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1.
Physiol Rev ; 2024 Mar 07.
Article in English | MEDLINE | ID: mdl-38451235

ABSTRACT

The endomembrane system consists of organellar membranes in the biosynthetic pathway: endoplasmic reticulum (ER), Golgi apparatus, and secretory vesicles, as well as those in the degradative pathway: early endosomes, macropinosomes, phagosomes, autophagosomes, late endosomes, and lysosomes. These endomembrane organelles/vesicles work together to synthesize, modify, package, transport, and degrade proteins, carbohydrates, and lipids, regulating the balance between cellular anabolism and catabolism. Large ion concentration gradients exist across endomembranes - Ca2+ gradients for most endomembrane organelles and H+ gradients for the acidic compartments. Ion (Na+, K+, H+, Ca2+, and Cl-) channels on the organellar membranes control ion flux in response to cellular cues, allowing rapid informational exchange between the cytosol and organelle lumen. Recent advances in organelle proteomics, organellar electrophysiology, luminal and juxta-organellar ion imaging have led to molecular identification and functional characterization of about two dozen endomembrane ion channels. For example, whereas IP3R1-3 channels mediate Ca2+ release from the ER in response to neurotransmitter and hormone stimulation, TRPML1-3 and TMEM175 channels mediate lysosomal Ca2+ and H+ release, respectively, in response to nutritional and trafficking cues. This review aims to summarize the current understandings of these endomembrane channels, with a focus on their subcellular localizations, ion permeation properties, gating mechanisms, cell biological functions, and disease relevance.

2.
Science ; 383(6685): eadj2609, 2024 Feb 23.
Article in English | MEDLINE | ID: mdl-38305684

ABSTRACT

Insects rely on a family of seven transmembrane proteins called gustatory receptors (GRs) to encode different taste modalities, such as sweet and bitter. We report structures of Drosophila sweet taste receptors GR43a and GR64a in the apo and sugar-bound states. Both GRs form tetrameric sugar-gated cation channels composed of one central pore domain (PD) and four peripheral ligand-binding domains (LBDs). Whereas GR43a is specifically activated by the monosaccharide fructose that binds to a narrow pocket in LBDs, disaccharides sucrose and maltose selectively activate GR64a by binding to a larger and flatter pocket in LBDs. Sugar binding to LBDs induces local conformational changes, which are subsequently transferred to the PD to cause channel opening. Our studies reveal a structural basis for sugar recognition and activation of GRs.


Subject(s)
Drosophila Proteins , Drosophila melanogaster , Sugars , Taste Perception , Taste , Animals , Taste/physiology , Taste Perception/physiology , Drosophila melanogaster/physiology , Drosophila Proteins/chemistry , Protein Conformation
3.
Br J Pharmacol ; 180 Suppl 2: S145-S222, 2023 10.
Article in English | MEDLINE | ID: mdl-38123150

ABSTRACT

The Concise Guide to PHARMACOLOGY 2023/24 is the sixth in this series of biennial publications. The Concise Guide provides concise overviews, mostly in tabular format, of the key properties of approximately 1800 drug targets, and over 6000 interactions with about 3900 ligands. There is an emphasis on selective pharmacology (where available), plus links to the open access knowledgebase source of drug targets and their ligands (https://www.guidetopharmacology.org/), which provides more detailed views of target and ligand properties. Although the Concise Guide constitutes almost 500 pages, the material presented is substantially reduced compared to information and links presented on the website. It provides a permanent, citable, point-in-time record that will survive database updates. The full contents of this section can be found at http://onlinelibrary.wiley.com/doi/10.1111/bph.16178. Ion channels are one of the six major pharmacological targets into which the Guide is divided, with the others being: G protein-coupled receptors, nuclear hormone receptors, catalytic receptors, enzymes and transporters. These are presented with nomenclature guidance and summary information on the best available pharmacological tools, alongside key references and suggestions for further reading. The landscape format of the Concise Guide is designed to facilitate comparison of related targets from material contemporary to mid-2023, and supersedes data presented in the 2021/22, 2019/20, 2017/18, 2015/16 and 2013/14 Concise Guides and previous Guides to Receptors and Channels. It is produced in close conjunction with the Nomenclature and Standards Committee of the International Union of Basic and Clinical Pharmacology (NC-IUPHAR), therefore, providing official IUPHAR classification and nomenclature for human drug targets, where appropriate.


