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The study focuses on the underlying regulatory mechanism of age-related hearing loss (ARHL), which results from autophagy dysregulation mediated by miR-130b-3p targeting PPARγ. We constructed miR-130b-3p knockout (antagomir) and PPARγ over-expression (OE-PPARγ) mice model by injecting mmu-miR-130b-3p antagomir and HBAAV2/Anc80-m-Pparg-T2A-mCHerry into the right ear' round window of each mouse, respectively. In vitro, we introduced oxidative stress within HEI-OC1 cells by H2O2 and exogenously changed the miR-130b-3p and PPARγ levels. MiRNA level was detected by RT-qPCR, proteins by western blotting and immunohistochemistry. Morphology of autophagosomes was observed by electron microscopy. In vivo, the cochlea of aged mice showed higher miR-130b-3p expression and lower PPARγ expression, while exogenous inhibition of miR-130b-3p up-regulated PPARγ expression. Autophagy-related biomarkers expression (ATG5, Beclin-1 and LC3B II/I) decreased in aged mice, which reversely increased after the inhibition of miR-130b-3p. The elevation of PPARγ demonstrated similar effects. Contrarily, exogenous overexpression of miR-130b-3p resulted in the decrease of ATG5, Beclin-1 and LC3B II/I. We created oxidative stress within HEI-OC1 by H2O2, subsequently observed the formation of autophagosomes under electron microscope, so as the elevated cell apoptosis rate and weakened cell viability. MiR-130b-3p/PPARγ contributed to the premature senescence of these H2O2-induced HEI-OC1 cells. MiR-130b-3p regulated HEI-OC1 cell growth by targeting PPARγ, thus leading to ARHL.
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Autofagia , Modelos Animales de Enfermedad , Ratones Noqueados , MicroARNs , Estrés Oxidativo , PPAR gamma , Presbiacusia , Animales , PPAR gamma/metabolismo , PPAR gamma/genética , MicroARNs/metabolismo , MicroARNs/genética , Ratones , Presbiacusia/genética , Presbiacusia/metabolismo , Presbiacusia/patología , Presbiacusia/fisiopatología , Línea Celular , Envejecimiento/metabolismo , Envejecimiento/patología , Ratones Endogámicos C57BL , Factores de Edad , Transducción de Señal , Audición/genética , Cóclea/metabolismo , Cóclea/patología , Apoptosis , Regulación de la Expresión GénicaRESUMEN
The aim of this study is to explore the function of USP14 on the sensitivity of retinoblastoma (RB) to cisplatin (DDP) and the underlying mechanism. USP14 was knockdown in Y79 cells by transfecting three siRNAs (si-USP14-1, si-USP14-2, and si-USP14-3), with si-USP14 NC as the negative control. si-USP14-3 was selected by results of Western blotting. The CCK-8 assay was used to detect the IC50 of Y79 cells and the growth curve. The cell cycle, cell apoptosis, and ROS level were measured by flow cytometry. The expression level of P-GP, ERCC1, survivin, GPX4, FTH1, ACSL4, NOX1, COX2, and FASN was determined by the Western blotting assay. CO-IP assay was utilized to evaluate the interaction between USP14 and FASN. The IC50 of DDP in Y79 cells and Y79/DDP cells was 7.83 µM and 24.67 µM, respectively. Compared to control and si-USP14 NC groups, increased apoptotic rate and ROS level, and arrested cell cycle in S phase were observed in USP14-knockdown Y79 cells. Compared to control and si-USP14 NC groups, increased apoptotic rate and arrested cell cycle in G0/G1 phase were observed in USP14-knockdown Y79/DDP cells. Compared to control, increased ROS level was observed in USP14-knockdown Y79/DDP cells. Compared to the si-USP14 NC groups, extremely downregulated P-GP, ERCC1, survivin, GPX4, FTH1, NOX1, COX2, and FASN were observed in USP14-knockdown Y79 cells or Y79/DDP cells, accompanied by the elevated expression of ACSL4. The interaction between USP14 and FASN was identified according to the result of CO-IP assay. By silencing USP14 in Y79 and Y79/DDP cells, levels of resistance-related proteins (P-GP, ERCC1, and survivin), ferroptosis-related proteins (FTH1 and GPX4), and lipid metabolism-related proteins (NOX1, COX2, and FASN) were dramatically reduced, accompanied by enhanced ROS level, increased apoptosis, and restrained DNA content, indicating that USP14 might suppress the DDP resistance in RB by mediating ferroptosis, which is an important target for treating RB.
