Neutrophil elastase selectively kills cancer cells and attenuates tumorigenesis.

Cancer cell genetic variability and similarity to host cells have stymied development of broad anti-cancer therapeutics. Our innate immune system evolved to clear genetically diverse pathogens and limit host toxicity; however, whether/how innate immunity can produce similar effects in cancer is unknown. Here, we show that human, but not murine, neutrophils release catalytically active neutrophil elastase (ELANE) to kill many cancer cell types while sparing non-cancer cells. ELANE proteolytically liberates the CD95 death domain, which interacts with histone H1 isoforms to selectively eradicate cancer cells. ELANE attenuates primary tumor growth and produces a CD8+T cell-mediated abscopal effect to attack distant metastases. Porcine pancreatic elastase (ELANE homolog) resists tumor-derived protease inhibitors and exhibits markedly improved therapeutic efficacy. Altogether, our studies suggest that ELANE kills genetically diverse cancer cells with minimal toxicity to non-cancer cells, raising the possibility of developing it as a broad anti-cancer therapy.

Ribonucleotide Reductases: Structure, Chemistry, and Metabolism Suggest New Therapeutic Targets.

Ribonucleotide reductases (RNRs) catalyze the de novo conversion of nucleotides to deoxynucleotides in all organisms, controlling their relative ratios and abundance. In doing so, they play an important role in fidelity of DNA replication and repair. RNRs' central role in nucleic acid metabolism has resulted in five therapeutics that inhibit human RNRs. In this review, we discuss the structural, dynamic, and mechanistic aspects of RNR activity and regulation, primarily for the human and Escherichia coli class Ia enzymes. The unusual radical-based organic chemistry of nucleotide reduction, the inorganic chemistry of the essential metallo-cofactor biosynthesis/maintenance, the transport of a radical over a long distance, and the dynamics of subunit interactions all present distinct entry points toward RNR inhibition that are relevant for drug discovery. We describe the current mechanistic understanding of small molecules that target different elements of RNR function, including downstream pathways that lead to cell cytotoxicity. We conclude by summarizing novel and emergent RNR targeting motifs for cancer and antibiotic therapeutics.

Homeostatic Control of Sebaceous Glands by Innate Lymphoid Cells Regulates Commensal Bacteria Equilibrium.

Immune cells and epithelium form sophisticated barrier systems in symbiotic relationships with microbiota. Evidence suggests that immune cells can sense microbes through intact barriers, but regulation of microbial commensalism remain largely unexplored. Here, we uncovered spatial compartmentalization of skin-resident innate lymphoid cells (ILCs) and modulation of sebaceous glands by a subset of RORγt+ ILCs residing within hair follicles in close proximity to sebaceous glands. Their persistence in skin required IL-7 and thymic stromal lymphopoietin, and localization was dependent on the chemokine receptor CCR6. ILC subsets expressed TNF receptor ligands, which limited sebocyte growth by repressing Notch signaling pathway. Consequently, loss of ILCs resulted in sebaceous hyperplasia with increased production of antimicrobial lipids and restricted commensalism of Gram-positive bacterial communities. Thus, epithelia-derived signals maintain skin-resident ILCs that regulate microbial commensalism through sebaceous gland-mediated tuning of the barrier surface, highlighting an immune-epithelia circuitry that facilitates host-microbe symbiosis.

A hyper-quiescent chromatin state formed during aging is reversed by regeneration.

Epigenetic alterations are a key hallmark of aging but have been limitedly explored in tissues. Here, using naturally aged murine liver as a model and extending to other quiescent tissues, we find that aging is driven by temporal chromatin alterations that promote a refractory cellular state and compromise cellular identity. Using an integrated multi-omics approach and the first direct visualization of aged chromatin, we find that globally, old cells show H3K27me3-driven broad heterochromatinization and transcriptional suppression. At the local level, site-specific loss of H3K27me3 over promoters of genes encoding developmental transcription factors leads to expression of otherwise non-hepatocyte markers. Interestingly, liver regeneration reverses H3K27me3 patterns and rejuvenates multiple molecular and physiological aspects of the aged liver.

