The positive feedback loop of the NAT10/Mybbp1a/p53 axis promotes cardiomyocyte ferroptosis to exacerbate cardiac I/R injury.

Ferroptosis is a nonapoptotic form of regulated cell death that has been reported to play a central role in cardiac ischemia‒reperfusion (I/R) injury. N-acetyltransferase 10 (NAT10) contributes to cardiomyocyte apoptosis by functioning as an RNA ac4c acetyltransferase, but its role in cardiomyocyte ferroptosis during I/R injury has not been determined. This study aimed to elucidate the role of NAT10 in cardiac ferroptosis as well as the underlying mechanism. The mRNA and protein levels of NAT10 were increased in mouse hearts after I/R and in cardiomyocytes that were exposed to hypoxia/reoxygenation. P53 acted as an endogenous activator of NAT10 during I/R in a transcription-dependent manner. Cardiac overexpression of NAT10 caused cardiomyocyte ferroptosis to exacerbate I/R injury, while cardiomyocyte-specific knockout of NAT10 or pharmacological inhibition of NAT10 with Remodelin had the opposite effects. The inhibition of cardiomyocyte ferroptosis by Fer-1 exerted superior cardioprotective effects against the NAT10-induced exacerbation of post-I/R cardiac damage than the inhibition of apoptosis by emricasan. Mechanistically, NAT10 induced the ac4C modification of Mybbp1a, increasing its stability, which in turn activated p53 and subsequently repressed the transcription of the anti-ferroptotic gene SLC7A11. Moreover, knockdown of Mybbp1a partially abolished the detrimental effects of NAT10 overexpression on cardiomyocyte ferroptosis and cardiac I/R injury. Collectively, our study revealed that p53 and NAT10 interdependently cooperate to form a positive feedback loop that promotes cardiomyocyte ferroptosis to exacerbate cardiac I/R injury, suggesting that targeting the NAT10/Mybbp1a/p53 axis may be a novel approach for treating cardiac I/R.

Heat sensitive E-helix cut ferritin nanocages for facile and high-efficiency loading of doxorubicin.

Ferritin possesses a stable and uniform cage structure, along with tumor-targeting properties and excellent biocompatibility, making it a promising drug delivery vehicle. However, the current ferritin drug loading strategy involves complex steps and harsh reaction conditions, resulting in low yield and recovery of drug loading, which limits the clinical application prospects of ferritin nanomedicine. In this study, we utilized the high-efficiency heat-sensitivity of the multiple channel switch structures of the E-helix-cut ferritin mutant (Ecut-HFn) and Cu2+ assistance to achieve high-efficiency loading of chemotherapeutic drugs in a one-step process at low temperatures. This method features mild reaction conditions (45 °C), high loading efficiency (about 110 doxorubicin (Dox) per Ecut-HFn), and improved protein and Dox recovery rates (with protein recovery rate around 94 % and Dox recovery rate reaching up to 45 %). The prepared ferritin-Dox particles (Ecut-HFn-Cu-Dox) exhibit a uniform size distribution, good stability, and retain the natural tumor targeting ability of ferritin. Overall, this temperature-controlled drug loading strategy utilizing heat-sensitivity ferritin mutants is energy-saving, environmentally friendly, efficient, and easy to operate, offering a new perspective for scaling up the industrial production of ferritin drug carriers.

Exosomes secreted from cardiomyocytes suppress the sensitivity of tumor ferroptosis in ischemic heart failure.

