Strategies for high cell density cultivation of Akkermansia muciniphila and its potential metabolism.

IMPORTANCE: Currently, there is significant interest in Akkermansia muciniphila as a promising next-generation probiotic, making it a hot topic in scientific research. However, to achieve efficient industrial production, there is an urgent need to develop an in vitro culture method to achieve high biomass using low-cost carbon sources such as glucose. This study aims to explore the high-density fermentation strategy of A. muciniphila by optimizing the culture process. This study also employs techniques such as LC-MS and RNA-Seq to explain the possible regulatory mechanism of high-density cell growth and increased cell surface hydrophobicity facilitating cell colonization of the gut in vitro culture. Overall, this research sheds light on the potential of A. muciniphila as a probiotic and provides valuable insights for future industrial production.

Japanese medaka Olpax6.1 mutant as a potential model for spondylo-ocular syndrome.

pax6 is a canonic master gene for eye formation. Knockout of pax6 affects the development of craniofacial skeleton and eye in mice. Whether pax6 affects the development of spinal bone has not been reported yet. In the present study, we used CRISPR/Cas9 system to generate Olpax6.1 mutant in Japanese medaka. Phenotype analysis showed that ocular mutation caused by the Olpax6.1 mutation occurred in the homozygous mutant. The phenotype of heterozygotes is not significantly different from that of wild-type. In addition, knockout Olpax6.1 resulted in severe curvature of the spine in the homozygous F2 generation. Comparative transcriptome analysis and qRT-PCR revealed that the defective Olpax6.1 protein caused a decrease in the expression level of sp7, col10a1a, and bglap, while the expression level of xylt2 did not change significantly. The functional enrichment of differentially expressed genes (DEGs) using the Kyoto Encyclopedia of Genes and Genomes database showed that the DEGs between Olpax6.1 mutation and wild-type were enriched in p53 signaling pathway, extracellular matrix (ECM) -receptor interaction, et al. Our results indicated that the defective Olpax6.1 protein results in the reduction of sp7 expression level and the activation of p53 signaling pathway, which leads to a decrease in the expression of genes encoding ECM protein, such as collagen protein family and bone gamma-carboxyglutamate protein, which further inhibits bone development. Based on the phenotype and molecular mechanism of ocular mutation and spinal curvature induced by Olpax6.1 knockout, we believe that the Olpax6.1-/- mutant could be a potential model for the study of spondylo-ocular syndrome.

Medaka (Oryzias latipes) Olpax6.2 acquires maternal inheritance and germ cells expression, but functionally degenerate in the eye.

Gene duplication provides raw material for the evolution of genetic and phenotypic complexity. It has remained a long-standing mystery how duplicated genes evolve into new genes by neofunctionalization via the acquisition of new expression and/or activity and simultaneous loss of the old expression and activity. Fishes have many gene duplicates from whole genome duplication, making them excellent for studying the evolution of gene duplicates. In the fish medaka (Oryzias latipes), an ancestral pax6 gene has given rise to Olpax6.1 and Olpax6.2. Here we report that medaka Olpax6.2 is evolving towards neofunctionalization. A chromosomal syntenic analysis indicated that Olpax6.1 and Olpax6.2 are structurally co-homologous to the single pax6 in other organisms. Interestingly, Olpax6.2 maintains all conserved coding exons but loses the non-coding exons of Olpax6.1, and has 4 promoters versus 8 in Olpax6.1. RT-PCR revealed that Olpax6.2 maintains expression in the brain eye, pancreas as Olpax6.1. Surprisingly, Olpax6.2 also exhibits maternal inheritance and gonadal expression by RT-PCR, in situ hybridization and RNA transcriptome analysis. The expression and distribution of Olpax6.2 is not different from Olpax6.1 in the adult brain, eye and pancreas, but exhibited overlapping and distinct expression in early embryogenesis. We show that ovarian Olpax6.2 expression occurs in female germ cells. Olpax6.2 knockout shows no obvious defect in eye development, while Olpax6.1 F0 mutant have severe defects in eye development. Thus, Olpax6.2 acquires maternal inheritance and germ cell expression, but functionally degenerates in the eye, making this gene as an excellent model to study the neofunctionalization of duplicated genes.

Efficient gene editing in a medaka (Oryzias latipes) cell line and embryos by SpCas9/tRNA-gRNA.

Generation of mutants with clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) is commonly carried out in fish species by co-injecting a mixture of Cas9 messenger RNA (mRNA) or protein and transcribed guide RNA (gRNA). However, the appropriate expression system to produce functional gRNAs in fish embryos and cells is rarely present. In this study, we employed a poly-transfer RNA (tRNA)-gRNA (PTG) system driven by cytomegalovirus (CMV) promoter to target the medaka (Oryzias latipes) endogenous gene tyrosinase(tyr) or paired box 6.1 (pax6.1) and illustrated its function in a medaka cell line and embryos. The PTG system was combined with the CRISPR/Cas9 system under high levels of promoter to successfully induce gene editing in medaka. This is a valuable step forward in potential application of the CRISPR/Cas9 system in medaka and other teleosts.

