Embryonic growth and effect of embryonic age on quantitative and functional characteristics of duck primary hepatocytes.

Primary hepatocytes (PH) have been widely used in metabolic and disease-resistance mechanism research. However, hepatocyte isolation (HI) remains challenging in ducks. This study aimed to explore embryonic growth and the effect of embryonic age (EA) on the quantitative and functional characteristics of PH in ducks. For embryonic growth, the size and weight of the embryo and liver were determined from 6 to 28 EA (E6-E28, similar below). As EA increased, the corresponding size and weight grew significantly. Specifically, embryonic length varied from 12.5 mm to 133.0 mm, and liver width varied from 2.0 mm to 26.2 mm. Embryonic weight ranged from 0.259 g to 53.58 g, and liver weight ranged from 0.007 g to 1.765 g. Liver index initially decreased and then increased with a ratio ranging from 1.06 to 3.29%. For quantitative and functional characteristics, they were determined from E6 to E22, as there were no obvious liver features before E6 and few cells obtained after E22. The number of cells isolated in liver increased from E6 to E16 and then sharply decreased from E16 to E22. The viability remained relatively stable from E6 to E10 and then decreased from E12 to E22. The comprehensive intensity of hepatic glycogen was stronger at E8 and E14. Albumin expression increased markedly from E6 to E18 by qPCR, and the overall albumin expression was stronger at E8 and E14 by immunofluorescence assay. Hepatocyte purity exceeded 90% except for E20 and E22. During culture, cell clusters appeared after 24-h culture, which were identified as nonhepatocytes. The growth curve showed an initial increase in cell quantity followed by a decrease, another increase, and then remaining stable. In conclusion, EA had a significant effect on the quantitative and functional characteristics of PH, and the suitable EA for HI were E8 and E14. Considering better operability and quantity, E14 was the optimal EA, laying a solid foundation for further hepatocyte purification, nutrient metabolism, and disease-resistance mechanism explorations in ducks.

Contrasting Iron Metabolism in Undifferentiated Versus Differentiated MO3.13 Oligodendrocytes via IL-1ß-Induced Iron Regulatory Protein 1.

Parkinson's disease (PD) is a prevalent neurodegenerative disorder characterized by the loss of dopaminergic neurons and the accumulation of iron in the substantia nigra. While iron accumulation and inflammation are implicated in PD pathogenesis, their impact on oligodendrocytes, the brain's myelin-forming cells, remains elusive. This study investigated the influence of interleukin-1ß (IL-1ß), an elevated proinflammatory cytokine in PD, on iron-related proteins in MO3.13 oligodendrocytes. We found that IL-1ß treatment in undifferentiated MO3.13 oligodendrocytes increased iron regulatory protein 1 and transferrin receptor 1 (TfR1) expression while decreasing ferroportin 1 (FPN1) expression. Consequently, iron uptake was enhanced, and iron release was reduced, leading to intracellular iron accumulation. Conversely, IL-1ß treatment in differentiated MO3.13 oligodendrocytes exhibited the opposite effect, with decreased TfR1 expression, increased FPN1 expression, and reduced iron uptake. These findings suggest that IL-1ß-induced dysregulation of iron metabolism in oligodendrocytes may contribute to the pathological processes observed in PD. IL-1ß can increase the iron content in undifferentiated oligodendrocytes, thus facilitating the differentiation of undifferentiated MO3.13 oligodendrocytes. In differentiated oligodendrocytes, IL-1ß may facilitate iron release, providing a potential source of iron for neighboring dopaminergic neurons, thereby initiating a cascade of deleterious events. This study provides valuable insights into the intricate interplay between inflammation, abnormal iron accumulation, and oligodendrocyte dysfunction in PD. Targeting the IL-1ß-mediated alterations in iron metabolism may hold therapeutic potential for mitigating neurodegeneration and preserving dopaminergic function in PD.

Research on preparation and antitumor activity of redox-responsive polymer micelles co-loaded with sorafenib and curcumin.

