Garciesculenxanthone B induces PINK1-Parkin-mediated mitophagy and prevents ischemia-reperfusion brain injury in mice.

Mitophagy is a selective form of autophagy involving the removal of damaged mitochondria via the autophagy-lysosome pathway. PINK1-Parkin-mediated mitophagy is one of the most important mechanisms in cardiovascular disease, cerebral ischemia-reperfusion (I/R) injury, and neurodegenerative diseases. In this study we conducted an image-based screening in YFP-Parkin HeLa cells to discover new mitophagy regulators from natural xanthone compounds. We found that garciesculenxanthone B (GeB), a new xanthone compound from Garcinia esculenta, induced the formation of YFP-Parkin puncta, a well known mitophagy marker. Furthermore, treatment with GeB dose-dependently promoted the degradation of mitochondrial proteins Tom20, Tim23, and MFN1 in YFP-Parkin HeLa cells and SH-SY5Y cells. We revealed that GeB stabilized PINK1 and triggered Parkin translocation to the impaired mitochondria to induce mitophagy, and these effects were abolished by knockdown of PINK1. Finally, in vivo experiments demonstrated that GeB partially rescued ischemia-reperfusion-induced brain injury in mice. Taken together, our findings demonstrate that the natural compound GeB can promote the PINK1-Parkin-mediated mitophagy pathway, which may be implicated in protection against I/R brain injury.

Oblongifolin C suppresses lysosomal function independently of TFEB nuclear translocation.

Lysosomes are the terminal organelles of the autophagic-endocytic pathway and play a key role in the degradation of autophagic contents. We previously reported that a natural compound oblongifolin C (OC) increased the number of autophagosomes and impaired the degradation of P62, most likely via suppression of lysosomal function and blockage of autophagosome-lysosome fusion. However, the precise mechanisms of how OC inhibits the lysosome-autophagy pathway remain unclear. In the present study, we investigated the effect of OC on transcription factor EB (TFEB), a master regulator of lysosomal biogenesis, lysosomal function and autophagy. We showed that treatment with OC (15 µM) markedly enhanced the nuclear translocation of TFEB in HeLa cells, concomitantly reduced the interaction of TFEB with 14-3-3 proteins. We further demonstrated that OC caused significant inhibition of mTORC1 along with TFEB nuclear translocation, and OC-mediated TFEB nuclear translocation was dependent on mTORC1 suppression. Intriguingly, this increased nuclear TFEB was accompanied by reduced TFEB luciferase activity, increased lysosomal pH and impaired cathepsin enzyme activities. In HeLa cells, treatment with OC (7.5 µM) resulted in about 30% of cell death, whereas treatment with hydroxycitrate, a caloric restriction mimetic (20 µM) did not affect the cell viability. However, cotreatment with OC and hydroxycitrate caused significantly great cytotoxicity (>50%). Taken together, these results demonstrate that inhibition of lysosome function is mediated by OC, despite evident TFEB nuclear translocation.

Cold block of in vitro eyeblink reflexes: evidence supporting the use of hypothermia as an anesthetic in pond turtles.

Use of hypothermia as a means of anesthesia for amphibians and reptiles is prohibited by agencies that establish veterinary guidelines. This has recently been called into question by members of the scientific community based on reviews of published literature. Using pond turtles (Trachemys scripta elegans), hypothermia as a method for anesthesia to precede euthanasia by decapitation was assessed. Turtles were subjected to hypothermia using a cooling followed by freezing protocol. Body temperature measurements ranged between -1 and -2°C while core body temperature was -1°C. Ice crystal formation was never observed. A protective reflex to noxious stimuli, the eyeblink response, was recorded from in vitro brainstem preparations subjected to cold. At 5-6°C, reflex responses were suppressed, demonstrating minimal synaptic transmission in brain circuits above temperatures used for hypothermia induction. These and previous data indicate that a re-evaluation of the use of hypothermia as an anesthetic in amphibians and reptiles is warranted.

Nujiangexathone A, a Novel Compound Derived from Garcinia nujiangensis, Induces Caspase-Dependent Apoptosis in Cervical Cancer through the ROS/JNK Pathway.

