A zincophilic separator with directional alignment and pore hydrophilicity towards stable aqueous zinc metal batteries.

A zincophilic PAN@Zn(OTF)2 (PZO) separator with an extremely thin thickness of 65.6 µm is introduced. This separator with a low cost of 6.1 $ m-2, exhibiting excellent mechanical and wettability properties. The cell with the PZO separator exhibits impressive electrochemical performances both in symmetrical Zn||Zn cell and Zn||NVO full cell.

PHLDA1 is a P53 target gene involved in P53-mediated cell apoptosis.

Pleckstrin homeolike domain, family A, member 1 (PHLDA1) is a multifunctional protein that plays diverse roles in A variety of biological processes, including cell death, and hence its altered expression has been found in different types of cancer. Although studies have shown a regulatory relationship between p53 and PHLDA1, the molecular mechanism is still unclear. Especially, the role of PHLDA1 in the process of apoptosis is still controversial. In this study, we found that the expression of PHLDA1 in human cervical cancer cell lines was correlated with the up-expression of p53 after treatment with apoptosis-inducing factors. Subsequently, the binding site and the binding effect of p53 on the promoter region of PHLDA1 were verified by our bioinformatics data analysis and luciferase reporter assay. Indeed, we used CRISPR-Cas9 to knockout the p53 gene in HeLa cells and further confirmed that p53 can bind to the promoter region of PHLDA1 gene, and then directly regulate the expression of PHLDA1 by recruiting P300 and CBP to change the acetylation and methylation levels in the promoter region. Finally, a series of gain-of-function experiments further confirmed that p53 re-expression in HeLap53-/- cell can up-regulate the reduction of PHLDA1 caused by p53 knockout, and affect cell apoptosis and proliferation. Our study is the first to explore the regulatory mechanism of p53 on PHLDA1 by using the p53 gene knockout cell model, which further proves that PHLDA1 is a target-gene in p53-mediated apoptosis, and reveals the important role of PHLDA1 in cell fate determination.

Implications of Activating the ANT2/mTOR/PGC-1α Feedback Loop: Insights into Mitochondria-Mediated Injury in Hypoxic Myocardial Cells.

Mitochondrial dysfunction is known to play a critical role in the development of cardiomyocyte death during acute myocardial infarction (AMI). However, the exact mechanisms underlying this dysfunction are still under investigation. Adenine nucleotide translocase 2 (ANT2) is a key functional protein in mitochondria. We aimed at exploring the potential benefits of ANT2 inhibition against AMI. We utilized an oxygen-glucose deprivation (OGD) cell model and an AMI mice model to detect cardiomyocyte injury. We observed elevated levels of reactive oxygen species (ROS), disrupted mitochondrial membrane potential (MMP), and increased apoptosis due to the overexpression of ANT2. Additionally, we discovered that ANT2 is involved in myocardial apoptosis by activating the mTOR (mechanistic target of rapamycin kinase)-dependent PGC-1α (PPARG coactivator 1 alpha) pathway, establishing a novel feedback loop during AMI. In our experiments with AC16 cells under OGD conditions, we observed protective effects when transfected with ANT2 siRNA and miR-1203. Importantly, the overexpression of ANT2 counteracted the protective effect resulting from miR-1203 upregulation in OGD-induced AC16 cells. All these results supported that the inhibition of ANT2 could alleviate myocardial cell injury under OGD conditions. Based on these findings, we propose that RNA interference (RNAi) technology, specifically miRNA and siRNA, holds therapeutic potential by activating the ANT2/mTOR/PGC-1α feedback loop. This activation could help mitigate mitochondria-mediated injury in the context of AMI. These insights may contribute to the development of future clinical strategies for AMI.

Three sesquiterpene lactones suppress lung adenocarcinoma by blocking TMEM16A-mediated Ca2+-activated Cl- channels.

