Achieving increasingly finely targeted drug delivery to organs,tissues,cells,and even to intracellular biomacromolecules is one of the core goals of nanomedicines.As the delivery destination is refined to cellular and...Achieving increasingly finely targeted drug delivery to organs,tissues,cells,and even to intracellular biomacromolecules is one of the core goals of nanomedicines.As the delivery destination is refined to cellular and subcellular targets,it is essential to explore the delivery of nanomedicines at the molecular level.However,due to the lack of technical methods,the molecular mechanism of the intracellular delivery of nanomedicines remains unclear to date.Here,we develop an enzyme-induced proximity labeling technology in nanoparticles(nano-EPL)for the real-time monitoring of proteins that interact with intracellular nanomedicines.Poly(lactic-co-glycolic acid)nanoparticles coupled with horseradish peroxidase(HRP)were fabricated as a model(HRP(+)-PNPs)to evaluate the molecular mechanism of nano delivery in macrophages.By adding the labeling probe biotin-phenol and the catalytic substrate H_(2)O_(2)at different time points in cellular delivery,nano-EPL technology was validated for the real-time in situ labeling of proteins interacting with nanoparticles.Nano-EPL achieves the dynamic molecular profiling of 740 proteins to map the intracellular delivery of HRP(+)-PNPs in macrophages over time.Based on dynamic clustering analysis of these proteins,we further discovered that different organelles,including endosomes,lysosomes,the endoplasmic reticulum,and the Golgi apparatus,are involved in delivery with distinct participation timelines.More importantly,the engagement of these organelles differentially affects the drug delivery efficiency,reflecting the spatial–temporal heterogeneity of nano delivery in cells.In summary,these findings highlight a significant methodological advance toward understanding the molecular mechanisms involved in the intracellular delivery of nanomedicines.展开更多
Background:Autophagy dysfunction plays a crucial role in tau accumulation and neurodegeneration in Alzheimer’s disease(AD).This study aimed to investigate whether and how the accumulating tau may in turn affect autop...Background:Autophagy dysfunction plays a crucial role in tau accumulation and neurodegeneration in Alzheimer’s disease(AD).This study aimed to investigate whether and how the accumulating tau may in turn affect autophagy.Methods:The primary hippocampal neurons,N2a and HEK293T cells with tau overexpression were respectively starved and treated with vinblastine to study the effects of tau on the initiating steps of autophagy,which was analysed by Student’s two-tailed t-test.The rapamycin and concanamycin A were employed to inhibit the mammalian target of rapamycin kinase complex 1(mTORC1)activity and the vacuolar H+-ATPase(v-ATPase)activity,respectively,which were analysed by One-way ANOVA with post hoc tests.The Western blotting,co-immunoprecipitation and immunofuorescence staining were conducted to gain insight into the mechanisms underlying the tau effects of mTORC1 signaling alterations,as analysed by Student’s two-tailed t-test or One-way ANOVA with post hoc tests.The autophagosome formation was detected by immunofuorescence staining and transmission electron microscopy.The amino acids(AA)levels were detected by high performance liquid chromatography(HPLC).Results:We observed that overexpressing human full-length wild-type tau to mimic AD-like tau accumulation induced autophagy deficits.Further studies revealed that the increased tau could bind to the prion-related domain of T cell intracellular antigen 1(PRD-TIA1)and this association significantly increased the intercellular level of amino acids(Leucine,P=0.0038;Glutamic acid,P=0.0348;Alanine,P=0.0037;Glycine,P=0.0104),with concordant upregulation of mTORC1 activity[phosphorylated eukaryotic translation initiation factor 4E-binding protein 1(p-4EBP1),P<0.0001;phosphorylated 70 kD ribosomal protein S6 kinase 1(p-p70S6K1),P=0.0001,phosphorylated unc-51-like autophagyactivating kinase 1(p-ULK1),P=0.0015]and inhibition of autophagosome formation[microtubuleassociated protein light chain 3 II(LC3 II),P=0.0073;LC3 puncta,P<0.0001].As expected,this tau-induced deficit of autophagosome formation in turn aggravated tau accumulation.Importantly,we also found that blocking TIA1 and tau interaction by overexpressing PRD-TIA1,downregulating the endogenous TIA1 expression by shRNA,or downregulating tau protein level by a small proteolysis targeting chimera(PROTAC)could remarkably attenuate tau-induced autophagy impairment.Conclusions:Our findings reveal that AD-like tau accumulation inhibits autophagosome formation and induces autophagy deficits by activating the TIA1/amino acid/mTORC1 pathway,and thus this work reveals new insight into tau-associated neurodegeneration and provides evidence supporting the use of new therapeutic targets for AD treat-ment and that of related tauopathies.展开更多
AIM:To investigate the mechanisms of chloride intracellular channel 1(CLIC1)in the metastasis of colon cancer under hypoxia-reoxygenation(H-R)conditions.METHODS:Fluorescent probes were used to detect reactive oxygen s...AIM:To investigate the mechanisms of chloride intracellular channel 1(CLIC1)in the metastasis of colon cancer under hypoxia-reoxygenation(H-R)conditions.METHODS:Fluorescent probes were used to detect reactive oxygen species(ROS)in LOVO cells.Wound healing assay and transwell assay were performed to examine the migration and invasion of LOVO cells.Expression of CLIC1 mRNA and protein,p-ERK,MMP-2and MMP-9 proteins was analyzed by reverse transcription-polymerase chain reaction and Western blot.METHODS:H-R treatment increased the intracellular ROS level in LOVO cells.The mRNA and protein expression of CLIC1 was elevated under H-R conditions.Functional inhibition of CLIC1 markedly decreased the H-R-enhanced ROS generation,cell migration,invasion and phosphorylation of ERK in treated LOVO cells.Additionally,the expression of MMP-2 and MMP-9 could be regulated by CLIC1-mediated ROS/ERK pathway.CONCLUSION:Our results suggest that CLIC1 protein is involved in the metastasis of colon cancer LOVO cells via regulating the ROS/ERK pathway in the H-R process.展开更多
