RESUMO
Nonenzymatic template-directed RNA copying requires catalysis by divalent metal ions. The primer extension reaction involves the attack of the primer 3'-hydroxyl on the adjacent phosphate of a 5'-5'-imidazolium-bridged dinucleotide substrate. However, the nature of the interaction of the catalytic metal ion with the reaction center remains unclear. To explore the coordination of the catalytic metal ion with the imidazolium-bridged dinucleotide substrate, we examined catalysis by oxophilic and thiophilic metal ions with both diastereomers of phosphorothioate-modified substrates. We show that Mg2+ and Cd2+ exhibit opposite preferences for the two phosphorothioate substrate diastereomers, indicating a stereospecific interaction of the divalent cation with one of the nonbridging phosphorus substituents. High-resolution X-ray crystal structures of the products of primer extension with phosphorothioate substrates reveal the absolute stereochemistry of this interaction and indicate that catalysis by Mg2+ involves inner-sphere coordination with the nonbridging phosphate oxygen in the pro-SP position, while thiophilic cadmium ions interact with sulfur in the same position, as in one of the two phosphorothioate substrates. These results collectively suggest that during nonenzymatic RNA primer extension with a 5'-5'-imidazolium-bridged dinucleotide substrate the interaction of the catalytic Mg2+ ion with the pro-SP oxygen of the reactive phosphate plays a crucial role in the metal-catalyzed SN2(P) reaction.
Assuntos
RNA Catalítico , RNA , RNA/química , Metais , Fosfatos de Dinucleosídeos , Fosfatos , Catálise , Oxigênio , Íons , RNA Catalítico/químicaRESUMO
In the RNA World before the emergence of an RNA polymerase, nonenzymatic template copying would have been essential for the transmission of genetic information. However, the products of chemical copying with the canonical nucleotides (A, U, C, and G) are heavily biased toward the incorporation of G and C, which form a more stable base pair than A and U. We therefore asked whether replacing adenine (A) with diaminopurine (D) might lead to more efficient and less biased nonenzymatic template copying by making a stronger version of the A:U pair. As expected, primer extension substrates containing D bound to U in the template more tightly than substrates containing A. However, primer extension with D exhibited elevated reaction rates on a C template, leading to concerns about fidelity. Our crystallographic studies revealed the nature of the D:C mismatch by showing that D can form a wobble-type base pair with C. We then asked whether competition with G would decrease the mismatched primer extension. We performed nonenzymatic primer extension with all four activated nucleotides on randomized RNA templates containing all four letters and used deep sequencing to analyze the products. We found that the DUCG genetic system exhibited a more even product distribution and a lower mismatch frequency than the canonical AUCG system. Furthermore, primer extension is greatly reduced following all mismatches, including the D:C mismatch. Our study suggests that D deserves further attention for its possible role in the RNA World and as a potentially useful component of artificial nonenzymatic RNA replication systems.
Assuntos
2-Aminopurina , RNA , RNA/química , 2-Aminopurina/química , 2-Aminopurina/análogos & derivados , Pareamento de Bases , Moldes Genéticos , Conformação de Ácido Nucleico , Modelos MolecularesRESUMO
2-Thiouridine (s2U) is a nucleobase modification that confers enhanced efficiency and fidelity both on modern tRNA codon translation and on nonenzymatic and ribozyme-catalyzed RNA copying. We have discovered an unusual base pair between two 2-thiouridines that stabilizes an RNA duplex to a degree that is comparable to that of a native A:U base pair. High-resolution crystal structures indicate similar base-pairing geometry and stacking interactions in duplexes containing s2U:s2U compared to those with U:U pairs. Notably, the CâO···H-N hydrogen bond in the U:U pair is replaced with a CâS···H-N hydrogen bond in the s2U:s2U base pair. The thermodynamic stability of the s2U:s2U base pair suggested that this self-pairing might lead to an increased error frequency during nonenzymatic RNA copying. However, competition experiments show that s2U:s2U base-pairing induces only a low level of misincorporation during nonenzymatic RNA template copying because the correct A:s2U base pair outcompetes the slightly weaker s2U:s2U base pair. In addition, even if an s2U is incorrectly incorporated, the addition of the next base is greatly hindered. This strong stalling effect would further increase the effective fidelity of nonenzymatic RNA copying with s2U. Our findings suggest that s2U may enhance the rate and extent of nonenzymatic copying with only a minimal cost in fidelity.
