Mining Salt Tolerance SNP Loci and Prediction of Candidate Genes in the Rice Bud Stage by Genome-Wide Association Analysis.

Mining salt tolerance genes is significant for breeding high-quality salt-tolerant rice varieties in order to improve the utilization of saline-alkaline land. In this study, 173 rice accessions were measured for their germination potential (GP), germination rate (GR), seedling length (SL), root length (RL), germination potential relative to salt damage rate (GPR), germination rate relative to salt damage rate (GRR), seedling length relative to salt damage rate (SLR), relative salt damage rate at the germination stage (RSD) and comprehensive relative salt damage rate in the early seedling stage (CRS) under normal and salt stress conditions. Genome-wide association analysis was performed with 1,322,884 high-quality SNPs obtained by resequencing. Eight quantitative trait loci (QTLs) related to salt tolerance traits at the germination stage were detected in 2020 and 2021. They were related to the GPR (qGPR2) and SLR (qSLR9), which were newly discovered in this study. Three genes were predicted as salt tolerance candidate genes: LOC_Os02g40664, LOC_Os02g40810, and LOC_Os09g28310. At present, marker-assisted selection (MAS) and gene-edited breeding are becoming more widespread. Our discovery of candidate genes provides a reference for research in this field. The elite alleles identified in this study may provide a molecular basis for cultivating salt-tolerant rice varieties.

Specific labeling and identification of bacteria based on concentration-dependent carbon dot staining combined with hyperspectral imaging.

Concentration-dependent carbon dot (CD) fluorescence was developed and utilized alongside hyperspectral microscopy as a specific labeling and identification technique for bacteria. Staining revealed that the CD concentration within cells depended on the characteristic intracellular environment of the species. Therefore, based on the concentration dependence of the CD fluorescence, different bacterial species were specifically labeled. Hyperspectral microscopy captured subtle fluorescence variations to identify bacteria. Method validation using Bacillus subtilis and Bacillus licheniformis succeeded with an identification accuracy of 99%. As a simple, rapid method for labeling and identifying bacterial species in mixtures, this technique has excellent potential for bacterial community studies.

Mapping the emergence of visual consciousness in the human brain via brain-wide intracranial electrophysiology.

Consciousness lies at the heart of our existence and experience. To probe how perceptual consciousness emerges in the brain, we recorded brain-wide intracranial electroencephalography signals from human patients while their perceptual consciousness was effectively manipulated using the continuous flash suppression paradigm. We observed substantial differences in brain activities when visual information gradually enters consciousness. Specifically, the functional connectivity first increases and then decreases, oscillations in the low-frequency band reduce in power, and those in the high-frequency band remain unchanged. We employed random forest-based classification to characterize the transitions from no perception to subconsciousness and then to consciousness, which showed an increase in signal variance at the second transition rather than the first. Further, the frontal-parietal junction dominates the first transition, whereas the temporal-frontal lobes dominate the second transition. Finally, we identified the most relevant neuronal features associated with consciousness. Altogether, these findings shed fresh light on the emergence of visual consciousness.

Fish-specific TLR18 in Nile tilapia (Oreochromis niloticus) recruits MyD88 and TRIF to induce expression of effectors in NF-κB and IFN pathways in melanomacrophages.

Toll-like receptors (TLRs) are evolutionarily conserved proteins of pattern recognition receptors (PRRs) and play a crucial role in innate immune systems recognition of conserved pathogen-related molecular samples (PAMPs). We identified and characterized TLR18 from Nile tilapia (Oreochromis niloticus), OnTLR18, to elucidate its role in tissue expression patterns, modulation of gene expression after microbial challenge and TLR ligands, subcellular localization in fish and human cells, and the possible effectors TLR18 induces in a melanomacrophage-like cell line (tilapia head kidney (THK) cells). OnTLR18 expression was detected in all tissues examined, with the highest levels in the intestine and the lowest in the liver. OnTLR18 transcript was up-regulated in immune-related organs after bacterial and polyinosinic-polycytidylic acid (poly I:C) challenges and in the THK cells after lipopolysaccharide (LPS) stimulation. In transfected THK and human embryonic kidney (HEK) 293 cells, OnTLR18 localizes in the intracellular compartment. OnMyD88 and OnTRIF, but not OnTIRAP, were co-immunoprecipitated with OnTLR18, suggesting that the former two molecules are recruited by OnTLR18 as adaptors. The constitutively active form of OnTLR18 induced the production of pro-inflammatory cytokines, type I interferon (IFN), and antimicrobial peptides such as tumor necrosis factor α, interferon (IFN) d2.13, tilapia piscidin (TP)2, TP3, TP4, and hepcidin in THK cells. Our results suggest that OnTLR18 plays an important role in innate immunity through initiating nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) and IFN signaling pathways via OnMyD88 and OnTRIF and induces the production of various effectors in melanomacrophages.

