Identification, expression and enzyme activity of the group III sPLA2 s in Cyprinus carpio L.

Five group III secreted phospholipase (pla2g3s) homologous genes located on different linkage groups were identified from common carp (Cyprinus carpio), which we named Ccpla2g3a1, Ccpla2g3a2, Ccpla2g3b, Ccpla2g3c1 and Ccpla2g3c2. The five genes encode 530, 525, 461, 752 and 753 amino acids, respectively. Sequence analysis showed that the Ccpla2g3as contain seven exons and the others contain four exons. Synteny analysis of fish pla2g3s indicated that pla2g3a and pla2g3b were from the same ancestor gene, and Ccpla2g3a1, Ccpla2g3a2, Ccpla2g3c1 and Ccpla2g3c2 were from the specific genome duplication of common carp. Due to the significant variation of the pla2g3bs from common carp and zebrafish (Danio rerio), they formed a separate group in the phylogenetic tree. The tissue distributions of Ccpla2g3s coincided with their expression profiles during the embryo stages. The expression levels of Ccpla2g3as and Ccpla2g3cs were low at the embryo stages, and they were abundant in the liver and brain, respectively, whereas the expression of Ccpla2g3b was high at 0.5 h after fertilization and in the ovary. We obtained three soluble recombinant proteins of the bee venom-like PLA2 (BVLP) from Ccpla2g3 and evaluated their PLA2 enzyme properties. The optimum pHs of MBP-a1-BVLP, MBP-b-BVLP and MBP-c1-BVLP were 7.5, 7.0 and 8.0, respectively, and specific activities were 7.68 ± 0.66, 4.155 ± 0.158 and 1.93 ± 0.05 U µmol-1 , respectively. The Kd for Ca2+ of MBP-b-BVLP was the lowest (2.6 µM), whereas the values for both MBP-a1-BVLP and MBP-c1-BVLP were about 15 µM. The Km values of three proteins ranged from 31.9 to 41.91 µM.

Mechanistic Study of Copper-Catalyzed C-H Hydroxylation/C-S Coupling by ESI-HR MS and DFT Calculations.

The reaction mechanism of Cu-catalyzed C-H hydroxylation/C-S coupling was studied using electrospray ionization high resolution mass spectrometry (ESI-HR MS) and density functional theory calculations (DFT). Notably, a series of CuI and CuIII complexes were observed as key intermediates and identified using ESI-HR MS. Furthermore, a catalyst cycle involving proton abstraction/oxidative addition/reductive elimination was proposed. This study is important and valuable with respect to C-H functionalization.

Mediation of association between semen microcystin exposure and semen quality by sex hormones in Chinese men.

Numerous studies have shown microcystins (MCs) inducing male reproductive toxicity, but the underlying mechanisms in humans are unclear. Therefore, this study aimed to evaluate the mediating role of serum sex hormones in the association between MC exposure and semen quality. In this study, we measured the levels of semen MCs and serum sex hormones in Chinese men [sample 1 (n = 649); sample 2 (n = 924)]. The results showed that there was a non-significant dose-dependent relationship between semen MCs and semen volume reduction (p for trend = 0.079) in sample 1, and semen MCs were significantly negatively associated with total motility, progressive motility, curvilinear velocity, mean angular displacement and acrosome integrity (p < 0.05) in sample 2. We also found that semen MCs were significantly positively associated with serum follicle stimulating hormone (FSH) (ß = 0.151; 95% CI: 0.065, 0.236), but negatively associated with serum inhibin B (INHB) (ß = -0.605; 95% CI: -0.944, -0.265), and these linear associations were confirmed in restricted cubic spline (RCS) models (all pnon-linearity > 0.1). Furthermore, mediation analysis revealed that serum INHB mediated 19.86% of the adverse effect of MC exposure on acrosome integrity. In conclusion, this study reveals the mediating roles of serum sex hormones in the relationship between MC exposure and decreased semen quality in men.

Exploring peptides from toad venom for source identification by LC-MS/MS using MRM method.