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N 6-Methyladenosine (m6A) is the most prevalent mRNA modification and is required for gene regulation in eukaryotes. ALKBH5, an m6A demethylase, is a promising target, particularly for anticancer drug discovery. However, the development of selective and potent inhibitors of ALKBH5 rather than FTO remains challenging. Herein, we used a targeted covalent inhibition strategy and identified a covalent inhibitor, TD19, which selectively inhibits ALKBH5 compared with FTO demethylase in protein-based and tumor cell-based assays. TD19 irreversibly modifies the residues C100 and C267, preventing ALKBH5 from binding to m6A-containing RNA. Moreover, TD19 displays good anticancer efficacy in acute myeloid leukemia and glioblastoma multiforme cell lines. Thus, the ALKBH5 inhibitor developed in this study, which selectively targets ALKBH5 compared with FTO, can potentially be used as a probe for investigating the biological functions of RNA demethylase and as a lead compound in anticancer research.
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Accurate indel calling plays an important role in precision medicine. A benchmarking indel set is essential for thoroughly evaluating the indel calling performance of bioinformatics pipelines. A reference sample with a set of known-positive variants was developed in the FDA-led Sequencing Quality Control Phase 2 (SEQC2) project, but the known indels in the known-positive set were limited. This project sought to provide an enriched set of known indels that would be more translationally relevant by focusing on additional cancer related regions. A thorough manual review process completed by 42 reviewers, two advisors, and a judging panel of three researchers significantly enriched the known indel set by an additional 516 indels. The extended benchmarking indel set has a large range of variant allele frequencies (VAFs), with 87% of them having a VAF below 20% in reference Sample A. The reference Sample A and the indel set can be used for comprehensive benchmarking of indel calling across a wider range of VAF values in the lower range. Indel length was also variable, but the majority were under 10 base pairs (bps). Most of the indels were within coding regions, with the remainder in the gene regulatory regions. Although high confidence can be derived from the robust study design and meticulous human review, this extensive indel set has not undergone orthogonal validation. The extended benchmarking indel set, along with the indels in the previously published known-positive set, was the truth set used to benchmark indel calling pipelines in a community challenge hosted on the precisionFDA platform. This benchmarking indel set and reference samples can be utilized for a comprehensive evaluation of indel calling pipelines. Additionally, the insights and solutions obtained during the manual review process can aid in improving the performance of these pipelines.
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Benchmarking , Secuenciación de Nucleótidos de Alto Rendimiento , Humanos , Biología Computacional , Control de Calidad , Mutación INDEL , Polimorfismo de Nucleótido SimpleRESUMEN
Hypoxia is an important tumor feature and hypoxia-inducible factor 1 (HIF-1) is a master regulator of cell response to hypoxia. Mouse double minute 2 homolog (MDM2) promotes cancer cell survival in retinoblastoma (RB), with the underlying mechanism remaining elusive. In this study, we investigated the role of MDM2 and its relation to HIF-1α in RB. Expression analysis on primary human RB samples showed that MDM2 expression was positively correlated with that of HIF-1α while negatively correlated with von Hippel-Lindau protein (pVHL), the regulator of HIF-1α. In agreement, RB cells with MDM2 overexpression showed increased expression of HIF-1α and decreased expression of pVHL, while cells with MDM2 siRNA knockdown or MDM2-specific inhibitor showed the opposite effect under hypoxia. Further immuno-precipitation analysis revealed that MDM2 could directly interact with pVHL and promotes its ubiquitination and degradation, which consequently led to the increase of HIF-1α. Inhibition of MDM2 and/or HIF-1α with specific inhibitors induced RB cell death and decreased the stem cell properties of primary RB cells. Taken together, our study has shown that MDM2 promotes RB survival through regulating the expression of pVHL and HIF-1α, and targeting MDM2 and/or HIF-1α represents a potential effective approach for RB treatment.
