Deubiquitylase USP7 plays a crucial role in the lineage differentiation of preimplantation blastocysts.

Preimplantation embryos undergo a series of important biological events, including epigenetic reprogramming and lineage differentiation, and the key genes and specific mechanisms that regulate these events are critical to reproductive success. USP7 is a deubiquitinase involved in the regulation of a variety of cellular functions, yet its precise function and mechanism in preimplantation embryonic development remain unknown. Our results showed that RNAi-mediated silencing of USP7 in mouse embryos or treatment with P5091, a small molecule inhibitor of USP7, significantly reduced blastocyst rate and blastocyst quality, and decreased total and TE cell numbers per blastocyst, as well as destroying normal lineage differentiation. The results of single-cell RNA-seq, RT-qPCR, western blot, and immunofluorescence staining indicated that interference with USP7 caused failure of the morula-to-blastocyst transition and was accompanied by abnormal expression of key genes (Cdx2, Oct4, Nanog, Sox2) for lineage differentiation, decreased transcript levels, increased global DNA methylation, elevated repressive histone marks (H3K27me3), and decreased active histone marks (H3K4me3 and H3K27ac). Notably, USP7 may regulate the transition from the morula to blastocyst by stabilizing the target protein YAP through the ubiquitin-proteasome pathway. In conclusion, our results suggest that USP7 may play a crucial role in preimplantation embryonic development by regulating lineage differentiation and key epigenetic modifications.

Single-cell RNA-seq and single-cell bisulfite-sequencing reveal insights into yak preimplantation embryogenesis.

Extensive epigenetic reprogramming occurs during preimplantation embryonic development. However, the impact of DNA methylation in plateau yak preimplantation embryos and how epigenetic reprogramming contributes to transcriptional regulatory networks are unclear. In this study, we quantified gene expression and DNA methylation in oocytes and a series of yak embryos at different developmental stages and at single-cell resolution using single-cell bisulfite-sequencing and RNA-seq. We characterized embryonic genome activation and maternal transcript degradation and mapped epigenetic reprogramming events critical for embryonic development. Through cross-species transcriptome analysis, we identified 31 conserved maternal hub genes and 39 conserved zygotic hub genes, including SIN3A, PRC1, HDAC1/2, and HSPD1. Notably, by combining single-cell DNA methylation and transcriptome analysis, we identified 43 candidate methylation driver genes, such as AURKA, NUSAP1, CENPF, and PLK1, that may be associated with embryonic development. Finally, using functional approaches, we further determined that the epigenetic modifications associated with the histone deacetylases HDAC1/2 are essential for embryonic development and that the deubiquitinating enzyme USP7 may affect embryonic development by regulating DNA methylation. Our data represent an extensive resource on the transcriptional dynamics of yak embryonic development and DNA methylation remodeling, and provide new insights into strategies for the conservation of germplasm resources, as well as a better understanding of mammalian early embryonic development that can be applied to investigate the causes of early developmental disorders.

Novel insights into toxin changes associated with the growth of Alexandrium pacificum: Revealing active toxin-secretion ability and toxin cell quota variation.

Paralytic shellfish toxins (PSTs) are widely distributed globally and are produced by Alexandrium pacificum in marine system. However, the characteristics of toxins producing and secreting associated with growth phases are still unclear, especially whether A. pacificum has the ability to actively secrete PSTs is controversial. In this study, variation characteristics of intracellular and extracellular PSTs contents associated with A. pacificum growth phases were investigated thoroughly. The results showed that intracellular and extracellular PSTs contents increased sharply during the exponential phase. But during the stationary phase, the intracellular PSTs content increased by only 26 %, and the extracellular PSTs content did not increase significantly. Since the increase in extracellular PSTs content mainly occurred at the exponential phase, when most cells were living, we speculated that active PSTs secretion of living cells might be an important production pathway of extracellular toxins besides leakage from dead cells. Furthermore, toxin cell quota variation associated with the growth phase was analysed. In the exponential phase, the toxin cell quota first increased and then decreased, with a maximum of 19.02 ± 1.80 fmol/cell at 6 d. However, after entering the stationary phase, this value slowly increased again, suggesting that vigilance should be raised for the plateau of Alexandrium blooms. In addition, cells in the exponential phase mainly produced O-sulfated components such as GTX1&4, cells in the stationary phase mainly produced O-sulfate-free components such as GTX5. In this study, the toxigenic rules of A. pacificum were comprehensively uncovered, which provided theoretical guidance for the prevention and mitigation of A. pacificum blooms.