Subject(s)
Databases, Pharmaceutical , Pharmacology , Humans , Ion Channels/chemistry , Ligands , Receptors, G-Protein-Coupled , Databases, Factual
4.
J Neurosci ; 43(36): 6230-6248, 2023 09 06.
Article in English | MEDLINE | ID: mdl-37474308

ABSTRACT

Synaptic vesicle (SV) endocytosis is a critical and well-regulated process for the maintenance of neurotransmission. We previously reported that synaptotagmin-11 (Syt11), an essential non-Ca2+-binding Syt associated with brain diseases, inhibits neuronal endocytosis (Wang et al., 2016). Here, we found that Syt11 deficiency caused accelerated SV endocytosis and vesicle recycling under sustained stimulation and led to the abnormal membrane partition of synaptic proteins in mouse hippocampal boutons of either sex. Furthermore, our study revealed that Syt11 has direct but Ca2+-independent binding with endophilin A1 (EndoA1), a membrane curvature sensor and endocytic protein recruiter, with high affinity. EndoA1-knockdown significantly reversed Syt11-KO phenotype, identifying EndoA1 as a main inhibitory target of Syt11 during SV endocytosis. The N-terminus of EndoA1 and the C2B domain of Syt11 were responsible for this interaction. A peptide (amino acids 314-336) derived from the Syt11 C2B efficiently blocked Syt11-EndoA1 binding both in vitro and in vivo Application of this peptide inhibited SV endocytosis in WT hippocampal neurons but not in EndoA1-knockdown neurons. Moreover, intracellular application of this peptide in mouse calyx of Held terminals of either sex effectively hampered both fast and slow SV endocytosis at physiological temperature. We thus propose that Syt11 ensures the precision of protein retrieval during SV endocytosis by inhibiting EndoA1 function at neuronal terminals.SIGNIFICANCE STATEMENT Endocytosis is a key stage of synaptic vesicle (SV) recycling. SV endocytosis retrieves vesicular membrane and protein components precisely to support sustained neurotransmission. However, the molecular mechanisms underlying the regulation of SV endocytosis remain elusive. Here, we reported that Syt11-KO accelerated SV endocytosis and impaired membrane partition of synaptic proteins. EndoA1 was identified as a main inhibitory target of Syt11 during SV endocytosis. Our study reveals a novel inhibitory mechanism of SV endocytosis in preventing hyperactivation of endocytosis, potentially safeguarding the recycling of synaptic proteins during sustained neurotransmission.


Subject(s)
Synaptic Transmission , Synaptic Vesicles , Animals , Mice , Endocytosis , Neurons/physiology , Synaptic Transmission/physiology , Synaptic Vesicles/metabolism , Synaptotagmins/genetics , Synaptotagmins/metabolism
5.
FEBS J ; 290(19): 4641-4659, 2023 Oct.
Article in English | MEDLINE | ID: mdl-37165739

ABSTRACT

Lysosome acidification is a dynamic equilibrium of H+ influx and efflux across the membrane, which is crucial for cell physiology. The vacuolar H+ ATPase (V-ATPase) is responsible for the H+ influx or refilling of lysosomes. TMEM175 was identified as a novel H+ permeable channel on lysosomal membranes, and it plays a critical role in lysosome acidification. However, how TMEM175 participates in lysosomal acidification remains unknown. Here, we present evidence that TMEM175 regulates lysosomal H+ influx and efflux in enlarged lysosomes isolated from COS1 treated with vacuolin-1. By utilizing the whole-endolysosome patch-clamp recording technique, a series of integrated lysosomal H+ influx and efflux signals in a ten-of-second time scale under the physiological pH gradient (luminal pH 4.60, and cytosolic pH 7.20) was recorded from this in vitro system. Lysosomal H+ fluxes constitute both the lysosomal H+ refilling and releasing, and they are asymmetrical processes with distinct featured kinetics for each of the H+ fluxes. Lysosomal H+ fluxes are entirely abolished when TMEM175 losses of function in the F39V mutant and is blocked by the antagonist (2-GBI). Meanwhile, lysosomal H+ fluxes are modulated by the pH-buffering capacity of the lumen and the lysosomal glycosylated membrane proteins, lysosome-associated membrane protein 1 (LAMP1). We propose that the TMEM175-mediated lysosomal H+ fluxes model would provide novel thoughts for studying the pathology of Parkinson's disease and lysosome storage disorders.