Nujiangexathone A (NJXA), a novel compound derived from Garcinia nujiangensis, has been demonstrated to inhibit the proliferation of several human cancer cell lines. This study is the first to demonstrate the apoptosis inductive activities of NJXA and the possible underlying mechanisms. Our results demonstrated that NJXA inhibited colony formation by HeLa and SiHa cells in a dose-dependent manner. An Annexin V-FITC/PI staining assay showed that NJXA strongly triggered apoptosis in a dose-dependent manner. Western blotting analyses showed that NJXA induced the caspase-dependent apoptosis of HeLa and SiHa cells by triggering a series of events, including changes in the levels of Bcl-2 family proteins, cytochrome c release, caspase-3 activation, and chromosome fragmentation. Furthermore, we demonstrated that NJXA induced cell apoptosis by activating the reactive oxygen species (ROS)-mediated JNK signaling pathway. Consistent with this finding, a ROS scavenger, N-acetyl-l-cysteine (NAC, 10 mM), hindered NJXA-induced apoptosis and attenuated the sensitivity of HeLa and SiHa cells to NJXA. In vivo results further confirmed that the tumor inhibitory effect of NJXA was partially through the induction of apoptosis. Taken together, our results demonstrated that NJXA induced the apoptosis of HeLa and SiHa cells through the ROS/JNK signaling pathway, indicating that NJXA could be important candidate for the clinical treatment of cervical cancer.

Coincidence detection in a neural correlate of classical conditioning is initiated by bidirectional 3-phosphoinositide-dependent kinase-1 signalling and modulated by adenosine receptors.

How the neural substrates for detection of paired stimuli are distinct from unpaired stimuli is poorly understood and a fundamental question for understanding the signalling mechanisms for coincidence detection during associative learning. To address this question, we used a neural correlate of eyeblink classical conditioning in an isolated brainstem from the turtle, in which the cranial nerves are directly stimulated in place of using a tone or airpuff. A bidirectional response is activated in <5 min of training, in which phosphorylated 3-phosphoinositide-dependent kinase-1 (p-PDK1) is increased in response to paired and decreased in response to unpaired nerve stimulation and is mediated by the opposing actions of neurotrophin receptors TrkB and p75(NTR) . Surprisingly, blockade of adenosine 2A (A2A ) receptors inhibits both of these responses. Pairing also induces substantially increased surface expression of TrkB that is inhibited by Src family tyrosine kinase and A2A receptor antagonists. Finally, the acquisition of conditioning is blocked by a PDK1 inhibitor. The unique action of A2A receptors to function directly as G proteins and in receptor transactivation to control distinct TrkB and p75(NTR) signalling pathways allows for convergent activation of PDK1 and protein kinase A during paired stimulation to initiate classical conditioning.

Sequential delivery of synaptic GluA1- and GluA4-containing AMPA receptors (AMPARs) by SAP97 anchored protein complexes in classical conditioning.

Multiple signaling pathways are involved in AMPAR trafficking to synapses during synaptic plasticity and learning. The mechanisms for how these pathways are coordinated in parallel but maintain their functional specificity involves subcellular compartmentalization of kinase function by scaffolding proteins, but how this is accomplished is not well understood. Here, we focused on characterizing the molecular machinery that functions in the sequential synaptic delivery of GluA1- and GluA4-containing AMPARs using an in vitro model of eyeblink classical conditioning. We show that conditioning induces the interaction of selective protein complexes with the key structural protein SAP97, which tightly regulates the synaptic delivery of GluA1 and GluA4 AMPAR subunits. The results demonstrate that in the early stages of conditioning the initial activation of PKA stimulates the formation of a SAP97-AKAP/PKA-GluA1 protein complex leading to synaptic delivery of GluA1-containing AMPARs through a SAP97-PSD95 interaction. This is followed shortly thereafter by generation of a SAP97-KSR1/PKC-GluA4 complex for GluA4 AMPAR subunit delivery again through a SAP97-PSD95 interaction. These data suggest that SAP97 forms the molecular backbone of a protein scaffold critical for delivery of AMPARs to the PSD during conditioning. Together, the findings reveal a cooperative interaction of multiple scaffolding proteins for appropriately timed delivery of subunit-specific AMPARs to synapses and support a sequential two-stage model of AMPAR synaptic delivery during classical conditioning.

