Chirality-induced supramolecular nanodishes: enantioselectivity and energy transfer.

Self-assembly is one of the most important issues of fabricating materials with precise chiral nanostructures. Herein, we constructed a chiral assembly system from amphiphiles containing hydrophobic/hydrophilic chiral coils bonded to hexabiphenyl, exhibiting controllable enantioselectivity over various aggregation behaviors. The chiral coils aroused various steric hindrances affecting intrinsic stacking tendency and compactness, leading to different aggregating behaviors, as concluded from the self-assembly investigation. The strong π-π stacking interaction between the long hexabiphenyl groups gave rise to a relatively compact arrangement in the aqueous solution, whereas the methyl side groups on the coil segments raised steric hindrance at the rigid-flexible interface, resulting in loose stacking and formation of nanostructures with a larger curvature. Compared with the achiral molecule 1 that formed micron-sized large sheets, molecules 2-4 containing chiral coils aggregated into nanodishes, which looked exactly like mosquito-repellent incense, to overcome surface tension. The helical structures effectively amplified chirality and exhibited strong circular dichroism (CD) signals, which indicate enantioselectivity. In addition, the relatively loose packing behavior permitted their co-assembly with a dye and aided efficient energy transfer, providing a foundation for the chiral application of supramolecules. Thus, by introducing a simple methyl side group in amphiphilic molecules, asymmetric synthesis and energy transfer efficiency can be realized.

miR-193b-5p promotes GCRV replication by inhibiting autophagy via targeting deptor in grass carp (Ctenopharyngodon idellus).

miRNAs are increasingly recognized for their crucial role in autophagy processes. Recent research has highlighted the significant function of autophagy in modulating immune responses. Within this context, specific miRNAs have been identified as indirect mediators of immune functions through their modulation of autophagy. In this study, we verified that miR-193b-5p simultaneously targeted the grass carp autophagy-related gene deptor, thereby reducing autophagy levels in CIK cells. Moreover, we found the expression levels of miR-193b-5p and deptor responding to pathogen infections in the GCRV-infected CIK cells. Notably, the overexpression of miR-193b-5p was found to induce the GCRV replication and reduce the irf3, irf7 and IFN1 expression. These findings also demonstrated that grass carp miR-193b-5p impacted the proliferation, migration, and antiapoptotic abilities of CIK cells. All the above results indicated that miR-193b-5p was linked to grass carp autophagy and played a vital role in antiviral immunity by targeting deptor. Our study may provide important insights into autophagy-related miRNAs and their roles in defense and immune mechanisms against pathogens in teleost.

Incorporating Lycium barbarum residue in diet boosts survival, growth, and liver health in juvenile grass carp (Ctenopharyngodon idellus).

This research elucidates the potential of Lycium barbarum residue (LBR), a by-product rich in bioactive substances, as a dietary supplement in aquaculture, especially for herbivorous fish like grass carp. In a detailed 120-day feeding trial, the impacts of varying LBR levels on juvenile grass carp were assessed, focusing on growth performance, survival rate, biochemical markers, and liver health. The study identified a 6% inclusion rate of LBR as optimal for enhancing survival and growth while mitigating hepatic lipid accumulation. Composition analysis of this diet revealed high concentrations of polysaccharides and flavonoids. Notably, the intake of LBR was found to enhance the antioxidant and immune-related enzymatic activities in the liver. Furthermore, it contributed to a reduction in hepatic fat deposition by decreasing the levels of triglycerides (TG) and total cholesterol (T-CHO) both in the liver and serum. Transcriptomic analysis of the liver highlighted LBR's substantial influence on lipid metabolism pathways, including the PPAR signaling pathway, primary bile acid biosynthesis, cholesterol metabolism, bile secretion, fat digestion and absorption, fatty acid degradation and fatty acid biosynthesis. Further, the expression level of genes pinpointed significant downregulation of fasn and dgat2, alongside upregulation of genes like pparda, cpt1b, cpt1ab and abca1b, in response to LBR supplementation. Overall, the findings present LBR as a promising enhancer of growth and survival in grass carp, with significant benefits in promoting fat metabolism and liver health, offering valuable insights for aquacultural nutrition strategies.