6.
Autophagy ; 19(4): 1368-1370, 2023 04.
Article in English | MEDLINE | ID: mdl-36120744

ABSTRACT

The acidic environment within lysosomes is maintained within a narrow pH range (pH 4.5-5.0) optimal for digesting autophagic cargo macromolecules so that the resulting building block metabolites can be reused. This pH homeostasis is a consequence of proton influx produced by a V-type H+-translocating ATPase (V-ATPase) and rapid proton efflux through an unidentified "leak" pathway. By performing a candidate expression screening, we discovered that the TMEM175 gene encodes a proton-activated, proton-selective channel (LyPAP) that is required for lysosomal H+ "leak" currents. The activity of LyPAP is most active when lysosomes are hyper-acidified, and cells lacking TMEM175 exhibit lysosomal hyper-acidification and impaired proteolytic degradation, both of which can be restored by optimizing lysosomal pH using pharmacological agents. Variants of TMEM175 that are associated with susceptibility to Parkinson disease (PD) cause a reduction in TMEM175-dependent LyPAP currents and lysosomal hyper-acidification. Hence, our studies not only reveal an essential H+-dissipating pathway in lysosomes, but also provide a molecular target to regulate pH-dependent lysosomal functions and associated pathologies.


Subject(s)
Vacuolar Proton-Translocating ATPases , Vacuolar Proton-Translocating ATPases/metabolism , Autophagy/genetics , Protons , Acids/metabolism , Lysosomes/metabolism , Hydrogen-Ion Concentration
7.
J Cell Biol ; 221(11)2022 11 07.
Article in English | MEDLINE | ID: mdl-36219209

ABSTRACT

Lysosomes mediate hydrolase-catalyzed macromolecule degradation to produce building block catabolites for reuse. Lysosome function requires an osmo-sensing machinery that regulates osmolytes (ions and organic solutes) and water flux. During hypoosmotic stress or when undigested materials accumulate, lysosomes become swollen and hypo-functional. As a membranous organelle filled with cargo macromolecules, catabolites, ions, and hydrolases, the lysosome must have mechanisms that regulate its shape and size while coordinating content exchange. In this review, we discussed the mechanisms that regulate lysosomal fusion and fission as well as swelling and condensation, with a focus on solute and water transport mechanisms across lysosomal membranes. Lysosomal H+, Na+, K+, Ca2+, and Cl- channels and transporters sense trafficking and osmotic cues to regulate both solute flux and membrane trafficking. We also provide perspectives on how lysosomes may adjust the volume of themselves, the cytosol, and the cytoplasm through the control of lysosomal solute and water transport.


Subject(s)
Ion Channels , Lysosomes , Water , Cytoplasm , Cytosol , Hydrolases/metabolism , Ion Channels/metabolism , Ions/metabolism , Lysosomes/metabolism , Water/metabolism
8.
STAR Protoc ; 3(2): 101453, 2022 06 17.
Article in English | MEDLINE | ID: mdl-35707683

ABSTRACT

Intracellular vesicles such as lysosomes contain micromolar to millimolar concentrations of Zn2+, and disturbing lysosomal Zn2+ homeostasis via lysosomal Zn2+ release leads to mitochondria damage and consequent lytic cell death. Methods have been developed to image cellular Zn2+ dynamics. Here, we present a protocol using GZnP3, a genetically encoded fluorescent Zn2+ indicator, to assess lysosomal Zn2+ release in cultured cells by fluorescence microscopy imaging. For complete details on the use and execution of this protocol, please refer to Du et al. (2021) or Minckley et al. (2019).


Subject(s)
Lysosomes , Zinc , Cell Death , Cells, Cultured , Lysosomes/genetics , Mitochondria/genetics , Zinc/metabolism
9.
Cell ; 185(13): 2292-2308.e20, 2022 06 23.
Article in English | MEDLINE | ID: mdl-35750034

ABSTRACT

Lysosomes require an acidic lumen between pH 4.5 and 5.0 for effective digestion of macromolecules. This pH optimum is maintained by proton influx produced by the V-ATPase and efflux through an unidentified "H+ leak" pathway. Here we show that TMEM175, a genetic risk factor for Parkinson's disease (PD), mediates the lysosomal H+ leak by acting as a proton-activated, proton-selective channel on the lysosomal membrane (LyPAP). Acidification beyond the normal range potently activated LyPAP to terminate further acidification of lysosomes. An endogenous polyunsaturated fatty acid and synthetic agonists also activated TMEM175 to trigger lysosomal proton release. TMEM175 deficiency caused lysosomal over-acidification, impaired proteolytic activity, and facilitated α-synuclein aggregation in vivo. Mutational and pH normalization analyses indicated that the channel's H+ conductance is essential for normal lysosome function. Thus, modulation of LyPAP by cellular cues may dynamically tune the pH optima of endosomes and lysosomes to regulate lysosomal degradation and PD pathology.


