Activation of FMS-like tyrosine kinase 3 protects against isoprenaline-induced cardiac hypertrophy by improving autophagy and mitochondrial dynamics.

FMS-like receptor tyrosine kinase 3 (Flt3) expression was reported to increase in the heart in response to pathological stress, but the role of Flt3 activation and its underlying mechanisms remain poorly elucidated. This study was designed to investigate the role of Flt3 activation in sympathetic hyperactivity-induced cardiac hypertrophy and its mechanisms through autophagy and mitochondrial dynamics. In vivo, cardiac hypertrophy was established by subcutaneous injection of isoprenaline (6 mg/kg·day) in C57BL/6 mice for 7 consecutive days. The Flt3-ligand intervention was launched 2 h prior to isoprenaline each day. In vitro, experiments of cardiomyocyte hypertrophy, autophagy, and mitochondrial dynamics were performed in neonatal rat cardiomyocytes (NRCMs). Our results revealed that the expression level of Flt3 protein was significantly increased in the hypertrophic myocardium provoked by isoprenaline administration. Flt3-ligand intervention alleviated isoprenaline-induced cardiac oxidative stress, hypertrophy, fibrosis, and contractile dysfunction. Isoprenaline stimulation impaired autophagic flux in hypertrophic mouse hearts, supported by the accumulation of LC3II and P62 proteins, while Flt3-ligand restored the impairment of autophagic flux. Flt3 activation normalized the imbalance of mitochondrial fission and fusion in the hearts of mice evoked by isoprenaline as evidenced by the neutralization of elevated mitochondrial fission markers and reduced mitochondrial fusion markers. In NRCMs, Flt3-ligand treatment attenuated isoprenaline-stimulated hypertrophy, which was abolished by a Flt3-specific blocker AC220. Activating Flt3 reversed isoprenaline-induced autophagosome accumulation and impairment of autophagic flux probably by enhancing SIRT1 expression and consequently TFEB nuclear translocation. Flt3 activation improved the imbalance of mitochondrial dynamics induced by isoprenaline in NRCMs through the SIRT1/P53 pathway. Activation of Flt3 mitigated ISO-stimulated hypertrophy probably involves the restoration of autophagic flux and balance of mitochondrial dynamics. Therefore, activation of Flt3 attenuates isoprenaline-induced cardiac hypertrophy in vivo and in vitro, the potential mechanism probably attributes to SIRT1/TFEB-mediated autophagy promotion and SIRT1/P53-mediated mitochondrial dynamics balance. These findings suggest that activation of Flt3 may be a novel target for protection against cardiac remodeling and heart failure during sympathetic hyperactivity.

Fluorescence Resonance Energy Transfer-Mediated Immunosensor Based on Design and Synthesis of the Substrate of Amp Cephalosporinase for Biosensing.

The traditional enzyme-linked immunosorbent assay (ELISA) has some disadvantages, such as insufficient sensitivity and low stability of the labeled enzyme, which limit its further applications. In this study, a more stable enzyme, Amp cephalosporinase (AmpC), was selected as the labeled enzyme, and its substrate was designed and synthesized. This substrate contained the cephalosporin ring core as the enzymatic recognition section and the structural motif of the 3-hydroxyflavone (3-HF) as the reporter molecule. AmpC can specifically catalyze the substrate and release 3-HF, which can enter the cavity of ß-cyclodextrin (ß-CD) on the surface of ZnS quantum dots and form a fluorescence resonance energy transfer (FRET) signal amplification system. An AmpC-catalyzed, FRET-mediated ultrasensitive immunosensor (ACF immunosensor) for procalcitonin (PCT) was developed by combining the signal amplification system of the polystyrene microspheres and effective immune-based magnetic separation. The ACF immunosensor has high sensitivity and specificity for the detection of PCT: its linear range is from 0.1 ng mL-1 to 70 ng mL-1, and the limit of detection can reach 0.03 ng mL-1. The spiking recoveries of PCT in human serum samples range from 98.3% to 107%, with relative standard deviations ranging from 2.14% to 12.0%. This approach was applied to detect PCT in real patient serum samples, and the results are consistent with those obtained with a commercial ELISA kit.

Phytic acid functionalized Fe3O4 nanoparticles loaded with Ti(IV) ions for phosphopeptide enrichment in mass spectrometric analysis.