Transmembrane protein TMEM16A, which encodes calcium-activated chloride channel has been implicated in tumorigenesis. Overexpression of TMEM16A is associated with poor prognosis and low overall survival in multiple cancers including lung adenocarcinoma, making it a promising biomarker and therapeutic target. In this study, three structure-related sesquiterpene lactones (mecheliolide, costunolide and dehydrocostus lactone) were extracted from the traditional Chinese medicine Aucklandiae Radix and identified as novel TMEM16A inhibitors with comparable inhibitory effects. Their effects on the proliferation and migration of lung adenocarcinoma cells were examined. Whole-cell patch clamp experiments showed that these sesquiterpene lactones potently inhibited recombinant TMEM16A currents in a concentration-dependent manner. The half-maximal concentration (IC50) values for three tested sesquiterpene lactones were 29.9 ± 1.1 µM, 19.7 ± 0.4 µM, and 24.5 ± 2.1 µM, while the maximal effect (Emax) values were 100.0% ± 2.8%, 85.8% ± 0.9%, and 88.3% ± 4.6%, respectively. These sesquiterpene lactones also significantly inhibited the endogenous TMEM16A currents and proliferation, and migration of LA795 lung cancer cells. These results demonstrate that mecheliolide, costunolide and dehydrocostus lactone are novel TMEM16A inhibitors and potential candidates for lung adenocarcinoma therapy.

Earth's evolving geodynamic regime recorded by titanium isotopes.

Earth's mantle has a two-layered structure, with the upper and lower mantle domains separated by a seismic discontinuity at about 660 km (refs. 1,2). The extent of mass transfer between these mantle domains throughout Earth's history is, however, poorly understood. Continental crust extraction results in Ti-stable isotopic fractionation, producing isotopically light melting residues3-7. Mantle recycling of these components can impart Ti isotope variability that is trackable in deep time. We report ultrahigh-precision 49Ti/47Ti ratios for chondrites, ancient terrestrial mantle-derived lavas ranging from 3.8 to 2.0 billion years ago (Ga) and modern ocean island basalts (OIBs). Our new Ti bulk silicate Earth (BSE) estimate based on chondrites is 0.052 ± 0.006‰ heavier than the modern upper mantle sampled by normal mid-ocean ridge basalts (N-MORBs). The 49Ti/47Ti ratio of Earth's upper mantle was chondritic before 3.5 Ga and evolved to a N-MORB-like composition between approximately 3.5 and 2.7 Ga, establishing that more continental crust was extracted during this epoch. The +0.052 ± 0.006‰ offset between BSE and N-MORBs requires that <30% of Earth's mantle equilibrated with recycled crustal material, implying limited mass exchange between the upper and lower mantle and, therefore, preservation of a primordial lower-mantle reservoir for most of Earth's geologic history. Modern OIBs record variable 49Ti/47Ti ratios ranging from chondritic to N-MORBs compositions, indicating continuing disruption of Earth's primordial mantle. Thus, modern-style plate tectonics with high mass transfer between the upper and lower mantle only represents a recent feature of Earth's history.

STAT3-Mediated Promoter-Enhancer Interaction Up-Regulates Inhibitor of DNA Binding 1 (ID1) to Promote Colon Cancer Progression.

BACKGROUND: High expression of inhibitor of DNA binding 1 (ID1) correlates with poor prognosis in colorectal cancer (CRC). Aberrant enhancer activation in regulating ID1 transcription is limited. METHODS: Immunohistochemistry (IHC), quantitative RT-PCR (RT-qPCR) and Western blotting (WB) were used to determine the expression of ID1. CRISPR-Cas9 was used to generate ID1 or enhancer E1 knockout cell lines. Dual-luciferase reporter assay, chromosome conformation capture assay and ChIP-qPCR were used to determine the active enhancers of ID1. Cell Counting Kit 8, colony-forming, transwell assays and tumorigenicity in nude mice were used to investigate the biological functions of ID1 and enhancer E1. RESULTS: Human CRC tissues and cell lines expressed a higher level of ID1 than normal controls. ID1 promoted CRC cell proliferation and colony formation. Enhancer E1 actively regulated ID1 promoter activity. Signal transducer and activator of transcription 3 (STAT3) bound to ID1 promoter and enhancer E1 to regulate their activity. The inhibitor of STAT3 Stattic attenuated ID1 promoter and enhancer E1 activity and the expression of ID1. Enhancer E1 knockout down-regulated ID1 expression level and cell proliferation in vitro and in vivo. CONCLUSIONS: Enhancer E1 is positively regulated by STAT3 and contributes to the regulation of ID1 to promote CRC cell progression and might be a potential target for anti-CRC drug studies.

