Efficient Modulation of Exon Skipping via Antisense Circular RNAs.

Splice-switching antisense oligonucleotides (ASOs) and engineered U7 small nuclear ribonucleoprotein (U7 Sm OPT) are the most commonly used methods for exon skipping. However, challenges remain, such as limited organ delivery and repeated dosing for ASOs and unknown risks of by-products produced by U7 Sm OPT. Here, we showed that antisense circular RNAs (AS-circRNAs) can effectively mediate exon skipping in both minigene and endogenous transcripts. We also showed a relatively higher exon skipping efficiency at the tested Dmd minigene than U7 Sm OPT. AS-circRNA specifically targets the precursor mRNA splicing without off-target effects. Moreover, AS-circRNAs with adeno-associated virus (AAV) delivery corrected the open reading frame and restored the dystrophin expression in a mouse model of Duchenne muscular dystrophy. In conclusion, we develop an alternative method for regulating RNA splicing, which might be served as a novel tool for genetic disease treatment.

Effect of DHA-Enriched Phospholipids from Fish Roe on Rat Fecal Metabolites: Untargeted Metabolomic Analysis.

Lipid metabolism disorder has become an important hidden danger threatening human health, and various supplements to treat lipid metabolism disorder have been studied. Our previous studies have shown that DHA-enriched phospholipids from large yellow croaker (Larimichthys Crocea) roe (LYCRPLs) have lipid-regulating effects. To better explain the effect of LYCRPLs on lipid regulation in rats, the fecal metabolites of rats were analyzed from the level of metabolomics in this study, and GC/MS metabolomics measurements were performed to figure out the effect of LYCRPLs on fecal metabolites in rats. Compared with the control (K) group, 101 metabolites were identified in the model (M) group. There were 54, 47, and 57 metabolites in the low-dose (GA), medium-dose (GB), and high-dose (GC) groups that were significantly different from that of group M, respectively. Eighteen potential biomarkers closely related to lipid metabolism were screened after intervention with different doses of LYCRPLs on rats, which were classified into several metabolic pathways in rats, including pyrimidine metabolism, the citric acid cycle (TCA cycle), the metabolism of L-cysteine, carnitine synthesis, pantothenate and CoA biosynthesis, glycolysis, and bile secretion. L-cysteine was speculated to be a useful biomarker of LYCRPLs acting on rat fecal metabolites. Our findings indicated that LYCRPLs may regulate lipid metabolism disorders in SD rats by activating these metabolic pathways.

An In Vitro Catalysis of Tea Polyphenols by Polyphenol Oxidase.

Tea polyphenol (TPs) oxidation caused by polyphenol oxidase (PPO) in manufacturing is responsible for the sensory characteristics and health function of fermented tea, therefore, this subject is rich in scientific and commercial interests. In this work, an in vitro catalysis of TPs in liquid nitrogen grinding of sun-dried green tea leaves by PPO was developed, and the changes in metabolites were analyzed by metabolomics. A total of 441 metabolites were identified in the catalyzed tea powder and control check samples, which were classified into 11 classes, including flavonoids (125 metabolites), phenolic acids (67 metabolites), and lipids (55 metabolites). The relative levels of 28 metabolites after catalysis were decreased significantly (variable importance in projection (VIP) > 1.0, p < 0.05, and fold change (FC) < 0.5)), while the relative levels of 45 metabolites, including theaflavin, theaflavin-3'-gallate, theaflavin-3-gallate, and theaflavin 3,3'-digallate were increased significantly (VIP > 1.0, p < 0.05, and FC > 2). The increase in theaflavins was associated with the polymerization of catechins catalyzed by PPO. This work provided an in vitro method for the study of the catalysis of enzymes in tea leaves.

Membrane-Bound EMC10 Is Required for Sperm Motility via Maintaining the Homeostasis of Cytoplasm Sodium in Sperm.

