Glutathione safeguards TET-dependent DNA demethylation and is critical for the acquisition of totipotency and pluripotency during preimplantation development.

During early development, both genome-wide epigenetic reprogramming and metabolic remodeling are hallmark changes of normal embryogenesis. However, little is known about their relationship and developmental functions during the preimplantation window, which is essential for the acquisition of totipotency and pluripotency. Herein, we reported that glutathione (GSH), a ubiquitous intracellular protective antioxidant that maintains mitochondrial function and redox homeostasis, plays a critical role in safeguarding postfertilization DNA demethylation and is essential for establishing developmental potential in preimplantation embryos. By profiling mitochondria-related transcriptome that coupled with different pluripotency, we found GSH is a potential marker that is tightly correlated with full pluripotency, and its beneficial effect on prompting developmental potential was functionally conformed using in vitro fertilized mouse and bovine embryos as the model. Mechanistic study based on preimplantation embryos and embryonic stem cells further revealed that GSH prompts the acquisition of totipotency and pluripotency by facilitating ten-eleven-translocation (TET)-dependent DNA demethylation, and ascorbic acid (AsA)-GSH cycle is implicated in the process. In addition, we also reported that GSH serves as an oviductal paracrine factor that supports development potential of preimplantation embryos. Thus, our results not only advance the current knowledge of functional links between epigenetic reprogramming and metabolic remodeling during preimplantation development but also provided a promising approach for improving current in vitro culture system for assisted reproductive technology.

Mitochondrial genome undergoes de novo DNA methylation that protects mtDNA against oxidative damage during the peri-implantation window.

Mitochondrial remodeling during the peri-implantation stage is the hallmark event essential for normal embryogenesis. Among the changes, enhanced oxidative phosphorylation is critical for supporting high energy demands of postimplantation embryos, but increases mitochondrial oxidative stress, which in turn threatens mitochondrial DNA (mtDNA) stability. However, how mitochondria protect their own histone-lacking mtDNA, during this stage remains unclear. Concurrently, the mitochondrial genome gain DNA methylation by this stage. Its spatiotemporal coincidence with enhanced mitochondrial stress led us to ask if mtDNA methylation has a role in maintaining mitochondrial genome stability. Herein, we report that mitochondrial genome undergoes de novo mtDNA methylation that can protect mtDNA against enhanced oxidative damage during the peri-implantation window. Mitochondrial genome gains extensive mtDNA methylation during transition from blastocysts to postimplantation embryos, thus establishing relatively hypermethylated mtDNA from hypomethylated state in blastocysts. Mechanistic study revealed that DNA methyltransferase 3A (DNMT3A) and DNMT3B enter mitochondria during this process and bind to mtDNA, via their unique mitochondrial targeting sequences. Importantly, loss- and gain-of-function analyses indicated that DNMT3A and DNMT3B are responsible for catalyzing de novo mtDNA methylation, in a synergistic manner. Finally, we proved, in vivo and in vitro, that increased mtDNA methylation functions to protect mitochondrial genome against mtDNA damage induced by increased mitochondrial oxidative stress. Together, we reveal mtDNA methylation dynamics and its underlying mechanism during the critical developmental window. We also provide the functional link between mitochondrial epigenetic remodeling and metabolic changes, which reveals a role for nuclear-mitochondrial crosstalk in establishing mitoepigenetics and maintaining mitochondrial homeostasis.

"Meiosis arrester" C-natriuretic peptide directly stimulates oocyte mtDNA accumulation and is implicated in aging-associated oocyte mtDNA loss.