Virus susceptibility of a new cell line derived from the fin of black carp Mylopharyngodon piceus.

A continuous cell line MPF derived from the fin of black carp Mylopharyngodon piceus was established and characterised in this study. Mylopharyngodon piceus fin (MPF) cells were subcultured for more than 80 passages with high viability recovery after long-term storage. The karyotyping analysis revealed that MPF had a modal diploid chromosome number (2n = 48) and identical ribosomal RNA sequence with black carp. In addition, the expression of pluripotency-associated markers including nanog, oct4 and vasa, were detected in MPF. The transient transfection efficiency of MPF reached 23% with a fluorescent reporter by modified electroporation and stable expression of red fluorescent MPF was established by the baculovirus system, indicating that MPF is an ideal platform for studying gene functions in vitro. Lastly, cytopathic effects were also observed and RNA transcripts of a viral gene increased after infection by spring viremia of carp virus (SVCV), suggesting that MPF could be an alternative tool for investigating pathogen-host interactions in black carp. In conclusion, a fin cell line that is susceptible to SVCV was established as a potential adult stem-cell line, providing a suitable tool for future genetic analyses and pathogen-host studies in black carp.

An overactive neddylation pathway serves as a therapeutic target and MLN4924 enhances the anticancer activity of cisplatin in pancreatic cancer.

The survival rate of patients with pancreatic cancer is between 3 and 5%. The neddylation pathway is overactive in multiple cancer types and is associated with poor prognosis. In recent years, the neddylation process has become a popular research target for the development of novel cancer therapies. However, the activation level of the pathway, and whether its targeting sensitizes pancreatic cancer cells to cisplatin treatment is currently unclear. In the present study, using reverse transcription-quantitative PCR and western blot analyses, the neddylation pathway was observed to be overactivated at the protein, but not the mRNA level. In addition, by analyzing The Cancer Genome Atlas data, it was demonstrated that high expression levels of NEDD8 activating enzyme E1 subunit 1 were observed to be a predictor of poor prognosis for patients with pancreatic cancer. Cisplatin enhanced the cytotoxic effects of MLN4924 both in vitro and in vivo according to Cell Counting kit-8 assays and an in vivo tumor model. Further mechanistic studies, including western blotting and immunohistochemistry assays, revealed that combined MLN4924 and cisplatin treatment induced higher levels of DNA damage by increasing the accumulation of well-defined cullin-ring ligase substrates, such as chromatin licensing and DNA replication factor 1, origin recognition complex subunit 1, p21, p27 and phosphorylated IκBα. The results of the present study support the clinical use of combined neddylation inhibitor and cisplatin treatment, which may improve the survival of, and impart other benefits for patients with pancreatic cancer.

Gonadal, Not Maternal, Acquisition of Duplicated pax6 Orthologs in Megalobrama Amblycephala.

: The highly conserved transcription factor Pax6 is involved in the development of the eyes, brain, and pancreas in vertebrates and invertebrates, whereas the additional expression pattern in other organs is still elusive. In this study, we cloned and characterized two pax6 homologs in blunt snout bream (Megalobrama amblycephala), named Mapax6a and Mapax6b. The protein alignment and phylogenetic tree showed that Mapax6a and Mapax6b were highly conserved compared with their counterparts in other species. Genomic information analysis revealed that the synteny conservation of Wilms tumor, Aniridia, genitourinary abnormalities, and mental retardation loci was also maintained in this species. By reverse transcription polymerase chain reaction, the expression of Mapax6a was later than that of Mapax6b which was found in the blastula stage, while the expression of Mapax6a started from the somite stage, and both of them persisted in a subsequent stage during the embryonic development. By RNA and protein detection, Mapax6a and Mapax6b were detected in the eye and brain as canonic patterns, and most importantly, they were also enriched in germ cells of the testis and ovary. Therefore, our findings validate the duplication of pax6 in fish, confirm the classical expression patterns in the brain and eye, and, for the first time, present a new acquisition of Mapax6a and Mapax6b in gonadal germ cells in particular. Therefore, our results enrich the expression pattern and evolutionary relationship of pax6 by suggesting that duplicated Mapax6 is involved in gametogenesis in Megalobrama amblycephala.

Efficient and Stable Delivery of Multiple Genes to Fish Cells by a Modified Recombinant Baculovirus System.