Recent studies show that ion channels/transporters play important roles in fundamental cellular functions. Several reports indicating the important roles of Cl- channels/transporters on cell proliferation suggest that...Recent studies show that ion channels/transporters play important roles in fundamental cellular functions. Several reports indicating the important roles of Cl- channels/transporters on cell proliferation suggest that the intracellular chloride concentration ([Cl-]i) regulated by them would be one of critical messengers. We investigated whether the [Cl-]i controls cell proliferation and cell cycle progression in human gastric cancer cells. Our studies indicated that furosemide, a blocker of Na+ /K+ /2Cl- cotransporter (NKCC), diminished cell growth by delaying the G1-S phase progression in gastric cancer cells with high expression and activity of NKCC. Furthermore, we found that the culture in the low Cl- medium (replacement of Cl- by NO3-) decreased the [Cl-]i and inhibited cell growth of gastric cancer cells and that this inhibition of cell growth was due to cell cycle arrest at the G0/G1 phase caused by diminutionof CDK2 and phosphorylated Rb. The culture of cells in the low Cl- medium significantly increased expressions of p21 mRNA and protein. In addition, the low Cl- medium induced phosphorylation of mitogen activated protein kinases (MAPKs). Treatment with an inhibitor of p38 or JNK significantly suppressed p21 upregulation caused by culture in a low Cl- medium and rescued gastric cancer cells from the low Cl- -induced G1 cell cycle arrest. These findings revealed that the [Cl-]i affects the cell proliferation via activation of MAPKs through upregulation of p21 in gastric cancer cells. Our results suggest that the [Cl-]i regulates important cellular functions in gastric cancer cells, leading to the development of novel therapeutic strategies.展开更多
Most of the conventional chemotherapeutic agents used for cancer chemotherapy suffer from multidrug resistance of tumor cells and poor antitumor efficacy.Based on physiological differences between the normal tissue an...Most of the conventional chemotherapeutic agents used for cancer chemotherapy suffer from multidrug resistance of tumor cells and poor antitumor efficacy.Based on physiological differences between the normal tissue and the tumor tissue,one effective approach to improve the efficacy of cancer chemotherapy is to develop pH-sensitive polymeric micellar delivery systems.The copolymers with reversible protonationedeprotonation core units or acid-liable bonds between the therapeutic agents and the micelle-forming copolymers can be used to form pH-sensitive polymeric micelles for extracellular and intracellular drug smart release.These systems can be triggered to release drug in response to the slightly acidic extracellular fluids of tumor tissue after accumulation in tumor tissues via the enhanced permeability and retention effect,or they can be triggered to release drug in endosomes or lysosomes by pH-controlled micelle hydrolysis or dissociation after uptake by cells via the endocytic pathway.The pH-sensitive micelles have been proved the specific tumor cell targeting,enhanced cellular internalization,rapid drug release,and multidrug resistance reversal.The multifunctional polymeric micelles combining extracellular pH-sensitivity with receptor-mediated active targeting strategies are of great interest for enhanced tumor targeting.The micelles with receptor-mediated and intracellular pH targeting functions are internalized via receptor-mediated endocytosis followed by endosomal-pH triggered drug release inside the cells,which reverses multidrug resistance.The pH sensitivity strategy of the polymeric micelles facilitates the specific drug delivery with reduced systemic side effects and improved chemotherapeutical efficacy,and is a novel promising platform for tumor-targeting drug delivery.展开更多
The advancement in the materials manufacturing at micrometer and nanometer scales has already enabled numerous applications in electronics, optics, chemistry, biology and medicine. Biomedical devices carrying micro-/n...The advancement in the materials manufacturing at micrometer and nanometer scales has already enabled numerous applications in electronics, optics, chemistry, biology and medicine. Biomedical devices carrying micro-/nanostructures are currently being widely used in drug delivery, drug release, biosensing and therapy. New clinical methods for disease diagnosis and treatments are being developed enabled by nanotech no logy. One-dimensional (ID) structures are playi ng an importa nt role in the direct drug delivery both in vivo and ex vivo among various micro-/nanostructures. Here, in this paper, we reviewed recent progresses made on next-generation intradermal and intracellular delivery strategies and applications with focus on ID microstructure-based approaches.展开更多
Lactic acid bacteria possess several interesting properties of great economic importance. Improvement and stabilization of these industrially important features are an active research area at the present time. The obj...Lactic acid bacteria possess several interesting properties of great economic importance. Improvement and stabilization of these industrially important features are an active research area at the present time. The objectives of this work are to study the effect of freezing and freeze-drying on the survival rate, autolytic activity and intracellular enzymatic activity of the main species of lactic acid bacteria used in the dairy industry. The article focused on several characteristics that were not well covered in the past. The obtained results revealed that both preservation methods have a significant effect on viability, autolytic activity and intracellular enzymatic activity. After six months of storage we found that frozen cultures exhibited higher survival rate, higher rate of intracellular enzymatic activity and lower rate of autolysis. The impact of conservation treatments was only strain specific in the case of survival rate. The results obtained lead to the selection of the best preservation method for the selected cultures based on survival rate, autolytic activity and intracellular enzymatic activity.展开更多