C-natriuretic peptide (CNP) is the central regulator of oocyte meiosis progression, thus coordinating synchronization of oocyte nuclear-cytoplasmic maturation. However, whether CNP can independently regulate cytoplasmic maturation has been long overlooked. Mitochondrial DNA (mtDNA) accumulation is the hallmark event of cytoplasmic maturation, but the mechanism underlying oocyte mtDNA replication remains largely elusive. Herein, we report that CNP can directly stimulate oocyte mtDNA replication at GV stage, and deficiency of follicular CNP may contribute largely to lower mtDNA copy number in in vitro matured oocytes. The mechanistic study showed that cAMP-PKA-CREB1 signaling cascade underlies the regulatory role of CNP in stimulating mtDNA replication and upregulating related genes. Of interest, we also report that CNP-NPR2 signaling is inhibited in aging follicles, and this inhibition is implicated in lower mtDNA copy number in oocytes from aging females. Together, our study provides the first direct functional link between follicular CNP and oocyte mtDNA replication, and identifies its involvement in aging-associated mtDNA loss in oocytes. These findings, not only update the current knowledge of the functions of CNP in coordinating oocyte maturation but also present a promising strategy for improving in vitro fertilization outcomes of aging females.

The mRNA-destabilizing protein Tristetraprolin targets "meiosis arrester" Nppc mRNA in mammalian preovulatory follicles.

C-natriuretic peptide (CNP) and its receptor guanylyl cyclase, natriuretic peptide receptor 2 (NPR2), are key regulators of cyclic guanosine monophosphate (cGMP) homeostasis. The CNP-NPR2-cGMP signaling cascade plays an important role in the progression of oocyte meiosis, which is essential for fertility in female mammals. In preovulatory ovarian follicles, the luteinizing hormone (LH)-induced decrease in CNP and its encoding messenger RNA (mRNA) natriuretic peptide precursor C (Nppc) are a prerequisite for oocyte meiotic resumption. However, it has never been determined how LH decreases CNP/Nppc In the present study, we identified that tristetraprolin (TTP), also known as zinc finger protein 36 (ZFP36), a ubiquitously expressed mRNA-destabilizing protein, is the critical mechanism that underlies the LH-induced decrease in Nppc mRNA. Zfp36 mRNA was transiently up-regulated in mural granulosa cells (MGCs) in response to the LH surge. Loss- and gain-of-function analyses indicated that TTP is required for Nppc mRNA degradation in preovulatory MGCs by targeting the rare noncanonical AU-rich element harbored in the Nppc 3' UTR. Moreover, MGC-specific knockout of Zfp36, as well as lentivirus-mediated knockdown in vivo, impaired the LH/hCG-induced Nppc mRNA decline and oocyte meiotic resumption. Furthermore, we found that LH/hCG activates Zfp36/TTP expression through the EGFR-ERK1/2-dependent pathway. Our findings reveal a functional role of TTP-induced mRNA degradation, a global posttranscriptional regulation mechanism, in orchestrating the progression of oocyte meiosis. We also provided a mechanism for understanding CNP-dependent cGMP homeostasis in diverse cellular processes.

A proteomic atlas of ligand-receptor interactions at the ovine maternal-fetal interface reveals the role of histone lactylation in uterine remodeling.

Well-orchestrated maternal-fetal cross talk occurs via secreted ligands, interacting receptors, and coupled intracellular pathways between the conceptus and endometrium and is essential for successful embryo implantation. However, previous studies mostly focus on either the conceptus or the endometrium in isolation. The lack of integrated analysis impedes our understanding of early maternal-fetal cross talk. Herein, focusing on ligand-receptor complexes and coupled pathways at the maternal-fetal interface in sheep, we provide the first comprehensive proteomic map of ligand-receptor pathway cascades essential for embryo implantation. We demonstrate that these cascades are associated with cell adhesion and invasion, redox homeostasis, and the immune response. Candidate interactions and their physiological roles were further validated by functional experiments. We reveal the physical interaction of albumin and claudin 4 and their roles in facilitating embryo attachment to endometrium. We also demonstrate a novel function of enhanced conceptus glycolysis in remodeling uterine receptivity by inducing endometrial histone lactylation, a newly identified histone modification. Results from in vitro and in vivo models supported the essential role of lactate in inducing endometrial H3K18 lactylation and in regulating redox homeostasis and apoptotic balance to ensure successful implantation. By reconstructing a map of potential ligand-receptor pathway cascades at the maternal-fetal interface, our study presents new concepts for understanding molecular and cellular mechanisms that fine-tune conceptus-endometrium cross talk during implantation. This provides more direct and accurate insights for developing potential clinical intervention strategies to improve pregnancy outcomes following both natural and assisted conception.