Subject(s)
Parkinson Disease , Endosomes/metabolism , Humans , Hydrogen-Ion Concentration , Intracellular Membranes/metabolism , Lysosomes/metabolism , Parkinson Disease/metabolism , Potassium Channels/metabolism , Protons
10.
Mol Neurobiol ; 59(1): 405-419, 2022 Jan.
Article in English | MEDLINE | ID: mdl-34705229

ABSTRACT

The cell-to-cell transmission of pathological α-synuclein (α-syn) has been proposed to be a critical event in the development of synucleinopathies. Recent studies have begun to reveal the underlying molecular mechanism of α-syn propagation. As one of the central steps, α-syn secretion is reported to be Ca2+-dependent and mediated by unconventional exocytosis. However, the soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNARE) requirement and vesicle identity of α-syn secretion remain elusive. Here we found that α-syn secretion is SNARE-dependent by systematically knocking down Q-SNAREs and R-SNAREs in exocytosis pathways. α-Syn secretion was mainly mediated by syntaxin 4 (STX4) and synaptosomal-associated protein 23 (SNAP23), but did not require STX1 and SNAP25, in differentiated SH-SY5Y cells. On the other hand, vesicle-associated membrane protein 3 (VAMP3), VAMP7, and VAMP8 were all involved in α-syn secretion, most likely in overlapping pathways. Application of super-resolution microscopy revealed localization of both endogenous and overexpressed α-syn in endosomes, lysosomes, and autophagosomes in rat primary cortical neurons. α-Syn co-localized with microtubule-associated protein 1 light chain 3 (LC3) most extensively, suggesting its tight association with the autophagy pathway. Consistently, α-syn secretion was regulated by the autophagy-lysosome pathway. Collectively, our data suggest that α-syn secretion is SNARE-dependent and is mediated by multiple vesicular pathways including exocytosis of recycling endosomes, multivesicular bodies, autophagosomes, and lysosomes.


Subject(s)
Exocytosis/physiology , Neurons/metabolism , SNARE Proteins/metabolism , Vesicular Transport Proteins/metabolism , alpha-Synuclein/metabolism , Animals , Autophagosomes/metabolism , Cell Line, Tumor , Endosomes/metabolism , Humans , Lysosomes/metabolism , Rats , Rats, Sprague-Dawley
11.
J Electrocardiol ; 70: 39-44, 2022.
Article in English | MEDLINE | ID: mdl-34902731

ABSTRACT

OBJECTIVE: This study aimed to analyze the application value of intracavitary electrocardiogram (ECG) classification in peripherally inserted central catheter (PICC) tip localization in patients with cancer. METHODS: Using a self-control study method, 325 patients with cancer underwent intracavitary ECGs to position the tip of a PICC catheter. The P wave, QRS wave amplitude, and waveform changes of each intracavitary ECG were recorded. Chest X-ray examination was performed after the catheterization to compare the results of different intracavity ECG maps with the results of the chest X-ray. RESULTS: The intracavitary ECG positioning maps of the 325 patients were divided into four categories: (1) increased P wave (293 cases), accounting for 90.15% (293/325) of all cases; compared with the positioning results of the chest X-rays, the placement rate was 98.98% (290/293); (2) negative deepening of the P wave (1 case), accounting for 0.31% (1/325) of all cases and with a placement rate of 100% (1/1); (3) no change in P wave (19 cases), accounting for 5.85% (19/325) of all cases and with a placement rate of 42.11% (8/19); (4) atrial fibrillation/atrial flutter (12 cases), accounting for 3.69% (12/325) of all cases and with a placement rate of 58.33% (7/12). The four types of intracavitary ECG positioning maps had statistically significant differences (χ2 = 133.924, P = 0.000). CONCLUSION: There are four types of intracavitary ECG localization maps: increased P wave, negative deepening of the P wave, no change in P wave, and atrial fibrillation/atrial flutter. The increased P wave pattern had the highest occurrence probability and high positioning accuracy. It therefore has strong clinical application value for PICC tip localization in patients with cancer.