Endoplasmic reticulum membrane protein complex subunit 10 (EMC10) is an evolutionarily conserved and multifunctional factor across species. We previously reported that Emc10 knockout (KO) leads to mouse male infertility. Emc10-null spermatozoa exhibit multiple aspects of dysfunction, including reduced sperm motility. Two subunits of a Na/K-ATPase, ATP1A4 and ATP1B3, are nearly absent in Emc10 KO spermatozoa. Here, two isoforms of EMC10 were characterized in the mouse testis and epididymis: the membrane-bound (mEMC10) and secreted (scEMC10) isoforms. We present evidence that mEMC10, rather than scEMC10, is required for cytoplasm sodium homeostasis by positively regulating ATP1B3 expression in germ cells. Intra-testis mEMC10 overexpression rescued the sperm motility defect caused by Emc10 KO, while exogenous recombinant scEMC10 protein could not improve the motility of spermatozoa from either Emc10 KO mouse or asthenospermic subjects. Clinically, there is a positive association between ATP1B3 and EMC10 protein levels in human spermatozoa, whereas no correlation was proven between seminal plasma scEMC10 levels and sperm motility. These results highlight the important role of the membrane-bound EMC10 isoform in maintaining cytoplasm sodium homeostasis and sperm motility. Based on the present results, the mEMC10-Na, K/ATPase α4ß3 axis is proposed as a novel mechanism underlying the regulation of cytoplasmic sodium and sperm motility, and its components seem to have therapeutic potential for asthenospermia.

Evidence-Based Nursing Model in Interventional Thrombolysis for Acute Lower Extremity Arterial Embolism.

Acute lower extremity arterial embolism (AE) is a serious clinical emergency, and, if not treated in time, it can easily lead to limb ischemia and necrosis and eventually facing amputation, which seriously damages patients' physical and mental health. In the past, the conventional drug thrombolytic therapy had slow and limited efficacy, and the best time for treatment is easily delayed, while arterial dissection and thrombectomy treatment, although fast, is traumatic and has many complications, which is not easily accepted by patients. The aim of this study was to investigate the value of evidence-based care model in the application of interventional thrombolysis for acute lower limb arterial embolism. Seventy-two patients with acute lower limb arterial embolism who underwent interventional thrombolysis treatment received by the Department of Vascular Surgery of our hospital from July 2016 to December 2021 were randomly divided into a control group (given conventional nursing services) and a quality group (given full quality nursing services) to compare the effect of nursing services in the two groups. The results showed that the postoperative psychological status of patients in the quality group was significantly better than that of patients in the control group (P < 0.05). The total incidence of postoperative adverse events and the total treatment efficiency of the quality group were better than those of the control group (P < 0.05). The efficacy of quality nursing care in patients with acute lower extremity arterial embolism is more desirable than conventional nursing care and is recommended. The site of vascular occlusion after bypass surgery can be clarified when angiography is performed after thrombolytic therapy, which can help secondary surgical intervention to prolong the time to patency. The efficacy of quality nursing care in patients with acute lower extremity arterial embolism is more desirable than that of conventional nursing care and is recommended.

Effect of inoculation with Penicillium chrysogenum on chemical components and fungal communities in fermentation of Pu-erh tea.