Objective To investigate the molecular characteristics and intracellular growth ability of Legionella pneumophila(L. pneumophila) strains from 1989 to 2016 in Sichuan Province, China. Methods Seventy-nine isolates of ...Objective To investigate the molecular characteristics and intracellular growth ability of Legionella pneumophila(L. pneumophila) strains from 1989 to 2016 in Sichuan Province, China. Methods Seventy-nine isolates of L. pneumophila were collected from environmental and clinical sources, including cooling towers, hot springs, bath water, fountains, and patients, and identified with 16 S rRNA gene analysis and serum agglutination assay. The isolates were then typed by Sequence-Based Typing(SBT), and Genotyping of forty-two LP1 strains were analyzed by means of multiple-locus VNTR analysis with 8 loci(MLVA-8). All strains were further analyzed for two virulence genes: Legionella vir homologue(lvh) and repeats in structural toxin(rtx A). The intracellular growth ability of 33 selected isolates was determined by examining their interaction with J774 cells. Results All isolates were identified to L. pneumophila including 11 serogroups, among which the main serogroup were LP1, accounting for 54.43%. Thirty-three different sequence types(STs) from five main clonal groups and five singletons were identified, along with 8 different MLVA patterns. Both the lvh and rtx A loci were found in all 79 strains. Thirty isolates showed high intracellular growth ability in J774 cells. Conclusion L. pneumophila is a potential threat to public health, and effective control and prevention strategies are urgently needed.展开更多
Proteins therapy is of great importance in the treatment of protein deficiency disease. Most human diseases are related to the malfunctioning of one or more proteins. The most effective and direct approach is protein ...Proteins therapy is of great importance in the treatment of protein deficiency disease. Most human diseases are related to the malfunctioning of one or more proteins. The most effective and direct approach is protein therapy, which delivers the proteins into the target cell to replace the dysfunction protein and maintain the balance of organism. However, clinical application is frequently hampered by various biological barriers to their successful delivery. This review aims to discuss the recent advances about microparticles and nanoparticles fabricated using micro and nanotechnology for intracellular delivery therapy protein and give some suggestions about the promising delivery system.展开更多
In this experiment the morphological changes of mouse peritoneal macrophages in the course of their conjugation with colloidal gold-labelled concanavalin A(ConA-Au) i by the surface receptor and then the endocytosis a...In this experiment the morphological changes of mouse peritoneal macrophages in the course of their conjugation with colloidal gold-labelled concanavalin A(ConA-Au) i by the surface receptor and then the endocytosis and transport of the ConA were observed展开更多
In this study, we designed and applied proteinmimicking nanoparticles(Protmin) as an intracellular nanosensor for in vivo detection of lead ions(Pb^(2+)).Monodispersed gold nanoparticles(Au NPs) of 13 nm in diameter w...In this study, we designed and applied proteinmimicking nanoparticles(Protmin) as an intracellular nanosensor for in vivo detection of lead ions(Pb^(2+)).Monodispersed gold nanoparticles(Au NPs) of 13 nm in diameter were modified using poly-adenine-tailed Pb^(2+)-specific 8–17 DNAzyme to form a spherical and functional Protmin. Substrate strands modified with a fluorophore at the 50 end and a quencher at the 30 end were bound to DNAzyme. Pb^(2+) facilitated cleavage of DNAzyme to release the fluorophore-modified short strands to generate fluorescence. We observed rapid kinetics of the Protmin nanosensor, for which the typical assay time was 10 min.Further, we demonstrated the Protmin nanosensor could readily enter living cells and respond to Pb^(2+) in the intracellular environment. The broad of range of Protmindesigns will be useful for advancing biological and medical applications.展开更多
AIM To establish a functional and molecular model of the intracellular pH(pH_i) regulatory mechanism in human induced pluripotent stem cells(hiPSCs).METHODS hiP SCs(HPS0077) were kindly provided by Dr. Dai from the Tr...AIM To establish a functional and molecular model of the intracellular pH(pH_i) regulatory mechanism in human induced pluripotent stem cells(hiPSCs).METHODS hiP SCs(HPS0077) were kindly provided by Dr. Dai from the Tri-Service General Hospital(IRB No. B-106-09). Changes in the pH_i were detected either by microspectrofluorimetry or by a multimode reader with a pH-sensitive fluorescent probe, BCECF, and the fluorescent ratio was calibrated by the high K^+/nigericin method. NH_4Cl and Na-acetate prepulse techniques were used to induce rapid intracellular acidosis and alkalization, respectively. The buffering power(β) was calculated from the ΔpH_i induced by perfusing different concentrations of(NH_4)_2SO_4. Western blot techniques and immunocytochemistry staining were used to detect the protein expression of pH_i regulators and pluripotency markers.RESULTS In this study, our results indicated that(1) the steadystate pH_i value was found to be 7.5 ± 0.01(n = 20) and 7.68 ± 0.01(n =20) in HEPES and 5% CO_2/HCO_3^- buffered systems, respectively, which were much greater than that in normal adult cells(7.2);(2) in a CO_2/HCO_3^--buffered system, the values of total intracellular buffering power(β) can be described by the following equation: β_(tot) = 107.79(pH_i)~2-1522.2(pH_i) + 5396.9(correlation coefficient R^2 = 0.85), in the estimated pH_i range of 7.1- 8.0;(3) the Na^+/H^+ exchanger(NHE) and the Na^+/HCO_3^- cotransporter(NBC) were found to be functionally activated for acid extrusion for pHi values less than 7.5 and 7.68, respectively;(4) V-ATPase and some other unknown Na^+-independent acid extruder(s) could only be functionally detected for pHi values less than 7.1;(5) the Cl^-/OH^- exchanger(CHE) and the Cl^- /HCO_3 anion exchanger(AE) were found to be responsible for the weakening of intracellular proton loading;(6) besides the CHE and the AE, a Cl^--independent acid loading mechanism was functionally identified; and(7) in hiPSCs, a strong positive correlation was observed between the loss of pluripotency and the weakening of the intracellular acid extrusion mechanism, which included a decrease in the steady-state pH i value and diminished the functional activity and protein expression of the NHE and the NBC.CONCLUSION For the first time, we established a functional and molecular model of a pHi regulatory mechanism and demonstrated its strong positive correlation with hiPSC pluripotency.展开更多