Deep sequencing identified miR-193b-3p as a positive regulator of autophagy targeting Akt3 in Ctenopharyngodon idella CIK cells during GCRV infection.

Recent research has highlighted complex and close interaction between miRNAs, autophagy, and viral infection. In this study, we observed the autophagy status in CIK cells infected with GCRV at various time points. We found that GCRV consistently induced cellar autophagy from 0 h to 12 h post infection. Subsequently, we performed deep sequencing on CIK cells infected with GCRV at 0 h and 12 h respectively, identifying 38 DEMs and predicting 9581 target genes. With the functional enrichment analyses of GO and KEGG, we identified 35 autophagy-related target genes of these DEMs, among which akt3 was pinpointed as the most central hub gene using module assay of the PPI network. Then employing the miRanda and Targetscan programs for prediction, and verification through a double fluorescent enzyme system and qPCR method, we confirmed that miR-193 b-3p could target the 3'-UTR of grass carp akt3, reducing its gene expression. Ultimately, we illustrated that grass carp miR-193 b-3p could promote autophagy in CIK cells. Above results collectively indicated that miRNAs might play a critical role in autophagy of grass carp during GCRV infection and contributed significantly to antiviral immunity by targeting autophagy-related genes. This study may provide new insights into the intricate mechanisms involved in virus, autophagy, and miRNAs.

MicroRNA-30e-3p regulates the inflammatory response by targeting the gimap8 gene in Ctenopharyngodon idella kidney (CIK) cells.

Recent studies have increasingly linked miRNAs with the modulation of inflammatory responses and immunosuppressive activities. This investigation reveals that mir-30e-3p selectively binds to and modulates gimap8, as demonstrated by luciferase reporter assays and qPCR analyses. Upon LPS stimulation of CIK cells, mir-30e-3p expression was notably elevated, inversely correlating with a decrease in gimap8 mRNA levels. Overexpression of mir-30e-3p attenuated the mRNA levels of pro-inflammatory cytokines beyond the effect of LPS alone, suggesting a regulatory role of mir-30e-3p in inflammation mediated by the gimap8 gene. These insights contribute to our understanding of the complex mechanisms governing inflammatory and immune responses.

miR-22a targets p62/SQSTM1 to negatively affect autophagy and disease resistance of grass carp (Ctenopharyngodon idella).

MicroRNAs (miRNAs) are integral to many biological functions, including autophagy, a process recently proven to be closely linked to innate immunity. In this study, we present findings on miR-22a, a teleost homolog of mammalian miR-22, illustrating its capacity to target the autophagy adaptor p62, subsequently inducing downregulation at both mRNA and protein levels. Utilizing Western blot analyses, we demonstrated that miR-22a inhibits the autophagy flux of CIK cells, correlated with an elevated presence of LC3 II. Additionally, the overexpression of miR-22a resulted in the suppression of NF-κB signaling, leading to reduced cellar antimicrobial abilities and increased apoptosis. These findings provide novel insights into the role of miR-22a as an autophagy-related miRNA and its immune mechanisms against pathogens via p62 in teleost, enriching our understanding of the interplay between autophagy and innate immunity.

miR-124 mediates the expression of ccBax to regulate Cyprinid herpesvirus 2 (CyHV-2)-induced apoptosis and viral replication.