Correlation between genetic alterations and clinicopathological features of papillary thyroid carcinomas.

OBJECTIVE: To investigate the correlations between multigene alterations and clinicopathological features in papillary thyroid carcinoma (PTC) samples. METHODS: In this retrospective study, 111 cytological specimens of thyroid nodules, including 74 PTC samples and 37 benign samples, were analyzed using a 22-gene mutation assay employing next-generation sequencing. Clinicopathological information was retrospectively collected and analyzed. RESULTS: Gene alterations were associated with a higher rate of lymph node metastasis (LNM) and thyroid capsular invasion, a lower rate of coexisting Hashimoto's thyroiditis, the classical PTC subtype, and younger age (<45 years). Among the 22 genes tested, the BRAF mutation rates showed a significant difference between the PTC and benign groups. In the subgroup analysis, younger age (odds ratio = 12.512, 95% confidence interval: 3.126-50.087) was an independent risk factor for LNM. In further analyses, BRAF mutation was significantly associated with LNM in the older subgroup (age ≥ 45 years), suggesting that the BRAF mutation test has greater value for determining PTC prognosis in the older age group. CONCLUSIONS: Our findings will provide a more comprehensive understanding of the relationship between gene mutations and PTC and may contribute to improved PTC management.

Correlation between NGS panel-based mutation results and clinical information in colorectal cancer patients.

Early mutation identification guides patients with colorectal cancer (CRC) toward targeted therapies. In the present study, 414 patients with CRC were enrolled, and amplicon-based targeted next-generation sequencing (NGS) was then performed to detect genomic alterations within the 73 cancer-related genes in the OncoAim panel. The overall mutation rate was 91.5 % (379/414). Gene mutations were detected in 38/73 genes tested. The most frequently mutated genes were TP53 (60.9 %), KRAS (46.6 %), APC (30.4 %), PIK3CA (15.9 %), FBXW7 (8.2 %), SMAD4 (6.8 %), BRAF (6.5 %), and NRAS (3.9 %). Compared with the wild type, TP53 mutations were associated with low microsatellite instability/microsatellite stability (MSI-L/MSS) (P = 0.007), tumor location (P = 0.043), and histological grade (P = 0.0009); KRAS mutations were associated with female gender (P = 0.026), distant metastasis (P = 0.023), TNM stage (P = 0.013), and histological grade (P = 0.004); APC mutations were associated with patients <64 years of age at diagnosis (P = 0.04); PIK3CA mutations were associated with tumor location (P = 4.97e-06) and female gender (P = 0.018); SMAD4 mutations were associated with tumor location (P = 0.033); BRAF mutations were associated with high MSI (MSI-H; P = 6.968e-07), tumor location (P = 1.58e-06), and histological grade (P = 0.04). Mutations in 164 individuals were found to be pathogenic or likely pathogenic. A total of 26 patients harbored MSI-H tumors and they all had at least one detected gene mutation. Mutated genes were enriched in signaling pathways associated with CRC. The present findings have important implications for improving the personalized treatment of patients with CRC in China.

Transsynaptic EphB/Ephrin-B signaling regulates growth of presynaptic boutons required for classical conditioning.

Learning-related presynaptic remodeling has been documented in only a few systems, and its molecular mechanisms are largely unknown. Here we describe a role for the bidirectional EphB/ephrin-B signaling system in structural plasticity of presynaptic nerve terminals using an in vitro model of classical conditioning. Conditioning or BDNF application induced significant growth of auditory nerve presynaptic boutons that convey the conditioned stimulus to abducens motor neurons. Interestingly, bouton enlargement occurred only for those synapses apposed to motor neuron dendrites rather than to somata. Phosphorylation of ephrin-B1, but not EphB2, was induced by both conditioning and BDNF application and was inhibited by postsynaptic injections of ephrin-B antibody. Finally, suppression of postsynaptic ephrin-B function inhibited presynaptic bouton enlargement that was rescued by activation of EphB2 by ephrin-B1-Fc. These data provide evidence for ephrin-B-induced EphB2 forward signaling in presynaptic structural plasticity during classical conditioning. They also reveal a functional interaction between BDNF/TrkB and the Eph/ephrin signaling systems in the coordination of presynaptic and postsynaptic modifications during conditioning.

