The Holothuria leucospilota genome elucidates sacrificial organ expulsion and bioadhesive trap enriched with amyloid-patterned proteins.

Some tropical sea cucumbers of the family Holothuriidae can efficiently repel or even fatally ensnare predators by sacrificially ejecting a bioadhesive matrix termed the Cuvierian organ (CO), so named by the French zoologist Georges Cuvier who first described it in 1831. Still, the precise mechanisms for how adhesiveness genetically arose in CO and how sea cucumbers perceive and transduce danger signals for CO expulsion during defense have remained unclear. Here, we report the first high-quality, chromosome-level genome assembly of Holothuria leucospilota, an ecologically significant sea cucumber with prototypical CO. The H. leucospilota genome reveals characteristic long-repeat signatures in CO-specific outer-layer proteins, analogous to fibrous proteins of disparate species origins, including spider spidroin and silkworm fibroin. Intriguingly, several CO-specific proteins occur with amyloid-like patterns featuring extensive intramolecular cross-ß structures readily stainable by amyloid indicator dyes. Distinct proteins within the CO connective tissue and outer surface cooperate to give the expelled matrix its apparent tenacity and adhesiveness, respectively. Genomic evidence offers further hints that H. leucospilota directly transduces predator-induced mechanical pressure onto the CO surface through mediation by transient receptor potential channels, which culminates in acetylcholine-triggered CO expulsion in part or in entirety. Evolutionarily, innovative events in two distinct regions of the H. leucospilota genome have apparently spurred CO's differentiation from the respiratory tree to a lethal defensive organ against predators.

Transcriptomic analysis reveals the early body wall regeneration mechanism of the sea cucumber Holothuria leucospilota after artificially induced transverse fission.

BACKGROUND: Sea cucumbers exhibit a remarkable ability to regenerate damaged or lost tissues and organs, making them an outstanding model system for investigating processes and mechanisms of regeneration. They can also reproduce asexually by transverse fission, whereby the anterior and posterior bodies can regenerate independently. Despite the recent focus on intestinal regeneration, the molecular mechanisms underlying body wall regeneration in sea cucumbers still remain unclear. RESULTS: In this study, transverse fission was induced in the tropical sea cucumber, Holothuria leucospilota, through constrainment using rubber bands. Histological examination revealed the degradation and loosening of collagen fibers on day-3, followed by increased density but disorganization of the connective tissue on day-7 of regeneration. An Illumina transcriptome analysis was performed on the H. leucospilota at 0-, 3- and 7-days after artificially induced fission. The differential expression genes were classified and enriched by GO terms and KEGG database, respectively. An upregulation of genes associated with extracellular matrix remodeling was observed, while a downregulation of pluripotency factors Myc, Klf2 and Oct1 was detected, although Sox2 showed an upregulation in expression. In addition, this study also identified progressively declining expression of transcription factors in the Wnt, Hippo, TGF-ß, and MAPK signaling pathways. Moreover, changes in genes related to development, stress response, apoptosis, and cytoskeleton formation were observed. The localization of the related genes was further confirmed through in situ hybridization. CONCLUSION: The early regeneration of H. leucospilota body wall is associated with the degradation and subsequent reconstruction of the extracellular matrix. Pluripotency factors participate in the regenerative process. Multiple transcription factors involved in regulating cell proliferation were found to be gradually downregulated, indicating reduced cell proliferation. Moreover, genes related to development, stress response, apoptosis, and cell cytoskeleton formation were also involved in this process. Overall, this study provides new insights into the mechanisms of whole-body regeneration and uncover potential cross-species regenerative-related genes.

Adenine base-editing-mediated exon skipping induces gene knockout in cultured pig cells.