Cyprinid herpesvirus 2 (CyHV-2), the etiological agent of herpesvirus haematopoietic necrosis (HVHN) in carp and goldfish, has caused significant economic losses in the aquaculture industry. During viral infection, the host initiates a series of active or passive defences to regulate the process of virus infection. Apoptosis is a key component of active cellular defence, and members of the Bcl-2 family have been shown to play a critical role in the apoptotic process. However, the mechanism of action of the Bcl-2 family in inducing apoptosis during CyHV-2 infection remains unclear. In this study, we revealed the molecular mechanism of miRNA-mediated silver crucian carp BAX (ccBax) in CyHV-2-induced apoptosis for the first time and demonstrated that the overexpression of miR-124 suppressed ccBax expression and significantly down-regulated apoptosis in caudal fin cells of Carassius auratus gibelio (GiCF), while miR-124 inhibitors were the opposite. These studies indicated that miR-124 inhibits CyHV-2-induced apoptosis by reducing the expression of ccBax. Furthermore, the fact that transfection of miR-124 mimics promoted CyHV-2 replication, whereas miR-124 inhibitors inhibited CyHV-2 replication, indicated that miR-124 inhibited CyHV-2-induced apoptosis and contributed to viral replication. All these results suggested that miR-124 suppresses virus-induced apoptosis and promotes viral replication by targeting and regulating ccBax expression.

Transcriptomic responses to low temperature stress in the Nile tilapia, Oreochromis niloticus.

The Nile tilapia, Oreochromis niloticus, is a species of high economic value and extensively cultured. The limited stress tolerance of this species to a low temperature usually leads to mass mortality and great loss. Nevertheless, there is limited information on the molecular mechanisms underlying the susceptibility to low temperature in the tilapia. In this study, tilapia was treated at 28 °C to a lethal temperature of 8 °C by a gradual decrement. Transcriptomic response of the immune organ, kidney, in tilapia was characterized using RNA-seq. In total, 2191 genes were annotated for significant expression, which were mainly associated with metabolism and immunity. Pathway analysis showed that immune-related pathways of phagosome and cell adhesion molecules (CAMs) pathway were significantly down-regulated under low temperature. Moreover, ferroptosis, a significantly changed pathway involved in tissue damage and acute renal failure, is reported here for the first time. The levels of serum parameters associated with kidney damage such as urea and uric acid (UA) increased significantly under low temperature. The immunofluorescence staining of the kidney showed that cell apoptosis occurred at low temperature. The results of the present study indicate that exposure to low temperature can cause kidney disfunction and down-regulate the immune-related pathway in the kidney of tilapia. This study provides new insight into the mechanism of kidney damage in fish under low temperature.

Two hepcidins from spotted scat (Scatophagus argus) possess antibacterial and antiviral functions in vitro.

Hepcidins are small cysteine-rich antimicrobial peptides that play an important role in host immunity against pathogenic organisms. In this study, two hepcidins, SA-hepcidin1 and SA-hepcidin2, were cloned from spotted scat (Scatophagus argus), and the tissue distributions of SA-hepcidins were determined. In addition, mature SA-hepcidin peptides were synthesized to allow evaluation of their antimicrobial and antiviral functions in vitro. SA-hepcidin1 belongs to the HAMP1 class and is widely expressed in all tested tissues from spotted scat, whereas SA-hepcidin2 belongs to the HAMP2 class and present only in the liver. The synthetic SA-hepcidins had similar levels of antibacterial activity against Gram-positive and Gram-negative bacteria; however, the antibacterial activity of SA-hepcidin1 was stronger than that of SA-hepcidin2. The antiviral activities of the synthetic SA-hepcidins were assessed against Siniperca chuatsi rhabdovirus (SCRV) and largemouth bass Micropterus salmoides reovirus (MsReV) in epithelioma papulosum cyprini (EPC) and grass carp fin (GCF) cells. SA-hepcidin2 had antiviral activity, but SA-hepcidin1 did not. The results of this study suggest that SA-hepcidins are important multifunctional proteins in the spotted scat immune system that are involved in resistance to various pathogens.

Mutations in the C-terminal region affect subcellular localization of crucian carp herpesvirus (CaHV) GPCR.