Downregulation of Claudin5 promotes malignant progression and radioresistance through Beclin1-mediated autophagy in esophageal squamous cell carcinoma.

BACKGROUND: Esophageal squamous cell carcinoma (ESCC) is a highly prevalent and aggressive cancer with poor treatment outcomes. Despite the critical role of tight junction proteins in tumorigenesis, the involvement of Claudin5 in ESCC remains poorly understood. Thus, this study aimed to investigate the role of Claudin5 in ESCC malignant progression and radioresistance, as well as the underlying regulatory mechanisms. METHODS: The expression of Claudin5 was evaluated in esophageal cancer tissue using both public databases and 123 clinical samples. CCK-8, transwell invasion, wound healing and clonogenic survival assays were used to examine the proliferation, invasion, migration and radiosensitivity of ESCC cells in vitro. Xenograft and animal lung metastasis experiments were conducted to examine the impact of Claudin5 on tumor growth and lung metastasis in vivo. The effect of Claudin5 on autophagy was detected via transmission electron microscopy, western blotting and autophagy flux. Immunohistochemical staining was used to detect Claudin5 expression in ESCC patient samples. The statistical difference was assessed with Student t test or one-way ANOVA. The correlation between Claudin5 expression and radiotherapy response rate was performed by the Chi-square test. The significance of Kaplan-Meier curves was evaluated by the Logrank test. RESULTS: Claudin5 expression was downregulated in ESCC tissues. Downregulation of Claudin5 promoted ESCC cell proliferation, invasion, and migration both in vitro and in vivo. Downregulation of Claudin5 decreased the radiosensitivity of ESCC cells. Moreover, downregulation of Claudin5 promoted autophagy and the expression of Beclin1. Beclin1 knockdown reversed the effect of Claudin5 downregulation on autophagy induction and the promotion of ESCC cell malignant progression and radioresistance. Additionally, low expression of Claudin5 in ESCC cancer tissues was associated with poor radiotherapy response and prognosis. CONCLUSIONS: In summary, these findings suggest that downregulation of Claudin5 promotes ESCC malignant progression and radioresistance via Beclin1-autophagy activation and may serve as a promising biomarker for predicting radiotherapy response and patient outcome in ESCC.

Six first-line tyrosine kinase inhibitors reveal novel inhibition potential for the EGFR S768I mutation.

Lung cancer, the leading cause of cancer-related mortality, is the most commonly diagnosed cancer. Tyrosine kinase inhibitors (TKIs) are considered a drug-targeted therapy for non-small cell lung cancers (NSCLCs) with epidermal growth factor receptor (EGFR) mutations. However, limited data are available involving the activity of EGFR TKIs against rare EGFR mutations. Here, based on an endogenous EGFR-depleted cell Line H3255 by CRISPR, H3255 cells with rare mutant EGFRS768I and compound mutations EGFRS768I+L858R were tested using cell proliferation assay, cytotoxicity, membrane potential, flow cytometry and Western blot analysis. We conducted cytotoxicity screening of EGFR mutations on six front-line TKIs based on first-, second-, and third-generation TKIs (afatinib, dacomitinib, osimertinib, erlotinib, gefitinib, and icotinib). The results showed that the sensitivity of these mutants containing rare variants EGFRS768I to six front-line TKIs was enriched in the irreversible TKI cytotoxicity assays by determining their change in cytotoxicity, apoptosis, cell proliferation and signal pathway factors. Importantly, the variants harboring EGFRL858R (H3255), EGFRS768I (H3255S768I) and EGFRS768I+L858R (H3255S768I+L858R) were sensitive to six TKIs and induced cytotoxicity through different pathways. Moreover, the compound mutations EGFRS768I+L858R showed more TKI resistance than EGFRS768I mutation and EGFRL858R mutation. We present a comprehensive reference for the sensitivity of EGFRS768I variants to six front-line TKIs. For patients with the EGFR S768I mutation and compound mutations EGFRS768I+L858R, six first-line TKIs appear to be reasonable therapeutic options.

Sulfone/Carbonyl-Based Donor-Acceptor Fluorescent Dyes: Synthesis, Structures, Photophysical Properties and Cell Imaging.