Gene-knockout pigs have important applications in agriculture and medicine. Compared with CRISPR/Cas9, Adenine base editor (ABE) convert single A·T pairs to G·C pairs in the genome without generating DNA double-strand breaks, and this method has higher accuracy and biosafety in pig genetic modification. However, the application of ABE in pig gene knockout is limited by protospacer-adjacent motif sequences and the base-editing window. Alternative mRNA splicing is an important mechanism underlying the formation of proteins with diverse functions in eukaryotes. Spliceosome recognizes the conservative sequences of splice donors and acceptors in a precursor mRNA. Mutations in these conservative sequences induce exon skipping, leading to proteins with novel functions or to gene inactivation due to frameshift mutations. In this study, adenine base-editing-mediated exon skipping was used to expand the application of ABE in the generation of gene knockout pigs. We first constructed a modified "all-in-one" ABE vector suitable for porcine somatic cell transfection that contained an ABE for single-base editing and an sgRNA expression cassette. The "all-in-one" ABE vector induced efficient sgRNA-dependent A-to-G conversions in porcine cells during single base-editing of multiple endogenous gene loci. Subsequently, an ABE system was designed for single adenine editing of the conservative splice acceptor site (AG sequence at the 3' end of the intron 5) and splice donor site (GT sequence at the 5' end of the intron 6) in the porcine gene GHR; this method achieved highly efficient A-to-G conversion at the cellular level. Then, porcine single-cell colonies carrying a biallelic A-to-G conversion in the splice acceptor site in the intron 5 of GHR were generated. RT-PCR indicated exon 6 skipped at the mRNA level. Western blotting revealed GHR protein loss, and gene sequencing showed no sgRNA-dependent off-target effects. These results demonstrate accurate adenine base-editing-mediated exon skipping and gene knockout in porcine cells. This is the first proof-of-concept study of adenine base-editing-mediated exon skipping for gene regulation in pigs, and this work provides a new strategy for accurate and safe genetic modification of pigs for agricultural and medical applications.

Comparative transcriptomic analysis of the different developmental stages of ovary in red swamp crayfish Procambarus clarkii.

BACKGROUND: The red swamp crayfish Procambarus clarkii is a freshwater species that possesses high adaptability, environmental tolerance, and fecundity. P. clarkii is artificially farmed on a large scale in China. However, the molecular mechanisms of ovarian development in P. clarkii remain largely unknown. In this study, we identified four stages of P. clarkii ovary development, the previtellogenic stage (stage I), early vitellogenic stage (stage II), middle vitellogenic stage (stage III), and mature stage (stage IV) and compared the transcriptomics among these four stages through next-generation sequencing (NGS). RESULTS: The total numbers of clean reads of the four stages ranged from 42,013,648 to 62,220,956. A total of 216,444 unigenes were obtained, and the GC content of most unigenes was slightly less than the AT content. Principal Component Analysis (PCA) and Anosim analysis demonstrated that the grouping of these four stages was feasible, and each stage could be distinguished from the others. In the expression pattern analysis, 2301 genes were continuously increase from stage I to stage IV, and 2660 genes were sharply decrease at stage IV compared to stages I-III. By comparing each of the stages at the same time, four clusters of differentially expressed genes (DEGs) were found to be uniquely highly expressed in stage I (136 genes), stage II (43 genes), stage III-IV (49 genes), and stage IV (22 genes), thus exhibiting developmental stage specificity. Moreover, in comparisons between adjacent stages, the number of DEGs between stage III and IV was the highest. GO enrichment analysis demonstrated that nutrient reservoir activity was highest at stage II and that this played a foreshadowing role in ovarian development, and the GO terms of cell, intracellular and organelle participated in the ovary maturation during later stages. In addition, KEGG pathway analysis revealed that the early development of the ovary was mainly associated with the PI3K-Akt signaling pathway and focal adhesion; the middle developmental period was related to apoptosis, lysine biosynthesis, and the NF-kappa B signaling pathway; the late developmental period was involved with the cell cycle and the p53 signaling pathway. CONCLUSION: These transcriptomic data provide insights into the molecular mechanisms of ovarian development in P. clarkii. The results will be helpful for improving the reproduction and development of this aquatic species.

Application of the modified cytosine base-editing in the cultured cells of bama minipig.