Heart failure (HF) patients in general have a higher risk of developing cancer. Several animal studies have indicated that cardiac remodeling and HF remarkably accelerate tumor progression, highlighting a cause-and-effect relationship between these two disease entities. Targeting ferroptosis, a prevailing form of non-apoptotic cell death, has been considered a promising therapeutic strategy for human cancers. Exosomes critically contribute to proximal and distant organ-organ communications and play crucial roles in regulating diseases in a paracrine manner. However, whether exosomes control the sensitivity of cancer to ferroptosis via regulating the cardiomyocyte-tumor cell crosstalk in ischemic HF has not yet been explored. Here, we demonstrate that myocardial infarction (MI) decreased the sensitivity of cancer cells to the canonical ferroptosis activator erastin or imidazole ketone erastin in a mouse model of xenograft tumor. Post-MI plasma exosomes potently blunted the sensitivity of tumor cells to ferroptosis inducers both in vitro in mouse Lewis lung carcinoma cell line LLC and osteosarcoma cell line K7M2 and in vivo with xenograft tumorigenesis model. The expression of miR-22-3p in cardiomyocytes and plasma-exosomes was significantly upregulated in the failing hearts of mice with chronic MI and of HF patients as well. Incubation of tumor cells with the exosomes isolated from post-MI mouse plasma or overexpression of miR-22-3p alone abrogated erastin-induced ferroptotic cell death in vitro. Cardiomyocyte-enriched miR-22-3p was packaged in exosomes and transferred into tumor cells. Inhibition of cardiomyocyte-specific miR-22-3p by AAV9 sponge increased the sensitivity of cancer cells to ferroptosis. ACSL4, a pro-ferroptotic gene, was experimentally established as a target of miR-22-3p in tumor cells. Taken together, our findings uncovered for the first time that MI suppresses erastin-induced ferroptosis through releasing miR-22-3p-enriched exosomes derived from cardiomyocytes. Therefore, targeting exosome-mediated cardiomyocyte/tumor pathological communication may offer a novel approach for the ferroptosis-based antitumor therapy.

Magnetosome-inspired synthesis of soft ferrimagnetic nanoparticles for magnetic tumor targeting.

Magnetic targeting is one of the most promising approaches for improving the targeting efficiency by which magnetic drug carriers are directed using external magnetic fields to reach their targets. As a natural magnetic nanoparticle (MNP) of biological origin, the magnetosome is a special "organelle" formed by biomineralization in magnetotactic bacteria (MTB) and is essential for MTB magnetic navigation to respond to geomagnetic fields. The magnetic targeting of magnetosomes, however, can be hindered by the aggregation and precipitation of magnetosomes in water and biological fluid environments due to the strong magnetic attraction between particles. In this study, we constructed a magnetosome-like nanoreactor by introducing MTB Mms6 protein into a reverse micelle system. MNPs synthesized by thermal decomposition exhibit the same crystal morphology and magnetism (high saturation magnetization and low coercivity) as natural magnetosomes but have a smaller particle size. The DSPE-mPEG-coated magnetosome-like MNPs exhibit good monodispersion, penetrating the lesion area of a tumor mouse model to achieve magnetic enrichment by an order of magnitude more than in the control groups, demonstrating great prospects for biomedical magnetic targeting applications.

Rational design of dual-functional surfaces on polypropylene with antifouling and antibacterial performances via a micropatterning strategy.

The hydrophobicity and inertness of the polypropylene (PP) material surface usually lead to serious biofouling and bacterial infections, which hamper its potential application as a biomedical polymer. Many strategies have been developed to improve its antifouling or antibacterial properties, yet designing a surface to achieve both antifouling and antibacterial performances simultaneously remains a challenge. Herein, we construct a dual-function micropatterned PP surface with antifouling and antibacterial properties through plasma activation, photomask technology and ultraviolet light-induced graft polymerization. Based on the antifouling agent poly(2-methacryloyloxyethyl phosphate choline) (PMPC) and the antibacterial agent quaternized poly(N,N-dimethylamino)ethyl methacrylate (QPDMAEMA), two different micropatterning structures have been successfully prepared: PP-PMPC-QPDMAEMA in which QPDMAEMA is the micropattern and PMPC is the coating polymer, and PP-QPDMAEMA-PMPC in which PMPC is the micropattern and QPDMAEMA is the coating polymer. The composition, elemental distribution and surface morphology of PP-PMPC-QPDMAEMA and PP-QPDMAEMA-PMPC have been thoroughly characterized by Fourier transform infrared (FT-IR) spectroscopy, X-ray photoelectron spectroscopy (XPS) and scanning electron microscopy (SEM), respectively. Compared with pristine PP, the two types of micropatterned PP films exhibit good surface hydrophilicity as characterized by water contact angle measurements. The results of anti-protein adsorption, platelet adhesion and antibacterial evaluation showed that PP-PMPC-QPDMAEMA and PP-QPDMAEMA-PMPC had good anti-protein adsorption properties, especially for lysozyme (Lyz). They can effectively prevent platelet adhesion, and the anti-platelet adhesion performance of PP-QPDMAEMA-PMPC is slightly better than that of the PP-PMPC-QPDMAEMA sample. The sterilization rate of S. aureus and E. coli is as high as 95% for the two types of micropatterned PP films. Due to the rational design of micropatterns on the PP surface, the two classes of dual-functional PP materials realize both the resistance of protein and platelet adhesion, and the killing of bacteria at the same time. We anticipate that this work could provide a design strategy for the construction of multifunctional biomedical polymer materials.