On the identification of hyperhydrated sodium chloride hydrates, stable at icy moon conditions.

Sodium chloride is expected to be found on many of the surfaces of icy moons like Europa and Ganymede. However, spectral identification remains elusive as the known NaCl-bearing phases cannot match current observations, which require higher number of water of hydration. Working at relevant conditions for icy worlds, we report the characterization of three "hyperhydrated" sodium chloride (SC) hydrates, and refined two crystal structures [2NaCl·17H2O (SC8.5); NaCl·13H2O (SC13)]. We found that the dissociation of Na+ and Cl- ions within these crystal lattices allows for the high incorporation of water molecules and thus explain their hyperhydration. This finding suggests that a great diversity of hyperhydrated crystalline phases of common salts might be found at similar conditions. Thermodynamic constraints indicate that SC8.5 is stable at room pressure below 235 K, and it could be the most abundant NaCl hydrate on icy moon surfaces like Europa, Titan, Ganymede, Callisto, Enceladus, or Ceres. The finding of these hyperhydrated structures represents a major update to the H2O-NaCl phase diagram. These hyperhydrated structures provide an explanation for the mismatch between the remote observations of the surface of Europa and Ganymede and previously available data on NaCl solids. It also underlines the urgent need for mineralogical exploration and spectral data on hyperhydrates at relevant conditions to help future icy world exploration by space missions.

Corrigendum: PHLDA1 Suppresses TLR4-Triggered Proinflammatory Cytokine Production by Interaction With Tollip.

[This corrects the article DOI: 10.3389/fimmu.2022.731500.].

PHLDA1 Suppresses TLR4-Triggered Proinflammatory Cytokine Production by Interaction With Tollip.

Pleckstrin homology-like domain, family A, member 1 (PHLDA1) has been reported to be expressed in many mammalian tissues and cells. However, the functions and exact mechanisms of PHLDA1 remain unclear. In this study, we found that PHLDA1 expression was significantly altered in macrophages after exposure to lipopolysaccharide (LPS) in vitro, suggesting that PHLDA1 may be involved in the regulation of TLR4 signaling pathway activated by LPS. PHLDA1 attenuated the production of LPS-stimulated proinflammatory cytokines (TNF-α, IL-6, and IL-1ß). Further research showed that the phosphorylation levels of some important signal molecules in TLR4/MyD88-mediated MAPK and NF-κB signaling pathways were reduced by PHLDA1, which in turn impaired the transcription factors NF-κB and AP1 nuclear translocation and their responsive element activities. Furthermore, we found that PHLDA1 repressed LPS-induced proinflammatory cytokine production via binding to Tollip which restrained TLR4 signaling pathway. A mouse model of endotoxemia was established to confirm the above similar results. In brief, our findings demonstrate that PHLDA1 is a negative regulator of LPS-induced proinflammatory cytokine production by Tollip, suggesting that PHLDA1 plays an anti-inflammatory role through inhibiting the TLR4/MyD88 signaling pathway with the help of Tollip. PHLDA1 may be a novel therapeutic target in treating endotoxemia.

Purine-Induced IFN-γ Promotes Uric Acid Production by Upregulating Xanthine Oxidoreductase Expression.