Molecular Cloning, Tissue Distribution, and Pharmacological Characterization of GPR84 in Grass Carp (Ctenopharyngodon Idella).

The G-protein-coupled receptor GPR84, activated by medium-chain fatty acids, primarily expressed in macrophages and microglia, is involved in inflammatory responses and retinal development in mammals and amphibians. However, our understanding of its structure, function, tissue expression, and signaling pathways in fish is limited. In this study, we cloned and characterized the coding sequence of GPR84 (ciGPR84) in grass carp. A phylogenetic analysis revealed its close relationship with bony fishes. High expression levels of GPR84 were observed in the liver and spleen. The transfection of HEK293T cells with ciGPR84 demonstrated its responsiveness to medium-chain fatty acids and diindolylmethane (DIM). Capric acid, undecanoic acid, and lauric acid activated ERK and inhibited cAMP signaling. Lauric acid showed the highest efficiency in activating the ERK pathway, while capric acid was the most effective in inhibiting cAMP signaling. Notably, DIM did not activate GPR84 in grass carp, unlike in mammals. These findings provide valuable insights for mitigating chronic inflammation in grass carp farming and warrant further exploration of the role of medium-chain fatty acids in inflammation regulation in this species.

Uncovering the regulation effect of modified clay on toxin production in Alexandrium pacificum: From physiological insights.

As a common dinoflagellate, Alexandrium pacificum can produce paralytic shellfish toxins (PSTs). It can be removed from water by Polyaluminium chloride modified clay (PAC-MC), but it is unclear whether PAC-MC can prevent PSTs content and toxicity from increasing and whether PAC-MC can stimulate PSTs biosynthesis by A. pacificum. Effect of PAC-MC on PSTs and the physiological mechanism were analysed here. The results showed total PSTs content and toxicity decreased respectively by 34.10 % and 48.59 % in 0.2 g/L PAC-MC group at 12 days compared with control group. And the restriction of total PSTs by PAC-MC was mainly achieved via inhibition of algal cell proliferation, by affecting A. pacificum physiological processes and changing phycosphere microbial community. Meanwhile, single-cell PSTs toxicity did not increase significantly throughout the experiment. Moreover, A. pacificum treated with PAC-MC tended to synthesize sulfated PSTs such as C1&2. Mechanistic analysis showed that PAC-MC induced upregulation of sulfotransferase sxtN (related to PSTs sulfation), and functional prediction of bacterial community also showed significant enrichment of "sulfur relay system" after PAC-MC treatment, which might also promote PSTs sulfation. The results will provide theoretical guidance for the application of PAC-MC to field control of toxic Alexandrium blooms.

Degradation of paralytic shellfish toxins during flocculation of Alexandrium pacificum by an oxidized modified clay: A laboratory experiment.