2.6-Å resolution cryo-EM structure of a class Ia ribonucleotide reductase trapped with mechanism-based inhibitor N3CDP.

Ribonucleotide reductases (RNRs) reduce ribonucleotides to deoxyribonucleotides using radical-based chemistry. For class Ia RNRs, the radical species is stored in a separate subunit (ß2) from the subunit housing the active site (α2), requiring the formation of a short-lived α2ß2 complex and long-range radical transfer (RT). RT occurs via proton-coupled electron transfer (PCET) over a long distance (~32-Å) and involves the formation and decay of multiple amino acid radical species. Here, we use cryogenic electron microscopy and a mechanism-based inhibitor 2'-azido-2'-deoxycytidine-5'-diphosphate (N3CDP) to trap a wild-type α2ß2 complex of Escherichia coli class Ia RNR. We find that one α subunit has turned over and that the other is trapped, bound to ß in a midturnover state. Instead of N3CDP in the active site, forward RT has resulted in N2 loss, migration of the third nitrogen from the ribose C2' to C3' positions, and attachment of this nitrogen to the sulfur of cysteine-225. In this study, an inhibitor has been visualized as an adduct to an RNR. Additionally, this structure reveals the positions of PCET residues following forward RT, complementing the previous structure that depicted a preturnover PCET pathway and suggesting how PCET is gated at the α-ß interface. This N3CDP-trapped structure is also of sufficient resolution (2.6 Å) to visualize water molecules, allowing us to evaluate the proposal that water molecules are proton acceptors and donors as part of the PCET process.

Metabolic regulation of gene expression by histone lactylation.

The Warburg effect, which originally described increased production of lactate in cancer, is associated with diverse cellular processes such as angiogenesis, hypoxia, polarization of macrophages and activation of T cells. This phenomenon is intimately linked to several diseases including neoplasia, sepsis and autoimmune diseases1,2. Lactate, which is converted from pyruvate in tumour cells, is widely known as an energy source and metabolic by-product. However, its non-metabolic functions in physiology and disease remain unknown. Here we show that lactate-derived lactylation of histone lysine residues serves as an epigenetic modification that directly stimulates gene transcription from chromatin. We identify 28 lactylation sites on core histones in human and mouse cells. Hypoxia and bacterial challenges induce the production of lactate by glycolysis, and this acts as a precursor that stimulates histone lactylation. Using M1 macrophages that have been exposed to bacteria as a model system, we show that histone lactylation has different temporal dynamics from acetylation. In the late phase of M1 macrophage polarization, increased histone lactylation induces homeostatic genes that are involved in wound healing, including Arg1. Collectively, our results suggest that an endogenous 'lactate clock' in bacterially challenged M1 macrophages turns on gene expression to promote homeostasis. Histone lactylation thus represents an opportunity to improve our understanding of the functions of lactate and its role in diverse pathophysiological conditions, including infection and cancer.

Isolation, identification, and pathogenicity of two novel goose astrovirus from goslings with severe gout in China.

Goose astroviruses (GAstVs) are important pathogens which can cause gout in goslings leading to huge economic losses for the goose farming industry in China. In 2023, an infectious disease characterized by visceral gout broke out in commercial goose farms in Guangxi and Guangdong provinces of China. In this study, two GAstV strains of GXNN and GDCS were successfully isolated from these two disease-ridden goose farms. The complete genomic lengths of these two strains were 7166 bp, and phylogenetic analysis showed that they were both GAstV-2 subtypes. The 3-dimensional structures of the capsid protein were predicted and six characteristic mutation sites at amino acid positions 60, 61, 228, 229, 456 and 523 were found within the strong antigenic regions. A recombination event occurred at 6833-7070 nt between the GAstV TZ03 and Turkey astrovirus CA/00 and this was detected in both the GXNN and GDCS strains. Another recombinant event occurred at 63-2747 nt between the GAstV XT1 and GAstV SDPY and this was detected in the GDCS strain. When 1-day-old goslings were infected with the novel GXNN and GDCS strains, they showed severe visceral gout. This was accompanied by enlarged spleens, liver hemorrhages and urate deposits in the kidneys and ureters and their blood urea nitrogen levels were significantly elevated. The mortality rates of the GXNN- and GDCS-infected groups were pathogenically high at 80 % and 60 %, respectively. These results will promote our understanding of the evolution and epidemic potential of GAstVs in China.