Objective: Limiting purine intake, inhibiting xanthine oxidoreductase (XOR) and inhibiting urate reabsorption in proximal tubule by uricosuric drugs, to reduce serum uric acid (UA) levels, are recognized treatments for gout. However, the mechanism of increased how XOR expression and activity in hyperuricemia and gout remains unclear. This study aims to explore whether exogenous purines are responsible for increased XOR expression and activity. Methods: HepG2 and Bel-7402 human hepatoma cells were stimulated with exogenous purine, or were exposed to conditioned growth medium of purine-stimulated Jurkat cells, followed by measurement of XOR expression and UA production to determine the effect of lymphocyte-secreted cytokines on XOR expression in hepatocytes. The expression of STAT1, IRF1 and CBP and their binding on the XDH promoter were detected by western blotting and ChIP-qPCR. The level of DNA methylation was determined by bisulfite sequencing PCR. Blood samples from 117 hyperuricemia patients and 119 healthy individuals were collected to analyze the correlation between purine, UA and IFN-γ concentrations. Results: Excess of purine was metabolized to UA in hepatocyte metabolism by XOR that was induced by IFN-γ secreted in the conditioned growth medium of Jurkat cells in response to exogenous purine, but it did not directly induce XOR expression. IFN-γ upregulated XOR expression due to the enhanced binding of STAT1 to IRF1 to further recruit CBP to the XDH promoter. Clinical data showed positive correlation of serum IFN-γ with both purine and UA, and associated risk of hyperuricemia. Conclusion: Purine not only acts as a metabolic substrate of XOR for UA production, but it induces inflammation through IFN-γ secretion that stimulates UA production through elevation of XOR expression.

Host-Guest Hydrogen Bonding in High-Pressure Acetone Clathrate Hydrates: In Situ Single-Crystal X-ray Diffraction Study.

The phenomenon of host-guest hydrogen bonding in clathrate hydrate crystal structures and its effect on physical and chemical properties have become subjects of extensive research. Hydrogen bonding has been studied for cubic (sI and sII) and hexagonal (sH) binary clathrates, while it has not been addressed for clathrate structures that exist at elevated pressures. Here, four acetone hydrate clathrates have been grown at high-pressure and low-temperature conditions. In situ single-crystal X-ray diffraction revealed that the synthesized phases possess already known trigonal (sTr), orthorhombic (sO), and tetragonal (sT) crystal structures as well as a previously unknown orthorhombic structure, so-called sO-II. Only sO and sII have previously been reported for acetone clathrates. Structural analysis suggests that acetone oxygens are hydrogen-bonded to the closest water oxygens of the host frameworks. Our discoveries show that clathrate hydrates hosting polar molecules are not as exotic as previously thought and could be stabilized at high-pressure conditions through hydrogen bonding.

Activation of parabrachial nucleus - ventral tegmental area pathway underlies the comorbid depression in chronic neuropathic pain in mice.

Depression symptoms are often found in patients suffering from chronic pain, a phenomenon that is yet to be understood mechanistically. Here, we systematically investigate the cellular mechanisms and circuits underlying the chronic-pain-induced depression behavior. We show that the development of chronic pain is accompanied by depressive-like behaviors in a mouse model of trigeminal neuralgia. In parallel, we observe increased activity of the dopaminergic (DA) neuron in the midbrain ventral tegmental area (VTA), and inhibition of this elevated VTA DA neuron activity reverses the behavioral manifestations of depression. Further studies establish a pathway of glutamatergic projections from the spinal trigeminal subnucleus caudalis (Sp5C) to the lateral parabrachial nucleus (LPBN) and then to the VTA. These glutamatergic projections form a direct circuit that controls the development of the depression-like behavior under the state of the chronic neuropathic pain.

Natural Antisense Transcript PEBP1P3 Regulates the RNA Expression, DNA Methylation and Histone Modification of CD45 Gene.

The leukocyte common antigen CD45 is a transmembrane phosphatase expressed on all nucleated hemopoietic cells, and the expression levels of its splicing isoforms are closely related to the development and function of lymphocytes. PEBP1P3 is a natural antisense transcript from the opposite strand of CD45 intron 2 and is predicted to be a noncoding RNA. The genotype-tissue expression and quantitative PCR data suggested that PEBP1P3 might be involved in the regulation of expression of CD45 splicing isoforms. To explore the regulatory mechanism of PEBP1P3 in CD45 expression, DNA methylation and histone modification were detected by bisulfate sequencing PCR and chromatin immunoprecipitation assays, respectively. The results showed that after the antisense RNA PEBP1P3 was knocked down by RNA interference, the DNA methylation of CD45 intron 2 was decreased and histone H3K9 and H3K36 trimethylation at the alternative splicing exons of CD45 DNA was increased. Knockdown of PEBP1P3 also increased the binding levels of chromatin conformation organizer CTCF at intron 2 and the alternative splicing exons of CD45. The present results indicate that the natural antisense RNA PEBP1P3 regulated the alternative splicing of CD45 RNA, and that might be correlated with the regulation of histone modification and DNA methylation.