Assuntos
RNA Catalítico , RNA , Tiouridina/análogos & derivados , RNA/química , Pareamento de Bases , Tiouridina/química , RNA Catalítico/química , Conformação de Ácido NucleicoRESUMO
Epidermal growth factor receptor variant III (EGFRvIII) is a mutant isoform of EGFR with a deletion of exons 2-7 making it insensitive to EGF stimulation and downstream signal constitutive activation. However, the mechanism underlying the stability of EGFRvIII remains unclear. Based on CRISPR-Cas9 library screening, we found that mucin1 (MUC1) is essential for EGFRvIII glioma cell survival and temozolomide (TMZ) resistance. We revealed that MUC1-C was upregulated in EGFRvIII-positive cells, where it enhanced the stability of EGFRvIII. Knockdown of MUC1-C increased the colocalization of EGFRvIII and lysosomes. Upregulation of MUC1 occurred in an NF-κB dependent manner, and inhibition of the NF-κB pathway could interrupt the EGFRvIII-MUC1 feedback loop by inhibiting MUC1-C. In a previous report, we identified AC1Q3QWB (AQB), a small molecule that could inhibit the phosphorylation of NF-κB. By screening the structural analogs of AQB, we obtained EPIC-1027, which could inhibit the NF-κB pathway more effectively. EPIC-1027 disrupted the EGFRvIII-MUC1-C positive feedback loop in vitro and in vivo, inhibited glioma progression, and promoted sensitization to TMZ. In conclusion, we revealed the pivotal role of MUC1-C in stabilizing EGFRvIII in glioblastoma (GBM) and identified a small molecule, EPIC-1027, with great potential in GBM treatment.
Assuntos
Neoplasias Encefálicas , Glioblastoma , Glioma , Humanos , Temozolomida/farmacologia , Glioblastoma/tratamento farmacológico , Glioblastoma/genética , Glioblastoma/metabolismo , NF-kappa B/metabolismo , Neoplasias Encefálicas/tratamento farmacológico , Neoplasias Encefálicas/genética , Neoplasias Encefálicas/metabolismo , Linhagem Celular Tumoral , Mucina-1/genéticaRESUMO
The understanding of sequence-specific DNA minor groove interactions has recently made major steps forward and as a result, the goal of development of compounds that target the minor groove is an active research area. In an effort to develop biologically active minor groove agents, we are preparing and exploring the DNA interactions of diverse diamidine derivatives with a 5'-GAATTC-3' binding site using a powerful array of methods including, biosensor-SPR methods, and X-ray crystallography. The benzimidazole-thiophene module provides an excellent minor groove recognition component. A central thiophene in a benzimidazole-thiophene-phenyl aromatic system provides essentially optimum curvature for matching the shape of the minor groove. Comparison of that structure to one with the benzimidazole replaced with an indole shows that the two structures are very similar, but have some interesting and important differences in electrostatic potential maps, the DNA minor groove binding structure based on x-ray crystallographic analysis, and inhibition of the major groove binding PU.1 transcription factor complex. The binding KD for both compounds is under 10 nM and both form amidine H-bonds to DNA bases. They both have bifurcated H-bonds from the benzimidazole or indole groups to bases at the center of the -AATT- binding site. Analysis of the comparative results provides an excellent understanding of how thiophene compounds recognize the minor groove and can act as transcription factor inhibitors.