Modulation of MagR magnetic properties via iron-sulfur cluster binding.

Iron-sulfur clusters are essential cofactors found in all kingdoms of life and play essential roles in fundamental processes, including but not limited to respiration, photosynthesis, and nitrogen fixation. The chemistry of iron-sulfur clusters makes them ideal for sensing various redox environmental signals, while the physics of iron-sulfur clusters and its host proteins have been long overlooked. One such protein, MagR, has been proposed as a putative animal magnetoreceptor. It forms a rod-like complex with cryptochromes (Cry) and possesses intrinsic magnetic moment. However, the magnetism modulation of MagR remains unknown. Here in this study, iron-sulfur cluster binding in MagR has been characterized. Three conserved cysteines of MagR play different roles in iron-sulfur cluster binding. Two forms of iron-sulfur clusters binding have been identified in pigeon MagR and showed different magnetic properties: [3Fe-4S]-MagR appears to be superparamagnetic and has saturation magnetization at 5 K but [2Fe-2S]-MagR is paramagnetic. While at 300 K, [2Fe-2S]-MagR is diamagnetic but [3Fe-4S]-MagR is paramagnetic. Together, the different types of iron-sulfur cluster binding in MagR attribute distinguished magnetic properties, which may provide a fascinating mechanism for animals to modulate the sensitivity in magnetic sensing.

Surface morphology and payload synergistically caused an enhancement of the longitudinal relaxivity of a Mn3O4/PtOx nanocomposite for magnetic resonance tumor imaging.

The construction of surface structures of manganese oxide nanoparticles (MONs) in order to promote their longitudinal relaxivity r1 to surpass those of commercially available Gd(iii) complexes is still a significant challenge. Herein, we successfully obtained Mn3O4/PtOx nanocomposites (NCs) with an r1 of 20.48 mM-1 s-1, four times higher than that of commercially available Gd-DTPA (5.11 mM-1 s-1). The r2/r1 ratio of these NCs is 1.46 lower than that of Gd-DTPA (2.38). This is the first time that such excellent T1 contrast performance has been achieved using MONs via synergistically utilizing the surface morphology and surface payload. These NCs are composed of porous Mn3O4"skeleton" nanostructures decorated with tiny PtOx nanoparticles (NPs) that are realized using laser ablation and irradiation in liquid and ion etching steps. Experimental results showed that the enlarged specific area of the porous Mn3O4/PtOx NCs and the payload of ultrafine PtOx NPs synergistically facilitated the T1 contrast capabilities. The former favors sufficient proton-electron interactions and the latter reduces the global molecular tumbling motion. These NCs also exhibit an evident computed tomography (CT) attenuation value of 24.13 HU L g-1, which is much better than that achieved using the commercial product iopromide (15.9 HU L g-1). The outstanding magnetic resonance (MR) imaging and CT imaging performances of the Mn3O4/PtOx NCs were proved through in vivo experiments. Histological examinations and blood circulation assays confirmed the good biosafety of the NCs. These novel findings showcase a brand-new strategy for fabricating excellent MON T1 contrast agents (CAs) on the basis of the surface structure and they pave the way for their practical clinical applications in dual-modal imaging.

Mettl14 Attenuates Cardiac Ischemia/Reperfusion Injury by Regulating Wnt1/ß-Catenin Signaling Pathway.