Alternative Splicing of Pre-mRNA in the Control of Immune Activity.

The human immune response is a complex process that responds to numerous exogenous antigens in preventing infection by microorganisms, as well as to endogenous components in the surveillance of tumors and autoimmune diseases, and a great number of molecules are necessary to carry the functional complexity of immune activity. Alternative splicing of pre-mRNA plays an important role in immune cell development and regulation of immune activity through yielding diverse transcriptional isoforms to supplement the function of limited genes associated with the immune reaction. In addition, multiple factors have been identified as being involved in the control of alternative splicing at the cis, trans, or co-transcriptional level, and the aberrant splicing of RNA leads to the abnormal modulation of immune activity in infections, immune diseases, and tumors. In this review, we summarize the recent discoveries on the generation of immune-associated alternative splice variants, clinical disorders, and possible regulatory mechanisms. We also discuss the immune responses to the neoantigens produced by alternative splicing, and finally, we issue some alternative splicing and immunity correlated questions based on our knowledge.

ARHGAP20 Expression Inhibited HCC Progression by Regulating the PI3K-AKT Signaling Pathway.

INTRODUCTION: One of the most common cancers is hepatocellular carcinoma (HCC), which is an aggressive cancer that is associated with high mortality. The expression and role of ARHGAP20 in HCC remain unclear. MATERIALS AND METHODS: The expression and clinical role of ARHGAP20 were investigated using online databases and HCC samples from Meizhou People's Hospital. Wound healing assays, transwell migration/invasion assays, and lung metastasis models were performed using nude mice. Gene set enrichment analyses were used to further explore the potential mechanisms. RESULTS: Inspired by expression analyses of three different public databases (ie, TIMER, Oncomine, and HCCDB database), we confirmed that ARHGAP20 was downregulated in clinical HCC tumors compared with normal controls. ARHGAP20 expression inhibited HCC migration and invasion in vitro and in vivo. Based on GSEA results, we tested markers of the PI3K-AKT signaling pathway. Interestingly, while ARHGAP20 upregulation suppressed HCC migration/invasion and phosphorylation of AKT/PI3K molecules, exposure to the PI3K-AKT pathway agonist rhIGF-1 partially rescued these phenomena. ARHGAP20 also showed a close correlation with certain components in the HCC immune microenvironment. Furthermore, we revealed that downregulated ARHGAP20 was significantly correlated with larger tumor size and vascular invasion, and could be used as an adverse independent prognostic factor for HCC OS but not RFS. CONCLUSION: ARHGAP20 was identified for the first time as a tumor suppressor gene that could inhibit HCC progression by regulating the PI3K-AKT signaling pathway and the immune microenvironment in HCC.

A 15-Gene Signature and Prognostic Nomogram for Predicting Overall Survival in Non-Distant Metastatic Oral Tongue Squamous Cell Carcinoma.

BACKGROUND: Oral tongue squamous cell carcinoma (OTSCC) is a devastating tumor with poor prognosis. There is an urgent need for reliable biomarkers to help predict prognosis and guide treatment for OTSCC. In the current study, we aimed to develop a robust multi-gene signature and prognostic nomogram to predict the prognosis of patients with non-distant metastatic OTSCC. METHODS: OTSCC-related differentially-expressed genes were screened from The Cancer Genome Atlas (TCGA) database. Univariate Cox regression based on 1,000 bootstrap replicates, LASSO regression and stepwise multivariate Cox regression were utilized to develop a novel multi-mRNA signature for predicting overall survival in OTSCC. The concordance index, area under receiver operating characteristic (ROC AUC) and calibration curve were employed to assess the prediction capacity of the novel multi-gene model. In addition, a prognostic nomogram was constructed to facilitate the clinical use of the fitted model. The Kaplan-Meier with log-rank test was employed to assess differences in overall survival. RESULTS: We successfully established a novel 15-mRNA prognostic model for predicting overall survival of non-distant metastatic OTSCC, involving ADTRP, ITGA3, RFC4, CCDC96, CYP2J2, NELL2, SPHK1, SPAG16, HBEGF, S100A9, EGFL6, ADGRG6, PDE4D, ABCA4, and CTTN. The prediction ability of this 15-gene signature was independent of other clinicopathological factors, with an HR of 11.5 (95% CI: 4.70-28.3). Moreover, internal validation by bootstrap analysis yielded a C-index of 0.849, with a 3-year AUC of 0.907 and 5-year AUC of 0.944, which implied excellent prediction accuracy of the fitted model. In addition, external validation by using the GEO dataset (GSE41116) yielded a C-index of 0.804, with a 3-year AUC of 0.868 and 5-year AUC of 0.855, which also indicated good prediction ability of the 15-gene model. Finally, a prognostic nomogram integrating risk group, grade, T stage and N stage was established. CONCLUSION: Our results demonstrate our 15-gene signature was independently associated with overall survival in non-distant metastatic OTSCC. Moreover, the prognostic nomogram integrating the 15-gene signature and clinicopathological factors has potential to be developed as a prognostic tool.