Remifentanil-induced inflammation in microglial cells: Activation of the PAK4-mediated NF-κB/NLRP3 pathway and onset of hyperalgesia.

BACKGROUND: The perioperative use of remifentanil is associated with postoperative hyperalgesia, which can impair recovery and extend hospitalization. Recent studies have revealed that microglia-mediated activation of the NLRP3 inflammasome plays a critical role in opioid-induced hyperalgesia, with NF-κB acting as a pivotal activation point for NLRP3. Despite these findings, the specific molecular mechanisms underlying remifentanil-induced postoperative hyperalgesia remain unclear. This study aims to develop a model of remifentanil-induced hyperalgesia and investigate the molecular mechanisms, focusing on the NF-κB/NLRP3 pathway, using both in vitro and in vivo approaches. METHOD: We established a remifentanil-induced hyperalgesia model and performed proteomic analysis to identify differential protein expression in the spinal cord tissue of rats. NLRP3 or PAK4 antagonists were administered intrathecally in vivo, and mechanical pain thresholds in the hind paws were measured using Von Frey testing. In vitro, we applied NLRP3 or PAK4 inhibitors or used lentivirus infection to silence PAK4, NF-κB, and NLRP3 genes. Protein expression was assessed through immunohistochemistry, immunofluorescence, and Western blotting. Additionally, ELISA was performed to measure IL-1ß and IL-18 levels, and RT-qPCR was conducted to evaluate the transcription of target genes. RESULTS: Proteomic analysis revealed that remifentanil upregulates PAK4 protein in spinal cord tissue two hours after the surgery. In addition, remifentanil induces morphological changes in the spinal cord dorsal horn, characterized by increased expression of PAK4, p-p65, NLRP3 and Iba-1 proteins, which in turn leads to elevated IL-1ß and IL-18 levels and an inflammatory response. Intrathecal injection of NLRP3 or PAK4 inhibitors mitigates remifentanil-induced hyperalgesia and associated changes. In vitro, downregulation of PAK4 inhibits the increase in PAK4, p-p65, NLRP3 and Caspase-1 induced by LPS. Conversely, the downregulation of NLRP3 does not impact the levels of PAK4 and p-p65 proteins, aligning with the in vivo results and suggesting that PAK4 acts as an upstream signaling molecule of NLRP3. CONCLUSION: Remifentanil can increase PAK4 expression in spinal cord dorsal horn cells by activating the NF-κB/NLRP3 pathway and mediating microglial activation, thereby contributing to postoperative hyperalgesia.

Piezo1 channel activation facilitates baroreflex afferent neurotransmission with subsequent blood pressure reduction in control and hypertension rats.