Downregulation of lncRNA MEG3 is involved in Parkinson's disease.

BACKGROUND: Long non-coding RNA (lncRNA) MEG3 regulates human cancers, while its role in Parkinson's disease (PD) is unknown. The present study explored the involvement of MEG3 in PD. METHODS: This study enrolled PD patients (n = 79) and healthy controls (n = 62) who were admitted to the Second Affiliated Hospital of Nanchang University from May 2016 to March 2018. RT-qPCR was performed to measure the expression of MEG3 and LRRK2. ROC curve analysis was performed for diagnostic analysis. Cell transfections were performed to analyze the interaction between MEG3 and LRRK2. Cell apoptosis and MTT assays were performed to evaluate the effect of cell transfections on cell apoptosis and viability. RESULTS: MEG3 was downregulated in PD patients compared to that in the healthy controls. ROC curve analysis showed altered expression of MEG3 in PD patients. MEG3 was also down-regulated in SH-SY5Y cells treated with MPP + . Overexpression of MEG3 increased the expression levels of leucine-rich repeat kinase 2 (LRRK2) in SH-SY5Y cells. In contrast, overexpression of LRRK2 showed no significant effects on MEG3. Overexpression of MEG3 improved the viability and inhibited the apoptosis of SH-SY5Y cells pretreated with MPP + . CONCLUSIONS: In conclusion, lncRNA MEG3 is downregulated in PD and may affect the expression of LRRK2 to regulate cell viability and apoptosis involved in PD.

Dietary Administration of Novel Multistrain Probiotics from Healthy Grouper Intestines Promotes the Intestinal Immune Response against NNV Infection.

Epinephelus lanceolatus (giant grouper) is a high-value cultured species in the Asia-Pacific region. However, nervous necrosis virus (NNV) is an infectious viral disease that affects over 120 species of marine cultured species and causes high mortality, ranging from 90-100% in the grouper industry. Probiotics isolated from the intestines of healthy individuals have provided insight into novel approaches involved in the defense against viral pathogens. In this study, we isolated three strains of bacteria as candidate probiotics from healthy grouper intestines and a 28-day feeding trial was performed. At day 21, the nervous necrosis virus (NNV) challenge test was conducted for 7 days to evaluate the antiviral effect of candidate probiotics. The results showed that candidate probiotics could improve growth conditions, such as weight gain (WG) and specific growth rate (SGR), and increase the utilization of feed. Furthermore, the candidate probiotic mixture had the ability to protect against NNV, which could decrease the mortality rate by 100% in giant grouper after NNV challenge. Subsequently, we analyzed the mechanism of the candidate probiotic mixture's defense against NNV. A volcano plot revealed 203 (control vs. NNV), 126 (NNV vs. probiotics - NNV), and 5 (control vs. probiotics - NNV) differentially expressed transcripts in intestinal tissue. Moreover, principal components analysis (PCA) and cluster analysis heatmap showed large differences among the three groups. Functional pathway analysis showed that the candidate probiotic mixture could induce the innate and adaptive immunity of the host to defend against virus pathogens. Therefore, we hope that potential candidate probiotics could be successfully applied to the industry to achieve sustainable aquaculture.

Poly(vinyl alcohol-co-itaconic acid) hydrogels grafted with several designed peptides for human pluripotent stem cell culture and differentiation into cardiomyocytes.