Subject(s)
Catheterization, Central Venous , Catheterization, Peripheral , Central Venous Catheters , Neoplasms , Catheterization, Central Venous/methods , Catheterization, Peripheral/methods , Electrocardiography/methods , Humans
12.
J Neurochem ; 159(4): 729-741, 2021 11.
Article in English | MEDLINE | ID: mdl-34599505

ABSTRACT

Recent work has revealed that spontaneous release plays critical roles in the central nervous system, but how it is regulated remains elusive. Here, we report that synaptotagmin-11 (Syt11), a Ca2+ -independent Syt isoform associated with schizophrenia and Parkinson's disease, suppressed spontaneous release. Syt11-knockout hippocampal neurons showed an increased frequency of miniature excitatory post-synaptic currents while over-expression of Syt11 inversely decreased the frequency. Neither knockout nor over-expression of Syt11 affected the average amplitude, suggesting the pre-synaptic regulation of spontaneous neurotransmission by Syt11. Glutathione S-transferase pull-down, co-immunoprecipitation, and affinity-purification experiments demonstrated a direct interaction of Syt11 with vps10p-tail-interactor-1a (vti1a), a non-canonical SNARE protein that maintains spontaneous release. Importantly, knockdown of vti1a reversed the phenotype of Syt11 knockout, identifying vti1a as the main target of Syt11 inhibition. Domain analysis revealed that the C2A domain of Syt11 bound vti1a with high affinity. Consistently, expression of the C2A domain alone rescued the phenotype of elevated spontaneous release in Syt11-knockout neurons similar to the full-length protein. Altogether, our results suggest that Syt11 inhibits vti1a-containing vesicles during spontaneous release.


Subject(s)
Qb-SNARE Proteins/drug effects , Synaptic Transmission/drug effects , Synaptotagmins/pharmacology , Animals , Electrophysiological Phenomena , Excitatory Postsynaptic Potentials , Gene Knock-In Techniques , Hippocampus/pathology , Immunoprecipitation , Mice , Mice, Inbred C57BL , Mice, Knockout , Neurons/pathology , Primary Cell Culture
13.
Cell Rep ; 37(3): 109848, 2021 10 19.
Article in English | MEDLINE | ID: mdl-34686351

ABSTRACT

During tumor progression, lysosome function is often maladaptively upregulated to match the high energy demand required for cancer cell hyper-proliferation and invasion. Here, we report that mucolipin TRP channel 1 (TRPML1), a lysosomal Ca2+ and Zn2+ release channel that regulates multiple aspects of lysosome function, is dramatically upregulated in metastatic melanoma cells compared with normal cells. TRPML-specific synthetic agonists (ML-SAs) are sufficient to induce rapid (within hours) lysosomal Zn2+-dependent necrotic cell death in metastatic melanoma cells while completely sparing normal cells. ML-SA-caused mitochondria swelling and dysfunction lead to cellular ATP depletion. While pharmacological inhibition or genetic silencing of TRPML1 in metastatic melanoma cells prevents such cell death, overexpression of TRPML1 in normal cells confers ML-SA vulnerability. In the melanoma mouse models, ML-SAs exhibit potent in vivo efficacy of suppressing tumor progression. Hence, targeting maladaptively upregulated lysosome machinery can selectively eradicate metastatic tumor cells in vitro and in vivo.


Subject(s)
Antineoplastic Agents/pharmacology , Lysosomes/drug effects , Melanocytes/drug effects , Melanoma/drug therapy , Mitochondria/drug effects , Skin Neoplasms/drug therapy , Transient Receptor Potential Channels/agonists , Zinc/metabolism , Animals , Cell Death , Cell Line, Tumor , Female , Gene Expression Regulation, Neoplastic , HEK293 Cells , Humans , Lysosomes/metabolism , Lysosomes/pathology , Melanocytes/metabolism , Melanocytes/pathology , Melanoma/genetics , Melanoma/metabolism , Melanoma/secondary , Mice, Nude , Mitochondria/metabolism , Mitochondria/pathology , Signal Transduction , Skin Neoplasms/genetics , Skin Neoplasms/metabolism , Skin Neoplasms/pathology , Time Factors , Transient Receptor Potential Channels/genetics , Transient Receptor Potential Channels/metabolism , Up-Regulation , Xenograft Model Antitumor Assays
14.
Proc Natl Acad Sci U S A ; 117(46): 29155-29165, 2020 11 17.
Article in English | MEDLINE | ID: mdl-33139539