A novel redox-responsive mPEG-SS-PLA (PSP) polymeric micelle was synthesized and prepared for the delivery of sorafenib (SAF) and curcumin (CUR). And a series of validations were conducted to confirm the structure of the synthesized polymer carriers. Using the Chou-Talalay approach, the combination indexes (CI) of SAF and CUR were determined, and explore the inhibitory effects of the two drugs on HepG2R cells at different ratios. SAF/CUR-PSP polymeric micelles were prepared by thin film hydration method, and the physicochemical properties of nanomicelles were evaluated. The biocompatibility, cell uptake, cell migration, and cytotoxicity assays were assessed in HepG2R cells. The expression of the phosphoinositol-3 kinase (PI3K)/serine/threonine kinase (Akt) signaling pathway was detected by Western blot assay. Additionally, the tumor suppressive effect of SAF/CUR-PSP micelles was clearly superior to free drug monotherapy or their physical combination in HepG2 cell-induced tumor xenografts. The current study revealed that mPEG-SS-PLA polymer micelles loaded with SAF and CUR showed the enhanced therapeutic effects against hepatocellular carcinoma in vitro and in vivo models. It has promising applications for cancer therapy.

Glucosamine-Modified Reduction-Responsive Polymeric Micelles for Liver Cancer Therapy.

In this work, glucose transporter-1 (GLUT-1) and glutathione (GSH) over-expression in liver cancer was utilized to design a reduction-responsive and active targeting drug delivery system AG-PEG-SS-PCL (APSP) for the delivery of sorafenib (SF). The SF-APSP micelles were prepared using the thin film hydration method and characterized by various techniques. In vitro release experiments showed that the cumulative release of SF-APSP micelles in the simulated tumor microenvironment (pH 7.4 with GSH) reached 94.76 ± 1.78% at 48 h, while it was only 20.32 ± 1.67% in the normal physiological environment (pH 7.4 without GSH). The in vitro study revealed that glucosamine (AG) enhanced the antitumor effects of SF, and SF-APSP micelles inhibited proliferation by targeting HepG2 cells and suppressing cyclin D1 expression. The in vivo antitumor efficacy study further confirmed that the SF-APSP micelles had excellent antitumor effects and better tolerance against nude mouse with HepG2 cells than other treatment groups. All in all, these results indicated that SF-APSP micelles could be a promising drug delivery system for anti-hepatoma treatment.

Nucleic acid-based scaffold systems and application in enzyme cascade catalysis.

Enzymes, as elements with catalytic functions, can be rationally designed and multiple assembled to form a composite catalytic system and achieve cascade catalytic functions. Enzyme cascade catalysis could produce various chemical products with high conversion rate in short time. With the development of DNA nanotechnology, assembling enzymes to different nucleic acid-based scaffolds in different spatial organizations could effectively improve the catalytic efficiency of enzymes. Herein, we review the construction and application of nucleic acid-based scaffold systems from the perspective of template assembly in three dimensions. The challenges and future outlooks in the development of enzyme cascades are also discussed. KEY POINTS: • The principles and construction of various nucleic acid scaffolds are summarized • The application of nucleic acid scaffolds in enzyme cascade catalysis is discussed.

Inactivation of the MSTN gene expression changes the composition and function of the gut microbiome in sheep.

BACKGROUND: Myostatin (MSTN) negatively regulates the muscle growth in animals and MSTN deficient sheep have been widely reported previously. The goal of this study was to explore how MSTN inactivation influences their gut microbiota composition and potential functions. RESULTS: We compared the slaughter parameters and meat quality of 3 MSTN-edited male sheep and 3 wild-type male sheep, and analyzed the gut microbiome of the MSTN-edited sheep (8 female and 8 male sheep) and wild-type sheep (8 female and 8 male sheep) through metagenomic sequencing. The results showed that the body weight, carcass weight and eye muscle area of MSTN-edited sheep were significantly higher, but there were no significant differences in the meat quality indexes. At the microbial level, the alpha diversity was significantly higher in the MSTN-edited sheep (P < 0.05), and the microbial composition was significantly different by PCoA analysis in the MSTN-edited and wild-type sheep. The abundance of Firmicutes significantly increased and Bacteroidota significantly decreased in the MSTN-edited sheep. At genus level, the abundance of Flavonifractor, Subdoligranulum, Ruthenibacterium, Agathobaculum, Anaerotignum, Oribacterium and Lactobacillus were significantly increased in the MSTN-edited sheep (P < 0.05). Further analysis of functional differences was found that the carotenoid biosynthesis was significantly increased and the peroxisome, apoptosis, ferroptosis, N-glycan biosynthesis, thermogenesis, and adipocytokines pathways were decreased in the MSTN-edited sheep (P < 0.05). Moreover, carbohydrate-active enzymes (CAZymes) results certified the abundance of the GH13_39, GH4, GH137, GH71 and PL17 were upregulated, and the GT41 and CBM20 were downregulated in the MSTN-edited sheep (P < 0.05). CONCLUSIONS: Our study suggested that MSTN inactivation remarkably influenced the composition and potential function of hindgut microbial communities of the sheep, and significantly promoted growth performance without affecting meat quality.