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Subunidad alfa del Factor 1 Inducible por Hipoxia , Proteínas Proto-Oncogénicas c-mdm2 , Neoplasias de la Retina , Retinoblastoma , Humanos , Hipoxia de la Célula/fisiología , Supervivencia Celular , Hipoxia , Subunidad alfa del Factor 1 Inducible por Hipoxia/genética , Proteínas Proto-Oncogénicas c-mdm2/genética , Neoplasias de la Retina/genética , UbiquitinaciónRESUMEN
Conductive hydrogels (CHs) are ideal electrolyte materials for the preparation of flexible supercapacitors (FSCs) due to their excellent electrochemical properties, mechanical properties, and deformation restorability. However, most of the reported CHs are prepared by the chemical crosslinking of synthetic polymers and thus usually display the disadvantages of poor self-healing abilities and nonadaptability at environmental temperatures, which greatly limits their application. To overcome these problems, in the present work, we constructed a sodium alginate-borax/gelatin double-network conductive hydrogel (CH) by a dynamic crosslinking between sodium alginate (SA) and borax via borate bonds and hydrogen bonding between amino acids in gelatin and SA chains. The CH displays an excellent elongation of 305.7% and fast self-healing behavior in 60 s. Furthermore, a phase-change material (PCM), Na2SO4·10H2O, was introduced into the CH, which, combined with the nucleation effect of borax, improved the ionic conductivity and temperature adaptability of the CH. The flexible supercapacitor (FSC) assembled with the obtained CH as the electrolyte exhibits a high specific capacitance of 185.3 F·g-1 at a current density of 0.25 A·g-1 and good stability with 84% capacitance retention after 10â¯000 cycles and excellent temperature tolerance with a resistance variation of 2.11 Ω in the temperature range of -20-60 °C. This green CH shows great application potential as an electrolyte for FSCs, and the preparation method can be potentially expanded to the fabrication of self-repairing FSCs with good temperature adaptabilities.
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Chemotherapy is the first-line treatment for human retinoblastoma (RB), but the occurrence of drug resistance greatly limited its efficacy in practice. RING-finger protein 6 (RNF6) is an E3 ubiquitin ligase that is aberrantly upregulated in a range of cancers and plays important roles in cancer progression. However, the role of RNF6 in RB is largely unknown. In this study, we investigated the role of RNF6 in RB drug resistance. Two carboplatin-resistant RB cells, Y-79/CR and SO-Rb50/CR, were generated based on Y-79 and SO-Rb50 cells. RT-PCR and western blot analyses showed that RNF6 expression on both mRNA and protein levels was significantly increased in Y-79/CR and SO-Rb50/CR cells comparing to their parental cells. Knockdown of RNF6 using siRNA in Y-79/CR and SO-Rb50/CR cells resulted in cells sensitive to carboplatin on a RNF6 siRNA dose dependent manner. Similarly, RNF6 overexpression in parental Y-79 and SO-Rb50 cells could help cells gain resistance to carboplatin on a RNF6 expression dependent manner. Signaling pathway analyses revealed that JAK2/STAT3 pathway was involved in the RNF6-induced carboplatin resistance in RB cells. We further revealed that RNF6 expression in both Y-79 and SO-Rb50 cells could render cells resistant to multiple anti-cancer drugs including carboplatin, vincristine and etoposide, an implication of RNF6 as a biomarker for RB drug resistance. Taken together, our study has revealed that RNF6 is upregulated in drug-resistant RB cells and RNF6 promotes drug resistance through JAK2/STAT3 signaling pathway. The importance of RNF6 in RB cells drug resistance may represent this protein as a potential biomarker and treatment target for drug resistance in RB.