We developed poly(vinyl alcohol-co-itaconic acid) (PV) hydrogels grafted with laminin-derived peptides that had different joint segments and several specific designs, including dual chain motifs. PV hydrogels grafted with a peptide derived from laminin-ß4 (PMQKMRGDVFSP) containing a joint segment, dual chain motif and cationic amino acid insertion could attach human pluripotent stem (hPS) cells and promoted high expansion folds in long-term culture (over 10 passages) with low differentiation rates, whereas hPS cells attached poorly on PV hydrogels grafted with laminin-α5 peptides that had joint segments with and without a cationic amino acid or on PV hydrogels grafted with laminin-ß4 peptides containing the joint segment only. The inclusion of a cationic amino acid in the laminin-ß4 peptide was critical for hPS cell attachment on PV hydrogels, which contributed to the zeta potential shifting to higher values (3-4 mV enhancement). The novel peptide segment-grafted PV hydrogels developed in this study supported hPS cell proliferation, which induced better hPS cell expansion than recombinant vitronectin-coated dishes (gold standard of hPS cell culture dishes) in xeno-free culture conditions. After long-term culture on peptide-grafted hydrogels, hPS cells could be induced to differentiate into specific lineages of cells, such as cardiomyocytes, with high efficiency.

The potential of the P2X7 receptor as a therapeutic target in a sub-chronic PCP-induced rodent model of schizophrenia.

OBJECTIVE: We studied the role of the P2X7 receptor on cognitive dysfunction in a mouse model of schizophrenia. METHODS: An adult mouse model was established by treatment with phencyclidine (PCP), an N-methyl-D-aspartate (NMDA) receptor antagonist. Young mice were divided into three groups: 1) the control (saline-injected) group; 2) experimental 5 mg/kg PCP-injected group; and 3) experimental 10 mg/kg PCP-injected group. The mice were subjected to the open-field and Morris water maze tests at 7 weeks. After intraperitoneal injection of the P2X7 receptor antagonist JNJ-47965567, the behaviour tests were performed again. Samples were taken after testing. The P2X7 receptor protein and mRNA expression levels were detected by immunohistochemistry, Western blotting and PCR. RESULTS: This study revealed that the infant sub-chronic PCP mice model showed severe spatial learning and memory impairment in the Morris water maze and schizophrenia-like symptoms (hypermotor behaviour) in the open-field test. The P2X7 receptor protein was highly expressed in the sub-chronic PCP mouse model and more highly expressed in the hippocampus than the prefrontal lobe. After the P2X7 receptor was blocked with JNJ-47965567, P2X7 receptor protein and mRNA expression in the frontal lobe were significantly increased, and the spatial memory impairment and hypermotor behaviour induced by PCP were reversed. CONCLUSION: PCP-induced cognitive impairment can be significantly improved by antagonizing the P2X7 receptor. Therefore, we believe that the P2X7 receptor plays an important role in the cognition of schizophrenic-like mice.

The Alteration of Intestinal Microbiota Profile and Immune Response in Epinephelus coioides during Pathogen Infection.

Epinephelus coioides, or grouper, is a high economic value fish species that plays an important role in the aquaculture industry in Asia. However, both viral and bacterial diseases have threatened grouper for many years, especially nervous necrosis virus, grouper iridovirus and Vibrio harveyi, which have caused a bottleneck in the grouper industry. Currently, intestinal microbiota can provide novel insights into the pathogenesis-related factors involved in pathogen infection. Hence, we investigated the comparison of intestinal microbiota communities in control group and pathogen-infected grouper through high-throughput sequencing of the 16S rRNA gene. Our results showed that microbial diversity was decreased, whereas microbial richness was increased during pathogen infection. The individuals in each group were distributed distinctly on the PLSDA diagram, especially the GIV group. Proteobacteria and Firmicutes were the most abundant bacterial phyla in all groups. Interestingly, beneficial genera, Faecalibacterium and Bifidobacterium, predominated in the intestines of the control group. In contrast, the intestines of pathogen-infected grouper had higher levels of harmful genera such as Sphingomonas, Atopostipes, Staphylococcus and Acinetobacter. Additionally, we investigated the expression levels of innate and adaptive immune-related genes after viral and bacterial infection. The results revealed that immunoglobulin T and proinflammatory cytokine levels in the intestine increased after pathogen infection. Through these unique bacterial compositions in diseased and uninfected fish, we could establish a novel therapeutic approach and bacterial marker for preventing and controlling these diseases.