Electron-accepting units play vital roles in constructing donor-acceptor (D-A) conjugated organic optoelectronic materials; the electronic structures and functions of the acceptors need to be carefully unveiled to controllably tailor the optoelectronic properties. We have synthesized two D-A conjugated organic fluorophores, TPA-SO and TPA-CO, with similar molecular skeletons based on sulfone- or carbonyl-containing polycyclic aromatic acceptors. Both TPA-SO and TPA-CO display obvious solvent polarity-dependent photophysical properties and large Stokes shift of over 100 nm for strong intramolecular charge transfer processes. Experimental evidence indicates that the sulfone group in TPA-SO merely serves as a strong electron-withdrawing unit. TPA-SO shows yellowish-green emission with a peak at 542 nm and an absolute photoluminescence quantum yield (PLQY) of 98 % in solution, whereas the carbonyl group in TPA-CO can act as both an electron-withdrawing unit and spin transition convertor, so TPA-CO displays red emission with a low absolute PLQY of 0.32 % in solution. Impressively, upon going from solution to aggregate state, TPA-SO nanoparticles keep a high PLQY of 9.5 % and moderate biocompatibility, thus they are good nano-agents for cellular fluorescence imaging. The results reveal that the inherent acceptor characteristic acts as a crucial effect in the photophysical properties and applications of the organic fluorophores.

Effectiveness of dual antiplatelet de-escalation therapy on the prognosis of patients with ST segment elevation myocardial infarction undergoing percutaneous coronary intervention.

AIM: To investigate the effectiveness of de-escalation of ticagrelor (from ticagrelor 90 mg to clopidogrel 75 mg or ticagrelor 60 mg) on the prognosis of patients with ST segment elevation myocardial infarction (STEMI) undergoing percutaneous coronary intervention (PCI) after 3 months of oral dual antiplatelet therapy (DAPT). METHODS: From March 2017 to August 2021, 1056 patients with STEMI in a single centre, through retrospective investigation and analysis, were divided into intensive (ticagrelor 90 mg), standard (clopidogrel 75 mg after PCI) and de-escalation groups (clopidogrel 75 mg or ticagrelor 60 mg after 3 months of treatment with 90 mg ticagrelor) based on the type and dose of P2Y12 inhibitor 3 months after PCI, and the patients had a ≥ 12-month history of oral DAPT. The primary end point was major adverse cardiovascular and cerebrovascular events (MACCEs) during the 12-month follow-up period, including composite end points of cardiac death, myocardial infarction, ischaemia-driven revascularization and stroke. The major safety endpoint was bleeding events. RESULTS: The results showed that during the follow-up period, there was no statistically significant difference in the incidence of MACCEs between the intensive and de-escalation groups (P > 0.05). The incidence of MACCEs in the standard treatment group was higher than that in the intensive treatment group (P = 0.014), but the incidence of bleeding events in the de-escalation group was significantly lower than that in the standard group (9.3% vs. 18.4%, χ²=7.191, P = 0.027). The Cox regression analysis showed that increases in haemoglobin (HGB) (HR = 0.986) and estimated glomerular filtration rate (eGFR) (HR = 0.983) could reduce the incidence of MACCEs, while old myocardial infarction (OMI) (P = 0.023) and hypertension (P = 0.013) were independent predictors of MACCEs. CONCLUSION: For STEMI patients undergoing PCI, the de-escalation scheme of ticagrelor to clopidogrel 75 mg or ticagrelor 60 mg at 3 months after PCI was related to the reduction of bleeding events, especially minor bleeding events, without an increase in ischaemic events.

Tunable Noninteger Flux Quantum of Vortices in Superconducting Strips.

Flux quantization has been widely regarded as the hallmark of the macroscopic quantum state of superconductivity. However, practical design of superconductor devices exploiting finite size confinement effects may induce exotic phenomena, including nonquantized vortices. In our research, the magnetic flux of vortices has been studied in a series of superconducting strips as a function of the strip width and the penetration depth. In both circumstances, the observation using scanning Hall probe microscope (SHPM) displays a controlled evolution from singly quantized vortices to nonquantized ones. It is also found that the magnetic flux is immune to the flowing supercurrent. The simulations based on Ginzburg-Landau theory agree well with experimental results. The observed behavior of the vortex flux may open new perspectives for fundamental research and applications based on vortex matter, such as vortex-memory devices and magnetic field traps for ultracold atoms.