ABSTRACT

LRRC8 family proteins on the plasma membrane play a critical role in cellular osmoregulation by forming volume-regulated anion channels (VRACs) necessary to prevent necrotic cell death. We demonstrate that intracellular LRRC8 proteins acting within lysosomes also play an essential role in cellular osmoregulation. LRRC8 proteins on lysosome membranes generate large lysosomal volume-regulated anion channel (Lyso-VRAC) currents in response to low cytoplasmic ionic strength conditions. When a double-leucine L706L707 motif at the C terminus of LRRC8A was mutated to alanines, normal plasma membrane VRAC currents were still observed, but Lyso-VRAC currents were absent. We used this targeting mutant, as well as pharmacological tools, to demonstrate that Lyso-VRAC currents are necessary for the formation of large lysosome-derived vacuoles, which store and then expel excess water to maintain cytosolic water homeostasis. Thus, Lyso-VRACs allow lysosomes of mammalian cells to act as the cell`s "bladder." When Lyso-VRAC current was selectively eliminated, the extent of necrotic cell death to sustained stress was greatly increased, not only in response to hypoosmotic stress, but also to hypoxic and hypothermic stresses. Thus Lyso-VRACs play an essential role in enabling cells to mount successful homeostatic responses to multiple stressors.


Subject(s)
Lysosomes/metabolism , Membrane Proteins/metabolism , Osmoregulation/physiology , Stress, Physiological/physiology , Animals , Anions , COS Cells , Cell Survival/physiology , Chlorocebus aethiops , Exocytosis , Gene Knockout Techniques , HEK293 Cells , Homeostasis , Humans , Membrane Proteins/genetics , Mice , Transcriptome , Vacuoles
15.
J Invest Dermatol ; 140(8): 1524-1532, 2020 08.
Article in English | MEDLINE | ID: mdl-32004565

ABSTRACT

Animal studies have suggested that transient receptor potential ion channels and G-protein coupled receptors play important roles in itch transmission. TRPV3 gain-of-function mutations have been identified in patients with Olmsted syndrome, which is associated with severe pruritus. However, the mechanisms causing itch remain poorly understood. Here, we show that keratinocytes lacking TRPV3 impair the function of protease-activated receptor 2 (PAR2), resulting in reduced neuronal activation and scratching behavior in response to PAR2 agonists. Moreover, we show that TRPV3 and PAR2 were upregulated in skin biopsies from patients and mice with atopic dermatitis, whereas their inhibition attenuated scratching and inflammatory responses in mouse atopic dermatitis models. These results reveal a previously unrecognized link between TRPV3 and PAR2 in keratinocytes to convey itch information and suggest that a blockade of PAR2 or TRPV3 individually or both may serve as a potential approach for antipruritic therapy in atopic dermatitis.


Subject(s)
Dermatitis, Atopic/complications , Pruritus/immunology , Receptor, PAR-2/metabolism , TRPV Cation Channels/metabolism , Animals , Antipruritics/pharmacology , Antipruritics/therapeutic use , Biopsy , Dermatitis, Atopic/drug therapy , Dermatitis, Atopic/immunology , Dermatitis, Atopic/pathology , Disease Models, Animal , Gain of Function Mutation , Humans , Keratinocytes/immunology , Keratinocytes/pathology , Male , Mice , Mice, Knockout , Pruritus/drug therapy , Pruritus/genetics , Pruritus/pathology , Receptor, PAR-2/agonists , Receptor, PAR-2/antagonists & inhibitors , Receptor, PAR-2/genetics , Signal Transduction/drug effects , Signal Transduction/immunology , Skin/cytology , Skin/immunology , Skin/pathology , TRPV Cation Channels/antagonists & inhibitors , TRPV Cation Channels/genetics , Up-Regulation
16.
J Chromatogr A ; 1614: 460715, 2020 Mar 15.
Article in English | MEDLINE | ID: mdl-31759640

ABSTRACT

It is challenging to greatly increase of the extraction selectivity and efficiency by stir bar sorptive extraction of ultra-trace polychlorinated biphenyls (PCBs) in complex environmental matrix, e.g., soils. To fulfill this purpose, one of the critical works is to prepare some coatings with high selectivity, adsorptive capacity and reusability. It is also important to develop some green, simple methods for preperation the coatings. In this work, a kind of highly efficient and bioactive coating based on hyperbranched aptamer (HB-Apt) was constructed via hybridization chain reaction (HCR). Then, the HB-Apt was coated on a magnetic stir bar and applied to headspace extraction of PCB72 and PCB106 in soils. The core-shell gold magnetic particles (Fe3O4@AuNPs, AuMNPs for short) was employed as the substrate to immobilize the HB-Apt. The extracted PCBs on the stir bar could be easily eluted in ethanol by stirring, and then sampled in gas chromatography-mass spectrometry (GC-MS) for qualification. The ultra-low detection limit (0.003-0.005 ng•g-1), good linearity (0.01-500 ng•g-1, R2≥0.994) and reproducibility (RSD: 4.58-6.53%) were obtained. Compared with the common aptamer coating, the HB-Apt coating exhibited good selectivity and higher extraction capacity. The results might be related to the fact that there are more aptamer fragments grated on the HB-Apt coating than those of the common aptamer coating. The magnetic stir bar can not only be employed for easy headspace extraction, but also facilitate separation and elution. Moreover, the coating could be recycled for at least 60 times before recoveries of the PCB72 and PCB106 in the spiked samples drop below 90%. All these indicated that the assay is simple, robust, environment friendly and promising for detection of trace PCBs in complex environmental samples.