Screening for the Proteins That Can Interact with Grouper Nervous Necrosis Virus Capsid Protein.

Nervous necrosis virus (NNV) can infect many species of fish and has an 80-100% mortality rate. NNV capsid protein (NNVCP) is the only structural protein of NNV, but there are few studies on the protein-protein interaction between NNVCP and the host cell. To investigate NNV morphogenesis, native NNV capsid protein (NNVCP) was used to screen for protein-protein interactions in this study. The results identified that 49 grouper optic nerve proteins can interact with NNVCP and may function as putative receptor or co-receptor, cytoskeleton, glucose metabolism and ATP generation, immunity, mitochondrial ion regulation, and ribosomal proteins. Creatine kinase B-type (CKB) is one of those 49 optic nerve proteins. CKB, a kind of enzyme of ATP generation, was confirmed to interact with NNVCP by far-Western blot and showed to colocalize with NNVCP in GF-1 cells. Compared to the control, the expression of CKB was significantly induced in the brain and eyes infected with NNV. Moreover, the amount of replication of NNV is relatively high in cells expressing CKB. In addition to providing the database of proteins that can interact with NNVCP for subsequent analysis, the results of this research also verified that CKB plays an important role in the morphogenesis of NNV.

Blast2Fish: a reference-based annotation web tool for transcriptome analysis of non-model teleost fish.

BACKGROUND: Transcriptome analysis by next-generation sequencing has become a popular technique in recent years. This approach is quite suitable for non-model organism study, as de novo assembly is independent of prior genomic sequences of organisms. De novo sequencing has benefited many studies on commercially important fish species. However, to understand the functions of these assembled sequences, they still need to be annotated with existing sequence databases. By combining Basic Local Alignment Search Tool (BLAST) and Gene Ontology analysis, we were able to identify homologous sequences of assembled sequences and describe their characteristics using pre-defined tags for each gene, though the above conventional annotation results obtained for non-model assembled sequences was still associated with a lack of pre-defined tags and poorly documented records in the database. RESULTS: We introduced Blast2Fish, a novel approach for performing functional enrichment analysis on non-model teleost fish transcriptome data. The Blast2Fish pipeline was designed to be a reference-based enrichment method. Instead of annotating the BLAST single top hit by a pre-defined gene-to-tag database, we included 500 hits to search related PubMed articles and parse biological terms. These descriptive terms were then sorted and recorded as annotations for the query. The results showed that Blast2Fish was capable of providing meaningful annotations on immunology topics for non-model fish transcriptome analysis. CONCLUSION: Blast2Fish provides a novel approach for annotating sequences of non-model fish. The reference-based strategy allows annotation to be performed without pre-defined tags for each gene. This method strongly benefits non-model teleost fish studies for gene functional enrichment analysis.

Secretory Production of Functional Grouper Type I Interferon from Epinephelus septemfasciatus in Escherichia coli and Bacillus subtilis.

Nervous necrosis virus (NNV) results in high mortality rates of infected marine fish worldwide. Interferons (IFNs) are cytokines in vertebrates that suppress viral replication and regulate immune responses. Heterologous overexpression of fish IFN in bacteria could be problematic because of protein solubility and loss of function due to protein misfolding. In this study, a protein model of the IFN-α of Epinephelus septemfasciatus was built based on comparative modeling. In addition, PelB and SacB signal peptides were fused to the N-terminus of E. septemfasciatus IFN-α for overexpression of soluble, secreted IFN in Escherichia coli (E-IFN) and Bacillus subtilis (B-IFN). Cytotoxicity tests indicated that neither recombinant grouper IFN-α were cytotoxic to a grouper head kidney cell line (GK). The GK cells stimulated with E-IFN and B-IFN exhibited elevated expression of antiviral Mx genes when compared with the control group. The NNV challenge experiments demonstrated that GK cells pretreated or co-treated with E-IFN and B-IFN individually had three times the cell survival rates of untreated cells, indicating the cytoprotective ability of our recombinant IFNs. These data provide a protocol for the production of soluble, secreted, and functional grouper IFN of high purity, which may be applied to aquaculture fisheries for antiviral infection.