N6-methyladenosine (m6A) methylation in RNA is a dynamic and reversible modification regulated by methyltransferases and demethylases, which has been reported to participate in many pathological processes of various diseases, including cardiac disorders. This study was designed to investigate an m6A writer Mettl14 on cardiac ischemia-reperfusion (I/R) injury and uncover the underlying mechanism. The m6A and Mettl14 protein levels were increased in I/R hearts and neonatal mouse cardiomyocytes upon oxidative stress. Mettl14 knockout (Mettl14+/-) mice showed pronounced increases in cardiac infarct size and LDH release and aggravation in cardiac dysfunction post-I/R. Conversely, adenovirus-mediated overexpression of Mettl14 markedly reduced infarct size and apoptosis and improved cardiac function during I/R injury. Silencing of Mettl14 alone significantly caused a decrease in cell viability and an increase in LDH release and further exacerbated these effects in the presence of H2O2, while overexpression of Mettl14 ameliorated cardiomyocyte injury in vitro. Mettl14 resulted in enhanced levels of Wnt1 m6A modification and Wnt1 protein but not its transcript level. Furthermore, Mettl14 overexpression blocked I/R-induced downregulation of Wnt1 and ß-catenin proteins, whereas Mettl14+/- hearts exhibited the opposite results. Knockdown of Wnt1 abrogated Mettl14-mediated upregulation of ß-catenin and protection against injury upon H2O2. Our study demonstrates that Mettl14 attenuates cardiac I/R injury by activating Wnt/ß-catenin in an m6A-dependent manner, providing a novel therapeutic target for ischemic heart disease.

Rational Design of PMPC/PDMC/PEGDA Hydrogel Micropatterns onto Polylactic Acid with Enhanced Biological Activity.

Polylactic acid (PLA) is one of the biodegradable materials that has been used in the areas of surgical healing lines, cancer treatment, and wound healing. However, the application of PLA is still rather limited due to its high hydrophobicity and poor antibacterial activity. In order to enhance the antifouling and antibacterial performances of PLA, here we modified the surface of PLA with various sizes of hydrogel micropatterns in negative or positive mode using plasma treatment, the photomask technique, and UV-graft polymerization. The hydrogel micropatterns consist of poly(ethylene glycol) diacrylate (PEGDA), poly(2-methacryloyloxyethylphosphorylcholine) (PMPC), and poly(methacryloyloxyethyltrimethylammonium chloride) (PDMC). Compared to PLA, the patterned PLA (PLA-PMPC/PDMC/PEGDA) shows obviously enhanced antifouling and antibacterial activities. For PLA-PMPC/PDMC/PEGDA with either positive or negative micropatterns, the antifouling and antibacterial properties are gradually increasing with decreasing the size of micropatterns. Compared with PLA-PMPC/PDMC/PEGDA bearing positive and negative micropatterns in the same size, the PLA-PMPC/PDMC/PEGDA with negative micropatterns exhibits slightly better biological activity and the PLA-PMPC/PDMC/PEGDA with 3 µm negative hydrogel micropatterns shows the best hydrophilicity, antifouling, and antibacterial properties. Combining the in vitro hemolysis assay, cytotoxicity, water absorption test, and degradation test results, it is suggested that the fabrication of hydrogel micropatterns onto the PLA surface could significantly improve biological activities of PLA. We expect that this work would provide a new strategy to potentially develop PLA as a promising wound dressing.

Highly Efficient Cyclic Dinucleotide Based Artificial Metalloribozymes for Enantioselective Friedel-Crafts Reactions in Water.

The diverse secondary structures of nucleic acids are emerging as attractive chiral scaffolds to construct artificial metalloenzymes (ArMs) for enantioselective catalysis. DNA-based ArMs containing duplex and G-quadruplex scaffolds have been widely investigated, yet RNA-based ArMs are scarce. Here we report that a cyclic dinucleotide of c-di-AMP and Cu2+ ions assemble into an artificial metalloribozyme (c-di-AMP⋅Cu2+ ) that enables catalysis of enantioselective Friedel-Crafts reactions in aqueous media with high reactivity and excellent enantioselectivity of up to 97 % ee. The assembly of c-di-AMP⋅Cu2+ gives rise to a 20-fold rate acceleration compared to Cu2+ ions. Based on various biophysical techniques and density function theory (DFT) calculations, a fine coordination structure of c-di-AMP⋅Cu2+ metalloribozyme is suggested in which two c-di-AMP form a dimer scaffold and the Cu2+ ion is located in the center of an adenine-adenine plane through binding to two N7 nitrogen atoms and one phosphate oxygen atom.