Mechanosensitive cation channels such as Piezo1 and Piezo2 are activated by mechanical force like a starched wall of the aorta while blood pressure (BP) rising, which helps to elucidate the underlying mechanism of mechanotransduction of baroreceptor endings. In this study we investigated how Piezo1 channel activation-mediated gender- and afferent-specific BP regulation in rats. We established high-fat diet and fructose drink-induced hypertension model rats (HFD-HTN) and deoxycorticosterone (DOCA)-sensitive hypertension model rats. We showed that the expression levels of Piezo1 and Piezo2 were significantly up-regulated in left ventricle of HFD and DOCA hypertensive rats, whereas the down-regulation of Piezo1 was likely to be compensated by Piezo2 up-regulation in the aorta. Likewise, down-regulated Piezo1 was observed in the nodose ganglion (NG), while up-regulated Piezo2 was found in the nucleus tractus solitarius (NTS), which might synergistically reduce the excitatory neurotransmitter release from the presynaptic membrane. Notably, microinjection of Yoda1 (0.025-2.5 mg/ml) into the NG concentration-dependently reduced BP in both hypertensive rat models as well as in control rats with similar EC50; the effect of Yoda1 was abolished by microinjection of a Piezo1 antagonist GsMTx4 (1.0 µM). Functional analysis in an in vitro aortic arch preparation showed that instantaneous firing frequency of single Ah-fiber of aortic depressor nerve was dramatically increased by Yoda1 (0.03-1.0 µM) and blocked by GsMTx4 (1.0 µM). Moreover, spontaneous synaptic currents recorded from identified 2nd-order Ah-type baroreceptive neurons in the NTS was also facilitated over 100% by Yoda1 (1.0 µM) and completely blocked by GsMTx4 (3.0 µM). These results demonstrate that Piezo1 expressed on Ah-type baroreceptor and baroreceptive neurons in the NG and NTS plays a key role in a sexual-dimorphic BP regulation under physiological and hypertensive condition through facilitation of baroreflex afferent neurotransmission, which is presumably collaborated by Piezo2 expression at different level of baroreflex afferent pathway via compensatory and synergistic mechanisms.

The crucial relationship between miRNA-27 and CSE/H2S, and the mechanism of action of GLP-1 in myocardial hypertrophy.

Cardiac hypertrophy is the most prevalent compensatory heart disease that ultimately leads to spontaneous heart failure. Mounting evidence suggests that microRNAs (miRs) and endogenous hydrogen sulfide (H2S) play a crucial role in the regulation of cardiac hypertrophy. In this study, we aimed to investigate whether inhibition of miR-27a could protect against cardiac hypertrophy by modulating H2S signaling. We established a model of cardiac hypertrophy by obtaining hypertrophic tissue from mice subjected to transverse aortic constriction (TAC) and from cells treated with angiotensin-II. Molecular alterations in the myocardium were quantified using quantitative real time PCR (qRT-PCR), Western blotting, and ELISA. Morphological changes were characterized by hematoxylin and eosin (HE) staining and Masson's trichrome staining. Functional myocardial changes were assessed using echocardiography. Our results demonstrated that miR-27a levels were elevated, while H2S levels were reduced in TAC mice and myocardial hypertrophy. Further luciferase and target scan assays confirmed that cystathionine-γ-lyase (CSE) was a direct target of miR-27a and was negatively regulated by it. Notably, enhancement of H2S expression in the heart was observed in mice injected with recombinant adeno-associated virus vector 9 (rAAV9)-anti-miR-27a and in cells transfected with a miR-27a inhibitor during cardiac hypertrophy. However, this effect was abolished by co-transfection with CSE siRNA and the miR-27a inhibitor. Conversely, injecting rAAV9-miR-27a yielded opposite results. Interestingly, our findings demonstrated that glucagon-like peptide-1 (GLP-1) agonists could mitigate myocardial damage by down-regulating miR-27a and up-regulating CSE. In summary, our study suggests that inhibition of miR-27a holds therapeutic promise for the treatment of cardiac hypertrophy by increasing H2S levels. Furthermore, our findings unveil a novel mechanism of GLP-1 agonists involving the miR-27a/H2S pathway in the management of cardiac hypertrophy.

Tubular lysosomes harbor active ion gradients and poise macrophages for phagocytosis.