Bama minipig is a unique miniature swine bred from China. Their favorable characteristics include delicious meat, strong adaptability, tolerance to rough feed, and high levels of stress tolerance. Unfavorable characteristics are their low lean meat percentage, high fat content, slow growth rate, and low feed conversion ratio. Genome-editing technology using CRISPR/Cas9 efficiently knocked out the myostatin gene (MSTN) that has a negative regulatory effect on muscle production, effectively promoting pig muscle growth and increasing lean meat percentage of the pigs. However, CRISPR/Cas9 genome editing technology is based on random mutations implemented by DNA double-strand breaks, which may trigger genomic off-target effects and chromosomal rearrangements. The application of CRISPR/Cas9 to improve economic traits in pigs has raised biosafety concerns. Base editor (BE) developed based on CRISPR/Cas9 such as cytosine base editor (CBE) effectively achieve targeted modification of a single base without relying on DNA double-strand breaks. Hence, the method has greater safety in the genetic improvement of pigs. The aim of the present study is to utilize a modified CBE to generate MSTN-knockout cells of Bama minipigs. Our results showed that the constructed "all-in-one"-modified CBE plasmid achieved directional conversion of a single C·G base pair to a T·A base pair of the MSTN target in Bama miniature pig fibroblast cells. We successfully constructed multiple single-cell colonies of Bama minipigs fibroblast cells carrying the MSTN premature termination and verified that there were no genomic off-target effects detected. This study provides a foundation for further application of somatic cell cloning to construct MSTN-edited Bama minipigs that carry only a single-base mutation and avoids biosafety risks to a large extent, thereby providing experience and a reference for the base editing of other genetic loci in Bama minipigs.

Efficient CRISPR/Cas9-mediated gene editing in Guangdong small-ear spotted pig cells using an optimized electrotransfection method.

OBJECTIVES: Guangdong Small-ear Spotted (GDSS) pigs are a pig breed native to China that possesses unfortunate disadvantages, such as slow growth rate, low lean-meat percentage, and reduced feed utilization. In contrast to traditional genetic breeding methods with long cycle time and high cost, CRISPR/Cas9-mediated gene editing for the modification of the pig genome can quickly improve production traits, and therefore this technique exhibits important potential in the genetic improvement and resource development of GDSS pigs. In the present study, we aimed to establish an efficient CRISPR/Cas9-mediated gene-editing system for GDSS pig cells by optimizing the electrotransfection parameters, and to realize efficient CRISPR/Cas9-mediated gene editing of GDSS pig cells. RESULTS: After optimization of electrotransfection parameters for the transfection of GDSS pig cells, we demonstrated that a voltage of 150 V and a single pulse with a pulse duration of 20 ms were the optimal electrotransfection parameters for gene editing in these cells. In addition, our study generated GDSS pig single-cell colonies with biallelic mutations in the myostatin (MSTN) gene and insulin-like growth factor 2 (IGF2) intron-3 locus, which play an important role in pig muscle growth and muscle development. The single-cell colonies showed no foreign gene integration or off-target effects, and maintained normal cell morphology and viability. These gene-edited, single-cell colonies can in the future be used as donor cells to generate MSTN- and IGF2-edited GDSS pigs using somatic cell nuclear transfer (SCNT). CONCLUSIONS: This study establishes the foundation for genetic improvement and resource development of GDSS pigs using CRISPR/Cas9-mediated gene editing combined with SCNT.

CRISPR/Cas9-mediated MSTN disruption accelerates the growth of Chinese Bama pigs.

CRISPR/Cas9-mediated genome editing technology is a simple and highly efficient and specific genome modification approach with wide applications in the animal industry. CRISPR/Cas9-mediated genome editing combined with somatic cell nuclear transfer rapidly constructs gene-edited somatic cell-cloned pigs for the genetic improvement of traits or simulation of human diseases. Chinese Bama pigs are an excellent indigenous minipig breed from Bama County of China. Research on genome editing of Chinese Bama pigs is of great significance in protecting its genetic resource, improving genetic traits and in creating disease models. This study aimed to address the disadvantages of slow growth and low percentage of lean meat in Chinese Bama pigs and to knock out the myostatin gene (MSTN) by genome editing to promote growth and increase lean meat production. We first used CRISPR/Cas9-mediated genome editing to conduct biallelic knockout of the MSTN, followed by somatic cell nuclear transfer to successfully generate MSTN biallelic knockout Chinese Bama pigs, which was confirmed to have significantly faster growth rate and showed myofibre hyperplasia when they reached sexual maturity. This study lays the foundation for the rapid improvement of production traits of Chinese Bama pigs and the generation of gene-edited disease models in this breed.

Two Distinct C-Type Lysozymes in Goldfish: Molecular Characterization, Antimicrobial Potential, and Transcriptional Regulation in Response to Opposing Effects of Bacteria/Lipopolysaccharide and Dexamethasone/Leptin.