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Neoplasias de la Retina , Retinoblastoma , Línea Celular Tumoral , Proteínas de Unión al ADN/genética , Resistencia a Medicamentos , Humanos , Janus Quinasa 2 , Neoplasias de la Retina/tratamiento farmacológico , Neoplasias de la Retina/genética , Retinoblastoma/tratamiento farmacológico , Retinoblastoma/genética , Factor de Transcripción STAT3/genética , Factor de Transcripción STAT3/metabolismo , Transducción de SeñalRESUMEN
Conductive hydrogels (CHs) are a potential material for flexible electronics. However, most of CHs display disadvantages of low ionic conductivities and intolerance to low temperatures. Herein, a novel physical CHs with salt contents as high as 30 wt% was prepared with chitosan (CTS) and sodium alginate (SA) by combining the anti-polyelectrolyte effect and semi-dissolution acidification sol-gel transition (SD-A-SGT) method. The obtained hydrogels show extremely high ionic conductivities up to 2.96 × 10-1 S·cm-1 at room temperature and 4.9 × 10-2 S·cm-1 at -20 °C. The effects of different salts on the ion mobility and electrochemical properties of CTS/SA CHs were predicted and analyzed. The flexible supercapacitor assembled using CTS/SA CHs as the electrolyte exhibits the specific capacitance as high as 405 F·g-1 at the current density of 0.25 A·g-1 and satisfying electrochemical stability with 74.91% capacitance retention in 1000 cycles. Our work has provided a new strategy for constructing green CHs with high ionic conductivities.
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Alginatos/química , Quitosano/química , Hidrogeles/química , Capacidad Eléctrica , Conductividad Eléctrica , Polielectrolitos/química , Sales (Química)/químicaRESUMEN
Exploration of the initial reactions of H-free and nitro-free energetic materials could enrich our understanding of the thermal decomposition mechanism of various energetic materials (EMs). In this work, two furoxan compounds, 3,4-dinitrofurazanfuroxan (DNTF) and benzotrifuroxan (BTF), were investigated to shed light on the decay mechanism of furoxan compounds based on the combination of self-consistent charge density functional tight binding and molecular dynamics simulations. The results show that DNTF and BTF decay via a unimolecular mechanism, and the transformation of the furoxan ring into a nitro group is suggested as a novel initial channel. Five initial steps of DNTF thermal decomposition are observed, including NO2 loss and the N(O)-O bond cleavage of the central and peripheral rings. The bond cleavage of peripheral rings dominates the decay at low temperatures, while the central ring opening and C-NO2 dissociation govern the high temperature decay. Besides, NO2, CO and NO fragments are mainly yielded at high temperatures, while CO3N2 is dominant at low temperatures. The three-stage characteristic of the exothermic BTF decay is described under programmed heating conditions for the first time. Four initial steps of BTF thermal decomposition were identified, including furoxan ring opening reactions and the breakage of the 6-membered ring C-C bond. The cleavage of the N(O)-O bond is dominant in the initial step of BTF decomposition under different heating conditions, and the frequency increases with increasing temperature. In addition, the amounts of CON, ON and CO are higher at high temperatures, while C2O2N2 shows an opposite trend. The findings of this work provide deep insights into the complicated sensitivity mechanism of EMs.