Measuring parameters of laser self-mixing interferometry sensor based on back propagation neural network.

Self-mixing interferometry (SMI) is a well-known non-destructive sensing technique that has been widely applied in both laboratory and engineering applications. In a laser SMI sensing system, there are two vital parameters, i.e., optical feedback factor C and line-width enhancement factor α, which influence the operation characteristics of the laser as well as the sensing performance. Therefore, many efforts have been made to determine them. Most of the existing methods of estimating these two parameters can often be operated in a certain feedback regime, e.g., weak or moderate feedback regime. In this paper, we propose a new method to estimate C and α based on back-propagation neural network for all feedback regimes. A parameter predicting model was trained and built. The performance of the proposed predicting model was tested using simulation and experiment data. The results show that the proposed method can estimate C and α with an average error of 2.76% and 2.99%, respectively. Additionally, the proposed method is noise-proof. The method and results are useful for extending the utilization of SMI technology in practical engineering fields.

Loss of diurnal behavioral rhythms and impaired lipid metabolism in growing pigs with mistimed feeding.

The misalignment of eating time and the endogenous circadian rhythm impairs the body's ability to maintain homeostasis. Although it is well established that children and growing animals differ from adults in their energy metabolism and behavioral patterns, little is known about how mistimed feeding disturbs the diurnal rhythms of behavior and metabolism in children and growing diurnal animals. In this study, growing pigs (diurnal animal) were randomly assigned to the daytime-restricted feeding (DRF) and nighttime-restricted feeding (NRF) groups for 5 weeks. Compared with observations in the DRF group, NRF disrupted the diurnal rhythm of behavior and clock genes and lowered the serum ghrelin, dopamine, and serotonin levels during the daytime and nighttime. Microbiome analysis results suggested that NRF altered the diurnal rhythm and composition of the gut microbiota, and increased log-ratios of Catenibacterium:Butyrivibrio and Streptococcus:Butyrivibrio. Based on the serum proteome, the results further revealed that rhythmic and upregulated proteins in NRF were mainly involved in oxidative stress, lipid metabolism, immunity, and cancer biological pathways. Serum physiological indicators further confirmed that NRF decreased the concentration of melatonin and fibroblast growth factor 21 during the daytime and nighttime, increased the diurnal amplitude and concentrations of very-low-density lipoprotein cholesterol, triglyceride, and total cholesterol, and increased the apolipoprotein B/ApoA1 ratio, which is a marker of metabolic syndrome. Taken together, this study is the first to reveal that mistimed feeding disrupts the behavioral rhythms of growing pigs, reprograms gut microbiota composition, reduces the serum levels of hormones associated with fighting depression and anxiety, and increases the risk of lipid metabolic dysregulation.

Night-restricted feeding improves locomotor activity rhythm and modulates nutrient utilization to accelerate growth in rabbits.

An unfavorable lifestyle disrupts the circadian rhythm, leading to metabolic dysfunction in adult humans and animals. Increasing evidence suggests that night-restricted feeding (NRF) can effectively prevent ectopic fat deposition caused by circadian rhythm disruption, and reduce the risk of metabolic diseases. However, previous studies have mainly focused on the prevention of obesity in adults by regulating dietary patterns, whereas limited attention has been paid to the effect of NRF on metabolism during growth and development. Here, we used weaning rabbits as models and found that NRF increased body weight gain without increasing feed intake, and promoted insulin-mediated protein synthesis through the mTOR/S6K pathway and muscle formation by upregulating MYOG. NRF improved the circadian clock, promoted PDH-regulated glycolysis and CPT1B-regulated fatty-acid ß-oxidation, and reduced fat content in the serum and muscles. In addition, NRF-induced body temperature oscillation might be partly responsible for the improvement in the circadian clock and insulin sensitivity. Time-restricted feeding could be used as a nondrug intervention to prevent obesity and accelerate growth in adolescents.