Two-stage AMPA receptor trafficking in classical conditioning and selective role for glutamate receptor subunit 4 (tGluA4) flop splice variant.

Previously, we proposed a two-stage model for an in vitro neural correlate of eyeblink classical conditioning involving the initial synaptic incorporation of glutamate receptor A1 (GluA1)-containing α-amino-3-hydroxy-5-methylisoxazole-4-propionic acid type receptors (AMPARs) followed by delivery of GluA4-containing AMPARs that support acquisition of conditioned responses. To test specific elements of our model for conditioning, selective knockdown of GluA4 AMPAR subunits was used using small-interfering RNAs (siRNAs). Recently, we sequenced and characterized the GluA4 subunit and its splice variants from pond turtles, Trachemys scripta elegans (tGluA4). Analysis of the relative abundance of mRNA expression by real-time RT-PCR showed that the flip/flop variants of tGluA4, tGluA4c, and a novel truncated variant tGluA4trc1 are major isoforms in the turtle brain. Here, transfection of in vitro brain stem preparations with anti-tGluA4 siRNA suppressed conditioning, tGluA4 mRNA and protein expression, and synaptic delivery of tGluA4-containing AMPARs but not tGluA1 subunits. Significantly, transfection of abducens motor neurons by nerve injections of tGluA4 flop rescue plasmid prior to anti-tGluA4 siRNA application restored conditioning and synaptic incorporation of tGluA4-containing AMPARs. In contrast, treatment with rescue plasmids for tGluA4 flip or tGluA4trc1 failed to rescue conditioning. Finally, treatment with a siRNA directed against GluA1 subunits inhibited conditioning and synaptic delivery of tGluA1-containing AMPARs and importantly, those containing tGluA4. These data strongly support our two-stage model of conditioning and our hypothesis that synaptic incorporation of tGluA4-containing AMPARs underlies the acquisition of in vitro classical conditioning. Furthermore, they suggest that tGluA4 flop may have a critical role in conditioning mechanisms compared with the other tGluA4 splice variants.

The natural compound neobractatin inhibits cell proliferation mainly by regulating the RNA binding protein CELF6.

The fruits of Garcinia bracteata can be eaten raw or processed into spices, which are considered to possess nutritional and medicinal value. Neobractatin (NBT) is a natural compound isolated from Garcinia bracteate. This study showed that NBT showed antitumor effect by upregulation of CELF6. CELF6, an RNA-binding protein of the CELF family, is involved in cancer cell proliferation. However, the role of CELF6 in human cervical cancer remains unknown. Here, we showed that CELF6 overexpression significantly suppressed HeLa cell proliferation. Mechanistically, the RNA immunoprecipitation sequencing (RIP-seq) results suggested that CELF6 physically targeted the cyclin D1 transcript, affecting protein stability. Overexpression of CELF6 increased the degradation of cyclin D1. Consistent results were obtained for the effect of NBT, which increased the expression of CELF6 at both the mRNA and protein levels. An in vivo study further confirmed the regulatory effect of NBT on CELF6 and cyclin D1 levels in a HeLa xenograft model. Similar effects of NBT on CELF6 were also shown in K562 cells in vitro and in vivo. In conclusion, our findings identified CELF6 as a tumor suppressor and a novel therapeutic target in cervical cancer. The upregulation of CELF6 expression by NBT and its antiproliferative effect on HeLa cells indicated that NBT from G. bracteata might be a small-molecule compound targeting CELF6.

Oligomeric amyloid-{beta} inhibits the proteolytic conversion of brain-derived neurotrophic factor (BDNF), AMPA receptor trafficking, and classical conditioning.