Lysozymes are key antimicrobial peptides in the host innate immune system that protect against pathogen infection. In this study, the full-length cDNAs of two c-type lysozymes (gfLyz-C1 and gfLyz-C2) were cloned from goldfish (Carassius auratus). The structural domains, three-dimensional structures, and amino acid sequences of gfLyz-C1 and gfLyz-C2 were highly comparable, as the two proteins shared 89.7% sequence identity. The gfLyz-C1 and gfLyz-C2 recombinant proteins were generated in the insoluble fractions of an Escherichia coli system. Based on the results of lysoplate and turbidimetric assays, gfLyz-C1 and gfLyz-C2 showed broad-spectrum antimicrobial properties with high levels of activity against Micrococcus lysodeikticus, Vibrio parahemolyticus, and Edwardsiella tarda, and relatively low activity against E. coli. Both gfLyz-C1 and gfLyz-C2 mRNAs were mainly expressed in the trunk kidney and head kidney, and gfLyz-C1 was expressed at much higher levels than gfLyz-C2 in the corresponding tissues. The expression of the gfLyz-C1 and gfLyz-C2 transcripts in the trunk kidney and head kidney was induced in these tissues by challenge with heat-inactivated E. coli and lipopolysaccharides (LPS), and the transcriptional responses of gfLyz-C1 were more intense. In goldfish primary trunk kidney cells, the levels of the gfLyz-C1 and gfLyz-C2 transcripts were upregulated by heat-inactivated E. coli, V. parahemolyticus, and E. tarda, as well as LPS, and downregulated by treatment with dexamethasone and leptins. Overall, this study may provide new insights that will improve our understanding of the roles of c-type lysozymes in the innate immunity of cyprinid fish, including the structural and phylogenetic characteristics, antimicrobial effects, and regulatory mechanism.

Suppression of leptin-AI/AII transcripts by insulin in goldfish liver: A fish specific response of leptin under food deprivation.

Leptin is primarily considered a peripheral satiety hormone and is also found to perform important roles in energy homeostasis in vertebrates ranging from fish to mammals. The liver is a major source of leptin production in teleost fish. Using goldfish as a model, a previous report by our group illustrated the positive regulation of leptin mRNA levels by treatment with the hyperglycemic hormone glucagon, and our present study provided evidence for the negative regulation of hepatic leptin-AI and leptin-AII transcripts through the administration of the hypoglycemic hormone insulin. This study is the first to demonstrate changes in the hepatopancreatic insulin, glucagon, leptin-AI and leptin-AII mRNA levels in goldfish during fasting and refeeding. Insulin was found to be effective in suppressing leptin-AI and leptin-AII transcript levels in goldfish liver via both in vivo intraperitoneal injection and in vitro cell incubation approaches. Only the insulin receptor, not the IGF-I receptor, was involved in insulin-inhibited leptin mRNA level. The suppression of leptin levels by insulin was caused by the activation of MKK3/6/p38MAPK and MEK1/2/Erk1/2 cascades. Insulin treatment could eliminate the stimulation of glucagon on leptin mRNA level. Our study describes the regulation and signal transduction mechanism of insulin on leptin mRNA levels in the goldfish liver, suggesting that the leptin function in fish is speculated to be not only an anorexigenic factor but also a metabolic mediator. This also supports the hypothesis that the poikilothermal fish use a passive survival strategy during the periods of food deprivation, which is mediated by the fish-specifically high leptin levels induced by the cooperation of insulin and glucagon.

A novel caspase-6 from sea cucumber Holothuria leucospilota: Molecular characterization, expression analysis and apoptosis detection.

In this study, a novel caspase-6 named HLcaspase-6 was identified from sea cucumber Holothuria leucospilota. The full-length cDNA of HLcaspase-6 is 2195 bp in size, containing a 126 bp 5'-untranslated region (UTR), a 1043 bp 3'-UTR and a 1026 bp open reading frame (ORF) encoding a protein of 341 amino acids with a deduced molecular weight of 38.57 kDa. HLcaspase-6 contains the common signatures of the caspase family, including the conserved pentapeptide motif QACRG, as well as the P20 and P10 domains. In addition, HLcaspase-6 contains a short pro-domain. HLcaspase-6 mRNA is ubiquitously expressed in all tissues examined, with the highest transcript level in the intestine, followed by coelomocytes. In in vitro experiments, the expression of HLcaspase-6 mRNA in coelomocytes was significantly up-regulated by lipopolysaccharides (LPS) or polyriboinosinic-polyribocytidylic acid [poly (I:C)] challenge, suggesting that HLcaspase-6 might play important roles in the innate immune defense of sea cucumber against bacterial and viral infections. Moreover, we further confirmed that overexpression of HLcaspase-6 could induce apoptosis and activate the p53 signal pathway.