Chemical synthesis, purification, and characterization of 3'-5'-linked canonical cyclic dinucleotides (CDNs).

So far, four cyclic dinucleotides (CDNs) have been discovered as important second messengers in nature, where three canonical CDNs of c-di-GMP, c-di-AMP and c-AMP-GMP were found in bacteria containing two 3'-5' phosphodiester linkages and one non-canonical CDN 2'3'-c-GMP-AMP was identified in mammals containing mixed 2'-5' and 3'-5' phosphodiester linkages. The CDNs are produced by specific cyclases and degraded by phosphodiesterases (PDEs). All of the known CDNs could bind to the stimulator of interferon genes (STING) to induce type I interferon (IFN) responses and the three bacterial CDNs are sensed by specific riboswitches to regulate gene expression. The emerging physiological functions of bacterial CDNs lead the motivation to investigate other possible canonical CDNs. In recent years, many endeavors have been devoted to develop fast, convenient and cheap strategies for chemically synthesizing CDNs and their analogues. The phosphoramidite approach using commercial starting materials has attracted much attention. Herein, we describe an adapted one-pot strategy that enables fast synthesis of crude 3'-5'-linked canonical CDNs followed by purification of the obtained CDNs using reversed phase high-performance of liquid chromatography (HPLC). Furthermore, we report the full characterization of CDNs by mass spectrometry (MS) and nuclear magnetic resonance (NMR) techniques.

Fabrication of PMPC/PTM/PEGDA micropatterns onto polypropylene films behaving with dual functions of antifouling and antimicrobial activities.

Polymer materials with high biocompatibility and versatile functions are urgently required in the biomedical field. The hydrophobic surface and inert traits of polymer materials usually encounter severe biofouling and bacterial infection which hinder the potential application of polymers as biomedical materials. Although many antifouling or antimicrobial coatings have been developed for modification of biomedical devices/implants, few can simultaneously fulfill the requirements for antimicrobial and antifouling activities. Herein, we constructed bifunctional micropatterns with antifouling and antimicrobial properties onto polypropylene (PP) films using argon plasma activation treatment, photomask technique and UV-initiated graft polymerization method. Different sizes of PMPC/PTM/PEGDA micropatterns were fabricated on PP films to yield patterned PP-PMPC/PTM/PEGDA as evidenced by infrared (IR) spectroscopy, scanning electron microscopy (SEM) and X-ray photoelectron spectroscopy (XPS), where PMPC is poly(2-methacryloyloxyethyl phosphorylcholine) for enhancement of hydrophilicity and biocompatibility, PTM is poly(methacryloyloxyethyltrimethylammonium chloride) for contribution to antimicrobial activity and PEGDA is poly(ethylene glycol diacrylate) as the crosslinker. The surface hydrophilicity of patterned PP-PMPC/PTM/PEGDA was characterized by the static water contact angle test. The results showed that the PP sample with a micropattern with the size of 5 µm exhibited the best hydrophilicity. For biological assays of patterned PP-PMPC/PTM/PEGDA, the micropattern size at 5 µm performed the best for both antiplatelet adhesion and antimicrobial activities. We anticipate that this work could provide a new method for building bifunctional biomedical materials to promote the application of PP in biomedical fields.

Widespread bacterial lysine degradation proceeding via glutarate and L-2-hydroxyglutarate.

Lysine degradation has remained elusive in many organisms including Escherichia coli. Here we report catabolism of lysine to succinate in E. coli involving glutarate and L-2-hydroxyglutarate as intermediates. We show that CsiD acts as an α-ketoglutarate-dependent dioxygenase catalysing hydroxylation of glutarate to L-2-hydroxyglutarate. CsiD is found widespread in bacteria. We present crystal structures of CsiD in complex with glutarate, succinate, and the inhibitor N-oxalyl-glycine, demonstrating strong discrimination between the structurally related ligands. We show that L-2-hydroxyglutarate is converted to α-ketoglutarate by LhgO acting as a membrane-bound, ubiquinone-linked dehydrogenase. Lysine enters the pathway via 5-aminovalerate by the promiscuous enzymes GabT and GabD. We demonstrate that repression of the pathway by CsiR is relieved upon glutarate binding. In conclusion, lysine degradation provides an important link in central metabolism. Our results imply the gut microbiome as a potential source of glutarate and L-2-hydroxyglutarate associated with human diseases such as cancer and organic acidurias.