Lysosomes adopt dynamic, tubular states that regulate antigen presentation, phagosome resolution, and autophagy. Tubular lysosomes are studied either by inducing autophagy or by activating immune cells, both of which lead to cell states where lysosomal gene expression differs from the resting state. Therefore, it has been challenging to pinpoint the biochemical properties lysosomes acquire upon tubulation that could drive their functionality. Here we describe a DNA-based assembly that tubulates lysosomes in macrophages without activating them. Proteolytic activity maps at single-lysosome resolution revealed that tubular lysosomes were less degradative and showed proximal to distal luminal pH and Ca2+ gradients. Such gradients had been predicted but never previously observed. We identify a role for tubular lysosomes in promoting phagocytosis and activating MMP9. The ability to tubulate lysosomes without starving or activating immune cells may help reveal new roles for tubular lysosomes.

The feasibility of atrial Fibrillatory wave amplitude in predicting ablation outcomes in persistent atrial fibrillation.

BACKGROUND: Atrial fibrosis has a significant impact on the success rate of catheter ablation (CA) treatment of atrial fibrillation (AF). The fibrotic tissues could be reflected by the amplitude of the fibrillatory wave (F-wave). METHODS AND RESULTS: 704 patients with persistent AF and at least 1-year follow-up after CA were included as the internal group. 101 patients from another hospital were used as the external validation cohort. A 12­lead ECG was performed before CA and the maximum FWA in three ECG leads (aVL, aVF, V1) were measured. The FWA score (0 to 6 points according to the amplitude range of the three leads) of each patients was calculated. Five models including clinical features, FWA score, CHA2DS2-VASc score, APPLE score and the fusion of clinical features and FWA score were built. The FWA score was superior to the model constructed by clinical variables, CHA2DS2-VASc score and APPLE score. It not only had good predictive performance for AF recurrence, with an AUC value of 0.812 (95% CI 0.724-0.900), but also showed a significant predictive value for the recurrence rate according to F-wave amplitude. In the external validation cohort, the FWA score showed similar results (AUC 0.768, 95% CI 0.672-0.865). CONCLUSIONS: The present study reveals the significant predictive value of the FWA score for persistent AF ablation recurrence.

Radical Transport Facilitated by a Proton Transfer Network at the Subunit Interface of Ribonucleotide Reductase.

Ribonucleotide reductases (RNRs) play an essential role in the conversion of nucleotides to deoxynucleotides in all organisms. The Escherichia coli class Ia RNR requires two homodimeric subunits, α and ß. The active form is an asymmetric αα'ßß' complex. The α subunit houses the site for nucleotide reduction initiated by a thiyl radical (C439•), and the ß subunit houses the diferric-tyrosyl radical (Y122•) that is essential for C439• formation. The reactions require a highly regulated and reversible long-range proton-coupled electron transfer pathway involving Y122•[ß] ↔ W48?[ß] ↔ Y356[ß] ↔ Y731[α] ↔ Y730[α] ↔ C439[α]. In a recent cryo-EM structure, Y356[ß] was revealed for the first time and it, along with Y731[α], spans the asymmetric α/ß interface. An E52[ß] residue, which is essential for Y356 oxidation, allows access to the interface and resides at the head of a polar region comprising R331[α], E326[α], and E326[α'] residues. Mutagenesis studies with canonical and unnatural amino acid substitutions now suggest that these ionizable residues are important in enzyme activity. To gain further insights into the roles of these residues, Y356• was photochemically generated using a photosensitizer covalently attached adjacent to Y356[ß]. Mutagenesis studies, transient absorption spectroscopy, and photochemical assays monitoring deoxynucleotide formation collectively indicate that the E52[ß], R331[α], E326[α], and E326[α'] network plays the essential role of shuttling protons associated with Y356 oxidation from the interface to bulk solvent.

Early onset atrial lesions in a patient with a novel LMNA frameshift mutation.