The first tropical sea cucumber caspase-8 from Holothuria leucospilota: Molecular characterization, involvement of apoptosis and inducible expression by immune challenge.

In this study, the first tropical sea cucumber caspase-8 named HLcaspase-8 was identified from Holothuria leucospilota. The full-length cDNA of HLcaspase-8 is 2293 bp in size, containing a 245 bp 5'-untranslated region (UTR), a 521 bp 3'-UTR and a 1527 bp open reading frame (ORF) encoding a protein of 508 amino acids with a deduced molecular weight of 57.47 kDa. Besides the common signatures of caspase family including conserved cysteine active site pentapeptide motif QACQG, P20 domain and P10 domain, HLcaspase-8 also contains a characteristic DED domain. The over-expression of HLcaspase-8 in HEK293T cells showed that HLcaspase-8 protein could induce apoptosis and the apoptosis could be promoted by TNF-α, indicating that the apoptosis induced by HLcaspase-8 might also be triggered via a receptor-mediated pathway. Moreover, the expression of HLcaspase-8 in in vitro experiments performed in coelomocytes was significantly up-regulated by lipopolysaccharides (LPS) or polyriboinosinic-polyribocytidylic Acid [poly (I:C)] challenge, suggesting that the sea cucumber caspase-8 might play some important roles in the innate immune defense against bacterial and viral infections.

[In vitro study of joint intervention of E-cad and Bmi-1 mediated by transcription activator-like effector nuclease in nasopharyngeal carcinoma].

OBJECTIVE: To explore the effect of intervention of E-cadherin (E-cad) and B-lymphoma Moloney murine leukemia virus insertion region-1 (Bmi-1) mediated by transcription activator-like effector nuclease (TALEN) on the biological behaviors of nasopharyngeal carcinoma cells.
 Methods: Multi-locus gene targeting vectors pUC-DS1-CMV-E-cad-2A-Neo-DS2 and pUC-DS1-Bmi-1 shRNA-Zeo-DS2 were constructed, and the E-cad and Bmi-1 targeting vectors were transferred with TALEN plasmids to CNE-2 cells individually or simultaneously. The integration of target genes were detected by PCR, the expressions of E-cad and Bmi-1 were detected by Western blot. The changes of cell proliferation were detected by cell counting kit-8 (CCK-8) assay. The cell cycle and apoptosis were detected by flow cytometry. The cell migration and invasion were detected by Transwell assay.
 Results: The E-cad and Bmi-1 shRNA expression elements were successfully integrated into the genome of CNE-2 cells, the protein expression level of E-cad was up-regulated, and the protein expression level of Bmi-1 was down-regulated. The intervention of E-cad and Bmi-1 didn't affect the proliferation, cell cycle and apoptosis of CNE-2 cells, but it significantly inhibited the migration and invasion ability of CNE-2 cells. Furthermore, the intervention of E-cad and Bmi-1 together significantly inhibited the migration ability of nasopharyngeal carcinoma cells compared with the intervention of E-cad or Bmi-1 alone (all P<0.01).
 Conclusion: The joint intervention of E-cad and Bmi-1 mediated by TALEN can effectively inhibit the migration and invasion of nasopharyngeal carcinoma cells in vitro, which may lay the preliminary experimental basis for gene therapy of human cancer.

Leptin Stimulates Prolactin mRNA Expression in the Goldfish Pituitary through a Combination of the PI3K/Akt/mTOR, MKK3/6/p38MAPK and MEK1/2/ERK1/2 Signalling Pathways.