Toad venom is a traditional Chinese medicine (TCM) with various sources and wide-ranging preparations. Previous quality assessment studies primarily concentrated on small molecular compounds like toad dienolactones and indole alkaloids, studies on macromolecular peptides and proteins as quality assessment standards remained at the qualitative stage, lacking the development of practical and convenient quantitative methods. In this study, to explore the peptides from toad venom as a new method for identifying and evaluating its source, a complete scan of the water extract of peptides from toad venom was conducted using HPLC-Quadrupole Time-of-Flight Mass Spectrometer (Q-TOF) 5600, leading to the identification of peptides based on mass spectrometry data. Subsequently, HPLC- Quadrupole-Linear Ion Trap Mass Spectrometer (Q-Trap) 5500 employing Multiple Reaction Monitoring (MRM) mode was utilized to quantitatively analyze peptides in various sources of toad venom, followed by Partial Least Squares Discriminant Analysis (PLS-DA) to further analyze the data and evaluate the effectiveness. This study highlights the importance of exploring macromolecular substance in natural products research and provides a foundation for further studies on toad venom.

Exposure to different surface-modified polystyrene nanoparticles caused anxiety, depression, and social deficit in mice via damaging mitochondria in neurons.

Nanoplastics (NPs) are unavoidable hazardous materials that result from the human production and use of plastics. While there is evidence that NPs can bioaccumulate in the brain, no enough research regarding the pathways by which NPs reach the brain was conducted, and it is also urgently needed to evaluate the health threat to the nervous system. Here, we observed accumulation of polystyrene nanoplastics (PS-NPs) with different surface modifications (PS, PS-COOH, and PS-NH2) in mouse brains. Further studies showed that PS-NPs disrupted the tight junctions between endothelial cells and transport into endothelial cells via the endocytosis and macropinocytosis pathways. Additionally, NPs exposure induced a series of alternations in behavioral tests, including anxiety- and depression-like changes and impaired social interaction performance. Further results identified that NPs could be internalized into neurons and localized in the mitochondria, bringing about mitochondrial dysfunction and a concurrent decline of ATP production, which might be associated with abnormal animal behaviors. The findings provide novel insights into the neurotoxicity of NPs and provide a basis for the formulation of policy on plastic production and usage by relevant government agencies.

Sertoli cell-derived extracellular vesicles traverse the blood-testis barrier and deliver miR-24-3p inhibitor into germ cells improving sperm mobility.

Asthenozoospermia, characterized by poor sperm motility, is a common cause of male infertility. Improving energy metabolism and alleviating oxidative stress through drug regimens are potential therapeutic strategies. In this study, we observed upregulated miR-24-3p levels in asthenozoospermia spermatozoa, contributing to energy metabolism disorder and oxidative stress by reducing GSK3ß expression. Thus, reducing miR-24-3p levels using drugs is expected to improve sperm motility. The blood-testis barrier (BTB) protects the testis from xenobiotics and drugs. In this study, we found that Sertoli cell-derived small extracellular vesicles (SC-sEV) can traverse the BTB and enter germ cells. We successfully loaded miR-24-3p inhibitor into SC-sEV, creating the nano-drug SC-sEV@miR-24-3p inhibitor, which effectively delivers miR-24-3p inhibitor into germ cells. In a gossypol-induced mouse asthenozoospermia model, administration of SC-sEV@miR-24-3p inhibitor significantly improved sperm motility, in vitro fertilization success, and blastocyst formation rates. As anticipated, it also improved the litter size of asthenozoospermia mice. These results suggest that SC-sEV@miR-24-3p inhibitor holds promise as a potential clinical treatment for asthenospermia.

Differently surface-labeled polystyrene nanoplastics at an environmentally relevant concentration induced Crohn's ileitis-like features via triggering intestinal epithelial cell necroptosis.