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Background: Acute infection, such as periprosthetic joint infection and superficial surgical site infection, after primary total joint arthroplasty (TJA) is a serious complication, and its risk factors remain controversial. This study aimed to identify the risk factors for acute infection after primary TJA, especially the serological indicators that reflect preoperative nutritional statuses, such as albumin level and albumin to fibrinogen ratio (AFR). Methods: We retrospectively reviewed patients who underwent elective primary hip or knee arthroplasty at our institution from 2009 to 2021. Potential risk factors of acute infection and demographic information were extracted from an electronic health record. Patients who suffered acute infection, such as PJI or SSI, after TJA were considered the study group. Non-infected patients were matched 1:2 with the study group according to sex, age, the involved joint (hip or knee), and year of surgery (control group). The variables of potential risk factors for acute postoperative infection (demographic characteristics, preoperative comorbidities and drug use, operative variables, and laboratory values) were collected and evaluated by regression analysis. Restrictive cubic spline regression analysis was also used to examine the relationship between preoperative serum albumin levels and acute postoperative infection. Results: We matched 162 non-infected patients with 81 patients who suffered from acute postoperative infection. Among the patients who suffered from acute infection within 90 days after TJA, 18 were diagnosed with periprosthetic joint infection and 63 with surgical site infection. Low albumin levels were strongly associated with acute postoperative infection (95% confidence interval, 0.822-0.980; P = 0.015). This risk increased as preoperative albumin levels decreased, with a negative dose-response relationship (P overall = 0.002; P nonlinear = 0.089). However, there was no significant association between the AFR and acute infection after primary TJA (P = 0.100). Conclusion: There is currently insufficient evidence to confirm the relationship between preoperative AFR and acute infection after elective primary TJA, while a lower preoperative albumin level is an independent risk factor for acute infection with a negative dose-response relationship. This suggests that optimal nutritional management may be benefited before elective primary TJA.
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The difference in the initial decomposition step of pyrazoles and imidazoles was explored using the M062X method for optimization and G4-MP2 and approximated CCSD(T) methods for energies. Laplacian bond order analysis was used to study the effect of the nitro group on the bond strength and predict the bond dissociation energy (BDE) of the ring. Thermochemistry results show that the most possible decay channel of 1H-pyrazole and 3-nitropyrazole is the N2 elimination, while the preferred initial step of 1H-imidazole is the CHN elimination. However, the nitro-nitrite isomerization dominates the decomposition of other nitro derivatives of 1H-pyrazole and 1H-imidazole. As for the formation of HO and HONO, the high energy barrier makes it difficult to take place. Based on the analysis of the lowest energy barrier and the BDE of NO2 loss, it can be concluded that imidazoles are more stable than pyrazoles. This work contributes to revealing the difference in the initial step of energetic isomers and the understanding of the decomposition mechanism of energetic azoles.
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Skeletal muscle possesses remarkable regenerative ability because of the resident muscle stem cells (MuSCs). A prominent feature of quiescent MuSCs is a high content of heterochromatin. However, little is known about the mechanisms by which heterochromatin is maintained in MuSCs. By comparing gene-expression profiles from quiescent and activated MuSCs, we found that the mammalian Hairless (Hr) gene is expressed in quiescent MuSCs and rapidly down-regulated upon MuSC activation. Using a mouse model in which Hr can be specifically ablated in MuSCs, we demonstrate that Hr expression is critical for MuSC function and muscle regeneration. In MuSCs, loss of Hr results in reduced trimethylated Histone 3 Lysine 9 (H3K9me3) levels, reduced heterochromatin, increased susceptibility to genotoxic stress, and the accumulation of DNA damage. Deletion of Hr leads to an acceleration of the age-related decline in MuSC numbers. We have also demonstrated that despite the fact that Hr is homologous to a family of histone demethylases and binds to di- and trimethylated H3K9, the expression of Hr does not lead to H3K9 demethylation. In contrast, we show that the expression of Hr leads to the inhibition of the H3K9 demethylase Jmjd1a and an increase in H3K9 methylation. Taking these data together, our study has established that Hr is a H3K9 demethylase antagonist specifically expressed in quiescent MuSCs.