Paralytic shellfish toxins (PSTs), produced by Alexandrium pacificum in the marine environment, are a group of potent neurotoxins which specifically block voltage-gated sodium channels in excitable cells. During the toxigenic A. pacificum blooms outbreaks, PSTs can be accumulated through the food chain and finally enter the human body, posing a significant threat to human health and safety. This study experimented with a novel type of oxidized modified clay, potassium peroxymonosulfate modified clay (PMPS-MC), which could remove A. pacificum cells as well as reduce intracellular and extracellular PSTs toxicity rapidly. For the extracellular PSTs, its content decreased to below the detection limit rapidly through oxidative degradation within 15 min of 10 mg/L PMPS-MC treatment. Whereafter, although the residual cells in water column and some viable cells in flocculated sediment continued to secrete toxins, the extracellular PSTs content and toxicity in the PMPS-MC treatment groups remained significantly lower than those in the control group. For the intracellular PSTs, PMPS-MC might induce the transformation of more toxic GTX1&4 to less toxic GTX2&3 and C1&2, resulting in intracellular PSTs toxicity reduced within 15 min. In addition, intracellular PSTs content and toxicity in the PMPS-MC treatment groups were consistently lower than the control group within 48 h, possibly by inhibiting the A. pacificum cells growth. These results will provide a scientific basis for the field application of modified clay to control A. pacificum blooms.

ZFP57 regulates DNA methylation of imprinted genes to facilitate embryonic development of somatic cell nuclear transfer embryos in Holstein cows.

Aberrant epigenetic nuclear reprogramming, especially imprinting pattern disorders, is one of the major causes of failure of clone development from somatic cell nuclear transfer (SCNT). Previous studies showed that ZFP57 is a key protein required for imprint maintenance after fertilization. In this study, we found that imprinting control regions in several imprinted genes were significantly hypomethylated in cloned embryos compared with in vitro fertilization embryos, indicating a loss of imprinted gene methylation. The ZFP57 expression was capable of maintaining the correct degree of methylation at several imprinting control regions and correcting abnormal hypomethylation. Moreover, we successfully obtained bovine fetal fibroblasts overexpressing ZFP57, which were used as donors for SCNT. Our results demonstrated that overexpression of ZFP57 increased total and trophectoderm cell numbers and the ratio of inner cell mass to total cells, reduced the apoptosis rate and significantly enhanced the development of SCNT blastocysts in vitro, ultimately achieving a degree of methylation similar to that in in vitro fertilization embryos. We concluded that overexpression of ZFP57 in donor cells provided an effective method for enhancing nuclear reprogramming and developmental potential in SCNT embryos. The ZFP57 protein played a key role in maintaining the methylation of imprinted genes during early embryonic development, which may be effective for enhanced SCNT in cattle.

A New Perspective: Revealing the Algicidal Properties of Bacillus subtilis to Alexandrium pacificum from Bacterial Communities and Toxins.

Algicidal bacteria are important in the control of toxic dinoflagellate blooms, but studies on the environmental behavior of related algal toxins are still lacking. In this study, Bacillus subtilis S3 (S3) showed the highest algicidal activity against Alexandrium pacificum (Group IV) out of six Bacillus strains. When treated with 0.5% (v/v) S3 bacterial culture and sterile supernatant, the algicidal rates were 69.74% and 70.22% at 12 h, respectively, and algicidal substances secreted by S3 were considered the mechanism of algicidal effect. During the algicidal process, the rapid proliferation of Alteromonas sp. in the phycosphere of A. pacificum may have accelerated the algal death. Moreover, the algicidal development of S3 released large amounts of intracellular paralytic shellfish toxins (PSTs) into the water, as the extracellular PSTs increased by 187.88% and 231.47% at 12 h, compared with the treatment of bacterial culture and sterile supernatant at 0 h, respectively. Although the total amount of PSTs increased slightly, the total toxicity of the algal sample decreased as GTX1/4 was transformed by S3 into GTX2/3 and GTX5. These results more comprehensively reveal the complex relationship between algicidal bacteria and microalgae, providing a potential source of biological control for harmful algal blooms and toxins.

Functional characterization of melanocortin-3 receptor in rainbow trout (Oncorhynchus mykiss).