G protein-coupled receptors (GPCRs) are known as seven transmembrane domain receptors and consequently can mediate diverse biological functions via regulation of their subcellular localization. Crucian carp herpesvirus (CaHV) was recently isolated from infected fish with acute gill hemorrhage. CaHV GPCR of 349 amino acids (aa) was identified based on amino acid identity. A series of variants with truncation/deletion/substitution mutation in the C-terminal (aa 315-349) were constructed and expressed in fathead minnow (FHM) cells. The roles of three key C-terminal regions in subcellular localization of CaHV GPCR were determined. Lysine-315 (K-315) directed the aggregation of the protein preferentially at the nuclear side. Predicted N-myristoylation site (GGGWTR, aa 335-340) was responsible for punctate distribution in periplasm or throughout the cytoplasm. Predicted phosphorylation site (SSR, aa 327-329) and GGGWTR together determined the punctate distribution in cytoplasm. Detection of organelles localization by specific markers showed that the protein retaining K-315 colocalized with the Golgi apparatus. These experiments provided first evidence that different mutations of CaHV GPCR C-terminals have different affects on the subcellular localization of fish herpesvirus-encoded GPCRs. The study provided valuable information and new insights into the precise interactions between herpesvirus and fish cells, and could also provide useful targets for antiviral agents in aquaculture.

Comparative renal gene expression in response to abrupt hypoosmotic shock in spotted scat (Scatophagus argus).

Scatophagus argus, a euryhaline fish, is notable for its ability to tolerate a wide range of environmental salinities and especially for its tolerance to a rapid, marked reduction in salinity. Therefore, S. argus is a good model for studying the molecular mechanisms mediating abrupt hyperosmoregulation. The serum osmotic pressure decreased steeply within one hour after transferring S. argus from seawater (SW) to freshwater (FW) and remained at new balance throughout the duration of one week. To explain this phenomenon and understand the molecular responses to an abrupt hypoosmotic shock, hypoosmotic stress responsive genes were identified by constructing two suppression subtractive hybridization (SSH) cDNA libraries from the kidneys of S. argus that had been transferred from SW to FW. After trimming and blasting, 52 ESTs were picked out from the subtractive library. Among them, 11 genes were significantly up-regulated (p < 0.05). The kinetics studies of gene expression levels were conducted for 1 week after the transfer using quantitative real-time PCR. A significant variation in the expression of these genes occurred within 12h after the hypoosmotic shock, except for growth hormone (GH) and polyadenylate binding protein 1 (PBP1), which were significantly up-regulated 2 days post-transfer. Our results suggest different functional roles for these genes in response to hypoosmotic stress during the stress response phase (1 hpt-12 hpt) and stable phase (12 hpt-7 dpt). Furthermore, the plasma growth hormone level was detected to be significantly elevated at 1 hpt and 24 hpt following abrupt hypoosmotic shock. Meanwhile, several hematological parameters, hemoglobin (HGB), red blood cell (RBC) and mean cellular hemoglobin concentration (MCHC), were observed to be significantly increased at 12 hpt and 2 dpt compared with that of control group. Our results provide a solid basis from which to conduct future studies on the osmoregulatory mechanisms in the euryhaline fish.

[A preliminary study of long-term mitochondrial dysfunction in rat brain caused by lipopolysaccharide-induced sepsis].

OBJECTIVE: To preliminarily investigate the long-term structural and functional injuries of mitochondria in rat brain caused by sepsis. METHODS: Wistar rats were randomly assigned into sepsis and control groups. A rat model of sepsis was prepared by an intraperitoneal injection of 10 mg/kg lipopolysaccharide (LPS) of gram-negative bacteria, and the survival assay was performed. Eight rats in the sepsis group were sacrificed at 12, 24, 48, or 72 hours after LPS injection, while rats in the control group were sacrificed after an intraperitoneal injection of an equal volume of normal saline. Mitochondria were extracted from rat brain tissue. Mitochondrial membrane potential (MMP) and mitochondrial swelling level were determined by flow cytometry, and the activities of electron transport chain complexes (I-V) were measured using enzyme assay kits. Hematoxylin-eosin (HE) staining and electron microscopy were used to observe morphological changes in brain tissue and mitochondria. RESULTS: The sepsis group had a significantly lower survival rate than the control group (P<0.01). The MMP and activities of electron transport chain complexes (I-V) in the sepsis group, which were significantly lower than those in the control group (P<0.05), were reduced to the lowest levels at 48 hours and partially recovered at 72 hours. The mitochondrial swelling level in the sepsis group, which was significantly higher than that in the control group (P<0.05), increased to the peak level at 48 hours and partially recovered at 72 hours. Hematoxylin and Eosin staining revealed substantial damages in the structure of brain tissue, and electron microscopy showed mitochondrial swelling, and vacuolization in a few mitochondria. CONCLUSIONS: In the rat model of LPS-induced sepsis, both structural and functional injuries are found in cerebral mitochondria, and achieve the peak levels probably at around 48 hours.