A novel magnetic nanomaterial for use in metal ion based affinity chromatography is described. It is based on the chelation between the phosphate groups of phytic acid (PA) and Ti(IV) ions. Due to the large number (6) of phosphate groups of PA, it has a large capacity for Ti(IV) ions. PA was first immobilized on magnetite nanoparticles (PA-MNPs) and then loaded with Ti(IV) ions to obtain the sorbent (Ti-PA-MNPs). The fraction of Ti(IV) ions on the surface of PA-MNPs that is exposed to the solution binds the phosphate groups of phosphopeptides. The bound phosphopeptides can then be magnetically separated. The method was applied to the enrichment of the phosphopeptides in a ß-casein tryptic digest. A tryptic digest of bovine serum albumin (BSA) was added at a molar ratio (ß-casein to BSA) of 1:2000 to study selectivity. The phosphopeptides were quantified by mass spectrometry. The limit of detection can be as low as 8 × 10-10 mol L-1. This sorbent has a high absorption capacity (53.5 µg mg-1) and shows good recoveries (90%). As many as 2145 phosphopeptides were isolated from 500 µg tryptic digest of a rat liver lysate after enrichment by Ti-PA-MNPs. This is superior to that (1568 phosphopeptides) of commercial TiO2 kit. Graphical abstract Schematic presentation of fabrication for a novel modified magnetic nanomaterial (Ti-PA-MNPs) based on the chelation of phytic acid (PA) with Ti(IV) ions. Ti-PA-MNPs were successfully applied to enriching low abundance phosphopeptides from biosamples in mass spectrometric analysis.

Determination of protein kinase A activity and inhibition by using hydroxyapatite nanoparticles as a fluorescent probe.

The authors describe a fluorometric method for the determination of the activity and inhibition of protein kinase A (PKA). In the presence of ATP, PKA catalyzes the transfer of phosphate groups from ATP to a peptide, and the generated phosphorylated peptide quenches the fluorescence (measured at excitation/emission peaks of 340/440 nm) of the hydroxyapatite nanoparticles (HAP-NPs). A linear logarithmic relationship of PKA concentrations with fluorescence intensity in the range from 1 to 50 U·L-1 was obtained, and the lower limit of detection (LOD) is 0.5 U·L-1. This is much lower than LODs reported in the literature. The PKA inhibitor H-89 was studied, and the inhibition plot has a sigmoidal shape with a half-maximal inhibitory concentration of around 750 nM of H-89. At a 4.5 nM level of H-89, fluorescence of HAP-NPs fell to levels of no PKA controls, demonstrating that the assay is a viable tool to screen for kinase inhibitors. An assay with Hela cell lysates in combination with forskolin (an activator of adenylyl cyclase) and IBMX (a phosphodiesterase inhibitor used to activate the cellular activity of PKA) resulted in decreased fluorescence of HAP-NPs. This suggests that the assay can be applied for testing in vitro cell kinase activity. In our perception, this method will enable high-throughput screening for kinase-related drugs and fluorometric enzymatic detection in various areas. Graphical abstract Fluorescence assay based on hydroxyapatite nanoparticles (HAP) fluorescence quenching was developed for analysis of the activity and inhibition of protein kinase A (PKA).

Upregulation of TRIM16 mitigates doxorubicin-induced cardiotoxicity by modulating TAK1 and YAP/Nrf2 pathways in mice.