Subject(s)
Polychlorinated Biphenyls/analysis , Soil Pollutants/analysis , Adsorption , Gas Chromatography-Mass Spectrometry , Gold , Limit of Detection , Magnetic Phenomena , Metal Nanoparticles , Polychlorinated Biphenyls/isolation & purification , Reproducibility of Results , Soil Pollutants/isolation & purification
17.
Elife ; 82019 09 17.
Article in English | MEDLINE | ID: mdl-31526479

ABSTRACT

Epithelial homeostasis and regeneration require a pool of quiescent cells. How the quiescent cells are established and maintained is poorly understood. Here, we report that Trpv6, a cation channel responsible for epithelial Ca2+ absorption, functions as a key regulator of cellular quiescence. Genetic deletion and pharmacological blockade of Trpv6 promoted zebrafish epithelial cells to exit from quiescence and re-enter the cell cycle. Reintroducing Trpv6, but not its channel dead mutant, restored the quiescent state. Ca2+ imaging showed that Trpv6 is constitutively open in vivo. Mechanistically, Trpv6-mediated Ca2+ influx maintained the quiescent state by suppressing insulin-like growth factor (IGF)-mediated Akt-Tor and Erk signaling. In zebrafish epithelia and human colon carcinoma cells, Trpv6/TRPV6 elevated intracellular Ca2+ levels and activated PP2A, which down-regulated IGF signaling and promoted the quiescent state. Our findings suggest that Trpv6 mediates constitutive Ca2+ influx into epithelial cells to continuously suppress growth factor signaling and maintain the quiescent state.


Subject(s)
Calcium Channels/metabolism , Cell Proliferation , Epithelial Cells/physiology , TRPV Cation Channels/metabolism , Animals , Calcium/metabolism , Gene Deletion , Humans , Intercellular Signaling Peptides and Proteins/metabolism , Signal Transduction , TRPV Cation Channels/deficiency , Zebrafish , Zebrafish Proteins/deficiency , Zebrafish Proteins/metabolism
18.
Medicine (Baltimore) ; 98(34): e16922, 2019 Aug.
Article in English | MEDLINE | ID: mdl-31441876

ABSTRACT

BACKGROUND: Circular RNAs (circRNAs) have displayed dysregulated expression in several types of cancer. Nevertheless, their function and underlying mechanisms in cervical cancer remains largely unknown. This study aimed to describe the regulatory mechanisms in cervical cancer. METHODS: We downloaded the circRNAs expression profiles from Gene Expression Omnibus database, and RNAs expression profiles from The Cancer Genome Atlas database. We established a circRNA-miRNA-mRNA and circRNA-miRNA-hubgene network. The interactions between proteins were analyzed using the STRING database and hubgenes were identified using MCODE plugin. Then, we conducted a circRNA-miRNA-hubgenes regulatory module. Functional and pathway enrichment analyses were conducted using R packages "Clusterprofile". RESULTS: Six circRNAs, 15 miRNAs, and 158 mRNAs were identified to construct the ceRNA network of cervical cancer. PPI (protein-protein interaction) network and module analysis identified 7 hubgenes. Then, a circRNA-miRNA-hubgene subnetwork was constructed based on the 1 DEcircRNAs, 3 DEmiRNAs, and 3 DEmRNAs. The KEGG pathway analysis indicated DEmRNAs are involved in progesterone-mediated oocyte maturation, cell cycle, and oocyte meiosis. CONCLUSION: These ceRNAs are critical in the pathogenesis of cervical and may serve as future therapeutic biomarkers.