Cytosine and adenine deaminase base-editors induce broad and nonspecific changes in gene expression and splicing.

Cytosine or adenine base editors (CBEs or ABEs) hold great promise in therapeutic applications because they enable the precise conversion of targeted base changes without generating of double-strand breaks. However, both CBEs and ABEs induce substantial off-target DNA editing, and extensive off-target RNA single nucleotide variations in transfected cells. Therefore, the potential effects of deaminases induced by DNA base editors are of great importance for their clinical applicability. Here, the transcriptome-wide deaminase effects on gene expression and splicing is examined. Differentially expressed genes (DEGs) and differential alternative splicing (DAS) events, induced by base editors, are identified. Both CBEs and ABEs generated thousands of DEGs and hundreds of DAS events. For engineered CBEs or ABEs, base editor-induced variants had little effect on the elimination of DEGs and DAS events. Interestingly, more DEGs and DAS events are observed as a result of over expressions of cytosine and adenine deaminases. This study reveals a previously overlooked aspect of deaminase effects in transcriptome-wide gene expression and splicing, and underscores the need to fully characterize such effects of deaminase enzymes in base editor platforms.

Circ_0081572 inhibits the progression of periodontitis through regulating the miR-378h/RORA axis.

OBJECTIVES: Revealing the role and mechanism of circ_0081572 in periodontitis progression. DESIGN: Quantitative real-time PCR (qRT-PCR) was applied to measure the expression of circ_0081572, microRNA (miR)-378h and retinoid acid-related orphan receptor A (RORA). Lipopolysaccharide (LPS) was used to treat periodontal ligament cells (PDLCs) to construct periodontitis cell model in vitro. Cell counting kit 8 (CCK8) assay and flow cytometry were used to measure cell viability and apoptosis. The caspase 3 activity was detected by Caspase 3 Activity Assay Kit. Western blot assay was performed to detect the expression of apoptosis-associated proteins and RORA. The inflammation response and oxidative stress were determined by detecting the levels of inflammatory cytokines and reactive oxygen species (ROS). The relationship between miR-378h and circ_0081572 or RORA was verified by dual-luciferase reporter assay, RNA immunoprecipitation (RIP) assay and biotin-labeled RNA pull-down assay. RESULTS: Circ_0081572 was a stability circRNA with downregulated expression in the gingival tissues of periodontitis patients. Overexpression of circ_0081572 could alleviate LPS-induced PDLCs injury. Circ_0081572 could serve as a sponge for miR-378h. Furthermore, miR-378h could reverse the inhibition of circ_0081572 on LPS-induced PDLCs injury. In addition, RORA could be targeted by miR-378h, and its silencing could reverse the suppressive effect of miR-378h inhibitor and circ_0081572 overexpression on LPS-induced PDLCs injury. CONCLUSIONS: Our results suggested that circ_0081572 might prevent periodontitis by regulating the miR-378h /RORA axis.

Diketopyrrolopyrrole-based fluorescence probes for the imaging of lysosomal Zn2+ and identification of prostate cancer in human tissue.

A series of diketopyrrolopyrrole-based fluorescent probes (DPP-C2, LysoDPP-C2, LysoDPP-C3, and LysoDPP-C4) have been developed for the detection of low pH and Zn2+ in an AND logic fashion. The chelation of Zn2+ or the protonation of a morpholine moiety within these probes results in a partial increase in the fluorescence intensity, an effect ascribed to suppression of one possible photo-induced electron transfer (PET) pathway. In contrast, a large increase in the observed fluorescence intensity is observed at low pH and in the presence of Zn2+; this is rationalized in terms of both possible PET pathways within the probes being blocked. Job plots, fluorescence titration curves, and isothermal titration calorimetry proved consistent with a 1 : 1 Zn2+ complexation stoichiometry. Each probe demonstrated an excellent selectivity towards Zn2+ and the resulting Zn2+ complexes demonstrated pH sensitivity over the 3.5-9 pH range. Fluorescence imaging experiments confirmed that LysoDPP-C4 was capable of imaging lysosomal Zn2+ in live cells. Little evidence of cytotoxicity was seen. LysoDPP-C4 was successfully applied to the bioimaging of nude mice, wherein it was shown capable of imaging the prostate. Histological studies using a human sample revealed that LysoDPP-C4 can discriminate cancerous prostate tissue from healthy prostate tissue.