The clinic application of doxorubicin (DOX) is severely limited by its severe cardiotoxicity. Tripartite motif-containing protein 16 (TRIM16) has E3 ubiquitin ligase activity and is upregulated in cardiomyocytes under pathological stress, yet its role in DOX-induced cardiotoxicity remains elusive. This study aims to investigate the role and mechanism of TRIM16 in DOX cardiotoxicity. Following TRIM16 overexpression in hearts with AAV9-TRIM16, mice were intravenously administered DOX at a dose of 4 mg/kg/week for 4 weeks to assess the impact of TRIM16 on doxorubicin-induced cardiotoxicity. Transfection of OE-TRIM16 plasmids and siRNA-TRIM16 was performed in neonatal rat cardiomyocytes (NRCMs). Our results revealed that DOX challenge elicited a significant upregulation of TRIM16 proteins in cardiomyocytes. TRIM16 overexpression efficiently ameliorated cardiac function while suppressing inflammation, ROS generation, apoptosis and fibrosis provoked by DOX in the myocardium. TRIM16 knockdown exacerbated these alterations caused by DOX in NRCMs. Mechanistically, OE-TRIM16 augmented the ubiquitination and degradation of p-TAK1, thereby arresting JNK and p38MAPK activation evoked by DOX in cardiomyocytes. Furthermore, DOX enhanced the interaction between p-TAK1 and YAP1 proteins, resulting in a reduction in YAP and Nrf2 proteins in cardiomyocytes. OE-TRIM16 elevated YAP levels and facilitated its nuclear translocation, thereby promoting Nrf2 expression and mitigating oxidative stress and inflammation. This effect was nullified by siTRIM16 or TAK1 inhibitor Takinib. Collectively, the current study elaborates that upregulating TRIM16 mitigates DOX-induced cardiotoxicity through anti-inflammation and anti-oxidative stress by modulating TAK1-mediated p38 and JNK as well as YAP/Nrf2 pathways, and targeting TRIM16 may provide a novel strategy to treat DOX-induced cardiotoxicity.

Flt3 Activation Mitigates Mitochondrial Fragmentation and Heart Dysfunction through Rebalanced L-OPA1 Processing by Hindering the Interaction between Acetylated p53 and PHB2 in Cardiac Remodeling.

Recent studies have shown that FMS-like receptor tyrosine kinase 3 (Flt3) has a beneficial effect on cardiac maladaptive remodeling. However, the role and mechanism of Flt3 in mitochondrial dynamic imbalance under cardiac stress remains poorly understood. This study aims to investigate how Flt3 regulates p53-mediated optic atrophy 1 (OPA1) processing and mitochondrial fragmentation to improve cardiac remodeling. Mitochondrial fragmentation in cardiomyocytes was induced by isoprenaline (ISO) and H2O2 challenge, respectively, in vitro. Cardiac remodeling in mice was established by ligating the left anterior descending coronary artery or by chronic ISO challenge, respectively, in vivo. Our results demonstrated that the protein expression of acetylated-p53 (ac-p53) in mitochondria was significantly increased under cell stress conditions, facilitating the dissociation of PHB2-OPA1 complex by binding to prohibitin 2 (PHB2), a molecular chaperone that stabilizes OPA1 in mitochondria. This led to the degradation of the long isoform of OPA1 (L-OPA1) that facilitates mitochondrial fusion and resultant mitochondrial network fragmentation. This effect was abolished by a p53 K371R mutant that failed to bind to PHB2 and impeded the formation of the ac-p53-PHB2 complex. The activation of Flt3 significantly reduced ac-p53 expression in mitochondria via SIRT1, thereby hindering the formation of the ac-p53-PHB2 complex and potentiating the stability of the PHB2-OPA1 complex. This ultimately inhibits L-OPA1 processing and leads to the balancing of mitochondrial dynamics. These findings highlight a novel mechanism by which Flt3 activation mitigates mitochondrial fragmentation and dysfunction through the reduction of L-OPA1 processing by dampening the interaction between ac-p53 and PHB2 in cardiac maladaptive remodeling.

Inhibition of the maize salt overly sensitive pathway by ZmSK3 and ZmSK4.

Soil salinity is a worldwide problem that adversely affects plant growth and crop productivity. The salt overly sensitive (SOS) pathway is evolutionarily conserved and essential for plant salt tolerance. In this study, we reveal how the maize shaggy/glycogen synthase kinase 3-like kinases ZmSK3 and ZmSK4, orthologs of brassinosteroid insensitive 2 in Arabidopsis thaliana, regulate the maize SOS pathway. ZmSK3 and ZmSK4 interact with and phosphorylate ZmSOS2, a core member of the maize SOS pathway. The mutants defective in ZmSK3 or ZmSK4 are hyposensitive to salt stress, with higher salt-induced activity of ZmSOS2 than that in the wild type. Furthermore, the Ca2+ sensors ZmSOS3 and ZmSOS3-like calcium binding protein 8 (ZmSCaBP8) activate ZmSOS2 to maintain Na+/K+ homeostasis under salt stress and may participate in the regulation of ZmSOS2 by ZmSK3 and ZmSK4. These findings discover the regulation of the maize SOS pathway and provide important gene targets for breeding salt-tolerant maize.