Junctophilin-4 facilitates inflammatory signalling at plasma membrane-endoplasmic reticulum junctions in sensory neurons.

KEY POINTS: Rat somatosensory neurons express a junctional protein, junctophilin-4 (JPH4) JPH4 is necessary for the formation of store operated Ca2+ entry (SOCE) complex at the junctions between plasma membrane and endoplasmic reticulum in these neurons. Knockdown of JPH4 impairs endoplasmic reticulum Ca2+ store refill and junctional Ca2+ signalling in sensory neurons. In vivo knockdown of JPH4 in the dorsal root ganglion (DRG) sensory neurons significantly attenuated experimentally induced inflammatory pain in rats. Junctional nanodomain Ca2+ signalling maintained by JPH4 is an important contributor to the inflammatory pain mechanisms. ABSTRACT: Junctions of endoplasmic reticulum and plasma membrane (ER-PM junctions) form signalling nanodomains in eukaryotic cells. ER-PM junctions are present in peripheral sensory neurons and are important for the fidelity of G protein coupled receptor (GPCR) signalling. Yet little is known about the assembly, maintenance and physiological role of these junctions in somatosensory transduction. Using fluorescence imaging, proximity ligation, super-resolution microscopy, in vitro and in vivo gene knockdown we demonstrate that a member of the junctophilin protein family, junctophilin-4 (JPH4), is necessary for the formation of store operated Ca2+ entry (SOCE) complex at the ER-PM junctions in rat somatosensory neurons. Thus we show that JPH4 localises to the ER-PM junctional areas and co-clusters with SOCE proteins STIM1 and Orai1 upon ER Ca2+ store depletion. Knockdown of JPH4 impairs SOCE and ER Ca2+ store refill in sensory neurons. Furthermore, we demonstrate a key role of the JPH4 and junctional nanodomain Ca2+ signalling in the pain-like response induced by the inflammatory mediator bradykinin. Indeed, an in vivo knockdown of JPH4 in the dorsal root ganglion (DRG) sensory neurons significantly shortened the duration of nocifensive behaviour induced by hindpaw injection of bradykinin in rats. Since the ER supplies Ca2+ for the excitatory action of multiple inflammatory mediators, we suggest that junctional nanodomain Ca2+ signalling maintained by JPH4 is an important contributor to the inflammatory pain mechanisms.

The niobium and tantalum concentration in the mantle constrains the composition of Earth's primordial magma ocean.

The bulk silicate Earth (BSE), and all its sampleable reservoirs, have a subchondritic niobium-to-tantalum ratio (Nb/Ta). Because both elements are refractory, and Nb/Ta is fairly constant across chondrite groups, this can only be explained by a preferential sequestration of Nb relative to Ta in a hidden (unsampled) reservoir. Experiments have shown that Nb becomes more siderophile than Ta under very reducing conditions, leading the way for the accepted hypothesis that Earth's core could have stripped sufficient amounts of Nb during its formation to account for the subchondritic signature of the BSE. Consequently, this suggestion has been used as an argument that Earth accreted and differentiated, for most of its history, under very reducing conditions. Here, we present a series of metal-silicate partitioning experiments of Nb and Ta in a laser-heated diamond anvil cell, at pressure and temperature conditions directly comparable to those of core formation; we find that Nb is more siderophile than Ta under any conditions relevant to a deep magma ocean, confirming that BSE's missing Nb is in the core. However, multistage core formation modeling only allows for moderately reducing or oxidizing accretionary conditions, ruling out the need for very reducing conditions, which lead to an overdepletion of Nb from the mantle (and a low Nb/Ta ratio) that is incompatible with geochemical observations. Earth's primordial magma ocean cannot have contained less than 2% or more than 18% FeO since the onset of core formation.