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Silenciador del Gen , Heterocromatina , Histona Demetilasas/antagonistas & inhibidores , Músculo Esquelético/fisiología , Células Madre/fisiología , Factores de Transcripción/metabolismo , Animales , Histonas/genética , Histonas/metabolismo , Metilación , Ratones , Ratones Pelados , Músculo Esquelético/citología , Células Madre/citología , Factores de Transcripción/genéticaRESUMEN
In order to resolve the existing discrepancies in the mechanism and key intermediates of oxadiazole thermolysis, the initial decomposition pathways of oxadiazoles have been studied comprehensively using the M062X method for optimization and CBS-QB3 and DLPNO-CCSD(T) methods for energies. The transformation from the furoxan ring to nitro group was suggested as a potential decay channel of furoxan compounds. Results of thermochemistry calculations showed that the preferred decomposition reaction of oxadiazoles is the ring-opening through the cleavage of the O-C or O-N bond. The introduction of the nitro group has little effect on the preferential path of oxadiazole thermal decomposition, but a great impact on the energy barrier. The lowest energy barrier and bond dissociation energy of NO2 loss of azoles were comprehensively studied based on the quantum chemistry calculations. The initial decay steps of 3,4-dinitrofurazanfuroxan and benzotrifuroxan were also studied to give insights into the mechanism of primary stages of thermal decomposition of oxadiazoles.
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Intramuscular fatty deposits, which are seen in muscular dystrophies and with aging, negatively affect muscle function. The cells of origin of adipocytes constituting these fatty deposits are mesenchymal stromal cells, fibroadipogenic progenitors (FAPs). We uncover a molecular fate switch, involving miR-206 and the transcription factor Runx1, that controls FAP differentiation to adipocytes. Mice deficient in miR-206 exhibit increased adipogenesis following muscle injury. Adipogenic differentiation of FAPs is abrogated by miR-206 mimics. Using a labeled microRNA (miRNA) pull-down and sequencing (LAMP-seq), we identified Runx1 as a miR-206 target, with miR-206 repressing Runx1 translation. In the absence of miR-206 in FAPs, Runx1 occupancy near transcriptional start sites of adipogenic genes and expression of these genes increase. We demonstrate that miR-206 mimicry in vivo limits intramuscular fatty infiltration. Our results provide insight into the underlying molecular mechanisms of FAP fate determination and formation of harmful fatty deposits in skeletal muscle.
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Células Madre Mesenquimatosas , MicroARNs , Adipocitos , Adipogénesis/genética , Animales , Diferenciación Celular , Ratones , MicroARNs/genética , Músculo EsqueléticoRESUMEN
A novel pH/glutathione (GSH) multi-responsive chitosan nanoparticles (NPs) material has been successfully designed and prepared by a self-assembly/self-crosslinking method for photodynamic therapy (PDT), which overcomes the shortcomings of traditional photosensitizer carriers, such as poor chemical stability, low loading efficiency and single-responsive photosensitizer release. Amphiphilic sulfhydryl chitosan (SA-CS-NAC) is first prepared by modifying chitosan (CS) with stearic acid (SA) and N-acetyl-L-cysteine (NAC), and then subject to self-assembly and self-crosslinking in the presence of photosensitizer, indocyanine green (ICG), to form the ICG-loaded amphiphilic sulfhydryl chitosan nanoparticles (SA-CS-NAC@ICG NPs). The ICG entrapment efficiency and loading efficiency of the NPs are found to be 95.2% and 27.6%, respectively. The multi-responsive ICG release of the NPs to the low pH and high GSH content of the microenvironment in tumor cells is successfully achieved. Under the laser irradiation, the SA-CS-NAC@ICG NPs produce the amount of reactive oxygen species (ROS) twice of that generated by free ICG under the same conditions. The in vitro cell experiment confirmed the strong cellular uptake ability, low biotoxicity and good tumor inhibition of the NPs. Our work has provided a new strategy for the targeted photosensitizer delivery for PDT.