Difference in sub-cellular trafficking of glycosylated and naked peptides, between normal and lung cancer cells, was established. Normal lung tissue discriminately sorted glycosylated from non-glycosylated peptides by...Difference in sub-cellular trafficking of glycosylated and naked peptides, between normal and lung cancer cells, was established. Normal lung tissue discriminately sorted glycosylated from non-glycosylated peptides by allowing golgi localization of the glycosylated peptides while restricting golgi entry of the naked peptides. This mechanism was surprisingly not observed in its cancer cell counterpart. Lung cancer cells tend to allow unrestricted localization of both glycosylated and naked peptides in the golgi apparatus. This newly discovered difference in sub-cellular trafficking between normal and lung cancer cells could potentially be used as an effective strategy in targeted intracellular delivery, especially targeting golgi-resident enzymes for possible treatment of diseases associated with glycans and glycoproteins, such as, congenital disease of glycosylation(CDG). This very important detail in intracellular trafficking inside normal and cancer cells is an indispensable part in nanoparticle-based intracellular drug delivery.展开更多
Objective To investigate the effect of oleanolic acid (OA) on apoptosis,correlation between apoptosis and intracellular calcium,and its mechanism in human lung adenocarcinoma cell line A549. Methods Human lung adenoca...Objective To investigate the effect of oleanolic acid (OA) on apoptosis,correlation between apoptosis and intracellular calcium,and its mechanism in human lung adenocarcinoma cell line A549. Methods Human lung adenocarcinoma A549 cells were incubated in vitro and assigned with OA concentrations of 0,10,20 and 40μg/mL. The apoptosis status of A549 cell line was detected with Annexin V-FITC/PI by flow cytometry (FCM); fluorescence intensity (FI) of A549 cells was assessed and the level of intracellular calcium was calculated at 24 hour of OA intervention. The relation between apoptosis and calcium FI was illustrated by curve fitting. Results FCM showed that 10,20 and 40μg/mL of OA could induce A549 cell apoptosis,which followed a concentration-effect pattern; 24-hour intervention with 20μg/mL and 40μg/mL OA showed increased A549 cell apoptosis,and was significantly different from that with 0μg/mL OA (P<0.01). The FI of intracellular calcium concentration in 10,20 and 40μg/mL OA groups was significantly higher than that in 0μg/mL group after 24 hours' intervention,and the FI showed a trend of increase with increased OA concentration (P<0.01). Curve fitting showed a significant correlation between apoptosis rate and intracellular calcium concentration in A549 cells (r=0.981,P<0.01). Regression equation was Y=0.508X-1.627. Conclusion OA plays a role in inducing apoptosis of human lung adenocarcinoma cells in a concentration-dependent manner. The OA-induced apoptosis is responsible for intracellular calcium overload of the tumor.展开更多
Osteocytes in vivo are embedded in the mineralized extracellular bone matrix,where their cell bodies reside in the lacunae and are interconnected to neighboring osteocytes through numerous intercellular processes.The ...Osteocytes in vivo are embedded in the mineralized extracellular bone matrix,where their cell bodies reside in the lacunae and are interconnected to neighboring osteocytes through numerous intercellular processes.The 3-dimensional（3D）osteocyte network positioning and ability to communicate with other bone cells make osteocytes ideal mechanosensors of bone.Thus the role of osteocyte network and intercellular communication between osteocytes in response to mechanical stimulation may clarify the mechanisms behind normal bone adaptation to mechanical loading.We have been using intracellular calcium([Ca<sup>2+</sup>]<sub>i</sub>)as a ubiquitous real-time signaling indicator for studying mechanotransduction in osteocytic network展开更多
This study measured platelet intracellular free calcium (PFCa 2+ ) of 1003 normal pregnancies in different trimester. The purpose of this study was to find the alteration of PFCa 2+ in normal pregnancy, to provide sci...This study measured platelet intracellular free calcium (PFCa 2+ ) of 1003 normal pregnancies in different trimester. The purpose of this study was to find the alteration of PFCa 2+ in normal pregnancy, to provide scientific basis for forecasting and preventing PIH (pregnancy induced hypertension). The results showed that the PFCa 2+ concentration was stable and slightly increased with the progression of gestational weeks.展开更多
Objectives: To construct retroviral vectorsexpressing antisense polymeric RNAs targeted at the coresequence (+13-+47) of HIV-l TAR RNA, and to testthe anti-HIV properties of this construct in transducedCD4^+T cells. M...Objectives: To construct retroviral vectorsexpressing antisense polymeric RNAs targeted at the coresequence (+13-+47) of HIV-l TAR RNA, and to testthe anti-HIV properties of this construct in transducedCD4^+T cells. Methods: The polymeric TAR-Core DNAwas amplified by the"self-primers-template PCR"展开更多
基金supported by Natural Science Foundation of Beijing Municipality(L212013)National Key Research and Development Program of China(No.2022YFA1206104)+2 种基金AI+Health Collaborative Innovation Cultivation Project(Z211100003521002)National Natural Science Foundation of China(81971718,82073786,81872809,U20A20412,81821004)Beijing Natural Science Foundation(7222020).