The melanocortin-3 receptor (MC3R) is an important regulator of energy homeostasis and inflammation in mammals. However, its function in teleost fish needs to be further explored. In this study, we characterized rainbow trout MC3R (rtMC3R), which encoded a putative protein of 331 amino acids. Phylogenetic and chromosomal synteny analyses showed that rtMC3R was closely related to bony fishes. Quantitative PCR (qPCR) revealed that the transcripts of rtMC3R were highly expressed in the brain and muscle. The cellular function of rtMC3R was further verified by the signal-pathway-specific luciferase reporter assays. Four agonists such as α-MSH, ß-MSH, ACTH (1-24), and NDP-MSH can active rtMC3R, increasing the production of intracellular cAMP and upregulating MAPK/ERK signals. Moreover, we found that rtMC3R stimulated with α-MSH and NDP-MSH can significantly inhibit the NF-κB signaling pathway. This research will be helpful for further studies on the function of MC3R in rainbow trout, especially the role of energy metabolism and immune regulation.

Profiling of Brevetoxin Metabolites Produced by Karenia brevis 165 Based on Liquid Chromatography-Mass Spectrometry.

In this study, Karenia brevis 165 (K. brevis 165), a Chinese strain, was used to research brevetoxin (BTX) metabolites. The sample pretreatment method for the enrichment of BTX metabolites in an algal culture medium was improved here. The method for screening and identifying intracellular and extracellular BTX metabolites was established based on liquid chromatography-time-of-flight mass spectrometry (LC-ToF-MS) and liquid chromatography triple quadrupole tandem mass spectrometry (LC-QqQ-MS/MS). The results show that the recovery rates for BTX toxins enriched by a hydrophilic-lipophilic balance (HLB) extraction column were higher than those with a C18 extraction column. This method was used to analyze the profiles of extracellular and intracellular BTX metabolites at different growth stages of K. brevis 165. This is the first time a Chinese strain of K. brevis has been reported that can produce toxic BTX metabolites. Five and eight kinds of BTX toxin metabolites were detected in the cell and culture media of K. brevis 165, respectively. Brevenal, a toxic BTX metabolite antagonist, was found for the first time in the culture media. The toxic BTX metabolites and brevenal in the K. brevis 165 cell and culture media were found to be fully proven in terms of the necessity of establishing a method for screening and identifying toxic BTX metabolites. The results found by qualitatively and quantitatively analyzing BTX metabolites produced by K. brevis 165 at different growth stages show that the total toxic BTX metabolite contents in single cells ranged between 6.78 and 21.53 pg/cell, and the total toxin concentration in culture media ranged between 10.27 and 449.11 µg/L. There were significant differences in the types and contents of toxic BTX metabolites with varying growth stages. Therefore, when harmful algal blooms occur, the accurate determination of BTX metabolite types and concentrations will be helpful to assess the ecological disaster risk in order to avoid hazards and provide appropriate disaster warnings.

Involvement of a short-type peptidoglycan recognition protein (PGRP) from Chinese giant salamanders Andrias davidianus in the immune response against bacterial infection.

PGRPs (Peptidoglycan recognition proteins) could recognize peptidoglycan and play vital roles in innate immunity among different animals. Till present, the functions of PGRP have been studied in various animals, but few reports have studied the amphibian PGRPs. In the current research, a short type PGRP was identified from Chinese giant salamander and its involvement in the innate immunity was studied. The ORF of AdPGRP-SC2 cDNA was 573 bp, which encoded 190 amino acids, and contained a PGRP and an amidase_2 domain. The qPCR analysis revealed that AdPGRP-SC2 mRNA transcripts expressed in different tissues, with the highest expression level in muscle, intestine and spleen. Results of immune challenges with peptidoglycan (PGN) demonstrated that expression patterns of AdPGRP-SC2 were significantly up-regulated in erythrocyte and spleen at the early injection stage. The recombinant AdPGRP-SC2 protein was successfully produced and purified, and it could show binding affinity to different bacteria. In the presence of Zn2+, the rAdPGRP-SC2 could exhibit a broad PAMPs binding activities, strongly agglutinate bacteria and exhibit amidase enzyme activity. Collectively, these data indicate AdPGRP-SC2 could act as PRR to recognize the invading microorganisms and as the antimicrobial effectors during the innate immune response of A. davidianus.