Genetic mutations in the lamin A/C gene (LMNA) have been linked to cardiomyopathy. Different mutational sites exhibit different clinical manifestations and prognoses. Herein, we identified a novel LMNA frameshift mutation, p.P485Tfs*67, from a patient with early-onset atrial disease. To verify the pathogenicity of this variation, a transgenic zebrafish model was constructed, which demonstrated that adult zebrafish with the LMNA mutation showed an abnormal ECG and impaired myocardial structure. Our study suggests the atrial pathogenicity of the LMNA-P485Tfs mutation, which is helpful to understand the function of the Ig-like domain of lamin A/C.

Texture analysis of SPECT myocardial perfusion provides prognostic value for dilated cardiomyopathy.

BACKGROUND: Texture analysis (TA) has demonstrated clinical values in extracting information, quantifying inhomogeneity, evaluating treatment outcomes, and predicting long-term prognosis for cardiac diseases. The aim of this study was to explore whether TA of SPECT myocardial perfusion could contribute to improving the prognosis of dilated cardiomyopathy (DCM) patients. METHODS: Eighty-eight patients were recruited in our study between 2009 and 2020 who were diagnosed with DCM and underwent single-photon emission tomography myocardial perfusion imaging (SPECT MPI). Forty TA features were obtained from quantitative analysis of SPECT imaging in subjects with myocardial perfusion at rest. All patients were divided into two groups: the all-cause death group and the survival group. The prognostic value of texture parameters was assessed by Cox regression and Kaplan-Meier analysis. RESULTS: Twenty-five all-cause deaths (28.4%) were observed during the follow-up (39.2±28.7 months). Compared with the survival group, NT-proBNP and total perfusion deficit (TPD) were higher and left ventricular ejection fraction (LVEF) was lower in the all-cause death group. In addition, 26 out of 40 texture parameters were significantly different between the two groups. Univariate Cox regression analysis revealed that NT-proBNP, LVEF, and 25 texture parameters were significantly associated with all-cause death. The multivariate Cox regression analysis showed that low gray-level emphasis (LGLE) (P = 0.010, HR = 4.698, 95% CI 1.457-15.145) and long-run low gray-level emphasis (LRLGE) (P =0.002, HR = 6.085, 95% CI 1.906-19.422) were independent predictors of the survival outcome. When added to clinical parameters, LVEF, TPD, and TA parameters, including LGLE and LRLGE, were incrementally associated with all-cause death (global chi-square statistic of 26.246 vs. 33.521; P = 0.028, global chi-square statistic of 26.246 vs. 34.711; P = 0.004). CONCLUSION: TA based on gated SPECT MPI could discover independent prognostic predictors of all-cause death in medically treated patients with DCM. Moreover, TA parameters, including LGLE and LRLGE, independent of the total perfusion deficit of the cardiac myocardium, appeared to provide incremental prognostic value for DCM patients.

Prevalence, antimicrobial susceptibility, and antibiotic resistance gene transfer of Bacillus strains isolated from pasteurized milk.

Pasteurization is carried out in dairy industries to kill harmful bacteria present in raw milk. However, endospore-forming bacteria, such as Bacillus, cannot be completely eliminated by pasteurization. In this study, a total of 114 Bacillus strains were isolated from 133 pasteurized milk samples. Antibiotic susceptibility tests showed that the percentage of Bacillus with intrinsic resistance to ampicillin and penicillin were 80 and 86%, respectively. Meanwhile, some Bacillus isolates had acquired resistance, including trimethoprim-sulfamethoxazole resistance (10 isolates), clindamycin resistance (8 isolates), erythromycin resistance (2 isolates), and tetracycline resistance (1 isolate). To further locate these acquired resistance genes, the plasmids were investigated in these 16 Bacillus strains. The plasmid profile indicated that Bacillus cereus BA008, BA117, and BA119 harbored plasmids, respectively. Subsequently, the Illumina Novaseq PE150 was applied for the genomic and plasmid DNA sequencing. Notably, the gene tetL encoding tetracycline efflux protein was found to be located on plasmid pBC46-TL of B. cereus BA117. In vitro conjugative transfer indicated that pBC46-TL can be transferred into Bacillus invictae BA142, Bacillus safensis BA143, and Bacillus licheniformis BA130. The frequencies were of 1.5 × 10-7 to 1.7 × 10-5 transconjugants per donor cells. Therefore, Bacillus strains with acquired antibiotic resistance may represent a potential risk for the spread of antibiotic resistance between Bacillus and other clinical pathogens via horizontal gene transfer.