文摘Achieving increasingly finely targeted drug delivery to organs,tissues,cells,and even to intracellular biomacromolecules is one of the core goals of nanomedicines.As the delivery destination is refined to cellular and subcellular targets,it is essential to explore the delivery of nanomedicines at the molecular level.However,due to the lack of technical methods,the molecular mechanism of the intracellular delivery of nanomedicines remains unclear to date.Here,we develop an enzyme-induced proximity labeling technology in nanoparticles(nano-EPL)for the real-time monitoring of proteins that interact with intracellular nanomedicines.Poly(lactic-co-glycolic acid)nanoparticles coupled with horseradish peroxidase(HRP)were fabricated as a model(HRP(+)-PNPs)to evaluate the molecular mechanism of nano delivery in macrophages.By adding the labeling probe biotin-phenol and the catalytic substrate H_(2)O_(2)at different time points in cellular delivery,nano-EPL technology was validated for the real-time in situ labeling of proteins interacting with nanoparticles.Nano-EPL achieves the dynamic molecular profiling of 740 proteins to map the intracellular delivery of HRP(+)-PNPs in macrophages over time.Based on dynamic clustering analysis of these proteins,we further discovered that different organelles,including endosomes,lysosomes,the endoplasmic reticulum,and the Golgi apparatus,are involved in delivery with distinct participation timelines.More importantly,the engagement of these organelles differentially affects the drug delivery efficiency,reflecting the spatial–temporal heterogeneity of nano delivery in cells.In summary,these findings highlight a significant methodological advance toward understanding the molecular mechanisms involved in the intracellular delivery of nanomedicines.
基金supported by grants from the Natural Science Foundation of China(91949205,31730035,81721005)the Science and Technology Committee of China(2016YFC1305800)+1 种基金the Special Project of Technological Innovation of Hubei Province(2018ACA142)Guangdong Provincial Key S&T Program(2018B030336001)。
文摘Background:Autophagy dysfunction plays a crucial role in tau accumulation and neurodegeneration in Alzheimer’s disease(AD).This study aimed to investigate whether and how the accumulating tau may in turn affect autophagy.Methods:The primary hippocampal neurons,N2a and HEK293T cells with tau overexpression were respectively starved and treated with vinblastine to study the effects of tau on the initiating steps of autophagy,which was analysed by Student’s two-tailed t-test.The rapamycin and concanamycin A were employed to inhibit the mammalian target of rapamycin kinase complex 1(mTORC1)activity and the vacuolar H+-ATPase(v-ATPase)activity,respectively,which were analysed by One-way ANOVA with post hoc tests.The Western blotting,co-immunoprecipitation and immunofuorescence staining were conducted to gain insight into the mechanisms underlying the tau effects of mTORC1 signaling alterations,as analysed by Student’s two-tailed t-test or One-way ANOVA with post hoc tests.The autophagosome formation was detected by immunofuorescence staining and transmission electron microscopy.The amino acids(AA)levels were detected by high performance liquid chromatography(HPLC).Results:We observed that overexpressing human full-length wild-type tau to mimic AD-like tau accumulation induced autophagy deficits.Further studies revealed that the increased tau could bind to the prion-related domain of T cell intracellular antigen 1(PRD-TIA1)and this association significantly increased the intercellular level of amino acids(Leucine,P=0.0038;Glutamic acid,P=0.0348;Alanine,P=0.0037;Glycine,P=0.0104),with concordant upregulation of mTORC1 activity[phosphorylated eukaryotic translation initiation factor 4E-binding protein 1(p-4EBP1),P<0.0001;phosphorylated 70 kD ribosomal protein S6 kinase 1(p-p70S6K1),P=0.0001,phosphorylated unc-51-like autophagyactivating kinase 1(p-ULK1),P=0.0015]and inhibition of autophagosome formation[microtubuleassociated protein light chain 3 II(LC3 II),P=0.0073;LC3 puncta,P<0.0001].As expected,this tau-induced deficit of autophagosome formation in turn aggravated tau accumulation.Importantly,we also found that blocking TIA1 and tau interaction by overexpressing PRD-TIA1,downregulating the endogenous TIA1 expression by shRNA,or downregulating tau protein level by a small proteolysis targeting chimera(PROTAC)could remarkably attenuate tau-induced autophagy impairment.Conclusions:Our findings reveal that AD-like tau accumulation inhibits autophagosome formation and induces autophagy deficits by activating the TIA1/amino acid/mTORC1 pathway,and thus this work reveals new insight into tau-associated neurodegeneration and provides evidence supporting the use of new therapeutic targets for AD treat-ment and that of related tauopathies.