The recombinant baculovirus has been widely used as an efficient tool to mediate gene delivery into mammalian cells but has barely been used in fish cells. In the present study, we constructed a recombinant baculovirus containing the dual-promoter cytomegalovirus (CMV) and white spot syndrome virus (WSSV) immediate-early gene 1 (ie1) (WSSV ie1), followed by a puromycin⁻green fluorescent protein (Puro-GFP, pf) or puromycin⁻red fluorescent protein (Puro-RFP, pr) cassette, which simultaneously allowed for easy observation, rapid titer determination, drug selection, and exogenous gene expression. This recombinant baculovirus was successfully transduced into fish cells, including Mylopharyngodon piceus bladder (MPB), fin (MPF), and kidney (MPK); Oryzias latipes spermatogonia (SG3); and Danio rerio embryonic fibroblast (ZF4) cells. Stable transgenic cell lines were generated after drug selection, which was further verified by Western blot. A cell monoclonal formation assay proved the stable heredity of transgenic MPB cells. In addition, a recombinant baculovirus containing a pr cassette and four transcription factors for induced pluripotent stem cells (iPSC) was constructed and transduced into ZF4 cells, and these exogenous genes were simultaneously delivered and transcribed efficiently in drug-selected ZF4 cells, proving the practicability of this modified recombinant baculovirus system. We also proved that the WSSV ie1 promoter had robust activity in fish cells in vitro and in vivo. Taken together, this modified recombinant baculovirus can be a favorable transgenic tool to obtain transient or stable transgenic fish cells.

Generation of albino medaka (Oryzias latipes) by CRISPR/Cas9.

CRISPR/Cas9 system is a powerful tool to produce the genetic modification in plants and animals such as mouse and zebrafish. However, this technique was less reported in fish model medaka (Oryzias latipes). Here, we describe an efficient and rapid procedure for genome editing in medaka tyr and generate a stable albino strain. The Cas9 mRNA and gRNA for tyr gene were injected into the embryos of orange-red medaka, and the tyr gene was disrupted in more than 90% of embryos in F0 and F1, which were validated by observation and sequencing of targeted locus. The pigment cells were largely decreased in the mutant medaka because open reading frames of tyr were shifted near the targeted locus, generating albino medaka. Taken together, this method provides a detailed procedure to generate the genetic modification medaka by using an optimized CRISPR/Cas9 system, and the new albino medaka provides an important research platform to study the pigmentation.

Maternal inheritance of Nanog ortholog in blunt-snout bream.

The homeodomain transcription factor Nanog plays an essential role in maintaining pluripotency and self-renewal of embryonic stem cells in mammals. However, the evolutionary conservation of its ortholog in teleosts remains elusive. Here we isolated and characterized a Nanog homolog named as Ma-Nanog in blunt-snout bream (Megalobrama amblycephala). The full-length genomic sequence is 3,326 bp in length and consists of four exons encoding a homeodomain protein of 386 amino acid residues. Comparison of protein sequences revealed that Ma-Nanog is highly homologous to those in other teleosts, particularly in the homeodomain region. During embryogenesis, RNA expression of Nanog was only detected in early developmental embryos, predominantly at the blastula stage, which suggested the transcripts were mainly present in pluripotent stem cells. RNA fluorescence in situ hybridization verified that the signal of the transcripts is present in the germ cells. RNA expression was observed in the oogonia and early stage of oocytes in the ovary, or in the spermatogonia, spermatocytes, and spermatids in the testis. Surprisingly, the transcripts were also detected in adult tissues such as in liver by RT-PCR or qRT-PCR. Subcellular localization of the Nanog protein was also verified in nuclei. Taken together, these results suggested that Ma-Nanog is maternally inherited with conserved features, thus can be potentially used as a marker for stem cells in blunt-snout bream.

Black carp vasa identifies embryonic and gonadal germ cells.

Identification of molecular markers is an essential step in the study of germ cells. Vasa is an RNA helicase and a well-known germ cell marker that plays a crucial role in germ cell development. Here, we identified the Vasa homolog termed Mpvasa as the first germ cell marker in black carp (Mylopharyngodon piceus). First, a 2819-bp full-length Mpvasa complementary DNA (cDNA) was cloned by PCR using degenerated primers of conserved sequences and gene-specific primers. The Mpvasa cDNA sequence encodes a 637-amino acid protein that contains eight conserved characteristic motifs of the DEAD box protein family, and shares high identity to grass carp (81%) and zebrafish (74%) vasa homologs. Second, Mpvasa expression was restricted to the gonad in adulthood by RT-PCR and Western blot analysis. The dynamic patterns of temporal-spatial expression of Mpvasa during gametogenesis were examined by in situ hybridization, and Mpvasa transcripts were strictly detected in gonadal germ cells throughout oogenesis, predominantly in immature oocytes (stage I, II, and III oocytes). Third, Mpvasa transcripts were highly detected in unfertilized eggs and early embryos, and the signal indicated a dynamic migration of the primordial germ cells during embryogenesis, suggesting that Mpvasa transcripts were maternally inherited and specifically distributed in germ cells. Taken together, these results demonstrated that Mpvasa is an applicable molecular marker for identification of gonadal and embryonic germ cells, which facilitates the isolation and utilization of germ cells in black carp.