Chitinophaga hostae sp. nov., isolated from the rhizosphere soil of Hosta plantaginea.

A Gram-negative, rod-shaped aerobic bacterium designated as strain 2R12T was isolated from the rhizosphere soil of Hosta plantaginea. Phylogenetic analyses based on the 16S rRNA gene revealed that strain 2R12T should be assigned to the genus Chitinophaga with the highest sequence similarity to Chitinophaga arvensicola DSM 3695T (99.1 %) and Chitinophaga ginsengisegetis DSM 18108T (98.6 %). The major fatty acids of strain 2R12T (>10 %) were iso-C15 : 0, C16 :1 ω5c and iso-C17 : 0 3-OH. The major polar lipids were phosphatidylethanolamine, two unidentified aminolipids and five unidentified lipids. The predominant respiratory quinone was MK-7. The genomic DNA G+C content was 46.1 mol%. The average nucleotide identity values of strain 2R12T with C. arvensicola DSM 3695T and C. ginsengisegetis DSM 18108T were 77.9 and 78.8 %, respectively, while in silico DNA-DNA hybridization values for strain 2R12T with these strains were 22.8 and 23.3 %, respectively. Based on comparative analysis of phylogenetic, phylogenomic, phenotypic and chemotaxonomic characteristics, strain 2R12T represents a novel species in the genus Chitinophaga, for which the name Chitinophaga hostae sp. nov. is proposed. The type strain is 2R12T (=ACCC 61757T=JCM 34719T).

Aluminium oxide nanoparticles compromise spatial memory performance and proBDNF-mediated neuronal function in the hippocampus of rats.

BACKGROUND: Alumina nanoparticles (aluminaNPs), which are widely used in a range of daily and medical fields, have been shown to penetrate blood-brain barrier, and distribute and accumulate in different brain areas. Although oral treatment of aluminaNPs induces hippocampus-dependent learning and memory impairments, characteristic effects and exact mechanisms have not been fully elucidated. Here, male adult rats received a single bilateral infusion of aluminaNPs (10 or 20 µg/kg of body weight) into the hippocampal region, and their behavioral performance and neural function were assessed. RESULTS: The results indicated that the intra-hippocampus infusions at both doses of aluminaNPs did not cause spatial learning inability but memory deficit in the water maze task. This impairment was attributed to the effects of aluminaNP on memory consolidation phase through activation of proBDNF/RhoA pathway. Inhibition of the increased proBDNF by hippocampal infusions of p75NTR antagonist could effectively rescue the memory impairment. Incubation of aluminaNPs exaggerated GluN2B-dependent LTD induction with no effects on LTD expression in hippocampal slices. AluminaNP could also depress the amplitude of NMDA-GluN2B EPSCs. Meanwhile, increased reactive oxygen specie production was reduced by blocking proBDNF-p75NTR pathway in the hippocampal homogenates. Furthermore, the neuronal correlate of memory behavior was drastically weakened in the aluminaNP-infused groups. The dysfunction of synaptic and neuronal could be obviously mitigated by blocking proBDNF receptor p75NTR, implying the involvement of proBDNF signaling in aluminaNP-impaired memory process. CONCLUSIONS: Taken together, our findings provide the first evidence that the accumulation of aluminaNPs in the hippocampus exaggeratedly activates proBDNF signaling, which leads to neural and memory impairments.

Co-exposure of melamine and cyanuric acid as a risk factor for oxidative stress and energy metabolism: Adverse effects on hippocampal neuronal and synaptic function induced by excessive ROS production.