Leptin actions at the pituitary level have been extensively investigated in mammalian species, but remain insufficiently characterized in lower vertebrates, especially in teleost fish. Prolactin (PRL) is a pituitary hormone of central importance to osmoregulation in fish. Using goldfish as a model, we examined the global and brain-pituitary distribution of a leptin receptor (lepR) and examined the relationship between expression of lepR and major pituitary hormones in different pituitary regions. The effects of recombinant goldfish leptin-AI and leptin-AII on PRL mRNA expression in the pituitary were further analysed, and the mechanisms underlying signal transduction for leptin-induced PRL expression were determined by pharmacological approaches. Our results showed that goldfish lepR is abundantly expressed in the brain-pituitary regions, with highly overlapping PRL transcripts within the pituitary. Recombinant goldfish leptin-AI and leptin-AII proteins could stimulate PRL mRNA expression in dose- and time-dependent manners in the goldfish pituitary, by both intraperitoneal injection and primary cell incubation approaches. Moreover, the PI3K/Akt/mTOR, MKK3/6/p38MAPK, and MEK1/2/ERK1/2-but not JAK2/STAT 1, 3 and 5 cascades-were involved in leptin-induced PRL mRNA expression in the goldfish pituitary.

Identification and functional characterization of a novel antistasin/WAP-like serine protease inhibitor from the tropical sea cucumber, Stichopus monotuberculatus.

A novel antistasin/WAP-like serine protease inhibitor, named as StmAW-SPI, was identified from sea cucumber (Stichopus monotuberculatus) and functionally characterized in this study. The full-length cDNA of StmAW-SPI is 1917 bp in length with a 72 bp 5'-untranslated region (UTR), a 294 bp 3'-UTR and a 1551 bp open reading frame (ORF) encoding a protein of 516 amino acids with a deduced molecular weight of 54.56 kDa. The StmAW-SPI protein has 5-fold internal repeats (IRs) of antistasin domain and 6-fold IRs of WAP domain. For the gene structure, StmAW-SPI contains 10 exons separated by 9 introns. The StmAW-SPI mRNA expression pattern was determined using quantitative real-time PCR. The highest level of StmAW-SPI was found in the intestine, followed by coelomocytes, gonad, body wall and respiratory tree. The StmAW-SPI expressions were significantly up-regulated after polyriboinosinic polyribocytidylic acid [Poly (I:C)] or lipopolysaccharides (LPS) challenge in in vitro experiments performed in primary coelomocytes. In addition, the serine protease inhibitory activity and bacterial protease inhibitory activity of StmAW-SPI were examined, and the antibacterial activity was also demonstrated in this study. Our study, as a whole, suggested that StmAW-SPI might play a critical role in the innate immune defense of sea cucumber against microbial infections, by not only inactivating the serine protease but also inhibiting the growth of pathogens.

Two isoforms of leptin in the White-clouds Mountain minnow (Tanichthys albonubes): Differential regulation by estrogen despite similar response to fasting.

Leptin has been well-established as a canonical anorexic peptide hormone in mammals, though much of its function in fish remains obscure. In this study, the cDNAs of two leptin isoforms (leptin-A and leptin-B) were cloned from the liver of a small cyprinid fish, Tanichthys albonubes. The two T. albonubes leptins, sharing low primary amino acid sequence homology with their mammalian counterparts, and between themselves, are highly conserved in three-dimensional protein structures and gene structures. Liver is a major source of leptin mRNA in T. albonubes with leptin-A being the dominant form. The expression of hepatic leptin-A but not leptin-B mRNA in female fish is significantly higher than in male fish. Transcriptional hepatic levels of leptin-A and leptin-B in both male and female fish were demonstrated to increase after long-term fasting (10-25days) but decline upon re-feeding (3days). Strikingly, estrogen (E2) administration induced only leptin-A but not leptin-B hepatic mRNA expression in both male and female fish. Our study here provides the first evidence for differential regulation of two leptins in fish, and sheds new light on the possible origin of leptin in lower vertebrates.

Goldfish Leptin-AI and Leptin-AII: Function and Central Mechanism in Feeding Control.

In mammals, leptin is a peripheral satiety factor that inhibits feeding by regulating a variety of appetite-related hormones in the brain. However, most of the previous studies examining leptin in fish feeding were performed with mammalian leptins, which share very low sequence homologies with fish leptins. To elucidate the function and mechanism of endogenous fish leptins in feeding regulation, recombinant goldfish leptin-AI and leptin-AII were expressed in methylotrophic yeast and purified by immobilized metal ion affinity chromatography (IMAC). By intraperitoneal (IP) injection, both leptin-AI and leptin-AII were shown to inhibit the feeding behavior and to reduce the food consumption of goldfish in 2 h. In addition, co-treatment of leptin-AI or leptin-AII could block the feeding behavior and reduce the food consumption induced by neuropeptide Y (NPY) injection. High levels of leptin receptor (lepR) mRNA were detected in the hypothalamus, telencephalon, optic tectum and cerebellum of the goldfish brain. The appetite inhibitory effects of leptins were mediated by downregulating the mRNA levels of orexigenic NPY, agouti-related peptide (AgRP) and orexin and upregulating the mRNA levels of anorexigenic cocaine-amphetamine-regulated transcript (CART), cholecystokinin (CCK), melanin-concentrating hormone (MCH) and proopiomelanocortin (POMC) in different areas of the goldfish brain. Our study, as a whole, provides new insights into the functions and mechanisms of leptins in appetite control in a fish model.