Nanoplastics (NPs), regarded as the emerging contaminants, can enter and be mostly accumulated in the digest tract, which pose the potential threat to intestinal health. In this study, mice were orally exposed to polystyrene (PS), PS-COOH and PS-NH2 NPs with the size of ∼100 nm at a human equivalent dose for 28 consecutive days. All three kinds of PS-NPs triggered Crohn's ileitis-like features, such as ileum structure impairment, increased proinflammatory cytokines and intestinal epithelial cell (IEC) necroptosis, and PS-COOH/PS-NH2 NPs exhibited higher adverse effects on ileum tissues. Furthermore, we found PS-NPs induced necroptosis rather than apoptosis via activating RIPK3/MLKL pathway in IECs. Mechanistically, we found that PS-NPs accumulated in the mitochondria and subsequently caused mitochondrial stress, which initiated PINK1/Parkin-mediated mitophagy. However, mitophagic flux was blocked due to lysosomal deacidification caused by PS-NPs, and thus led to IEC necroptosis. We further found that mitophagic flux recovery by rapamycin can alleviate NP-induced IEC necroptosis. Our findings revealed the underlying mechanisms concerning NP-triggered Crohn's ileitis-like features and might provide new insights for the further safety assessment of NPs.

Male reproductive toxicity induced by Microcystin-leucine-arginine (MC-LR).

Microcystin-LR (MC-LR) has been identified to pose an increasing threat to the male reproductive system in vivo and in vitro studies with the objects like mammal animals, amphibians, aquatic organisms, etc. This review demonstrates the latest research advances of the male reproductive toxicity induced by MC-LR in detail, which mainly consists of two aspects, namely pathological injuries to testis and prostate, as well as the endocrine disruption. Apart from the direct toxicity to the male reproductive system, we also underline the transgenerational reproductive toxicity that prenatal exposure may pass on to male offspring. This review also demonstrates the interactive effects between MC-LR and other compounds, including synergistic effects with some toxicants and antagonistic effects with some medicine or chemical modification. In terms of the mechanisms of MC-LR-induced toxicity, we mainly focus on the epigenetic modification and non-coding RNAs (ncRNAs)-related mechanisms which have provided a new perspective.

Systematic toxicity evaluation of polystyrene nanoplastics on mice and molecular mechanism investigation about their internalization into Caco-2 cells.

There is a growing concern regarding the toxic effects of nanoplastics (NPs) on aquatic and marine organism, while relatively few studies about their toxicity evaluation on mammals are conducted. In the present study, we observed accumulation of polystyrene NPs (PS NPs) in mice spleen, lung, kidney, small intestine, large intestine, testis, and brain after oral exposure to PS NPs (~100 nm, 10 mg/mL, 100 µL) for 28 days, and NPs were identified to induce cell apoptosis, inflammation, and structure disorder in these tissues. We also found that PS NPs could bring about hematological system injury and lipid metabolism disorder. Further in vitro studies identified that PS NPs could be absorbed by the intestinal epithelial Caco-2 cells by macropinocytosis and clathrin-mediated endocytosis, and induced disruption of tight junction between Caco-2 cells. Moreover, we found that it was easier for PS-NH2 and PS-COOH to enter into Caco-2 cells, which may be associated with observed stronger toxicity of PS-NH2 and PS-COOH NPs. In summary, this study demonstrated that NPs exposure brings about toxic effects to mice. This study could provide new insights regarding the distribution of NPs in humans, and helps us to evaluate the potential physiological risks of NPs to human beings.

Chronic MC-LR exposure promoted Aß and p-tau accumulation via regulating Akt/GSK-3ß signal pathway.

It has been reported that microcystin-leucine-arginine (MC-LR) can enter into the brain and demonstrate neurotoxicity resulting in learning and memory deficits. While, there is still a lack of clear understanding of the related molecular mechanisms. In this study, we observed ß-amyloid (Aß) accumulation and tau hyperphosphorylation (p-tau) at sites of Ser396 and Thr205 in mouse hippocampus and cortex, Alzheimer's disease (AD) like changes, after chronic exposure to MC-LR at different concentrations (1, 7.5, 15 and 30 µg/L) for 180 days. The hallmarks of AD are characterized by senile plaques and neurofibrillary tangles (NFT), with associated loss of neurons, resulting in cognitive impairment and dementia. Similarly, the production of Aß and tau hyperphosphorylation was also detected in HT-22 cells treated with MC-LR. In addition, MC-LR promoted increased expressions of BACE1 and PS1, but reduced mRNA expressions of ADAM family members both in vivo and in vitro, promoting the Aß production. Moreover, we identified Akt/GSK-3ß signal pathway mediated the Aß and p-tau accumulation, bringing about Alzheimer's disease-like changes. Furthermore, microglial cells were activated in those mice exposed to MC-LR. Inflammatory cytokines were also found being activated to release in vitro. In conclusion, this study could provide a clue for MC-LR-induced neurotoxicity, which gave insights into the environmental risks of Alzheimer's disease.