Subject(s)
Gene Expression Regulation, Neoplastic/genetics , Gene Regulatory Networks/genetics , RNA/genetics , Uterine Cervical Neoplasms/genetics , Biomarkers, Tumor/genetics , Female , Humans , Protein Interaction Maps , RNA/metabolism , RNA, Circular
19.
Zhongguo Gu Shang ; 32(7): 647-652, 2019 Jul 25.
Article in Chinese | MEDLINE | ID: mdl-31382724

ABSTRACT

OBJECTIVE: To evaluate the mid-term efficacy of radiofrequency ablation of nucleus pulposus by intervertebral foramen endoscopy BEIS technique in the treatment of lumbar spine surgery failure syndrome over 60 years old. METHODS: The clinical data of 40 patients over 60 years old with lumbar spine surgery failure syndrome admitted from January 2010 to January 2015 were retrospectively analyzed. Among them, there were 34 males and 6 females, aged from 60 to 76 years old with an average of 66 years, the courses of disease ranged from 10 months to 4 years. The patients were divided into two groups (BEIS group and revision group) according to the different surgery. The intervertebral foramen endoscopy BEIS technique and the transforaminal lumbar interbody fusion (TLIF) were performed in BEIS group and revision group respectively. There was no significant difference in general data such as sex, age, course of disease, surgical segment between two groups(P>0.05). The operation time, intraoperative bleeding volume, bed rest time after operation and hospitalization time were observed between two groups. At preoperative, postoperative 1 month, 1 year, 3 years, visual analogue scale(VAS) and Japanese Orthopaedic Association Score(JOA) were used to compare the efficacy. RESULTS: The operation time, intraoperative bleeding volume, bed rest time after operation and hospitalization time in BEIS group were (60.2±10.3) min, (60.1±4.5) ml, (2.2±1.5) d, (4.04±1.40) d, respectively, which were significantly lower than those of revision group (P<0.05). The VAS and JOA scores of the two groups at different time after operation were significantly improved (P<0.05), and there was statistically significant difference between two groups (P<0.05). CONCLUSIONS: Radiofrequency ablation of nucleus pulposus by intervertebral foramen endoscopy BEIS technique is more effective than TLIF revision in the treatment of lumbar spine surgery failure syndrome over 60 years old. It has advantages of shorter operation time, less bleeding, shorter bed rest after operation and hospitalization time, and is worthy of clinical promotion.


Subject(s)
Spinal Fusion , Aged , Endoscopy , Female , Humans , Lumbar Vertebrae , Male , Middle Aged , Retrospective Studies , Treatment Outcome
20.
Cell Metab ; 30(3): 508-524.e12, 2019 09 03.
Article in English | MEDLINE | ID: mdl-31204282

ABSTRACT

Fructose-1,6-bisphosphate (FBP) aldolase links sensing of declining glucose availability to AMPK activation via the lysosomal pathway. However, how aldolase transmits lack of occupancy by FBP to AMPK activation remains unclear. Here, we show that FBP-unoccupied aldolase interacts with and inhibits endoplasmic reticulum (ER)-localized transient receptor potential channel subfamily V, inhibiting calcium release in low glucose. The decrease of calcium at contact sites between ER and lysosome renders the inhibited TRPV accessible to bind the lysosomal v-ATPase that then recruits AXIN:LKB1 to activate AMPK independently of AMP. Genetic depletion of TRPVs blocks glucose starvation-induced AMPK activation in cells and liver of mice, and in nematodes, indicative of physical requirement of TRPVs. Pharmacological inhibition of TRPVs activates AMPK and elevates NAD+ levels in aged muscles, rejuvenating the animals' running capacity. Our study elucidates that TRPVs relay the FBP-free status of aldolase to the reconfiguration of v-ATPase, leading to AMPK activation in low glucose.


Subject(s)
AMP-Activated Protein Kinases/metabolism , Fructose-Bisphosphate Aldolase/metabolism , Glucose/metabolism , TRPV Cation Channels/metabolism , Acrylamides/pharmacology , Adenosine Triphosphatases/metabolism , Animals , Bridged Bicyclo Compounds, Heterocyclic/pharmacology , Caenorhabditis elegans/metabolism , Calcium/metabolism , Calcium Channels/metabolism , Endoplasmic Reticulum/metabolism , Enzyme Activation/drug effects , Enzyme Activation/genetics , Gene Knockout Techniques , HEK293 Cells , Humans , Lysosomes/metabolism , Male , Mice , Signal Transduction/drug effects , Signal Transduction/genetics , TRPV Cation Channels/antagonists & inhibitors , TRPV Cation Channels/genetics , Transfection
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