Impact of cadmium and diclofenac exposure on biochemical responses, transcriptome, gut microflora, and growth performance in grass carp (Ctenopharyngodonidella).

In recent years, the concentrations of cadmium (Cd) and diclofenac (DCF) in water have frequently exceeded the standard; however, the toxic effects of these two pollutants on grass carp under single and combined exposure are unknown. In this study, the concentrations of pollutants in different tissues were detected, and the toxicities of the two pollutants to grass carp under different exposure conditions were compared based on growth traits, biochemical responses, gut microbiome, and transcriptomes. Based on these findings, the brain showed the lowest levels of Cd and DCF accumulation. Oxidative stress and pathological damage were observed in the brain and intestines. Changes in the structure and abundance of the gut microflora affect the synthesis of neurotransmitters, such as GABA and steroids. Differentially expressed genes in the brain were enriched in circadian rhythm functions. The expression of PER, CLOCK,1L-1ß, 1L-17, and other genes are related to the abundance of Akkermansia, which indicates that the disorder of gut microflora will affect the normal circadian rhythm of the brain. All indices in the recovery group showed an increasing trend. Overall, the toxicity of Cd and DCF showed antagonism, and a single exposure had a stronger effect on gut microorganisms and circadian rhythm, which provided a scientific basis for exploring the comprehensive effects of different pollutants.

Chromosome-scale genome assemblies of sexually dimorphic male and female Acrossocheilus fasciatus.

Acrossocheilus fasciatus is a stream-dwelling fish species of the Barbinae subfamily. It is valued for its colorfully striped appearance and delicious meat. This species is also characterized by apparent sexual dimorphism and toxic ovum. Biology and aquaculture researches of A. fasciatus are hindered by the lack of a high-quality reference genome. Here, we report chromosome-level genome assemblies of the male and female A. fasciatus. The HiFi-only genome assemblies for both female and male individuals were 899.13 Mb (N50 length of 32.58 Mb) and 885.68 Mb (N50 length of 33.06 Mb), respectively. Notably, a substantial proportion of the assembled sequences, accounting for 96.15% and 98.35% for female and male genomes, respectively, were successfully anchored onto 25 chromosomes utilizing Hi-C data. We annotated the female assembly as a reference genome and identified a total of 400.62 Mb (44.56%) repetitive sequences, 27,392 protein-coding genes, and 35,869 ncRNAs. The high-quality male and female reference genomes will provide genomic resources for developing sex-specific molecular markers, inform single-sex breeding, and elucidate genetic mechanisms of sexual dimorphism.

Identification of Genes Related to Resistance to Ichthyophthirius multifiliis Based on Co-expression Network Analysis in Grass Carp.