Increasing Reduction Potentials of Type 1 Copper Center and Catalytic Efficiency of Small Laccase from Streptomyces coelicolor through Secondary Coordination Sphere Mutations.

The key to type 1 copper (T1Cu) function lies in the fine tuning of the CuII/I reduction potential (E°'T1Cu ) to match those of its redox partners, enabling efficient electron transfer in a wide range of biological systems. While the secondary coordination sphere (SCS) effects have been used to tune E°'T1Cu in azurin over a wide range, these principles are yet to be generalized to other T1Cu-containing proteins to tune catalytic properties. To this end, we have examined the effects of Y229F, V290N and S292F mutations around the T1Cu of small laccase (SLAC) from Streptomyces coelicolor to match the high E°'T1Cu of fungal laccases. Using ultraviolet-visible absorption and electron paramagnetic resonance spectroscopies, together with X-ray crystallography and redox titrations, we have probed the influence of SCS mutations on the T1Cu and corresponding E°'T1Cu . While minimal and small E°'T1Cu increases are observed in Y229F- and S292F-SLAC, the V290N mutant exhibits a major E°'T1Cu increase. Moreover, the influence of these mutations on E°'T1Cu is additive, culminating in a triple mutant Y229F/V290N/S292F-SLAC with the highest E°'T1Cu of 556 mV vs. SHE reported to date. Further activity assays indicate that all mutants retain oxygen reduction reaction activity, and display improved catalytic efficiencies (kcat /KM ) relative to WT-SLAC.

19F Electron-Nuclear Double Resonance Reveals Interaction between Redox-Active Tyrosines across the α/ß Interface of E. coli Ribonucleotide Reductase.

Ribonucleotide reductases (RNRs) catalyze the reduction of ribonucleotides to deoxyribonucleotides, thereby playing a key role in DNA replication and repair. Escherichia coli class Ia RNR is an α2ß2 enzyme complex that uses a reversible multistep radical transfer (RT) over 32 Å across its two subunits, α and ß, to initiate, using its metallo-cofactor in ß2, nucleotide reduction in α2. Each step is proposed to involve a distinct proton-coupled electron-transfer (PCET) process. An unresolved step is the RT involving Y356(ß) and Y731(α) across the α/ß interface. Using 2,3,5-F3Y122-ß2 with 3,5-F2Y731-α2, GDP (substrate) and TTP (allosteric effector), a Y356• intermediate was trapped and its identity was verified by 263 GHz electron paramagnetic resonance (EPR) and 34 GHz pulse electron-electron double resonance spectroscopies. 94 GHz 19F electron-nuclear double resonance spectroscopy allowed measuring the interspin distances between Y356• and the 19F nuclei of 3,5-F2Y731 in this RNR mutant. Similar experiments with the double mutant E52Q/F3Y122-ß2 were carried out for comparison to the recently published cryo-EM structure of a holo RNR complex. For both mutant combinations, the distance measurements reveal two conformations of 3,5-F2Y731. Remarkably, one conformation is consistent with 3,5-F2Y731 within the H-bond distance to Y356•, whereas the second one is consistent with the conformation observed in the cryo-EM structure. The observations unexpectedly suggest the possibility of a colinear PCET, in which electron and proton are transferred from the same donor to the same acceptor between Y356 and Y731. The results highlight the important role of state-of-the-art EPR spectroscopy to decipher this mechanism.

Predictive values of left ventricular mechanical dyssynchrony for CRT response in heart failure patients with different pathophysiology.