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Quitosano/química , Glutatión/química , Concentración de Iones de Hidrógeno , Nanopartículas/química , Fármacos Fotosensibilizantes/química , Animales , Apoptosis/efectos de los fármacos , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Técnicas de Química Sintética , Reactivos de Enlaces Cruzados , Modelos Animales de Enfermedad , Estabilidad de Medicamentos , Humanos , Espectroscopía de Resonancia Magnética , Ratones , Micelas , Peso Molecular , Nanopartículas/ultraestructura , Fotoquimioterapia , Especies Reactivas de Oxígeno/metabolismo , Espectroscopía Infrarroja por Transformada de Fourier , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Adult stem cells are essential for tissue homeostasis. In skeletal muscle, muscle stem cells (MuSCs) reside in a quiescent state, but little is known about the mechanisms that control homeostatic turnover. Here we show that, in mice, the variation in MuSC activation rate among different muscles (for example, limb versus diaphragm muscles) is determined by the levels of the transcription factor Pax3. We further show that Pax3 levels are controlled by alternative polyadenylation of its transcript, which is regulated by the small nucleolar RNA U1. Isoforms of the Pax3 messenger RNA that differ in their 3' untranslated regions are differentially susceptible to regulation by microRNA miR206, which results in varying levels of the Pax3 protein in vivo. These findings highlight a previously unrecognized mechanism of the homeostatic regulation of stem cell fate by multiple RNA species.
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Músculo Esquelético/fisiología , Mioblastos Esqueléticos/metabolismo , Factor de Transcripción PAX3/genética , Poliadenilación , Regiones no Traducidas 3' , Animales , Técnicas de Silenciamiento del Gen , Ratones , Ratones Mutantes , MicroARNs/metabolismo , ARN Mensajero/metabolismo , Ribonucleoproteína Nuclear Pequeña U1/genética , Ribonucleoproteína Nuclear Pequeña U1/metabolismoRESUMEN
The multimolecular complexes formed between 2,4,6,8,10,12-hexanitro-2,4,6,6,8,10,12-hexaazaisowurtzitane (CL-20) and nitropyrazole compounds were investigated using B3LYP-D3/6-311G(d,p) and B97-3c methods. CL-20 in these complexes was surrounded by methyl, nitro, and amino derivatives of 4-nitropyrazole. The influence of substituents on the molecular electrostatic potential distribution of nitropyrazoles was investigated to figure out the potential electrostatic interaction sites. For the complex, the O···H hydrogen bond was popular in the intermolecular interactions, and dispersion interaction played an essential role, especially in Cx/CL-20 multimolecular complexes. Trigger bond analysis showed that their strength increased upon the formation of intermolecular weak interactions. Nitro group charge calculations stated that the negative charge on almost all nitro groups showed a significant increase. Therefore, the sensitivity of CL-20 seemed to be lower than the original. In addition, the transfer of electron density between CL-20 and nitropyrzoles in complexes was investigated, revealing the influence of weak interactions on the electron density of CL-20.
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The necessity of mesenchymal stromal cells, called fibroadipogenic progenitors (FAPs), in skeletal muscle regeneration and maintenance remains unestablished. We report the generation of a PDGFRαCreER knockin mouse model that provides a specific means of labeling and targeting FAPs. Depletion of FAPs using Cre-dependent diphtheria toxin expression results in loss of expansion of muscle stem cells (MuSCs) and CD45+ hematopoietic cells after injury and impaired skeletal muscle regeneration. Furthermore, FAP-depleted mice under homeostatic conditions exhibit muscle atrophy and loss of MuSCs, revealing that FAPs are required for the maintenance of both skeletal muscle and the MuSC pool. We also report that local tamoxifen metabolite delivery to target CreER activity in a single muscle, removing potentially confounding systemic effects of ablating PDGFRα+ cells distantly, also causes muscle atrophy. These data establish a critical role of FAPs in skeletal muscle regeneration and maintenance.