The ciliate protozoan Ichthyophthirius multifiliis is an essential parasite causing white spot disease in grass carp, leading to significant economic losses. Understanding the molecular basis of grass carp's response to I. multifiliis has important scientific and environmental values. The transcriptional network analysis offers a valuable strategy to decipher the changes in gene expression in grass carp infected with I. multifiliis. Our goal was to screen the genes and pathways involved in resistance to I. multifiliis in grass carp. The different traits exhibited by grass carp infected with I. multifiliis may be caused by the differences in gene expression among grass carp individuals. Herein, to reveal those resistance-associated genes against I. multifiliis infection, we performed RNA sequencing using weighted gene co-expression network analysis (WGCNA). The biological function analysis and hub gene annotation for highly relevant modules revealed that different pathogen recognition and clearance responses resulted in different resistance to I. multifiliis infection. Furthermore, gene enrichment analysis revealed that I. multifiliis invasion in the disease-resistant group mainly activated immune pathways, including scavenger receptor activity and kappa B kinase/NF-kappa B signaling. By the annotation of the highly correlated module of the hub gene, we revealed that the apoptosis and ribosome biogenesis-related genes were enriched in the disease-resistant grass carp. The results of the dark grey module showed that several genes were mainly enriched in the two-component system (ko02020) and steroid biosynthesis (ko00100), suggesting that they are resistance-associated and energy metabolism-associated genes. In the disease resistance group, hub genes mainly included Nlrc3, fos, AAP8, HAP2, HAX, cho2, and zgc:113,036. This study revealed the gene network associated with disease resistance after I. multifiliis infection. The disease resistance-related pathways and central genes identified in this study are candidate references for breeders breeding disease-resistant. The results of this study may also provide some references for the development of drugs to antagonize I. multifiliis infection.

Full-Length Transcriptomes and Sex-Based Differentially Expressed Genes in the Brain and Ganglia of Giant River Prawn Macrobrachium rosenbergii.

Macrobrachium rosenbergii is an important aquaculture prawn that exhibits sexual dimorphism in growth, with males growing much faster than females. However, the mechanisms controlling these complex traits are not well understood. The nervous system plays an important role in regulating life functions. In the present work, we applied PacBio RNA-seq to obtain and characterize the full-length transcriptomes of the brains and thoracic ganglia of female and male prawns, and we performed comparative transcriptome analysis of female and male prawns. A total of 159.1-Gb of subreads were obtained with an average length of 2175 bp and 93.2% completeness. A total of 84,627 high-quality unigenes were generated and annotated with functional databases. A total of 6367 transcript factors and 6287 LncRNAs were predicted. In total, 5287 and 6211 significantly differentially expressed genes (DEGs) were found in the brain and thoracic ganglion, respectively, and confirmed by qRT-PCR. Of the 435 genes associated with protein processing pathways in the endoplasmic reticula, 42 DEGs were detected, and 21/26 DEGs with upregulated expression in the male brain/thoracic ganglion. The DEGs in this pathway are regulated by multiple LncRNAs in polypeptide folding and misfolded protein degradation in the different organs and sexes of the prawn. Our results provide novel theories and insights for studying the nervous system, sexual control, and growth dimorphism.

The identification of viral ribonucleotide reductase encoded by ORF23 and ORF141 genes and effect on CyHV-2 replication.

Introduction: Ribonucleotide reductase (RR) is essential for the replication of the double-stranded DNA virus CyHV-2 due to its ability to catalyze the conversion of ribonucleotides to deoxyribonucleotides, and is a potential target for the development of antiviral drugs to control CyHV-2 infection. Methods: Bioinformatic analysis was conducted to identify potential homologues of RR in CyHV-2. The transcription and translation levels of ORF23 and ORF141, which showed high homology to RR, were measured during CyHV-2 replication in GICF. Co-localization experiments and immunoprecipitation were performed to investigate the interaction between ORF23 and ORF141. siRNA interference experiments were conducted to evaluate the effect of silencing ORF23 and ORF141 on CyHV-2 replication. The inhibitory effect of hydroxyurea, a nucleotide reductase inhibitor, on CyHV-2 replication in GICF cells and RR enzymatic activity in vitro was also evaluated. Results: ORF23 and ORF141 were identified as potential viral ribonucleotide reductase homologues in CyHV-2, and their transcription and translation levels increased with CyHV-2 replication. Co-localization experiments and immunoprecipitation suggested an interaction between the two proteins. Simultaneous silencing of ORF23 and ORF141 effectively inhibited the replication of CyHV-2. Additionally, hydroxyurea inhibited the replication of CyHV-2 in GICF cells and the in vitro enzymatic activity of RR. Conclusion: These results suggest that the CyHV-2 proteins ORF23 and ORF141 function as viral ribonucleotide reductase and their function makes an effect to CyHV-2 replication. Targeting ribonucleotide reductase could be a crucial strategy for developing new antiviral drugs against CyHV-2 and other herpesviruses.