Amyloid-ß (Aß) peptide is thought to have a significant role in the progressive memory loss observed in patients with Alzheimer disease and inhibits synaptic plasticity in animal models of learning. We previously demonstrated that brain-derived neurotrophic factor (BDNF) is critical for synaptic AMPA receptor delivery in an in vitro model of eyeblink classical conditioning. Here, we report that acquisition of conditioned responses was significantly attenuated by bath application of oligomeric (200 nm), but not fibrillar, Aß peptide. Western blotting revealed that BDNF protein expression during conditioning is significantly reduced by treatment with oligomeric Aß, as were phosphorylation levels of cAMP-response element-binding protein (CREB), Ca(2+)/calmodulin-dependent protein kinase II (CaMKII), Ca(2+)/calmodulin-dependent protein kinase IV (CaMKIV), and ERK. However, levels of PKA and PKCζ/λ were unaffected, as was PDK-1. Protein localization studies using confocal imaging indicate that oligomeric Aß, but not fibrillar or scrambled forms, suppresses colocalization of GluR1 and GluR4 AMPA receptor subunits with synaptophysin, indicating that trafficking of these subunits to synapses during the conditioning procedure is blocked. In contrast, coapplication of BDNF with oligomeric Aß significantly reversed these findings. Interestingly, a tolloid-like metalloproteinase in turtle, tTLLs (turtle tolloid-like protein), which normally processes the precursor proBDNF into mature BDNF, was found to degrade oligomeric Aß into small fragments. These data suggest that an Aß-induced reduction in BDNF, perhaps due to interference in the proteolytic conversion of proBDNF to BDNF, results in inhibition of synaptic AMPA receptor delivery and suppression of the acquisition of conditioning.

Learning-Dependent Transcriptional Regulation of BDNF by its Truncated Protein Isoform in Turtle.

The vertebrate brain-derived neurotrophic factor (BDNF) gene produces a number of alternatively spliced transcripts only some of which generate the BDNF protein required for synaptic plasticity and learning. Many of the transcripts are uncharacterized and are of unknown biological significance. Previously, we described alternative splicing within the protein-coding sequence of the BDNF gene in the pond turtle (tBDNF) that generates a functionally distinct truncated protein isoform (trcBDNF) that is regulated during a neural correlate of eyeblink classical conditioning in ex vivo brainstem preparations. We hypothesized that trcBDNF has a dominant negative function because of its anticorrelated expression pattern compared to its full-length BDNF counterpart. The data presented here suggests that trcBDNF functions as a transcriptional repressor of a conditioning-inducible downstream tBDNF promoter that controls expression of full-length BDNF required for learning. First, expression of full-length transcripts is negatively correlated with trcBDNF; transcripts are inhibited when endogenous trcBDNF is ectopically induced and expressed when trcBDNF is inhibited. Second, ChIP-qPCR assays of a recombinant trcBDNF protein, RtrcBDNF, show strong binding to the downstream tBDNF exon III promoter that corresponds with inhibition of conditioning. Third, deletions of the C-terminus of RtrcBDNF result in inhibition of promoter binding and conditioning acquisition when a tropomyosin receptor kinase B (TrkB) binding site is accounted for. Finally, microinjection of RtrcBDNF directly into brainstem preparations inhibits conditioning. These data reveal a new mechanism of activity-dependent BDNF transcriptional regulation and suggest that BDNF is an autoregulatory gene. How generalizable this mechanism is across plasticity genes remains to be elucidated.

Cleavage of proBDNF to BDNF by a tolloid-like metalloproteinase is required for acquisition of in vitro eyeblink classical conditioning.

The tolloid/bone morphogenetic protein-1 family of metalloproteinases have an important role in the regulation of embryonic pattern formation and tissue morphogenesis. Studies suggest that they participate in mechanisms of synaptic plasticity in adults, but very little is known about their function. Recently, we isolated a reptilian ortholog of the tolloid gene family designated turtle tolloid-like gene (tTll). Here, we examined the role of tTLL in an in vitro model of eyeblink classical conditioning using an isolated brainstem preparation to assess its role in synaptic plasticity during conditioning. Analysis by real-time reverse transcription-PCR shows that an extracellularly secreted form of tTLL, tTLLs, is transiently expressed in the early stages of conditioning during conditioned response acquisition, whereas a cytosolic form, tTLLc, is not. Short interfering RNA (siRNA)-directed gene knockdown and rescue of tTLL expression demonstrate that it is required for conditioning. Significantly, we show that tTLLs cleaves the precursor proBDNF into mature BDNF in cleavage assay studies, and application of recombinant tTLLs protein alone to preparations results in induction of mature BDNF expression. The mature form of BDNF is minimally expressed in preparations treated with anti-tTLL siRNA, and the synaptic incorporation of both GluR1- and GluR4-containing AMPA receptors is significantly reduced, resulting in suppression of conditioning. This is the first study to demonstrate that expression of an extracellularly secreted tolloid-like metalloproteinase is regulated in the early stages of classical conditioning and functions in the conversion of proBDNF to mature BDNF. The mature form of BDNF is required for synaptic delivery of AMPA receptors and acquisition of conditioned responses.

