaxation profiles of both complexes curiously exhibit three distinct exponential regimes, and hold the largest effective energy barriers for any reported d-f SMMs up to 625 cm-1 .

Human amniotic mesenchymal stem cells inhibit hepatocellular carcinoma in tumour-bearing mice.

Hepatocellular carcinoma (HCC) is the third leading cause of the cancer-related death in the world. Human amniotic mesenchymal stem cells (hAMSCs) have been characterized with a pluripotency, low immunogenicity and no tumorigenicity. Especially, the immunosuppressive and anti-inflammatory effects of hAMSCs make them suitable for treating HCC. Here, we reported that hAMSCs administrated by intravenous injection significantly inhibited HCC through suppressing cell proliferation and inducing cell apoptosis in tumour-bearing mice with Hepg2 cells. Cell tracking experiments with GFP-labelled hAMSCs showed that the stem cells possessed the ability of migrating to the tumorigenic sites for suppressing tumour growth. Importantly, both hAMSCs and the conditional media (hAMSC-CM) have the similar antitumour effects in vitro, suggesting that hAMSCs-derived cytokines might be involved in their antitumour effects. Antibody array assay showed that hAMSCs highly expressed dickkopf-3 (DKK-3), dickkopf-1 (DKK-1) and insulin-like growth factor-binding protein 3 (IGFBP-3). Furthermore, the antitumour effects of hAMSCs were further confirmed by applications of the antibodies or the specific siRNAs of DKK-3, DKK-1 and IGFBP-3 in vitro. Mechanically, hAMSCs-derived DKK-3, DKK-1 and IGFBP-3 markedly inhibited cell proliferation and promoted apoptosis of Hepg2 cells through suppressing the Wnt/ß-catenin signalling pathway and IGF-1R-mediated PI3K/AKT signalling pathway, respectively. Taken together, our study demonstrated that hAMSCs possess significant antitumour effects in vivo and in vitro and might provide a novel strategy for HCC treatment clinically.

F-box proteins Pof3 and Pof1 regulate Wee1 degradation and mitotic entry in fission yeast.

The key cyclin-dependent kinase Cdk1 (Cdc2) promotes irreversible mitotic entry, mainly by activating the phosphatase Cdc25 while suppressing the tyrosine kinase Wee1. Wee1 needs to be downregulated at the onset of mitosis to ensure rapid activation of Cdk1. In human somatic cells, one mechanism of suppressing Wee1 activity is mediated by ubiquitylation-dependent proteolysis through the Skp1/Cul1/F-box protein (SCF) ubiquitin E3 ligase complex. This mechanism is believed to be conserved from yeasts to humans. So far, the best-characterized human F-box proteins involved in recognition of Wee1 are ß-TrCP (BTRCP) and Tome-1 (CDCA3). Although fission yeast Wee1 was the first identified member of its conserved kinase family, the F-box proteins involved in recognition and ubiquitylation of Wee1 have not been identified in this organism. In this study, our screen using Wee1-Renilla luciferase as the reporter revealed that two F-box proteins, Pof1 and Pof3, are required for downregulating Wee1 and are possibly responsible for recruiting Wee1 to SCF. Our genetic analyses supported a functional relevance between Pof1 and Pof3 and the rate of mitotic entry, and Pof3 might play a major role in this process.

Facile manipulation of protein localization in fission yeast through binding of GFP-binding protein to GFP.

GFP-binding protein (or GBP) has been recently developed in various systems and organisms as an efficient tool to purify GFP-fusion proteins. Due to the high affinity between GBP and GFP or GFP variants, this GBP-based approach is also ideally suited to alter the localization of functional proteins in live cells. In order to facilitate the wide use of the GBP-targeting approach in the fission yeast Schizosaccharomyces pombe, we developed a set of pFA6a-, pJK148- and pUC119-based vectors containing GBP- or GBP-mCherry-coding sequences and variants of inducible nmt1 or constitutive adh1 promoters that result in different levels of expression. The GBP or GBP-mCherry fragments can serve as cassettes for N- or C-terminal genomic tagging of genes of interest. We illustrated the application of these vectors in the construction of yeast strains with Dma1 or Cdc7 tagged with GBP-mCherry and efficient targeting of Dma1- or Cdc7-GBP-mCherry to the spindle pole body by Sid4-GFP. This series of vectors should help to facilitate the application of the GBP-targeting approach in manipulating protein localization and the analysis of gene function in fission yeast, at the level of single genes, as well as at a systematic scale.

Naloxone attenuates ischemic brain injury in rats through suppressing the NIK/IKKα/NF-κB and neuronal apoptotic pathways.

Although naloxone has been documented to exert neuroprotection in animal model of cerebral ischemia, the mechanism is not well understood. In this present study we investigated whether naloxone affected the mitochondrial apoptotic pathway in ischemic brain injury of rats. SD rats were subjected to a permanent middle cerebral artery occlusion surgery, and received naloxone (0.5, 1, 2 mg/kg, i.v.) immediately after ischemia. Neurological deficits were evaluated 24 h after ischemia using the McGraw Stroke Index, and then the rats were killed, and the brains were collected for further analyses. We show that naloxone treatment dose-dependently decreased the infarction volume and morphological injury, improved motor behavioral function, and markedly curtailed brain edema. Furthermore, naloxone administration significantly inhibited the nuclear translocation of NF-κB p65 and decreased the levels of nuclear NF-κB p65 in the ischemic penumbra. Naloxone administration also dose-dependently increased the NF-κB inhibitory protein (IκBα) levels and attenuated phosphorylated NIK and IKKα levels in the ischemic penumbra. In addition, naloxone administration dose-dependently increased Bcl-2 levels, decreased Bax levels, stabilized the mitochondrial transmembrane potential, and inhibited cytochrome c release and caspase 3 and caspase 9 activation. These results indicate that the neuroprotective effects of naloxone against ischemic brain injury involve the inhibition of NF-κB activation via the suppression of the NIK/IKKα/IκBα pathway and the obstruction of the mitochondrial apoptotic pathway in neurons.