Developing an effective method to improve the quality of Pu-erh tea is of great scientific and commercial interest. In this work, Penicillium chrysogenum P1 isolated from Pu-erh tea was inoculated in sterilized or unsterilized sun-dreid green tea leaves to develop pure-culture fermentation (PF) and enhanced fermentation (EF) of Pu-erh tea. Spectrophotometry and high performance liquid chromatography determined that contents of free amino acids (FAA), total tea polyphenols and eight polyphenolic compounds in PF were significantly lower than these in non-inoculation control test (CK) (P < 0.05), whereas the contents of soluble sugars and theabrownins (TB) in PF were significantly higher (P < 0.05) than in CK. A total of 416 volatile compounds were detected by headspace solid-phase micro-extraction combined with gas chromatography-mass spectrometry. Comparison to CK, 124 compounds in PF were degraded or decreased significantly [Variable importance in projection [(VIP) > 1.0, P < 0.05, fold change (FC) < 0.5], whereas 110 compounds in PF were formed or increased significantly (VIP > 1.0, P < 0.05, FC > 2). Compared with normal fermentation (NF), the levels of gallic acid, (+)-catechin, (-)-epicatechin and 64 volatile compounds in EF were significantly lower (VIP > 1.0, P < 0.05, FC < 0.5), whereas the levels of FAA and 39 volatile compounds were significantly higher (VIP > 1.0, P < 0.05, FC > 2). Amplicon sequencing of fungal internal transcribed spacer 1 (ITS1) revealed that P. chrysogenum P1 didn't become the dominant fungus in EF; while the fungal communities in EF were different from those in NF, in that the relative abundances of Blastobotrys bambusae and P. chrysogenum in EF were higher, and the relative abundances of Aspergillus niger and Kluyveromyces marxianus in EF were lower. Overall, inoculation of P. chrysogenum in unsterilized sun-dreid green tea leaves changed the the fungal communities in fermentation of Pu-erh tea, and chemical compounds in fermented tea leaves, i.e., the levels of TB and the compounds responsible for the stale flavor, e.g., 2-amino-4-methoxybenzothiazole were increased, resulting in improvement of the sensory quality, including mellower taste and stronger stale flavor.

Quality assessment of large yellow croaker (Larimichthys crocea) roe oil before and after refining.

This research aimed to assess the quality of the large yellow croaker (Larimichthys crocea) roe oil before and after refining. The crude and refined L. crocea roe oils were compared based on their peroxide value (PV), acid value (AV), iodine value (IV), saponification value (SV), and fatty acid composition. Furthermore, the volatile compounds were identified and analyzed via gas chromatography-mass spectroscopy (GC-MS) and electronic nose (E-nose) analysis. Meanwhile, the flavor fingerprint was established via headspace-gas chromatography-ion mobility spectrometry (HS-GC-IMS). The results showed that the PV, AV, IV, and SV of the refined oil were 4.44 ± 0.04 mmol kg-1, 2.86 ± 0.01 mgKOH g-1, 163.1 ± 0.8 g/100 g, and 222.9 ± 0.7 mg g-1, respectively. The docosahexaenoic acids (DHAs) content in the total polyunsaturated fatty acids (PUFAs) was increased. Moreover, 55 volatile compounds were identified in the refined oil; among these compounds, the contents of carboxylic acids, aldehydes, alcohols, ketones, and esters were reasonably increased, while the hydrocarbon and heterocyclic compound contents were decreased. The flavor fingerprints of the crude and refined L. crocea roe oils were established by HS-GC-IMS. The results demonstrated that the refining improved the quality of L. crocea roe oil.

The protective mechanisms of macroalgae Laminaria japonica consumption against lipid metabolism disorders in high-fat diet-induced hyperlipidemic rats.

Macroalgae Laminaria japonica (MLJ) has been reported to exhibit various biological activities including improving immunity, anti-aging, anti-tumor, anti-atherosclerosis and anti-diabetic, but the protective mechanisms of MLJ consumption against non-alcoholic fatty liver disease (NAFLD) associated with hyperlipidemia remain poorly understood. This study demonstrated that MLJ consumption prevented high-fat diet (HFD)-induced NAFLD associated with hyperlipidemia in a rat model, and improved hyperlipidemia-related parameters, e.g. serum and hepatic lipid profiles. Moreover, histological analysis showed that MLJ reduced lipid deposition in adipocytes and hepatocytes compared with the HFD group. Such beneficial effects may be associated with the modulation of the intestinal microbiota, especially some key microbial phylotypes involved in lipid metabolism homeostasis. The underlying protective mechanisms of MLJ consumption against HFD-induced NAFLD associated with hyperlipidemia were also studied by ultra-high performance liquid chromatography with quadruple-time of flight mass spectrometry (UPLC-QTOF/MS)-based liver metabolomics coupled with pathway analysis. The metabolic pathway enrichment analysis of the differentially abundant hepatic metabolites indicated that primary bile acid biosynthesis metabolism and cysteine and methionine metabolism were the two main metabolic pathways altered by MLJ consumption when compared with the model group. The analysis of the transcription levels of liver-related genes by RT-qPCR and the expressions of liver-related proteins by immunohistochemistry (IHC) showed that MLJ consumption could regulate the levels of mRNA transcription and protein expression related to hepatic lipid metabolism. In short, this study indicates that MLJ could be developed as functional food supplement for the prevention or treatment of NAFLD associated with hyperlipidemia.