Melamine (MEL) and cyanuric acid (CA) alone have relatively low toxicity, but together they may cause serious damage to multiple organs, including the central nervous system, however, the underlying mechanism is unknown. This study aimed to determine and compare the neurotoxic effects of MEL (20 µg/mL), CA (20 µg/mL) and their combination (10 µg/mL MEL and 10 µg/mL CA) on cultured hippocampal neurons. The cell viability, apoptosis, anti-oxidative and energy metabolic indices were detected following 24 h of incubations. The miniature excitatory postsynaptic currents (mEPSCs), miniature inhibitory postsynaptic currents (mIPSCs) and synaptic plasticity in the hippocampal CA1 neurons were recorded. Moreover, ROS scavenger NAC was co-infused to investigate the potential mechanism. We found the complex of MEL and CA but not their alone caused severe cell death and disturbed energy production through activation caspase-3-mediated apoptosis. Meanwhile, the combination significantly reduced the amplitude, decay time and frequency of mEPSCs but not mIPSCs, indicating the pre- and post-synaptic inhibitory actions on neuronal activity. Paired-pulsed ratio (PPR) and long-term potentiation (LTP) at the Schaffer collateral-CA1 synapses were critically depressed. However, the co-application of NAC could effectively mitigate the cellular apoptosis, energy metabolism dysfunction and the impairments in neuronal and synaptic function. Our findings provide the first evidence that the combination of MEL and CA can exert more prominently neurotoxic effects than their alone and certify that one of the potential mechanisms for neuronal and synaptic dysfunction is the ROS-mediated signaling pathway.

Optimization of cationic polymer-mediated transfection for RNA interference.

Transfection efficiency was estimated to optimize the conditions for RNA interference (RNAi), including transfection time, validity, and nucleic acid concentration and type, using the EZ Trans Cell Reagent, a cationic polymer. An shRNA against GFP was designed and transfected into cells using the EZ transfection reagent. The shRNA significantly decreased the expression of GFP. In addition, pre-diluted transfection reagent at room temperature and small nucleic acids increased the transfection efficiency, which peaked at 24 h. Compared with circular nucleic acids, linear nucleic acids showed higher transfection efficiency and a higher genome integration rate. We optimized cationic polymer-mediated RNAi conditions, and our data will be useful for future RNAi studies.

[Mechanism of Zixin Biqiu Granules in treatment of allergic rhinitis based on network pharmacology and molecular docking technology].

Allergic rhinitis(AR) is a chronic inflammatory disorder of nasal mucosa induced by allergen exposure triggering IgE-mediated inflammation. With frequent recurrence, it is difficult to be cured. Zixin Biqiu Granules is developed based on the 60-year clinical experience of CHAO En-xiang, a master of national medicine of China-Japan Friendship Hospital. At present, phase Ⅲ multicenter clinical trial is being prepared, but the mechanism is unclear. Therefore, this study explored the mechanism of Zixin Biqiu Granules against AR based on network pharmacology and molecular docking. First, the chemical components and targets of Zixin Biqiu Granules were retrieved from Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform(TCMSP), and the targets of AR from GeneCards and Online Mendelian Inheritance in Man(OMIM). Then the common targets of the two were screened out and the "Chinese medicine-component-target" network was constructed. Afterward, the common targets were imported into the STRING to obtain the interaction of them, and Cytoscape was employed to establish the protein-protein interaction(PPI) network. Through topological analysis, the core targets were obtained. DAVID was used for Gene Ontology(GO) term enrichment and Kyoto Encyclopedia of Genes and Genomes(KEGG) pathway enrichment. The "key target-pathway-biological process" network was constructed to explore the anti-AR mechanism of Zixin Biqiu Granules. Finally, the core targets were selected for molecular docking with the key components of Zixin Biqiu Granules to verify the mechanism. The results showed that there were 151 components and 97 targets in the "Chinese medicine-component-target" network. We obtained 20 core targets, 488 biological processes and 147 pathways in topological, GO, and KEGG enrichment analysis of the protein interaction network, and in the comprehensive analysis, it was found that Zixin Biqiu Granules mainly exerted the therapeutic effect through anti-inflammation and immunoregulation. Serine/threonine-protein kinase 1(AKT1) and tumor necrosis factor(TNF) were docked with the key components of Zixin Biqiu Granules, and the results showed that the key components of Zixin Biqiu Granules had high binding affinity to the core targets. This study preliminarily discussed the anti-AR mechanism of Zixin Biqiu Granules, which laid a scientific basis for its clinical application.

Mitochondrial transfer from induced pluripotent stem cells rescues developmental potential of in vitro fertilized embryos from aging females†.