A novel fluorescent probe for the early detection of prostate cancer based on endogenous zinc sensing.

BACKGROUND: The early detection of prostate cancer can significantly optimize the prognosis, prolong patient lifespan, and improve quality of life. It has been well documented that prostate cancer tissues have lower zinc content than normal prostate tissues due to an impairment of the zinc accumulation mechanism. METHODS: A novel diketopyrrolopyrrole (DPP)-based fluorescent zinc ion probe named DPP-C2 was prepared. The fluorescent intensity of this novel molecule is in direct proportion to environmental zinc concentration. Malignant (DU145 and PC3 cells) and normal prostate epithelial RWPE-1 cells were tested. Prostate cancer tissues were also cultured and observed as tissue sections. The probe was also intravenously administered to tumor-bearing (DU145 and PC3 cells) nude mice and observed under a whole-body fluorescence live-imaging system. RESULTS: The probe showed minimal cytotoxicity to malignant and normal prostate cells. The RWPE-1 cells showed not only stronger baseline fluorescence but also a significant increase in signal intensity after culturing in a zinc-supplemented medium. In human prostate sections, the pathologically confirmed cancer tissues exhibited weaker fluorescence signals than normal and benign hyperplastic tissues. With proper excitation, prostate tissues revealed more intense fluorescence signals than tumor tissues, whereas other surrounding tissues showed almost no fluorescence. CONCLUSIONS: The novel zinc ion fluorescent probe DPP-C2 is low toxic and showed potential application for the early detection of prostate cancer based on endogenous zinc sensing.

Downregulation of cystathionine ß-synthase/hydrogen sulfide contributes to rotenone-induced microglia polarization toward M1 type.

Microglia-mediated neuroinflammation is implicated in the pathogenesis of several neurodegenerative disorders. Microglia can be activated and polarized to exert pro- or anti-inflammatory roles in response to specific stimulus. Rotenone is an environmental toxin that has been shown to activate microglia and neuroinflammation. However, the effects and mechanisms of rotenone on microglia polarization are poorly studied. In the present study, we demonstrated that rotenone enhanced the levels of M1 phenotypic genes including TNF-α, iNOS and COX-2/PGE2 but reduced that of M2 markers such as Ym1/2 and IL-10 in mouse primary and immortalized microglia. Moreover, the transcription and protein expression of cystathionine-ß-synthase (CBS), as well as hydrogen sulfide (H2S) production were decreased in rotenone-treated primary microglia. Elevating endogenous H2S via CBS over-expression in immortalized microglia not only reduced the expression of pro-inflammatory M1 genes, but also enhanced the anti-inflammatory M2 marker IL-10 production in response to rotenone stimulation as compared to vector-transfected cells. Similarly, pretreatment with H2S donor NaHS (50, 100 and 500µmol/L) attenuated the increases of M1 gene expression triggered by rotenone treatment, and enhanced the M2 gene Ym1/2 expression in mouse primary microglia. In addition, we observed reactive oxygen species (ROS) scavenger N-acetyl-l-cysteine reversed the down-regulation of CBS and H2S generation caused by rotenone in microglia. NaHS pretreatment also decreased the ROS formation in rotenone-stimulated microglia. Taken together, these results reveal that probably via triggering ROS formation, rotenone suppressed the CBS-H2S pathway and thus promoted microglia polarization toward M1 pro-inflammatory phenotype.

Antidepressant-like effects of auraptenol in mice.

Depression is a major psychiatric disorder affecting nearly 21% of the world population and imposes a substantial health burden on society. Current available antidepressants are not adequate to meet the clinical needs. Here we report that auraptenol, an active component of the traditional Chinese medicine, angelicae dahuricae radix, had antidepressant-like effects in mice models of depression. In mouse forced swimming test and tail suspension test, two validated models of depression, auraptenol dose-dependently decreased the immobility duration within the dose range of 0.05-0.4 mg/kg. In addition, the antidepressant-like effects of auraptenol was significantly averted by a selective serotonin 5-HT1A receptor antagonist WAY100635 (1 mg/kg). These doses that affected the immobile response did not affect locomotor activity. In summary, this study for the first time identified an active component from the herbal medicine angelicae dahuricae radix that possesses robust antidepressant-like efficacy in mice. These data support further exploration for the possibility of developing auraptenol as a novel antidepressant agent in the treatment of major depression disorders.