Microcystin-leucine-arginine induces apical ectoplasmic specialization disassembly.

Microcystin-leucine-arginine (MC-LR) has been identified to be a hazardous material to cause hepatotoxicity. In this study, mice were exposed to MC-LR dissolved in drinking water at doses of 1, 10, 20 and 30 µg/L for 90 and 180 days, respectively. We validated MC-LR accelerated spermatid exfoliation and caused large vacuoles in testes, reducing sperm count and increasing percentage of morphologically abnormal sperm. Furthermore, we found MC-LR induced the apical ectoplasmic specialization (ES) disassembly by disrupting F-actin organization. Further studies identified that downregulation of Palladin, the actin crosslinking protein, might be associated with disassembly of the apical ES in mice testis following MC-LR exposure. We also confirmed that MC-LR disrupted the interaction between Palladin and other actin-related proteins and thus impeded the F-actin organization. Additionally, we found that autophagy initiated by AMPK/ULK1 signaling pathway mediated the degradation of Palladin in Sertoli cells challenged with MC-LR. Following exposure to MC-LR, reduced PP2A activity and upregulated expression of LKB1 and CAMKK2 could activate AMPK. In conclusion, these results revealed MC-LR induced the degradation of Palladin via AMPK/ULK1-mediated autophagy, which might result in the apical ES disorder and spermatid exfoliation from spermatogenic epithelium. Our work may provide a new perspective to understand MC-LR-induced male infertility.

Ultrafiltration strategy combined with nanoLC-MS/MS based proteomics for monitoring potential residual proteins in TCMIs.

Traditional Chinese medicine injections (TCMIs) containing complex constituents frequently cause unpredictable adverse reactions. The residual heterologous proteins in TCMIs may be one kind of the sensitized constituents. However, few methods were developed to identify and monitor the residual proteins of TCMIs in industry. Here, we described a method combining the advantages of ultrafiltration and mass spectrometry-based proteomics for monitoring the potential residual proteins in Re Du Ning injection (RDNI) intermediates and preparations. We identified and quantified both de novo peptides and the proteins matched against databases of three raw plants by using PEAKS software. Interesting, we found there was a significant decrease of peptides and proteins in No. 3-5 of RDNI intermediates and some even disappeared. Besides, we found this method could greatly reduce the interference of contaminants in proteomics experiments. The rapid and accurate method proposed in this paper could be used for monitoring potential residual proteins in TCMIs to guarantee their quality and safety.

Analyzing liver protein-bound DMAV by using size exclusion and ion exchange HPLC combined with ICP-MS and MRM mode in rats exposed to AS4S4.

Long-term exposure to high levels of arsenic (As) will result in damage to organs. Compared with free arsenic, protein-bound arsenic are more difficult to be excreted from the bodies due to their complexation with biological macromolecules. We developed a method of size exclusion chromatography (SEC) and ion exchange chromatography (IEC) combined with inductively coupled plasma-mass spectrometry (ICP-MS) and multiple reaction monitoring (MRM) mode, which was used to determine bound-arsenic species. DMAV was identified as bound arsenic species in rat livers after As4S4 overexposure. Subsequent proteomics analysis showed the potential binding partners included hemoglobin, glutathione S-transferases, superoxide dismutase [Cu-Zn] & [Mn], thiosulfate sulfurtransferase, and metallothionein-2. The method developed here was sensitive, repeatable, and conducive to arsenic analysis, especially for toxicity evaluation of arsenic-containing substances in vivo.

Anti-inflammatory and analgesic actions of bufotenine through inhibiting lipid metabolism pathway.