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Homeostasis , Células Madre Mesenquimatosas/metabolismo , Desarrollo de Músculos , Regeneración , Células 3T3 , Animales , Ratones , Ratones Transgénicos , Músculo EsqueléticoRESUMEN
Protein tyrosine phosphatase 1B (PTP1B) is considered a potential target for the treatment of type II diabetes and obesity due to its critical negative role in the insulin signaling pathway. However, improving the selectivity of PTP1B inhibitors over the most closely related T-cell protein tyrosine phosphatase (TCPTP) remains a major challenge for inhibitor development. Lys120 at the active site and Ser27 at the second pTyr binding site are distinct in PTP1B and TCPTP, which may bring differences in binding affinity. To explore the determinant of selective binding of inhibitor, molecular dynamics simulations with binding free energy calculations were performed on K120A and A27S mutated PTP1B, and the internal changes induced by mutations were investigated. Results reveal that the presence of Lys120 induces a conformational change in the WPD-loop and YRD-motif and has a certain effect on the selective binding at the active site. Ser27 weakens the stability of the inhibitor at the second pTyr binding site by altering the orientation of the Arg24 and Arg254 side chains via hydrogen bonds. Further comparison of alanine scanning demonstrates that the reduction in the energy contribution of Arg254 caused by A27S mutation leads to a different inhibitory activity. These observations provide novel insights into the selective binding mechanism of PTP1B inhibitors to TCPTP.
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Diabetes Mellitus Tipo 2/tratamiento farmacológico , Inhibidores Enzimáticos/química , Proteína Tirosina Fosfatasa no Receptora Tipo 1/química , Proteína Tirosina Fosfatasa no Receptora Tipo 2/química , Sitios de Unión , Dominio Catalítico , Diabetes Mellitus Tipo 2/genética , Diabetes Mellitus Tipo 2/patología , Inhibidores Enzimáticos/uso terapéutico , Humanos , Enlace de Hidrógeno , Insulina/metabolismo , Simulación del Acoplamiento Molecular , Simulación de Dinámica Molecular , Terapia Molecular Dirigida , Unión Proteica , Conformación Proteica/efectos de los fármacos , Proteína Tirosina Fosfatasa no Receptora Tipo 1/antagonistas & inhibidores , Proteína Tirosina Fosfatasa no Receptora Tipo 2/antagonistas & inhibidores , Transducción de Señal/efectos de los fármacosRESUMEN
Protein tyrosine phosphatase 1B (PTP1B), a key negative regulator in insulin signaling pathways, is regarded as a potential target for the treatment of type II diabetes and obesity. However, the mechanism underlying the selectivity of PTP1B inhibitors against T-cell protein tyrosine phosphatase (TCPTP) remains controversial, which is due to the high similarity between PTP1B and TCPTP sequence and the fact that no ligand-protein complex of TCPTP has been established yet. Here, the accelerated molecular dynamics (aMD) method was used to investigate the structural dynamics of PTP1B and TCPTP that are bound by two chemically similar inhibitors with distinct selectivity. The conformational transitions during the "open" to "close" states of four complexes were captured, and free energy profiles of important residue pairs were analyzed in detail. Additional MM-PBSA calculations confirmed that the binding free energies of final states were consistent with the experimental results, and the energetic contributions of important residues were further investigated by alanine scanning mutagenesis. By comparing the four complexes, the different conformational behavior of WPD-loop, R-loop, and the second pTyr binding site induced by inhibitors were featured and found to be crucial for the selectivity of inhibitors. This study provides new mechanistic insights of specific binding of inhibitors to PTP1B and TCPTP, which can be exploited to the further structural-based inhibitor design. Communicated by Ramaswamy H. Sarma.