Omega-3 polyunsaturated fatty acids suppress metastatic features of human cholangiocarcinoma cells by suppressing twist.

Cholangiocarcinoma (CCA) is a highly malignant cancer of the bile duct, which has a five-year survival rate less than 5% due to a high metastasis rate and lack of therapeutic options. Although omega-3 polyunsaturated fatty acids (n-3 PUFAs) have been shown to inhibit the proliferation of CCA cells, the effects on CCA metastasis have not been previously reported. In this study, we first assessed the proliferation, migration and invasion effects of n-3 PUFA-based fish oil on human CCA cells. Then, we investigated PUFA effects on metastasis in vivo by xenografting CCA cells into zebrafish larvae that overexpress a critical n-3 PUFA synthesis gene, Δ6 fatty acid desaturase. The results indicated that n-3 PUFA-based fish oil suppresses CCA cell growth, potentially by blocking the cell cycle at G2/M phase, and it inhibits migration and invasion potential with coincident downregulation of migration-related genes. Furthermore, zebrafish endogenous n-3 PUFAs appear to suppress CCA metastasis by inhibiting the expression of twist, a key regulator of tumor metastasis. Interestingly, only long chain n-3 PUFAs could inhibit the expression of twist in CCA cells. Together, our results suggest that n-3 PUFAs, especially DHA, may inhibit proliferation and metastasis of CCA cells by inhibiting the expression of twist.

The design of a thermoresponsive surface for the continuous culture of human pluripotent stem cells.

Commonly, stem cell culture is based on batch-type culture, which is laborious and expensive. We continuously cultured human pluripotent stem cells (hPSCs) on thermoresponsive dish surfaces, where hPSCs were partially detached on the same thermoresponsive dish by decreasing the temperature of the thermoresponsive dish to be below the lower critical solution temperature for only 30 min. Then, the remaining cells were continuously cultured in fresh culture medium, and the detached stem cells were harvested in the exchanged culture medium. hPSCs were continuously cultured for ten cycles on the thermoresponsive dish surface, which was prepared by coating the surface with poly(N-isopropylacrylamide-co-styrene) and oligovitronectin-grafted poly(acrylic acid-co-styrene) or recombinant vitronectin for hPSC binding sites to maintain hPSC pluripotency. After ten cycles of continuous culture on the thermoresponsive dish surface, the detached cells expressed pluripotency proteins and had the ability to differentiate into cells derived from the three germ layers in vitro and in vivo. Furthermore, the detached cells differentiated into specific cell lineages, such as cardiomyocytes, with high efficiency.

The microbiota profile and transcriptome analysis of immune response during metamorphosis stages in orange spotted grouper (Epinephelus coioides).

Metamorphosis is a transformation process in larval development associated with changes in morphological and physiological features, including the immune system. The gastrointestinal tract harbors a plethora of bacteria, which might affect the digestion and absorption of nutrients, immunity, and gut-brain crosstalk in the host. In this study, we have performed metagenomic and transcriptomic analyses on the intestines of grouper at the pre-, mid- and post-metamorphosis stages. The sequencing data of 16S rRNA gene showed drastic changes in the microbial communities at different developmental stages. The transcriptomic data revealed that the leukocyte transendothelial migration and the phagosome pathways might play important roles in mediating immunity in grouper at the three developmental stages. This information will increase our understanding of the metamorphosis process in grouper larvae, and shed light on the development of antimicrobial strategy during larval development.

Overexpression of Anti-müllerian Hormone Gene in vivo Affects Gonad Sex Differentiation in Undifferentiated Orange-Spotted Groupers (Epinephelus coioides).