Genomic and Transcriptional Analysis of the Necroptosis Pathway Elements RIPK and MLKL in Sea Cucumber, Holothuria leucospilota.

Background: Receptor-interacting protein kinases (RIPKs) and mixed-lineage kinase domain-like protein (MLKL) are crucial in regulating innate immune responses and cell death signaling (necroptosis and apoptosis), and are potential candidates for genetic improvement in breeding programs. Knowledge about the RIPK family and MLKL in sea cucumber remains limited. Methods: We searched the genomes of sea cucumber Holothuria leucospilota for genes encoding RIPKs and MLKL, performed phylogenetic tree, motif and functional domain analyses, and examined tissue distribution and embryonic development patterns using qPCR. Results: RIPK5 (Hl-RIPK5), RIPK7 (Hl-RIPK7) and MLKL (Hl-MLKL) were identified in sea cucumber H. leucospilota. Hl-RIPK5 and Hl-RIPK7 were mainly expressed in coelomocytes, suggesting that they play a role in innate immunity, whereas Hl-MLKL exhibited relatively low expression across tissues. During embryonic development, Hl-MLKL was highly expressed from the 2-cell stage to the morula stage, while Hl-RIPK5 and Hl-RIPK7 were primarily expressed after the morula stage, indicating different roles in embryonic development. In primary coelomocytes, Hl-RIPK5 transcriptional activity was significantly depressed by LPS, poly(I:C), or pathogen Vibrio harveyi. Hl-RIPK7 expression levels were unchanged following the same challenges. Hl-MLKL mRNA levels were significantly decreased with poly(I:C) or V. harveyi, but did not change with LPS. Conclusions: These findings provide valuable insights into the evolutionary tree and characterization of RIPK and MLKL genes in sea cucumber, contributing to the broader understanding of the RIPK gene family and MLKL in ancient echinoderms.

Production of MSTN knockout porcine cells using adenine base-editing-mediated exon skipping.

Gene-knockout pigs have important applications in agriculture and medicine. Compared with CRISPR/Cas9 and cytosine base editing (CBE) technologies, adenine base editing (ABE) shows better safety and accuracy in gene modification. However, because of the characteristics of gene sequences, the ABE system cannot be widely used in gene knockout. Alternative splicing of mRNA is an important biological mechanism in eukaryotes for the formation of proteins with different functional activities. The splicing apparatus recognizes conserved sequences of the 5' end splice donor and 3' end splice acceptor motifs of introns in pre-mRNA that can trigger exon skipping, leading to the production of new functional proteins, or causing gene inactivation through frameshift mutations. This study aimed to construct a MSTN knockout pig by inducing exon skipping with the aid of the ABE system to expand the application of the ABE system for the preparation of knockout pigs. In this study, first, we constructed ABEmaxAW and ABE8eV106W plasmid vectors and found that their editing efficiencies at the targets were at least sixfold and even 260-fold higher than that of ABEmaxAW by contrasting the editing efficiencies at the gene targets of endogenous CD163, IGF2, and MSTN in pigs. Subsequently, we used the ABE8eV106W system to realize adenine base (the base of the antisense strand is thymine) editing of the conserved splice donor sequence (5'-GT) of intron 2 of the porcine MSTN gene. A porcine single-cell clone carrying a homozygous mutation (5'-GC) in the conserved sequence (5'-GT) of the intron 2 splice donor of the MSTN gene was successfully generated after drug selection. Unfortunately, the MSTN gene was not expressed and, therefore, could not be characterized at this level. No detectable genomic off-target edits were identified by Sanger sequencing. In this study, we verified that the ABE8eV106W vector had higher editing efficiency and could expand the editing scope of ABE. Additionally, we successfully achieved the precise modification of the alternative splice acceptor of intron 2 of the porcine MSTN gene, which may provide a new strategy for gene knockout in pigs.