Gerontoxanthone I and Macluraxanthone Induce Mitophagy and Attenuate Ischemia/Reperfusion Injury.

Mitophagy is a crucial process in controlling mitochondrial biogenesis. Balancing mitophagy and mitochondrial functions is required for maintaining cellular homeostasis. In this study, we found that Gerontoxanthone I (GeX1) and Macluraxanthone (McX), xanthone derivatives isolated from Garcinia bracteata C. Y. Wu ex Y. H. Li, induced Parkin puncta accumulation and promoted mitophagy. GeX1 and McX treatment induced the degradation of mitophagy-related proteins such as Tom20 and Tim23. GeX1 and McX directly stabilized PTEN-induced putative kinase 1 (PINK1) on the outer membrane of the mitochondria, and then recruited Parkin to mitochondria. This significantly induced phosphorylation and ubiquitination of Parkin, suggesting that GeX1 and McX mediate mitophagy through the PINK1-Parkin pathway. Transfecting ParkinS65A or pretreated MG132 abolished the induction effects of GeX1 and McX on mitophagy. Furthermore, GeX1 and McX treatment decreased cell death and the level of ROS in an ischemia/reperfusion (IR) injury model in H9c2 cells compared to a control group. Taken together, our data suggested that GeX1 and McX induce PINK1-Parkin-mediated mitophagy and attenuate myocardial IR injury in vitro.

Characterization and Transcriptional Activation of the Immediate Early Gene ARC During a Neural Correlate of Classical Conditioning.

Plasticity and learning genes require regulatory mechanisms that have the flexibility to respond to a variety of sensory stimuli to generate adaptive behavioral responses. The immediate early gene (IEG) activity-regulated cytoskeleton-associated protein (ARC) is rapidly induced not only by neuronal stimulation but also during a variety of learning tasks. How ARC is regulated in response to complex stimuli during associative learning remains to be fully detailed. Here, we characterized the structure of the ARC gene in the pond turtle and mechanisms of its transcriptional activation during a neural correlate of eyeblink classical conditioning. The tARC gene is regulated in part by the presence of paused polymerase (RNAPII) that is poised at the promoter for rapid gene induction. Conditioning induces permissive chromatin modifications in the tARC promoter that allows binding by the transcription factor cAMP response element-binding protein (CREB) within 5 min of training. During learning acquisition, the pausing factor negative elongation factor (NELF) dissociates from the promoter thereby releasing RNAPII for active transcription. Data additionally suggest that the DNA insulator protein CCCTC-binding factor (CTCF) is required for transcription by mediating a learning-induced interaction of the ARC promoter with an enhancer element. Our study suggests that the learning-inducible IEG tARC utilizes both paused RNAPII and rapid chromatin modifications that allow for dynamic gene responsiveness required when an organism is presented with a variety of environmental stimuli.

The natural compound neobractatin inhibits tumor metastasis by upregulating the RNA-binding-protein MBNL2.