Assuntos
Pentamidina , Tiofenos , Benzimidazóis/química , Sítios de Ligação , DNA/química , Desenho de Fármacos , Indóis/farmacologia , Modelos Moleculares , Conformação de Ácido Nucleico , Pentamidina/química , Ressonância de Plasmônio de Superfície , Tiofenos/química , Tiofenos/farmacologia , Fatores de TranscriçãoRESUMO
The template-directed synthesis of RNA played an important role in the transition from prebiotic chemistry to the beginnings of RNA based life, but the mechanism of RNA copying chemistry is incompletely understood. We measured the kinetics of template copying with a set of primers with modified 3'-nucleotides and determined the crystal structures of these modified nucleotides in the context of a primer/template/substrate-analog complex. pH-rate profiles and solvent isotope effects show that deprotonation of the primer 3'-hydroxyl occurs prior to the rate limiting step, the attack of the alkoxide on the activated phosphate of the incoming nucleotide. The analogs with a 3 E ribose conformation show the fastest formation of 3'-5' phosphodiester bonds. Among those derivatives, the reaction rate is strongly correlated with the electronegativity of the 2'-substituent. We interpret our results in terms of differences in steric bulk and charge distribution in the ground vs. transition states.
Assuntos
RNA/metabolismo , Arabinose/química , Cristalografia por Raios X , Primers do DNA/metabolismo , Óxido de Deutério/química , Imidazóis/química , Cinética , Conformação de Ácido Nucleico , Nucleotídeos/química , RNA/química , Relação Estrutura-Atividade , Moldes Genéticos , Água/químicaRESUMO
High oxidative stress and inflammatory cell infiltration are major causes of the persistent bone erosion and difficult tissue regeneration in rheumatoid arthritis (RA). Triptolide (TPL) has become a highly anticipated anti-rheumatic drug due to its excellent immunomodulatory and anti-inflammatory effects. However, the sudden drug accumulation caused by the binding of "stimulus-response" and "drug release" in a general smart delivery system is difficult to meet the shortcoming of extreme toxicity and the demand for long-term administration of TPL. Herein, we developed a dual dynamically cross-linked hydrogel (SPT@TPL), which demonstrated sensitive RA microenvironment regulation and microenvironment modulation-independent TPL release for 30 days. The abundant borate ester/tea polyphenol units in SPT@TPL possessed the capability to respond and regulate high reactive oxygen species (ROS) levels on-demand. Meanwhile, based on its dense dual crosslinked structure as well as the spontaneous healing behavior of numerous intermolecular hydrogen bonds formed after the breakage of borate ester, TPL could remain stable and slowly release under high ROS environments of RA, which dramatically reduced the risk of TPL exerting toxicity while maximized its long-term efficacy. Through the dual effects of ROS regulation and TPL sustained-release, SPT@TPL alleviated oxidative stress and reprogrammed macrophages into M2 phenotype, showing marked inhibition of inflammation and optimal regeneration of articular cartilage in RA rat model. In conclusion, this hydrogel platform with both microenvironment initiative regulation and TPL long-term sustained release provides a potential scheme for rheumatoid arthritis.
RESUMO
Articular cartilage and subchondral bone defects have always been problematic because the osteochondral tissue plays a crucial role in the movement of the body and does not recover spontaneously. Here, an injectable hydrogel composed of oxidized sodium alginate/gelatin/chondroitin sulfate (OSAGC) was designed for the minimally invasive treatment and promotion of osteochondral regeneration. The OSAGC hydrogel had a double network based on dynamic covalent bonds, demonstrating commendable injectability and self-healing properties. Chondroitin sulfate was organically bound to the hydrogel network, retaining its own activity and gradually releasing during the degradation process as well as improving mechanical properties. The compressive strength could be increased up to 3 MPa by regulating the concentration of chondroitin sulphate and the oxidation level, and this mechanical stimulation could help repair injured tissue. The OSAGC hydrogel had a favourable affinity to articular cartilage and was able to release active ingredients in a sustained manner over 3 months. The OSAGC showed no cytotoxic effects. Results from animal studies demonstrated its capacity to regenerate new bone tissue in four weeks and new cartilage tissue in twelve weeks. The OSAGC hydrogel represented a promising approach to simplify bone surgery and repair damaged osteochondral tissue.