Inhibition Effect of Triglyceride Accumulation by Large Yellow Croaker Roe DHA-PC in HepG2 Cells.

The phospholipids (PLs) of large yellow croaker (Pseudosciaena crocea, P. crocea) roe contain a high level of polyunsaturated fatty acids, especially docosahexaenoic acid (DHA), which can lower blood lipid levels. In previous research, PLs of P. crocea roe were found able to regulate the accumulation of triglycerides. However, none of these involve the function of DHA-containing phosphatidylcholine (DHA-PC), which is the main component of PLs derived from P. crocea roe. The function by which DHA-PC from P. crocea roe exerts its effects has not yet been clarified. Herein, we used purified DHA-PC and oleic acid (OA) induced HepG2 cells to establish a high-fat model, and the cell activity and intracellular lipid levels were then measured. The mRNA and protein expression of Fatty Acid Synthase (FAS), Carnitine Palmitoyl Transferase 1A (CPT1A) and Peroxisome Proliferator-Activated Receptor α (PPARα) in HepG2 cells were detected via RT-qPCR and western blot as well. It was found that DHA-PC can significantly regulate triglyceride accumulation in HepG2 cells, the effect of which was related to the activation of PPARα receptor activity, upregulation of CPT1A, and downregulation of FAS expression. These results can improve the understanding of the biofunction of hyperlipidemia mediated by DHA-PC from P. crocea roe, as well as provide a theoretical basis for the utilization of DHA-PC from P. crocea roe as a functional food additive.

A high performance liquid chromatography method for simultaneous detection of 20 bioactive components in tea extracts.

Tea is the second most widely consumed beverage and contains various bioactive compounds. A simple method to analyze these compounds is of great scientific and commercial interest. In this work, a 30 min HPLC method was developed using a simple gradient elution system, and the mobile phases and elution gradients were optimized. This method separated 17 polyphenols and three alkaloid compounds in tea extracts, including catechins, alkaloids, phenolic acids, flavonols, and flavone, which are responsible for the bioactivity and flavor of tea. Excellent linearity was observed for all standard calibration curves, and correlation coefficients were above 0.9994. Heatmap analysis demonstrated significant separation between green, black, and pu-erh tea samples. The method described here is accurate and sensitive enough for the determination of active components in tea and could potentially be applied to other food products for the comprehensive investigation of their quality.

Combined effects of proinflammatory cytokines and intermittent cyclic mechanical strain in inhibiting osteogenicity in human periodontal ligament cells.

Mechanical strain plays an important role in bone formation and resorption during orthodontic tooth movement. The mechanism has not been fully studied, and the process becomes complex with increased amounts of periodontal patients seeking orthodontic care. Our aims were to elucidate the combined effects of proinflammatory cytokines and intermittent cyclic strain (ICS) on the osteogenic capacity of human periodontal ligament cells. Cultured human periodontal ligament cells were exposed to proinflammatory cytokines (interleukin-1ß 5 ng/mL and tumor necrosis factor-α 10 ng/mL) for 1 and 5 days, and ICS (0.5 Hz, 12% elongation) was applied for 4 h per day. The autocrine of inflammatory cytokines was measured by enzyme-linked immunosorbent assay. The expression of osteoblast markers runt-related transcription factor 2 and rabbit collagen type I was determined using real-time polymerase chain reaction and Western blot. The osteogenic capacity was also detected by alkaline phosphatase (ALP) staining, ALP activity, and alizarin red staining. We demonstrated that ICS impaired the osteogenic capacity of human periodontal ligament cells when incubated with proinflammatory cytokines, as evidenced by the low expression of ALP staining, low ALP activity, reduced alizarin red staining, and reduced osteoblast markers. These data, for the first time, suggest that ICS has a negative effect on the inductive inhibition of osteogenicity in human PDL cells mediated by proinflammatory cytokines.