Inflammation is a primary defense and immune response. However, under pathological conditions, the inflammation processes always become uncontrolled and lead to chronic diseases. Bufotenine, as a natural component from toad venom, showed great potential for development as a novel anti-inflammation and analgesia agent. This study aimed to investigate the therapeutic effects of bufotenine against inflammation and pain on animal models with a focus on lipid metabolism. In pharmacological studies, bufotenine significantly inhibited the swelling rates on formalin-induced paw edema model, and increased paw withdrawal mechanical thresholds (PWMTs) in von Frey test and thermal pain thresholds (TPTs) in hot-plate test. High-sensitivity lipidomics analysis revealed the effects might be related to the down-regulation of inflammatory mediators from cyclooxygenase (COX), lipoxygenase (LOX), cytochrome P450 (CYP450), linoleic acid (LA), docosahexaenoic acid (DHA) and other pathways. The activities might result from the binding of bufotenine and its receptors, including sigma-1 receptor and 5-Hydroxytryptamine receptor 3A, thus regulating lipid metabolism pathway. The research provided a systemic evidence for the actions and mechanism of bufotenine. It suggested that the natural compound might be a potential candidate for reducing inflammatory pain disorders.

Silencing of METTL3 effectively hinders invasion and metastasis of prostate cancer cells.

Background: Since primary prostate cancer (PCa) can advance to the life-threatening metastatic PCa, exploring the molecular mechanisms underlying PCa metastasis is crucial for developing the novel targeted preventive strategies for decreasing the mortality of PCa. RNA N6-methyladenosine (m6A) is an emerging regulatory mechanism for gene expression and its specific roles in PCa progression remains elusive. Methods: Western blotting, quantitative real-time PCR and immunohistochemical analyses were used to detect target gene expression in PCa cells in vitro and prostate tissues from patients. RNA immunoprecipitation was conducted to analyze the specific binding of mRNA to the target protein. Migration and invasion assays were used to assess the migratory capacities of cancer cells. The correlation between target gene expression and survival rate of PCa patients was analyzed based the TCGA database. Results: We found that total RNA N6-methyladenosine (m6A) modification levels were markedly upregulated in human PCa tissues due to increased expression of methyltransferase like 3 (METTL3). Further studies revealed that the migratory and invasive capacities of PCa cells were markedly suppressed upon METTL3 knockdown. Mechanistically, METTL3 mediates m6A modification of USP4 mRNA at A2696, and m6A reader protein YTHDF2 binds to and induces degradation of USP4 mRNA by recruiting RNA-binding protein HNRNPD to the mRNA. Decrease of USP4 fails to remove the ubiquitin group from ELAVL1 protein, resulting in a reduction of ELAVL1 protein. Lastly, downregulation of ELAVL1 in turn increases ARHGDIA expression, promoting migration and invasion of PCa cells. Conclusions: Our findings highlight the role of METTL3 in modulating invasion and metastasis of PCa cells, providing insight into promising therapeutic strategies for hindering PCa progressing to deadly metastases.

m6A mRNA methylation regulates testosterone synthesis through modulating autophagy in Leydig cells.

Macroautophagy/autophagy is indispensable for testosterone synthesis in Leydig cells (LCs), and here we report a negative association between m6A modification and autophagy in LCs during testosterone synthesis. A gradual decrease of METTL14 (methyltransferase like 14) and an increase of ALKBH5 (alkB homolog 5, RNA demethylase) were observed in LCs during their differentiation from stem LCs to adult LCs. These events led to reduced mRNA methylation levels of N6-methyladenosine (m6A) and enhanced autophagy in LCs. Similar regulation of METTL14, ALKBH5, and m6A was also observed in LCs upon treatment with human chorionic gonadotropin (HsCG). Mechanistically, m6A modification promoted translation of PPM1A (protein phosphatase 1A, magnesium dependent, alpha isoform), a negative AMP-activated protein kinase (AMPK) regulator, but decreased expression of CAMKK2 (calcium/calmodulin-dependent protein kinase kinase 2, beta), a positive AMPK regulator, by reducing its RNA stability. Thus, m6A modification resulted in reduced AMPK activity and subsequent autophagy inhibition. We further demonstrated that ALKBH5 upregulation by HsCG was dependent on enhanced binding of the transcriptional factor CEBPB (CCAAT/enhancer binding protein [C/EBP], beta) and the TFEB (transcription factor EB) to its gene promoter. Moreover, HsCG treatment decreased METTL14 by reducing its stability. Collectively, this study highlights a vital role of m6A RNA methylation in the modulation of testosterone synthesis in LCs, providing insight into novel therapeutic strategies by exploiting m6A RNA methylation as targets for treating azoospermatism and oligospermatism patients with reduction in serum testosterone.Abbreviations: 3-MA: 3-methyladenine; ACTB: Actin, beta; ALKBH5: alkB homolog 5, RNA demethylase; AMPK: AMP-activated protein kinase; BafA1: bafilomycin A1; CAMKK2: calcium/calmodulin-dependent protein kinase kinase 2, beta; CEBPB: CCAAT/enhancer-binding protein (C/EBP), beta; ChIP: chromatin immunoprecipitation; FTO: fat mass and obesity associated; HsCG: human chorionic gonadotropin; HSD3B: 3ß-hydroxysteroid dehydrogenase; LCs: Leydig cells; m6A: N6-methyladenosine; METTL14: methyltransferase like 14; METTL3: methyltransferase like 3; MTOR: mechanistic target of rapamycin kinase; PPM1A: protein phosphatase 1A, magnesium dependent, alpha isoform; PRKAA: 5'-AMP-activated protein kinase catalytic subunit alpha; SQSTM1: sequestosome 1; STK11/LKB1: serine/threonine kinase 11; TFEB: transcription factor EB; ULK1: unc-51-like kinase 1; WTAP: Wilms tumor 1-associating protein; YTHDF: YTH N6-methyladenosine RNA binding protein.