BACKGROUND: Cardiac resynchronization therapy (CRT) patients with different pathophysiology may influence mechanical dyssynchrony and get different ventricular resynchronization and clinical outcomes. METHODS: Ninety-two dilated cardiomyopathy (DCM) and fifty ischemic cardiomyopathy (ICM) patients with gated single-photon emission computed tomography (SPECT) myocardial perfusion imaging (MPI) were included in this retrospective study. Patients were classified based on the concordance between the left ventricular (LV) lead and the latest contraction or relaxation position. If the LV lead was located on or adjacent to both the latest contraction and relaxation position, the patient was categorized into the both match group; if the LV lead was located on or adjacent to the latest contraction or relaxation position, the patient was classified into the one match group; if the LV lead was located on or adjacent to neither the latest contraction nor relaxation position, the patient was categorized to the neither group. CRT response was defined as [Formula: see text] improvement of LV ejection fraction at the 6-month follow-up. Variables with P < .05 in the univariate analysis were included in the stepwise multivariate model. RESULTS: During the follow-up period, 58.7% (54 of 92) for DCM patients and 54% (27 of 50) for ICM patients were CRT responders. The univariate analysis and stepwise multivariate analysis showed that QRS duration, systolic phase bandwidth (PBW), diastolic PBW, diastolic phase histogram standard deviation (PSD), and left ventricular mechanical dyssynchrony (LVMD) concordance were independent predictors of CRT response in DCM patients; diabetes mellitus and left ventricular end-systolic volume were significantly associated with CRT response in ICM patients. The intra-group comparison revealed that the CRT response rate was significantly different in the both match group of DCM (N = 18, 94%) and ICM (N = 24, 62%) patients (P = .016). However, there was no significant difference between DCM and ICM in the one match and neither group. For the inter-group comparison, Kruskal-Wallis H-test revealed that CRT response was significantly different in all the groups of DCM patients (P < .001), but not in ICM patients (P = .383). CONCLUSIONS: Compared with ICM patients, systolic PBW, diastolic PBW and PSD have better predictive and prognostic values for the CRT response in DCM patients. Placing the LV lead in or adjacent to the latest contraction and relaxation position can improve the clinical outcomes of DCM patients, but it does not apply to ICM patients.

Endovascular thrombectomy for acute ischemic stroke in elderly patients with atrial fibrillation.

BACKGROUND: To assess the clinical outcomes after endovascular thrombectomy (EVT) in elderly large vessel occlusion (LVO)-related acute ischemic stroke (AIS) patients with atrial fibrillation (AF). METHODS: Between January 2019 and December 2020, consecutive AF patients who received EVT due to anterior-circulation stroke were enrolled. The primary outcome was modified Rankin scale (mRS) score at 90 days. Secondary outcomes included all-cause mortality, the recanalization status after EVT (assessed using modified thrombolysis in cerebral infarction scale, mTICI) and any intracranial hemorrhage (ICH). A multivariate logistic regression model was performed to identify predictors of the functional outcome. RESULTS: A total of 148 eligible patients were finally enrolled. Among them, 42 were ≥ 80 years old. Compared to their younger counterparts, patients aged ≥80 years had lower likelihood of good functional outcome (mRS score 0-2) at 90 days (26.2% vs. 48.1%, P = 0.015), less satisfied recanalization (mTICI, 2b-3) (78.6% vs. 94.3%, P = 0.004) and higher all-cause mortality rate (35.7% vs. 14.2%, P = 0.003). A multivariable logistic regression analysis showed that age ≥ 80 years at baseline were the significant predictors for a poor functional outcome (OR: 3.72, 95% CI: 1.17-11.89, p = 0.027). Intravenous thrombolysis (IVT) prior to EVT and longer time intervals from onset of symptoms to EVT tended to be associated with poor functional outcome in patients ≥80 years old. CONCLUSIONS: Age ≥ 80 years was a significant predictor of unfavorable outcomes after EVT for AIS patients with AF. An increased risk of adverse events must be balanced against the benefit from EVT in elderly patients with AF.