Identification and characterization of a novel goose-type and chicken-type lysozyme genes in Chinese rare minnow (Gobiocypris rarus) with potent antimicrobial activity.

Lysozymes act as important innate immunity molecule against various pathogen infections. In the present study, a new c-type and g-type lysozymes were isolated from rare minnow Gobiocypris rarus. The deduced amino acid sequence of c-type lysozyme contained 145 residues and possessed conserved catalytic residues (Glu53 and Asp72). The deduced g-type lysozyme contained 185 residues and possessed three conserved catalytic residues (Glu73, Asp86 and Asp97). qPCR analysis revealed that GrlysG and GrlysC were constitutively expressed in all examined tissues. GrlysG was most abundant in kidney and liver, while GrlysC was predominantly expressed in liver. The transcripts of GrlysG and GrlysC genes could be significantly up-regulated after Aeromonas hydrophila and poly I:C infection in the kidney and liver. The recombinant GrlysG and GrlysC proteins were successfully produced and purified. The optimal pH and temperature for rGrlysG and rGrlysC protein lytic activities was determined to be 6.5 and 32 °C, respectively. From the minimal inhibitory concentration test, the rGrlysG and rGrlysC exhibited apparent antibacterial activities against both Gram-positive and Gram-negative bacteria at different concentrations. In conclusion, the present study indicated that c-type and g-type lysozymes participated the innate immune response to A. hydrophila and virus infections in fish.

A simple way to synthesize large-scale Cu2O/Ag nanoflowers for ultrasensitive surface-enhanced Raman scattering detection.

Here, we report a simple strategy to prepare highly sensitive surface-enhanced Raman spectroscopy (SERS) substrates based on Ag decorated Cu2O nanoparticles by combining two common techniques, viz, thermal oxidation growth of Cu2O nanoparticles and magnetron sputtering fabrication of a Ag nanoparticle film. Methylene blue is used as the Raman analyte for the SERS study, and the substrates fabricated under optimized conditions have very good sensitivity (analytical enhancement factor ∼108), stability, and reproducibility. A linear dependence of the SERS intensities with the concentration was obtained with an R 2 value >0.9. These excellent properties indicate that the substrate has great potential in the detection of biological and chemical substances.

Identification of the first cathelicidin gene from skin of Chinese giant salamanders Andrias davidianus with its potent antimicrobial activity.

Cathelicidins, as effector molecules, play important roles against infections and represent a crucial component of the innate immune system in vertebrates. They are widely studied in mammals, but little is known in amphibians. In the present study, we report the identification and characterization of a novel cathelicidin from Chinese giant salamander Andrias davidianus, which is the first study in Caudata amphibian. The cDNA sequence encodes a predicted 148-amino-acid polypeptide, which composed of a 20-residue signal peptide, a 94-residue conserved cathelin domain and a 34-residue mature peptide. From the multiple sequence alignments and phylogenetic analysis, AdCath shared conserved structure with other orthologs and clustered with other amphibian peptides. The tissue expression profiles revealed AdCath was highly expressed in skin. To study the function of AdCath gene, the AdCath precursor protein and mature peptide were recombinantly expressed and chemical synthesized respectively. The rAdCath protein could bind to LPS in a dose-dependent manner. When the concentrations of rAdCath protein and mature peptide were up to 22 µg/mL, they showed significantly cytotoxicity to human 293T cell lines. The rAdCath protein and synthetic peptide could exhibit antibacterial activities detected by the minimum inhibitory concentrations assay. From the SEM assay, the synthetic mature peptide could destroy the membrane of bacteria and cause loss of membrane integrity. Collectively, these findings characterized the first cathelicidin from A. davidianus, and highlighted its potential antimicrobial activities, indicating its important roles in the skin immune response against different bacteria.