Sex differentiation in teleost fishes occurs in response to sex determination signals, which induce the gonad to develop as either an ovary or testis. However, sex differentiation mechanisms in fishes are diverse, and information on gonad differentiation in sex changing fishes remains limited. The orange-spotted grouper (Epinephelus coioides) is a protogynous hermaphroditic fish that provides an ideal model for investigating gonad differentiation in vertebrates. In this study, Transcriptome data showed that expression levels of amh and amhrII in gonads were increased during sex differentiation. Then we investigated the effect of overexpression anti-Müllerian hormone (Amh) on gonad development in juvenile orange-spotted groupers. Expression levels of female-related genes and serum 17ß-estradiol levels were decreased, while expression of male-related genes and serum 11-ketotestosterone levels were increased in fish fed with amh-plasmid. Overexpression of Amh was also promoted the spermatogonia proliferation and induced the development of male gonads in undifferentiated orange-spotted groupers, but that this male tendency was preceded by female differentiation. In summary, these results illustrated that Amh overexpression by amh-plasmid feeding induced male gonad development in undifferentiated groupers.

Female-to-male sex reversal in orange-spotted grouper (Epinephelus coioides) caused by overexpressing of Amh in vivo.

A variety of mechanisms are involved in sex determination in vertebrates. The orange-spotted grouper (Epinephelus coioides), a teleost fish, functions first as females and later as a male and is an ideal model to investigate the regulation of sexual fate. Here, we report female-to-male sex reversal in juvenile orange-spotted groupers caused by overexpressing anti-Müllerian hormone (Amh). Tissue distribution analyses showed that amh and amhrII primarily expressed in the gonad, and expression level in the testis was much higher than that in the ovary. In gonads, the expression of amh was located in the Sertoli cells around spermatogonia of the testis and in the zona pellucida of the mature ovary, and the expression of amhrII was located in the Sertoli cells of the testis and in the oocytes of the ovary. Decrease in female-related genes and serum 17ß-estradiol level, increase in male-related genes and serum 11-ketotestosterone, ovarian regression, and spermatogonia proliferation were observed during plasmid feeding experiment. These results illustrate that amh overexpression plasmid feeding can induce a female-to-male transition in grouper.

A transcriptomic investigation of digestive processes in orange-spotted grouper, Epinephelus coioides, before, during, and after metamorphic development.

The orange-spotted grouper (OG), Epinephelus coioides, is an ecologically and economically important species with strong market demand. However, larval rearing for this species is especially difficult, with mass mortality occurring at multiple stages including the period coinciding with metamorphic development. The aim of the present study was to characterise the molecular ontogenesis of genes that influence appetite, feeding, and digestion in OG larvae head and body tissue at 12, 18, and 50 days post hatch (dph), which coincides with the beginning and end of metamorphic development. The sequences of many transcripts involved in the regulation of appetite, feeding and digestive processes were detected from 12 dph in OG larvae, including those that were differentially expressed in body tissue in fish at different stages of development such as cholecystokinin, peptide Y, and meprin A. Of the transcripts encoding digestive enzymes, only the expression level of bile salt-activated lipase decreased as development progressed. In contrast, a dramatic increase in expression for other body-expressed transcripts encoding digestive enzymes and a proton pump subunit was observed at 50 dph, which is indicative of an increase in digestive capacity. In addition, we have provided evidence suggesting that various trypsinogen isoforms are present, and have differing expression patterns throughout larval development in whole body tissue. We also report on the presence of a prey-specific transcript encoding α-amylase that was present in the body-transcriptome. Taken together, these results give insight into the processes underpinning attainment of digestive capacity, and form the basis of a new transcriptomic database that will aid further study into the digestive development and dietary requirements of orange-spotted grouper larvae.

Transcriptome profiling analysis of grouper during nervous necrosis virus persistent infection.

Nervous necrosis virus (NNV) infection has been considered a serious disease in farmed grouper. Particularly, the persistent infection model conducts the grouper into a carrier state that continues to spread the virus through spawning. This particular model makes disease control more difficult in the aquaculture industry. In the present study, we used RNA-Seq, a high-throughput method based on next-generation sequencing, to profile the expression of genes during the period of NNV persistent infection. We evaluated the transcriptomic changes in the brain tissue of grouper. The inactivated-NNV vaccine was used as a comparison group. Based on the differentially expressed genes, highly immune cell active signaling and surface receptor expression were triggered during persistent infection. The interferon-induced response was also highly expressed in the infected brain tissue. However, critical negative regulatory factors of T-cells, such as PD-L1 and LAG3, were up-regulated. The present transcriptome study revealed a comprehensive view of the state of NNV persistent infection and provided insights into the state of impaired NNV clearance in the grouper.