Synthesis of All Possible Canonical (3'-5'-Linked) Cyclic Dinucleotides and Evaluation of Riboswitch Interactions and Immune-Stimulatory Effects.

The cyclic dinucleotides (CDNs) c-di-GMP, c-di-AMP, and c-AMP-GMP are widely utilized as second messengers in bacteria, where they signal lifestyle changes such as motility and biofilm formation, cell wall and membrane homeostasis, virulence, and exo-electrogenesis. For all known bacterial CDNs, specific riboswitches have been identified that alter gene expression in response to the second messengers. In addition, bacterial CDNs trigger potent immune responses, making them attractive as adjuvants in immune therapies. Besides the three naturally occurring CDNs, seven further CDNs containing canonical 3'-5'-linkages are possible by combining the four natural ribonucleotides. Herein, we have synthesized all ten possible combinations of 3'-5'-linked CDNs. The binding affinity of novel CDNs and GEMM riboswitch variants was assessed utilizing a spinach aptamer fluorescence assay and in-line probing assays. The immune-stimulatory effect of CDNs was evaluated by induction of type I interferons (IFNs), and a novel CDN c-AMP-CMP was identified as a new immune-stimulatory agent.

Identification of New FLT3 Inhibitors That Potently Inhibit AML Cell Lines via an Azo Click-It/Staple-It Approach.

Acute myeloid leukemia (AML) is an aggressive malignancy with only a handful of therapeutic options. About 30% of AML patients harbor mutated FLT3 kinase, and thus, this cancer-driver has become a hotly pursued AML target. Herein we report a new class of FLT3 inhibitors, which potently inhibit the proliferation of acute myeloid leukemia (AML) cells at nanomolar concentrations.

Alkyne-substituted diminazene as G-quadruplex binders with anticancer activities.

G-quadruplex ligands have been touted as potential anticancer agents, however, none of the reported G-quadruplex-interactive small molecules have gone past phase II clinical trials. Recently it was revealed that diminazene (berenil, DMZ) actually binds to G-quadruplexes 1000 times better than DNA duplexes, with dissociation constants approaching 1 nM. DMZ however does not have strong anticancer activities. In this paper, using a panel of biophysical tools, including NMR, FRET melting assay and FRET competition assay, we discovered that monoamidine analogues of DMZ bearing alkyne substitutes selectively bind to G-quadruplexes. The lead DMZ analogues were shown to be able to target c-MYC G-quadruplex both in vitro and in vivo. Alkyne DMZ analogues display respectable anticancer activities (single digit micromolar GI50) against ovarian (OVCAR-3), prostate (PC-3) and triple negative breast (MDA-MB-231) cancer cell lines and represent interesting new leads to develop anticancer agents.

Terpyridine-Cu(ii) targeting human telomeric DNA to produce highly stereospecific G-quadruplex DNA metalloenzyme.

The cofactors commonly involved in natural enzymes have provided the inspiration for numerous advances in the creation of artificial metalloenzymes. Nevertheless, to design an appropriate cofactor for a given biomolecular scaffold or vice versa remains a challenge in developing efficient catalysts in biochemistry. Herein, we extend the idea of G-quadruplex-targeting anticancer drug design to construct a G-quadruplex DNA metalloenzyme. We found that a series of terpyridine-Cu(ii) complexes (CuLn) can serve as excellent cofactors to dock with human telemetric G-quadruplex DNA. The resulting G-quadruplex DNA metalloenzyme utilising CuL1 catalyzes an enantioselective Diels-Alder reaction with enantioselectivity of >99% enantiomeric excess and about 73-fold rate acceleration compared to CuL1 alone. The terpyridine-Cu(ii) complex cofactors demonstrate dual functions, both as an active site to perform catalysis and as a structural regulator to promote the folding of human telemetric G-quadruplex DNA towards excellent catalysts.