Assuntos
Alginatos , Cartilagem Articular , Sulfatos de Condroitina , Hidrogéis , Alginatos/química , Alginatos/farmacologia , Animais , Sulfatos de Condroitina/química , Sulfatos de Condroitina/farmacologia , Cartilagem Articular/efeitos dos fármacos , Hidrogéis/química , Hidrogéis/farmacologia , Regeneração Óssea/efeitos dos fármacos , Gelatina/química , Coelhos , Força Compressiva , Engenharia Tecidual/métodos , Injeções , Condrócitos/efeitos dos fármacos , Condrócitos/citologia , Alicerces Teciduais/química , Regeneração/efeitos dos fármacosRESUMO
A DNA polymerase with a single mutation and a divalent calcium cofactor catalyzes the synthesis of unnatural N3'âP5' phosphoramidate (NP) bonds to form NP-DNA. However, this template-directed phosphoryl transfer activity remains orders of magnitude slower than native phosphodiester synthesis. Here, we used time-resolved x-ray crystallography to show that NP-DNA synthesis proceeds with a single detectable calcium ion in the active site. Using insights from isotopic and elemental effects, we propose that one-metal-ion electrophilic substrate activation is inferior to the native two-metal-ion mechanism. We found that this deficiency in divalent activation could be ameliorated by trivalent rare earth and post-transition metal cations, substantially enhancing NP-DNA synthesis. Scandium(III), in particular, confers highly specific NP activity with kinetics enhanced by more than 100-fold over calcium(II), yielding NP-DNA strands up to 100 nucleotides in length.
Assuntos
Proteínas de Bactérias , Cálcio , Coenzimas , DNA Polimerase Dirigida por DNA , DNA , Geobacillus stearothermophilus , Cálcio/química , DNA/biossíntese , DNA Polimerase Dirigida por DNA/química , Nucleotídeos/química , Coenzimas/química , Geobacillus stearothermophilus/enzimologia , Proteínas de Bactérias/química , Ativação Enzimática , Cristalografia por Raios X , Conformação Proteica , BiocatáliseRESUMO
Chemically modified antisense oligonucleotides (ASO) currently in pre-clinical and clinical experiments mainly focus on the 2'-position derivatizations to enhance stability and targeting affinity. Considering the possible incompatibility of 2'-modifications with RNase H stimulation and activity, we have hypothesized that the atom specific modifications on nucleobases can retain the complex structure and RNase H activity, while enhancing ASO's binding affinity, specificity, and stability against nucleases. Herein we report a novel strategy to explore our hypothesis by synthesizing the deoxynucleoside phosphoramidite building block with the seleno-modification at 5-position of thymidine, as well as its Se-oligonucleotides. Via X-ray crystal structural study, we found that the Se-modification was located in the major groove of nucleic acid duplex and didn't cause the thermal and structural perturbations. Surprisingly, our nucleobase-modified Se-DNAs were exceptionally resistant to nuclease digestion, while compatible with RNase H activity. This affords a novel avenue for potential antisense modification in the form of Se-antisense oligonucleotides (Se-ASO).
RESUMO
Traditional treatment methods for irreversible endometrial damage face a number of challenges in clinical practice, the most important of which are bacterial infection and the inability to restore endometrial function. By modifying glucan, oxidized dextran (OD) with multifunctional aldehyde groups was obtained in this study. Based on the dynamic Schiff base reaction between gelatin (GA) and OD, a GA-OD adaptive membrane with good biocompatibility, self-healing, biodegradability, and antimicrobial properties was created. In vitro studies revealed that GA and OD cross-linking overcame GA's low gel temperature, accelerated gelling, and improved mechanical properties, hydrophilicity, and degradability. The dynamic bond formed by the reaction between GA and OD caused the GA-OD film to self-heal. Meanwhile, the GA-OD membrane had antibacterial properties. To assess the repair effect of GA-OD film, an in vivo rat endometrial injury model filled with GA-OD adaptive membrane was created. According to the results of the study, the GA-OD membrane was biocompatible, and the uterine tissue did not have edema and inflammation. Further study on the postoperative endometrial regeneration effect of GA-OD material showed that it had an excellent ability for epithelial reconstruction and cell proliferation. As a result, the use of GA-OD composite film in endometrial repair has promising therapeutic implications.