基金Supported by The "Eleventh Five-year Plan" for Medical Sci-ence Development of PLA,No.06MB243the National Natural Science Foundation of China,No.81101101 and No.51273165+1 种基金the Key Project of Chinese Ministry of Education,No.212149the Projects of Sichuan Province,No.2010SZ0294,No.2011JQ0032 and No.12ZB038
文摘AIM:To investigate the mechanisms of chloride intracellular channel 1(CLIC1)in the metastasis of colon cancer under hypoxia-reoxygenation(H-R)conditions.METHODS:Fluorescent probes were used to detect reactive oxygen species(ROS)in LOVO cells.Wound healing assay and transwell assay were performed to examine the migration and invasion of LOVO cells.Expression of CLIC1 mRNA and protein,p-ERK,MMP-2and MMP-9 proteins was analyzed by reverse transcription-polymerase chain reaction and Western blot.METHODS:H-R treatment increased the intracellular ROS level in LOVO cells.The mRNA and protein expression of CLIC1 was elevated under H-R conditions.Functional inhibition of CLIC1 markedly decreased the H-R-enhanced ROS generation,cell migration,invasion and phosphorylation of ERK in treated LOVO cells.Additionally,the expression of MMP-2 and MMP-9 could be regulated by CLIC1-mediated ROS/ERK pathway.CONCLUSION:Our results suggest that CLIC1 protein is involved in the metastasis of colon cancer LOVO cells via regulating the ROS/ERK pathway in the H-R process.
文摘Recent studies show that ion channels/transporters play important roles in fundamental cellular functions. Several reports indicating the important roles of Cl- channels/transporters on cell proliferation suggest that the intracellular chloride concentration ([Cl-]i) regulated by them would be one of critical messengers. We investigated whether the [Cl-]i controls cell proliferation and cell cycle progression in human gastric cancer cells. Our studies indicated that furosemide, a blocker of Na+ /K+ /2Cl- cotransporter (NKCC), diminished cell growth by delaying the G1-S phase progression in gastric cancer cells with high expression and activity of NKCC. Furthermore, we found that the culture in the low Cl- medium (replacement of Cl- by NO3-) decreased the [Cl-]i and inhibited cell growth of gastric cancer cells and that this inhibition of cell growth was due to cell cycle arrest at the G0/G1 phase caused by diminutionof CDK2 and phosphorylated Rb. The culture of cells in the low Cl- medium significantly increased expressions of p21 mRNA and protein. In addition, the low Cl- medium induced phosphorylation of mitogen activated protein kinases (MAPKs). Treatment with an inhibitor of p38 or JNK significantly suppressed p21 upregulation caused by culture in a low Cl- medium and rescued gastric cancer cells from the low Cl- -induced G1 cell cycle arrest. These findings revealed that the [Cl-]i affects the cell proliferation via activation of MAPKs through upregulation of p21 in gastric cancer cells. Our results suggest that the [Cl-]i regulates important cellular functions in gastric cancer cells, leading to the development of novel therapeutic strategies.
基金This work was financially supported from the National Nature Science Foundation of China(NO.81360483)from the Nature Science Foundation of Ningxia(No.NZ12193).
文摘Most of the conventional chemotherapeutic agents used for cancer chemotherapy suffer from multidrug resistance of tumor cells and poor antitumor efficacy.Based on physiological differences between the normal tissue and the tumor tissue,one effective approach to improve the efficacy of cancer chemotherapy is to develop pH-sensitive polymeric micellar delivery systems.The copolymers with reversible protonationedeprotonation core units or acid-liable bonds between the therapeutic agents and the micelle-forming copolymers can be used to form pH-sensitive polymeric micelles for extracellular and intracellular drug smart release.These systems can be triggered to release drug in response to the slightly acidic extracellular fluids of tumor tissue after accumulation in tumor tissues via the enhanced permeability and retention effect,or they can be triggered to release drug in endosomes or lysosomes by pH-controlled micelle hydrolysis or dissociation after uptake by cells via the endocytic pathway.The pH-sensitive micelles have been proved the specific tumor cell targeting,enhanced cellular internalization,rapid drug release,and multidrug resistance reversal.The multifunctional polymeric micelles combining extracellular pH-sensitivity with receptor-mediated active targeting strategies are of great interest for enhanced tumor targeting.The micelles with receptor-mediated and intracellular pH targeting functions are internalized via receptor-mediated endocytosis followed by endosomal-pH triggered drug release inside the cells,which reverses multidrug resistance.The pH sensitivity strategy of the polymeric micelles facilitates the specific drug delivery with reduced systemic side effects and improved chemotherapeutical efficacy,and is a novel promising platform for tumor-targeting drug delivery.
基金National Natural Science Foundation of China under Grants 51875518,81501607 and 51475419Key Research and Develop mem Projects of Zhejiang Province under Grant 2017C01054.
文摘The advancement in the materials manufacturing at micrometer and nanometer scales has already enabled numerous applications in electronics, optics, chemistry, biology and medicine. Biomedical devices carrying micro-/nanostructures are currently being widely used in drug delivery, drug release, biosensing and therapy. New clinical methods for disease diagnosis and treatments are being developed enabled by nanotech no logy. One-dimensional (ID) structures are playi ng an importa nt role in the direct drug delivery both in vivo and ex vivo among various micro-/nanostructures. Here, in this paper, we reviewed recent progresses made on next-generation intradermal and intracellular delivery strategies and applications with focus on ID microstructure-based approaches.
文摘Lactic acid bacteria possess several interesting properties of great economic importance. Improvement and stabilization of these industrially important features are an active research area at the present time. The objectives of this work are to study the effect of freezing and freeze-drying on the survival rate, autolytic activity and intracellular enzymatic activity of the main species of lactic acid bacteria used in the dairy industry. The article focused on several characteristics that were not well covered in the past. The obtained results revealed that both preservation methods have a significant effect on viability, autolytic activity and intracellular enzymatic activity. After six months of storage we found that frozen cultures exhibited higher survival rate, higher rate of intracellular enzymatic activity and lower rate of autolysis. The impact of conservation treatments was only strain specific in the case of survival rate. The results obtained lead to the selection of the best preservation method for the selected cultures based on survival rate, autolytic activity and intracellular enzymatic activity.