Endogenous hydrogen sulfide is involved in osteogenic differentiation in human periodontal ligament cells.

OBJECTIVE: Endogenous hydrogen sulfide (H2S) has recently emerged as an important intracellular gaseous signaling molecule within cellular systems. Endogenous H2S is synthesized from l-cysteine via cystathionine ß-synthase and cystathionine γ-lyase and it regulates multiple signaling pathways in mammalian cells. Indeed, aberrant H2S levels have been linked to defects in bone formation in experimental mice. The aim of this study was to examine the potential production mechanism and function of endogenous H2S within primary human periodontal ligament cells (PDLCs). DESIGN: Primary human PDLCs were obtained from donor molars with volunteer permission. Immunofluorescent labeling determined expression of the H2S synthetase enzymes. These enzymes were inhibited with D,L-propargylglycine or hydroxylamine to examine the effects of H2S signaling upon the osteogenic differentiation of PDLCs. Gene and protein expression levels of osteogenic markers in conjunction with ALP staining and activity and alizarin red S staining of calcium deposition were used to assay the progression of osteogenesis under different treatment conditions. Cultures were exposed to Wnt3a treatment to assess downstream signaling mechanisms. RESULTS: In this study, we show that H2S is produced by human PDLCs via the cystathionine ß-synthase/cystathionine γ-lyase pathway to promote their osteogenic differentiation. These levels must be carefully maintained as excessive or deficient H2S levels temper the observed osteogenic effect by inhibiting Wnt/ß-catenin signaling. CONCLUSIONS: These results demonstrate that optimal concentrations of endogenous H2S must be maintained within PDLCs to promote osteogenic differentiation by activating the Wnt/ß-catenin signaling cascade.

The 5-HT2A receptor potassium-chloride cotransporter 2 signaling pathway in a rat incision pain model.

Postoperative pain is a critical problem in clinical pain administration. Due to the unclear formation mechanism of postoperative pain, the treatment of postoperative pain is still in the symptomatic treatment stage and lacking satisfactory analgesic effect. Postoperative pain can be simulated by using a rat incision pain model. We observed changes in pain-related behavior of rats affected by the 5-hydroxytryptamine 2A receptor (5-HT2AR) agonist, TCB-2, and antagonist, ketanserin, through intrathecal delivery. The transcription and translation level of potassium-chloride cotransporter 2 (KCC2) in the spinal cord was also measured to investigate the role of the 5-HT2AR-KCC2 pathway in the mechanism of incision pain. Compared with the control group, rats in the incision pain group had decreased mechanical withdrawal threshold (MWT), with significant differences on day 1-7 after surgery, and significant decreases in thermal withdrawal latency (TWL) on days 1, 2, 3 and 6 (P<0.05). Compared with the incision + dimethyl sulfoxide (DMSO) group, MWT and TWL decreased in the incision + ketanserin group on day 1 and 2 (P<0.05). There was no significant difference detected in TWL of incision + TCB-2 group on day 1, while MWT increased significantly compared to the incision + DMSO group (P<0.05). Furthermore, the transcription and translation levels of KCC2 in the incision + ketanserin group decreased significantly in comparison to the incision + DMSO group (P<0.05), while an increase was detected in the incision + TCB-2 group (P<0.05). MWT and TWL decreased in the incision pain rats, accompanied with a decreased transcription and translation level of KCC2. Intrathecal delivery of the 5-HT2AR agonist, TCB-2, alleviated the decreased WMT and inhibited the decreased transcription and translation level of KCC2, while intrathecal delivery of the 5-HT2AR antagonist, ketanserin, aggravated the decreased WMT and transcription and translation levels of KCC2, suggesting the involement of the 5-HT2AR-KCC2 pathway in the formation mechanism of incision pain in rats.