Assuntos
Aldeídos , Anti-Infecciosos , Ratos , Animais , Anti-Infecciosos/farmacologia , Antibacterianos/farmacologia , Gelatina/química , Hidrogéis/químicaRESUMO
Excessive infiltration of activated neutrophils is regarded as a predominant cause of tissue injury in neutrophilic inflammation. Although programmed cell death like apoptosis maintains the homeostasis of activated neutrophils, this process is disrupted by an abnormal inflammatory response. Unlike endogenous calreticulin exposed during apoptosis, exogenous calreticulin acts as an "aged" signal and initiates premature macrophage-mediated programmed cell removal (PrCR), which is independent of apoptosis. Here, we report a nano-mediated strategy to stimulate the precise clearance of activated neutrophils initiated with artificial aged signal and alleviated inflammation. Polymeric nanoparticles PC@PLGA were fabricated by cloaking poly(lactic-co-glycolic acid) (PLGA) with a hybrid membrane derived from platelet-derived extracellular vesicles (PEVs, denoted by P) and the calreticulin-expressed membrane obtained from doxorubicin-treated cells (denoted by C). P-selectin in PEVs favors PC@PLGA to anchor activated neutrophils, while calreticulin mimics exogenous "aged" signal secreted by macrophages to trigger PrCR. We showed that PC@PLGA specifically targeted activated neutrophils and misled macrophages to recognize them as "aged" neutrophils and then initiated premature PrCR and prevented proinflammatory response and tissue damage in a mouse model of acute lung injury and severe acute pancreatitis. The collective findings indicate the efficiency of specific elimination of activated neutrophils with exogenous aged signal in improving inflammation therapy.
Assuntos
Nanopartículas , Pancreatite , Camundongos , Animais , Neutrófilos , Calreticulina , Doença Aguda , Macrófagos/metabolismo , Inflamação/metabolismoRESUMO
The disadvantages of mainstream therapies for endometrial injury are difficult to resolve, herein, we suggest an omnibearing improvement strategy by introducing an injectable multifunctional self-assembled dual-crosslinked sodium alginate/recombinant collagen hydrogel. The hydrogel possessed a reversible and dynamic double network based on dynamic covalent bonds and ionic interactions, which also contributed to excellent capability in viscosity and injectability. Moreover, it was also biodegradable with a suitable speed, giving off active ingredients during the degradation process and eventually disappearing completely. In vitro tests exhibited that the hydrogel was biocompatible and able to enhance endometrial stromal cells viability. These features synergistically promoted cell multiplication and maintenance of endometrial hormone homeostasis, which accelerated endometrial matrix regeneration and structural reconstruction after severe injury in vivo. Furthermore, we explored the interrelation between the hydrogel characteristics, endometrial structure, and postoperative uterine recovery, which would benefit deep research on regulation of uterine repair mechanism and optimization of hydrogel materials. The injectable hydrogel could achieve favourable therapeutic efficacy without the need of exogenous hormones or cells, which would be of clinical value in endometrium regeneration.