基金supported by grants from the National Natural Science Foundation of China [81672048]the Youth Innovation Project of Sichuan Medical Association Foundation [Q15081]the Priority Project on Infectious Disease Control and Prevention [2018ZX10714002]
文摘Objective To investigate the molecular characteristics and intracellular growth ability of Legionella pneumophila(L. pneumophila) strains from 1989 to 2016 in Sichuan Province, China. Methods Seventy-nine isolates of L. pneumophila were collected from environmental and clinical sources, including cooling towers, hot springs, bath water, fountains, and patients, and identified with 16 S rRNA gene analysis and serum agglutination assay. The isolates were then typed by Sequence-Based Typing(SBT), and Genotyping of forty-two LP1 strains were analyzed by means of multiple-locus VNTR analysis with 8 loci(MLVA-8). All strains were further analyzed for two virulence genes: Legionella vir homologue(lvh) and repeats in structural toxin(rtx A). The intracellular growth ability of 33 selected isolates was determined by examining their interaction with J774 cells. Results All isolates were identified to L. pneumophila including 11 serogroups, among which the main serogroup were LP1, accounting for 54.43%. Thirty-three different sequence types(STs) from five main clonal groups and five singletons were identified, along with 8 different MLVA patterns. Both the lvh and rtx A loci were found in all 79 strains. Thirty isolates showed high intracellular growth ability in J774 cells. Conclusion L. pneumophila is a potential threat to public health, and effective control and prevention strategies are urgently needed.
基金supported by the program of Ma jor scientific and technological specialized project for "significant new formulation of new drugs" (No. 2009ZX09310-007 and No. 2009ZX09301007)National Science Foundation of China Committee (No. 30873180)+2 种基金Foundation of Ministry of Education of China (No. 20090073120085)the Shanghai Science and Technology Committee (No. 0952nm03700 and No. 0952nm03700)The authors thank the Analytical Center of Shanghai JiaoTong University for Technical Support
文摘Proteins therapy is of great importance in the treatment of protein deficiency disease. Most human diseases are related to the malfunctioning of one or more proteins. The most effective and direct approach is protein therapy, which delivers the proteins into the target cell to replace the dysfunction protein and maintain the balance of organism. However, clinical application is frequently hampered by various biological barriers to their successful delivery. This review aims to discuss the recent advances about microparticles and nanoparticles fabricated using micro and nanotechnology for intracellular delivery therapy protein and give some suggestions about the promising delivery system.
文摘In this experiment the morphological changes of mouse peritoneal macrophages in the course of their conjugation with colloidal gold-labelled concanavalin A(ConA-Au) i by the surface receptor and then the endocytosis and transport of the ConA were observed
基金supported by the National Natural Science Foundation of China(Nos.21390414 and 21605087)the Chinese Academy of Sciences(No.QYZDJ-SSW-SLH031)+2 种基金the China Postdoctoral Science Foundation funded project(No.BX201700123)the Scientific Research Foundation of Nanjing University of Posts and Telecommunications(No.NY215058)the Natural Science Fund for Colleges and Universities in Jiangsu Province(16KJB150032)
文摘In this study, we designed and applied proteinmimicking nanoparticles(Protmin) as an intracellular nanosensor for in vivo detection of lead ions(Pb^(2+)).Monodispersed gold nanoparticles(Au NPs) of 13 nm in diameter were modified using poly-adenine-tailed Pb^(2+)-specific 8–17 DNAzyme to form a spherical and functional Protmin. Substrate strands modified with a fluorophore at the 50 end and a quencher at the 30 end were bound to DNAzyme. Pb^(2+) facilitated cleavage of DNAzyme to release the fluorophore-modified short strands to generate fluorescence. We observed rapid kinetics of the Protmin nanosensor, for which the typical assay time was 10 min.Further, we demonstrated the Protmin nanosensor could readily enter living cells and respond to Pb^(2+) in the intracellular environment. The broad of range of Protmindesigns will be useful for advancing biological and medical applications.