Evaluation of analgesic and anti-inflammatory actions of indolealkylamines from toad venom in mice using lipidomics and molecular docking.

ETHNOPHARMACOLOGICAL RELEVANCE: Toad venom is one of widely used traditional Chinese medicines due to its analgesic and anti-inflammatory activities. However, hydrophilic alkaloids from toad venom, which may have certain pharmacological activities, have not been systematic studied. AIM OF THE STUDY: The aim of the study was to identify the indolealkylamines (IAAs) from toad venom and investigate the analgesic and anti-inflammatory actions. MATERIALS AND METHODS: The alkaloids were extracted and identified by high-resolution mass spectrometry. The analgesic abilities were determined using hot-plate test, formalin test and von Frey test. High-sensitivity lipidomics was used to investigate the regulatory function of IAAs on inflammatory eicosanoids. Besides, network pharmacology and molecular docking were used to demonstrate the candidate targets of IAAs. RESULTS: 22 constituents have been characterized by high performance liquid chromatography (HPLC)-Triple TOF 5600, including six specific IAAs (serotonin, N-methyl serotonin, bufotenine, bufotenidine, bufothionine and dehydrobufotenine). Pharmacological studies showed that the IAAs from toad venom exerted significant analgesic activities at doses of 5, 15 and 45 mg/kg in vivo. Moreover, lipids analysis revealed IAAs might down-regulate inflammatory mediators from COX, LOX, DHA and LA pathways in formalin models, thus showing anti-inflammatory effect. The potent pharmacological function might because of the binding of IAAs and protein targets, such as sigma-1 receptor. CONCLUSION: The studies provided a systemic evidence for the analgesic and anti-inflammatory activities of IAAs from toad venom. It suggested that IAAs might be a potential candidate to reduce inflammatory pain disorders.

Cell affinity screening combined with nanoLC-MS/MS based peptidomics for identifying cancer cell binding peptides from Bufo Bufo gargarizans.

Animal venoms contain many peptides with high specificity and selectivity against their protein targets, a characteristic which makes venoms an invaluable source of potential drugs. High-sensitivity mass spectrometry (MS)- based peptidomic platform has evolved as a predominant method for natural peptide drug discovery due to its strength for direct and rapid identification of peptides and peptide-associated post-translational modifications (PTMs). In this study, we used cell-affinity assays combined with nanoLC-MS/MS based peptidomics to identify cancer cell binding peptides (CBPs) from Bufo Bufo gargarizans. We identified 76 potential cell binding peptides and 237 non-affinity peptides in venom extracts from Asiatic toads, and some were verified with MS-parallel reaction monitoring (PRM) mode. These peptides were further analyzed and internalized within human cells and some demonstrated anti-tumor properties in vitro. These specific peptides might be used as templates for peptide-based drug design or optimization.