Assuntos
Alginatos , Hidrogéis , Feminino , Humanos , Hidrogéis/farmacologia , Hidrogéis/química , Alginatos/química , Endométrio , Colágeno , ÚteroRESUMO
Cellulose/chitosan composite, as a mature commercial antibacterial dressing, is an important type of wound repair material. However, how to achieve the perfect compound of two components and improve antibacterial activity is a major, lingering issue. In this study, a bifunctional group modified bacterial cellulose (DCBC) was prepared by carboxymethylation and selective oxidation. Further, the chitosan (CS) was compounded in the network of DCBC by self-crosslinking to form dialdehyde carboxymethyl bacterial cellulose/chitosan composites (S-DCBC/CS). The aldehyde group can react with amino of CS by Schiff base reaction. The carboxyl group of DCBC and the amorphous distribution of CS molecular chains increase the antimicrobial properties of composites. The bacteriostatic rate of composites could be higher than 95%. Bacteria can be attracted onto the surface of composites, what we call it "directional adhesion antibacterial effects". In particular, a kind of large animal wound model, deep â ¡ degree infected scald of Bama miniature pig, was used to research the antimicrobial and healing properties of materials. The S-DCBC/CS can effectively inhibit bacterial proliferation of wound and kill the bacteria. The wound healing rate of S-DCBC/CS was up to 80% after three weeks. The composites show better antibacterial and promoting concrescence effects than traditional chitosan dressings.
RESUMO
Osteomyelitis is deep tissue inflammation caused by bacterial infection. If such an infection persists, it can lead to dissolution and necrosis of the bone tissue. As a result of the extensive use of antibiotics, drug-resistant bacteria are an increasingly common cause of osteomyelitis, limiting the treatment options available to surgeons. Photodynamic antibacterial chemotherapy has attracted increasing attention as a potential alternative treatment. Its advantages are a broad antibacterial spectrum, lack of drug resistance, and lack of toxic side effects. In this study, we explored the impact of the new photosensitizer LD4 in photodynamic antimicrobial chemotherapy (PACT), both alone and in combination with an antibiotic, on osteomyelitis. A rabbit tibial osteomyelitis model was employed and microbiological, histological, and radiological studies were performed. New Zealand white rabbits (n = 36) were randomly divided into a control group, antibiotic group, PACT group and PACT + antibiotic group for treatment. In microbiological analysis, a reduction in bacterial numbers of more than 99.9% was recorded in the PACT group and the PACT + antibiotic group 5 weeks after treatment (p < 0.01). In histological analysis, repair of the damaged bone tissue was observed in the PACT group, and bone repair in the PACT + antibiotic group was even more significant. In radiological analysis, the X-ray Norden score showed that the severity of bone tissue defects or destruction followed the pattern: PACT + antibiotic group < PACT group < antibiotic group < control group.
RESUMO
The postoperative recurrence and metastasis of triple negative breast cancer (TNBC) remain one fatal reason for the failure of clinical treatments. Although the rise of immunotherapy has brought hopes for reducing postoperative recurrence and potential metastasis, the low immune response and immunosuppression of tumor microenvironment (TME) still restrain its extensive application. Herein, we reported a boosting strategy by improving immunogenicity and reversing suppressive TME to realize efficient immunotherapy of TNBC. In this work, a CaCO3 biomineralized hydrogel DC vaccine was synthesized by fixing the membrane proteins of 4T1 cells-DCs fusion cells (FP) into biomineralized silk fibroin hydrogel. On one side, the FP-containing biomineralized hydrogel vaccine (SH@FP@CaCO3) has increased immunogenicity by providing a wide variety of tumor-associated antigens (TAAs) and realizing long-term protein release for DCs maturation and T cell activation. On the other side, the introduction of CaCO3 would increase the pH of TME and promote the polarization of M2-type macrophages to M1-type macrophages, thus reversing the immune-inhibitory microenvironment and relieving the immunosuppressive effect on T cells. The results indicate that the biomineralized hydrogel vaccine shows excellent immune activation effects by simultaneously enhancing the immunogenicity and reversing the immunosuppression TME, which provides a promising strategy for cancer immunotherapy.