Tumor metastasis is the predominant cause of lethality in cancer. We found that Neobractatin (NBT), a natural compound isolated from Garcinia bracteata, could efficiently inhibit breast and lung cancer cells metastasis. However, the mechanisms of NBT inhibiting cancer metastasis remain unclear. Based on the RNA-sequencing result and transcriptome analysis, Muscleblind-like 2 (MBNL2) was found to be significantly upregulated in the cells treated with NBT. The Cancer Genome Atlas (TCGA) database analysis indicated that the expression of MBNL2 in breast and lung carcinoma tumor tissues was significantly lower compared to normal tissues. We thus conducted to investigate the antimetastatic role of MBNL2. MBNL2 overexpression mimicked the effect of NBT on breast cancer and lung cancer cell motility and metastasis, in addition significantly enhanced the inhibition effect of NBT. MBNL2 knockdown furthermore partially eliminated the inhibitory effect of NBT on metastasis. Mechanistically, we demonstrated that NBT- and MBNL2-mediated antimetastasis regulation significantly correlated with the pAKT/epithelial-mesenchymal transition (EMT) pathway. Subsequent in vivo study showed the same metastasis inhibition effect in NBT and MBNL2 in MDA-MB-231 xenografts mouse model. This study suggest that NBT possesses significant antitumor activity in breast and lung cancer cells that is partly mediated through the MBNL2 expression and enhancement in metastasis via the pAKT/EMT signaling pathway.

The Natural Compound Neobractatin Induces Cell Cycle Arrest by Regulating E2F1 and Gadd45α.

The complexity and multi-target feature of natural compounds have made it difficult to elucidate their mechanism of action (MoA), which hindered the development of lead anticancer compounds to some extent. In this study, we applied RNA-Seq and GSEA transcriptome analysis to rapidly and efficiently evaluate the anticancer mechanisms of neobractatin (NBT), a caged prenylxanthone isolated from the Chinese herb Garcinia bracteata. We found that NBT exerted anti-proliferative effect on various cancer cells and caused both G1/S and G2/M arrest in synchronized cancer cells through its effects on the expression of E2F1 and GADD45α. The in vivo animal study further suggested that NBT could reduce tumor burden in HeLa xenograft model with no apparent toxicity. By demonstrating the biological effect of NBT, we provided evidences for further investigations of this novel natural compound with anticancer potential.

Synaptic localization of GluR4-containing AMPARs and Arc during acquisition, extinction, and reacquisition of in vitro classical conditioning.

Data suggest that modulation of synaptic strength by incorporation of GluR4-containing AMPARs occurs during conditioned response (CR) acquisition in an in vitro model of classical conditioning. Here we extend these findings by showing that synaptically localized GluR4 subunits parallels the expression of CRs during conditioning training in which there is differential expression of CRs, such as during acquisition, extinction, and reacquisition. Moreover, colocalization and coimmunoprecipitation data suggest that Arc associates with GluR4-containing AMPARs during these different training procedures. Once induced, Arc remains present in synapses during these phases of conditioning. The results are consistent with the interpretation that synaptic incorporation of GluR4-containing AMPARs supports the expression of CRs in this preparation, and that Arc may be involved in trafficking of GluR4 subunits during conditioning. Moreover, the maintained presence of synaptically localized Arc during all phases of conditioning examined indicates that synapses do not return to their naïve state after extinction and that, given the potential trafficking function of Arc, may facilitate relearning after extinction.

MeCP2 regulates Tet1-catalyzed demethylation, CTCF binding, and learning-dependent alternative splicing of the BDNF gene in Turtle.

MECP2 mutations underlying Rett syndrome cause widespread misregulation of gene expression. Functions for MeCP2 other than transcriptional are not well understood. In an ex vivo brain preparation from the pond turtle Trachemys scripta elegans, an intraexonic splicing event in the brain-derived neurotrophic factor (BDNF) gene generates a truncated mRNA transcript in naïve brain that is suppressed upon classical conditioning. MeCP2 and its partners, splicing factor Y-box binding protein 1 (YB-1) and methylcytosine dioxygenase 1 (Tet1), bind to BDNF chromatin in naïve but dissociate during conditioning; the dissociation correlating with decreased DNA methylation. Surprisingly, conditioning results in new occupancy of BDNF chromatin by DNA insulator protein CCCTC-binding factor (CTCF), which is associated with suppression of splicing in conditioning. Knockdown of MeCP2 shows it is instrumental for splicing and inhibits Tet1 and CTCF binding thereby negatively impacting DNA methylation and conditioning-dependent splicing regulation. Thus, mutations in MECP2 can have secondary effects on DNA methylation and alternative splicing.