Interleukin-2 enhancer binding factor 2 (ILF2) in pacific white shrimp (Litopenaeus vannamei): Alternatively spliced isoforms with different responses in the immune defenses against vibrio infection.

Alternative splicing is an essential molecular mechanism that increase the protein diversity of a species to regulate important biological processes. As a transcription factor, Interleukin-2 enhancer binding factor 2 (ILF2) regulates the functions of interleukin-2 (IL-2) at the levels of transcription, splicing and translation, and plays other critical roles in the immune system. ILF2 is well-documented in vertebrates, while little is currently known in crustacean species such as the Pacific white shrimp (Litopenaeus vannamei). In the present study, five cDNA for spliced isoforms of Lv-ILF2 were identified, in which four of them are the full-length long isoforms (Lv-ILF2-L1, Lv-ILF2-L2, Lv-ILF2-L3 and Lv-ILF2-L4) and one of them is a truncated short isoform (Lv-ILF2-S). The whole sequence of ILF2 gene from L. vannamei was obtained, which is 11,680 bp in length with 9 exons separated by 8 introns. All five isoforms contain a domain associated with zinc fingers (DZF). Two alternative splicing types (alternative 5' splice site and alternative 3' splice site) were identified in the five isoforms. The Lv-ILF2 mRNA showed a broad distribution in all detected tissues, and the Lv-ILF2-L transcript levels were higher than those of Lv-ILF2-S in corresponding tissues. The mRNA levels of Lv-ILF2-S in the hepatopancreas, heart, muscle and stomach, but not in the eyestalk, were significantly increased after challenges with Vibrio harveyi or lipopolysaccharide (LPS), while no significant changes were observed for the transcript levels of Lv-ILF2-L in these tissues under the same immune stimulants. On the contrary, the transcript levels of neither Lv-ILF2-S nor Lv-ILF2-L were affected by challenges of polyinosinic: polycytidylic acid [Poly (I:C)]. In addition, after knockdown of the Lv-ILF2 mRNA level by siRNA, the mortality of shrimp and the hepatopancreatic bacterial numbers were significantly increased under V. harveyi challenge, indicating that Lv-ILF2 might participate in the immune defenses against V. harveyi invasion. Collectively, our study here supplied the first evidence for a novel splicing mechanism of ILF2 transcripts, and provided a functional link between the Lv-ILF2 isoforms and the capacity against pathogenic Vibrio in penaeid shrimp.

CRISPR/Cas9-Mediated Biallelic Knockout of IRX3 Reduces the Production and Survival of Somatic Cell-Cloned Bama Minipigs.

Bama minipigs are a local pig breed that is unique to China and has a high development and utilization value. However, its high fat content, low feed utilization rate, and slow growth rate have limited its popularity and utilization. Compared with the long breeding cycle and high cost of traditional genetic breeding of pigs, clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated (Cas) endonuclease 9 system (CRISPR/Cas9)-mediated gene editing can cost-effectively implement targeted mutations in animal genomes, thereby providing a powerful tool for rapid improvement of the economic traits of Bama minipigs. The iroquois homeobox 3 (IRX3) gene has been implicated in human obesity. Mouse experiments have shown that knocking out IRX3 significantly enhances basal metabolism, reduces fat content, and controls body mass and composition. This study aimed to knock out IRX3 using the CRISPR/Cas9 gene editing method to breed Bama minipigs with significantly reduced fat content. First, the CRISPR/Cas9 gene editing method was used to efficiently obtain IRX3-/- cells. Then, the gene-edited cells were used as donor cells to produce surviving IRX3-/- Bama minipigs using somatic cell cloning. The results show that the use of IRX3-/- cells as donor cells for the production of somatic cell-cloned pigs results in a significant decrease in the average live litter size and a significant increase in the average number of stillbirths. Moreover, the birth weight of surviving IRX3-/- somatic cell-cloned pigs is significantly lower, and viability is poor such that all piglets die shortly after birth. Therefore, the preliminary results of this study suggest that IRX3 may have important biological functions in pigs, and IRX3 should not be used as a gene editing target to reduce fat content in Bama minipigs. Moreover, this study shows that knocking out IRX3 does not favor the survival of pigs, and whether targeted regulation of IRX3 in the treatment of human obesity will also induce severe adverse consequences requires further investigation.