Impaired autophagy and mitochondrial dynamics are involved in Sorafenib-induced cardiomyocyte apoptosis.

Sorafenib (Sor), a novel multi-target anticancer drug also induces severe toxicity in heart, while the mechanism of its cardiotoxicity remains to be fully elucidated. Dysregulation of autophagy and mitochondrial dynamics imbalance have been implicated in cardiomyocyte death. The aim of this study is to test the hypothesis that Sor disrupts autophagy and mitochondrial dynamics, thereby aggravating Sor-induced oxidative stress damage to cardiomyocytes. Our results revealed that Sor (≥ 5 µM) concentration- and time-dependently reduced cell viability and induced apoptosis in H9c2 myoblasts. Sor treatment promoted intracellular reactive oxygen species (ROS) generation, and subsequent Ca2+ overload as well as apoptosis, which were abolished by the ROS scavenger MPG. Sor inhibited the basal autophagy activity of cells, as supported by the fact that ERK1/2 inhibition-dependent decreases of autophagosomes and autolysosomes, and p62 accumulation in a concentration- and time-dependent manner. Improving autophagy with rapamycin abrogated Sor-induced ROS and Ca2+ overloads, and cell apoptosis. Furthermore, Sor compromised mitochondrial morphology and caused excessive mitochondrial fragmentation in cells. The imbalance of mitochondrial dynamics was attributed to ROS-mediated CaMKII overactivity, and increased phosphorylation of dynamin-related protein 1 (phosph-Drp1). Suppression of CaMKII with KN-93 or mitochondrial fission with mitochondrial division inhibitor-1 (Mdivi-1) attenuated Sor-induced ROS and Ca2+ overloads as well as apoptosis. In conclusion, these results provide the first evidence that impairments in autophagy and mitochondrial dynamics are involved in Sor-induced cardiomyocyte apoptosis. The present study may provide a potential strategy for preventing or reducing cardiotoxicity of Sor.

Development of dual-enhancer biocatalyst with photothermal property for the degradation of cephalosporin.

The abuse of cephalosporins poses a serious threat to human health and the ecological environment. In this work, cephalosporinase (AmpC enzyme) and Prussian blue (PB) crystals were encapsulated into ZIF-8 metal-organic frameworks (MOFs), and a photothermal AmpC/PB@ZIF-8 MOFs (APZ) nanocatalyst was prepared for the catalytic degradation of cephalosporin. The temperature of the APZ catalytic degradation system can be regulated by irradiation with near infrared light due to the photothermal effect of PB, and then, the activity of the APZ biocatalyst is significantly enhanced. Thereby, the degradation efficiency of cefuroxime can reach to 96%, and the degradation kinetic rate of cefuroxime augmented 4.5-fold comparing with that catalyzed by free enzyme. Moreover, encapsulation of the enzyme and PB can increase the affinity and charge transfer efficiency between APZ and substrate molecules, which can also improve the degradation efficiency of cephalosporins. Catalytic degradation pathways for three generations of cephalosporins were proposed based on their degradation products. The dual-enhancer biocatalyst based on the photothermal effect and immobilization of the PB and enzyme can significantly enhance the activity and stability of the enzyme, and it can also be recycled. Therefore, the biocatalyst has potential applications for the effective degradation of cephalosporins in the environment.

QKI-Regulated Alternative Splicing Events in Cervical Cancer: Pivotal Mechanism and Potential Therapeutic Strategy.

QKI is a vital regulator in RNA splicing and maturation, but its role in cervical cancer (CC) is little known. In this study, we found that QKI is decreased in human CC, and overexpression of QKI inhibits HeLa cell proliferation and promotes the apoptosis of cancer cells. We identified hundreds of endogenous QKI-regulated alternative splicing events (ASEs) and differentially expressed genes (DEGs) in QKI-overexpressed HeLa cells by RNA-seq and selectively validated their expression by quantitative reverse-transcription polymerase chain reaction. The gene ontology and Kyoto encyclopedia of genes and genomes (KEGG) enrichment analysis showed that QKI-regulated ASEs and DEGs were closely related to cancer, apoptosis, and transcriptional regulatory functions. In short, QKI may affect the occurrence and development of CC by regulating gene expression through AS.