Berberine inhibits cancer cells growth by suppressing fatty acid synthesis and biogenesis of extracellular vesicles.

AIMS: Berberine is an isoquinoline alkaloid extracted from the root, rhizome and stem bark of Coptidis Rhizoma. Previous studies have revealed the anti-tumor potential of berberine against various types of cancer cells. However, the underlying mechanisms are not yet fully understood. In this study, we focused on the effects of berberine on fatty acid synthesis and extracellular vesicles formation in cancer cells, and revealed the internal mechanism of berberine inhibition on cancer cell proliferation. MATERIALS AND METHODS: Anti-proliferative activity of berberine was determined by cell counting and microscope observation and cell cycle analysis. Activities of AMPK and ACC, expression of extracellular vesicles markers were detected by western blotting. 13C labeling metabolic flux analysis was used for determination of de novo synthesis of fatty acids. The excreted extracellular vesicles in culture mediums were separated by both polyethylene glycol enrichment of extracellular vesicles and differential centrifugation separation. KEY FINDINGS: Among our early experiments, 5-10 µmol/L berberine exhibited the substantial anti-proliferative effect against human colon cancer cell line HCT116, cervical cancer cell line HeLa and other cancer cells. It was also revealed that, through activating AMPK, berberine inhibited ACC activity then suppressed intracellular fatty acid synthesis, finally decreased the biogenesis of extracellular vesicles. Moreover, supplement with citrate acid, palmitic acid, as well as exogenous extracellular vesicles, could rescue the inhibitory effect of berberine on cell proliferation, suggesting that inhibited ACC activity, suppressed fatty acid synthesis and decreased extracellular vesicles production were important mechanisms account for berberine inhibiting cancer cell proliferation. SIGNIFICANCE: Our study indicates that berberine suppresses cancer cell proliferation through inhibiting the synthesis of fatty acids and decreasing biogenesis and secretion of extracellular vesicles, suggests that berberine is a promising candidate for the development of new therapies for cancer.

Delineating an extracellular redox-sensitive module in T-type Ca2+ channels.

T-type (Cav3) Ca2+ channels are important regulators of excitability and rhythmic activity of excitable cells. Among other voltage-gated Ca2+ channels, Cav3 channels are uniquely sensitive to oxidation and zinc. Using recombinant protein expression in HEK293 cells, patch clamp electrophysiology, site-directed mutagenesis, and homology modeling, we report here that modulation of Cav3.2 by redox agents and zinc is mediated by a unique extracellular module containing a high-affinity metal-binding site formed by the extracellular IS1-IS2 and IS3-IS4 loops of domain I and a cluster of extracellular cysteines in the IS1-IS2 loop. Patch clamp recording of recombinant Cav3.2 currents revealed that two cysteine-modifying agents, sodium (2-sulfonatoethyl) methanethiosulfonate (MTSES) and N-ethylmaleimide, as well as a reactive oxygen species-producing neuropeptide, substance P (SP), inhibit Cav3.2 current to similar degrees and that this inhibition is reversed by a reducing agent and a zinc chelator. Pre-application of MTSES prevented further SP-mediated current inhibition. Substitution of the zinc-binding residue His191 in Cav3.2 reduced the channel's sensitivity to MTSES, and introduction of the corresponding histidine into Cav3.1 sensitized it to MTSES. Removal of extracellular cysteines from the IS1-IS2 loop of Cav3.2 reduced its sensitivity to MTSES and SP. We hypothesize that oxidative modification of IS1-IS2 loop cysteines induces allosteric changes in the zinc-binding site of Cav3.2 so that it becomes sensitive to ambient zinc.