Conventional heterologous mitochondrial replacement therapy is clinically complicated by "tri-parental" ethical concerns and limited source of healthy donor oocytes or zygotes. Autologous mitochondrial transfer is a promising alternative in rescuing poor oocyte quality and impaired embryo developmental potential associated with mitochondrial disorders, including aging. However, the efficacy and safety of mitochondrial transfer from somatic cells remains largely controversial, and unsatisfying outcomes may be due to distinct mitochondrial state in somatic cells from that in oocytes. Here, we propose a potential strategy for improving in vitro fertilization (IVF) outcomes of aging female patients via mitochondrial transfer from induced pluripotent stem (iPS) cells. Using naturally aging mice and well-established cell lines as models, we found iPS cells and oocytes share similar mitochondrial morphology and functions, whereas the mitochondrial state in differentiated somatic cells is substantially different. By microinjection of isolated mitochondria into fertilized oocytes following IVF, our results indicate that mitochondrial transfer from iPS, but not MEF cells, can rescue the impaired developmental potential of embryos from aging female mice and obtain an enhanced implantation rate following embryo transfer. The beneficial effect may be explained by the fact that mitochondrial transfer from iPS cells not only compensates for aging-associated loss of mtDNA, but also rescues mitochondrial metabolism of subsequent preimplantation embryos. Using mitochondria from iPS cells as the donor, our study not only proposes a promising strategy for improving IVF outcomes of aging females, but also highlights the importance of synchronous mitochondrial state in supporting embryo developmental potential.

Warming Shapes nirS- and nosZ-Type Denitrifier Communities and Stimulates N2O Emission in Acidic Paddy Soil.

Warming strongly stimulates soil nitrous oxide (N2O) emission, contributing to the global warming trend. Submerged paddy soils exhibit huge N2O emission potential; however, the N2O emission pathway and underlying mechanisms for warming are not clearly understood. We conducted an incubation experiment using 15N to investigate the dynamics of N2O emission at controlled temperatures (5, 15, 25, and 35°C) in 125% water-filled pore space. The community structures of nitrifiers and denitrifiers were determined via high-throughput sequencing of functional genes. Our results showed that elevated temperature sharply enhanced soil N2O emission from submerged paddy soil. Denitrification was the main contributor, accounting for more than 90% of total N2O emission at all treatment temperatures. N2O flux was coordinatively regulated by nirK-, nirS-, and nosZ-containing denitrifiers but not ammonia-oxidizing archaea or ammonia-oxidizing bacteria. The nirS-containing denitrifiers were more sensitive to temperature shifts, especially at a lower temperature range (5 to 25°C), and showed a stronger correlation with N2O flux than that of nirK-containing denitrifiers. In contrast, nosZ-containing denitrifiers exhibited substantial variation at higher temperatures (15 to 35°C), thereby playing an important role in N2O consumption. Certain taxa of nirS- and nosZ-containing denitrifiers regulated N2O flux, including nirS-containing denitrifiers affiliated with Rhodanobacter and Cupriavidus as well as nosZ-containing denitrifiers affiliated with Azoarcus and Azospirillum. Together, these findings suggest that elevated temperature can significantly increase N2O emission from denitrification in submerged paddy soils by shifting the overall community structures and enriching some indigenous taxa of nirS- and nosZ-containing denitrifiers. IMPORTANCE The interdependence between global warming and greenhouse gas N2O has always been the hot spot. However, information on factors contributing to N2O and temperature-dependent community structure changes is scarce. This study demonstrated high-temperature-induced N2O emission from submerged paddy soils, mainly via stimulating denitrification. Further, we speculate that key functional denitrifiers drive N2O emission. This study showed that denitrifiers were more sensitive to temperature rise than nitrifiers, and the temperature sensitivity differed among denitrifier communities. N2O-consuming denitrifiers (nosZ-containing denitrifiers) were more sensitive at a higher temperature range than N2O-producing denitrifiers (nirS-containing denitrifiers). This study's findings help predict N2O fluxes under different degrees of warming and develop strategies to mitigate N2O emissions from paddy fields based on microbial community regulation.