Assuntos
Vacinas Anticâncer , Neoplasias de Mama Triplo Negativas , Linhagem Celular Tumoral , Humanos , Hidrogéis , Fatores Imunológicos/uso terapêutico , Imunoterapia/métodos , Neoplasias de Mama Triplo Negativas/patologia , Microambiente TumoralRESUMO
Electro-responsive Graphene oxide-poly(acrylic acid) (GO-PAA) nanocomposite hydrogels with different concentrations of GO were successfully fabricated via in situ polymerization. The covalently crosslinked PAA network is intertwined with GO sheets by the bridging of hydrogen-bond interactions thus resulting in an integrated and stable hydrogel network. The swelling, mechanical and conductivity properties of the hydrogel are impacted as a result. The influences of different factors on the electro-response behavior of the hydrogels were deeply explored. Because of electrostatic double layer of the GO, the response properties of hydrogels in different voltage, pH, and ionic strength improved significantly. Meanwhile, with the addition of GO, the response performance of hydrogel in biological applications was greatly expanded. Furthermore, GO-PAA hydrogel shows a good compatibility with bone marrow-derived mesenchymal stem cells (BMSCs). The electro-mechanical coupling of the hydrogel can change the morphology of the adhesive cells, and regulate the cytoskeleton of the cell under the condition of electrical stimulation, which can further promote the differentiation of neural stem cells. This electro-responsive hydrogel could be widely used in many fields of biomedical application such as artificial muscle and tissue engineering scaffold.
Assuntos
Células da Medula Óssea/metabolismo , Diferenciação Celular , Condutividade Elétrica , Grafite/química , Hidrogéis/química , Células-Tronco Mesenquimais/metabolismo , Animais , Células da Medula Óssea/citologia , Células-Tronco Mesenquimais/citologia , RatosRESUMO
A new separation system of capillary electrophoresis (CE1) for the highly sensitive determination of copper was established by using ethylenediaminetetraacetic acid (EDTA) as a complexing agent and employing cetyltrimethylammonium chloride (CTAC) as a capillary inner wall modifier. Benefitted from the combination of field-enhanced sample injection (FESI) method, a limit of detection (LOD) of 2.7 nM was obtained, which was much lower than that of the conventional methods. This made it possible to determine trace copper in HeLa cell only by a simple cell extraction (CE2) treatment. Two copper-extraction methods-acid-hydrolysis and freeze-thaw-were compared. Limited by the requirement of low ion strength in FESI, only the extract using freeze-thaw could be successfully applied in the determination. The effectiveness assessment of this CE(2)-FESI method was adopted by inductively coupled plasma-atomic emission spectrometry (ICP-AES) as a gold standard.
Assuntos
Cobre/análise , Eletroforese Capilar/métodos , Calibragem , Células HeLa , Humanos , Concentração de Íons de Hidrogênio , Limite de DetecçãoRESUMO
Monitoring autophagic flux is important for the analysis of autophagy. Tandem fluorescent-tagged LC3 (mRFP-EGFP-LC3) is a convenient assay for monitoring autophagic flux based on different pH stability of EGFP and mRFP fluorescent proteins. However, it has been reported that there is still weak fluorescence of EGFP in acidic environments (pH between 4 and 5) or acidic lysosomes. So it is possible that autolysosomes are labeled with yellow signals (GFP(+)RFP(+) puncta), which results in misinterpreting autophagic flux results. Therefore, it is desirable to choose a monomeric green fluorescent protein that is more acid sensitive than EGFP in the assay of autophagic flux. Here, we report on an mTagRFP-mWasabi-LC3 reporter, in which mWasabi is more acid sensitive than EGFP and has no fluorescence in acidic lysosomes. Meanwhile, mTagRFP-mWasabi-LC3ΔG was constructed as the negative control for this assay. Compared with mRFP-EGFP-LC3, our results showed that this reporter is more sensitive and accurate in detecting the accumulation of autophagosomes and autolysosomes. Using this reporter, we find that high-dose rapamycin (30 µM) will impair autophagic flux, inducing many more autophagosomes than autolysosomes in HeLa cells, while low-dose rapamycin (500 nM) has an opposite effect. In addition, other chemical autophagy inducers (cisplatin, staurosporine and Z18) also elicit much more autophagosomes at high doses than those at low doses. Our results suggest that the dosage of chemical autophagy inducers would obviously influence autophagic flux in cells.