Low particle concentrations of nanoplastics impair the gut health of medaka.

The environmental occurrence of nanoplastics (NPs) is now evident but their long-term impacts on organisms are unclear, limiting ecological and health risk assessment. We hypothesized that chronic exposure to low particle concentrations of NPs can result in gut-associated toxicity, and subsequently affect survival of fish. Japanese medaka Oryzias latipes were exposed to polystyrene NPs (diameter 100 nm; 0, 10, 104, and 106 items/L) for 3 months, and histopathology, digestive and antioxidant enzymes, immunity, intestinal permeability, gut microbiota, and mortality were assessed. NP exposures caused intestinal lesions, and increased intestinal permeability of the gut. The trypsin, lipase, and chymotrypsin activities were increased, but the amylase activity was decreased. Oxidative damage was reflected by the decreased superoxide dismutase and alkaline phosphatase and increased malondialdehyde, catalase, and lysozyme. The integrated biomarkers response index values of all NP-exposed medaka were significantly increased compared to the control group. Moreover, NP exposures resulted in a decrease of diversity and changed the intestinal microbiota composition. Our results provide new evidence that long-term NPs exposure impaired the health of fish at extremely low particle concentrations, suggesting the need for long-term toxicological studies resembling environmental particle concentrations when assessing the risk of NPs.

Establishment of a Coilia nasus Spermatogonial Stem Cell Line Capable of Spermatogenesis In Vitro.

The process by which spermatogonial stem cells (SSCs) continuously go through mitosis, meiosis, and differentiation to produce gametes that transmit genetic information is known as spermatogenesis. Recapitulation of spermatogenesis in vitro is hindered by the challenge of collecting spermatogonial stem cells under long-term in vitro culture conditions. Coilia nasus is a commercially valuable anadromous migrant fish found in the Yangtze River in China. In the past few decades, exploitation and a deteriorating ecological environment have nearly caused the extinction of C. nasus's natural resources. In the present study, we established a stable spermatogonial stem cell line (CnSSC) from the gonadal tissue of the endangered species C. nasus. The cell line continued to proliferate and maintain stable cell morphology, a normal diploid karyotype, and gene expression patterns after more than one year of cell culture (>80 passages). Additionally, CnSSC cells could successfully differentiate into sperm cells through a coculture system. Therefore, the establishment of endangered species spermatogonial stem cell lines is a model for studying spermatogenesis in vitro and a feasible way to preserve germplasm resources.

Generation and Characterization of ORF55/ORF57-Deleted Recombinant Cyprinid herpesvirus 2 Mutants with Chimeric Capsid Protein Gene of Grouper Nervous Necrosis Virus.

Cyprinid herpesvirus 2 (CyHV-2) is a pathogen that causes significant losses to the global aquaculture industry due to mass mortality in crucian carp and goldfish. This study demonstrates that the ORF55/ORF57 deletion mutants CyHV-2-Δ55-CP and CyHV-2-Δ57-CP obtained through homologous recombination replicate effectively within the caudal fin of Carassius auratus gibelio (GiCF) cells and exhibit morphologies similar to the CyHV-2 wild-type strain. Both mutants demonstrated a decrease in virulence, with CyHV-2-Δ57-CP exhibiting a more significant reduction. This serves as a reference for the subsequent development of recombinant attenuated vaccines against CyHV-2. Additionally, both mutants expressed the inserted RGNNV-CP (capsid protein of Redspotted grouper nervous necrosis virus) fusion protein gene, and inoculation with CyHV-2-Δ57-CP-infected GiCF cell lysates elicited an antibody response in the grouper. These results indicate that, while ORF55 and ORF57 genes of CyHV-2 are not required for viral replication in vitro, they do play a role in virulence in vivo. Additionally, expression of foreign protein in CyHV-2 suggests that the fully attenuated mutant of CyHV-2 could potentially function as a viral vector for developing subunit vaccines or multivalent recombinant attenuated vaccines.