基金Supported by Ministry of Science and Technology Grants of Taiwan,No.MOST 106-2320-B-016-003-MY2(to Loh SH)and No.MOST 106-2314-B-016-037-MY3(to Tsai YT)National Defense Medical Center Grants of Taiwan,No.MAB-106-033(to Loh SH),No.MAB-105-043 and No.MAB-106-034(to Dai NZ)Teh-Tzer Study Group for Human Medical Research Foundation of Taiwan,No.A1061037 and No.A1061054(to Loh SH)
文摘AIM To establish a functional and molecular model of the intracellular pH(pH_i) regulatory mechanism in human induced pluripotent stem cells(hiPSCs).METHODS hiP SCs(HPS0077) were kindly provided by Dr. Dai from the Tri-Service General Hospital(IRB No. B-106-09). Changes in the pH_i were detected either by microspectrofluorimetry or by a multimode reader with a pH-sensitive fluorescent probe, BCECF, and the fluorescent ratio was calibrated by the high K^+/nigericin method. NH_4Cl and Na-acetate prepulse techniques were used to induce rapid intracellular acidosis and alkalization, respectively. The buffering power(β) was calculated from the ΔpH_i induced by perfusing different concentrations of(NH_4)_2SO_4. Western blot techniques and immunocytochemistry staining were used to detect the protein expression of pH_i regulators and pluripotency markers.RESULTS In this study, our results indicated that(1) the steadystate pH_i value was found to be 7.5 ± 0.01(n = 20) and 7.68 ± 0.01(n =20) in HEPES and 5% CO_2/HCO_3^- buffered systems, respectively, which were much greater than that in normal adult cells(7.2);(2) in a CO_2/HCO_3^--buffered system, the values of total intracellular buffering power(β) can be described by the following equation: β_(tot) = 107.79(pH_i)~2-1522.2(pH_i) + 5396.9(correlation coefficient R^2 = 0.85), in the estimated pH_i range of 7.1- 8.0;(3) the Na^+/H^+ exchanger(NHE) and the Na^+/HCO_3^- cotransporter(NBC) were found to be functionally activated for acid extrusion for pHi values less than 7.5 and 7.68, respectively;(4) V-ATPase and some other unknown Na^+-independent acid extruder(s) could only be functionally detected for pHi values less than 7.1;(5) the Cl^-/OH^- exchanger(CHE) and the Cl^- /HCO_3 anion exchanger(AE) were found to be responsible for the weakening of intracellular proton loading;(6) besides the CHE and the AE, a Cl^--independent acid loading mechanism was functionally identified; and(7) in hiPSCs, a strong positive correlation was observed between the loss of pluripotency and the weakening of the intracellular acid extrusion mechanism, which included a decrease in the steady-state pH i value and diminished the functional activity and protein expression of the NHE and the NBC.CONCLUSION For the first time, we established a functional and molecular model of a pHi regulatory mechanism and demonstrated its strong positive correlation with hiPSC pluripotency.
文摘Difference in sub-cellular trafficking of glycosylated and naked peptides, between normal and lung cancer cells, was established. Normal lung tissue discriminately sorted glycosylated from non-glycosylated peptides by allowing golgi localization of the glycosylated peptides while restricting golgi entry of the naked peptides. This mechanism was surprisingly not observed in its cancer cell counterpart. Lung cancer cells tend to allow unrestricted localization of both glycosylated and naked peptides in the golgi apparatus. This newly discovered difference in sub-cellular trafficking between normal and lung cancer cells could potentially be used as an effective strategy in targeted intracellular delivery, especially targeting golgi-resident enzymes for possible treatment of diseases associated with glycans and glycoproteins, such as, congenital disease of glycosylation(CDG). This very important detail in intracellular trafficking inside normal and cancer cells is an indispensable part in nanoparticle-based intracellular drug delivery.
文摘Objective To investigate the effect of oleanolic acid (OA) on apoptosis,correlation between apoptosis and intracellular calcium,and its mechanism in human lung adenocarcinoma cell line A549. Methods Human lung adenocarcinoma A549 cells were incubated in vitro and assigned with OA concentrations of 0,10,20 and 40μg/mL. The apoptosis status of A549 cell line was detected with Annexin V-FITC/PI by flow cytometry (FCM); fluorescence intensity (FI) of A549 cells was assessed and the level of intracellular calcium was calculated at 24 hour of OA intervention. The relation between apoptosis and calcium FI was illustrated by curve fitting. Results FCM showed that 10,20 and 40μg/mL of OA could induce A549 cell apoptosis,which followed a concentration-effect pattern; 24-hour intervention with 20μg/mL and 40μg/mL OA showed increased A549 cell apoptosis,and was significantly different from that with 0μg/mL OA (P<0.01). The FI of intracellular calcium concentration in 10,20 and 40μg/mL OA groups was significantly higher than that in 0μg/mL group after 24 hours' intervention,and the FI showed a trend of increase with increased OA concentration (P<0.01). Curve fitting showed a significant correlation between apoptosis rate and intracellular calcium concentration in A549 cells (r=0.981,P<0.01). Regression equation was Y=0.508X-1.627. Conclusion OA plays a role in inducing apoptosis of human lung adenocarcinoma cells in a concentration-dependent manner. The OA-induced apoptosis is responsible for intracellular calcium overload of the tumor.
基金supported by the US National Institutes of Health grants R21 AR052417,R01 AR052461,RC1 AR058453(XEG),and R01 AR054385(LW)
文摘Osteocytes in vivo are embedded in the mineralized extracellular bone matrix,where their cell bodies reside in the lacunae and are interconnected to neighboring osteocytes through numerous intercellular processes.The 3-dimensional（3D）osteocyte network positioning and ability to communicate with other bone cells make osteocytes ideal mechanosensors of bone.Thus the role of osteocyte network and intercellular communication between osteocytes in response to mechanical stimulation may clarify the mechanisms behind normal bone adaptation to mechanical loading.We have been using intracellular calcium([Ca<sup>2+</sup>]<sub>i</sub>)as a ubiquitous real-time signaling indicator for studying mechanotransduction in osteocytic network
文摘This study measured platelet intracellular free calcium (PFCa 2+ ) of 1003 normal pregnancies in different trimester. The purpose of this study was to find the alteration of PFCa 2+ in normal pregnancy, to provide scientific basis for forecasting and preventing PIH (pregnancy induced hypertension). The results showed that the PFCa 2+ concentration was stable and slightly increased with the progression of gestational weeks.
文摘Objectives: To construct retroviral vectorsexpressing antisense polymeric RNAs targeted at the coresequence (+13-+47) of HIV-l TAR RNA, and to testthe anti-HIV properties of this construct in transducedCD4^+T cells. Methods: The polymeric TAR-Core DNAwas amplified by the"self-primers-template PCR"
