Evolutionary and immune-activating character analyses of NLR genes in algae suggest the ancient origin of plant intracellular immune receptors.

Nucleotide-binding leucine-rich repeat (NLR) proteins are crucial intracellular immune receptors in plants, responsible for detecting invading pathogens and initiating defense responses. While previous studies on the evolution and function of NLR genes were mainly limited to land plants, the evolutionary trajectory and immune-activating character of NLR genes in algae remain less explored. In this study, genome-wide NLR gene analysis was conducted on 44 chlorophyte species across seven classes and seven charophyte species across five classes. A few but variable number of NLR genes, ranging from one to 20, were identified in five chlorophytes and three charophytes, whereas no NLR gene was identified from the remaining algal genomes. Compared with land plants, algal genomes possess fewer or usually no NLR genes, implying that the expansion of NLR genes in land plants can be attributed to their adaptation to the more complex terrestrial pathogen environments. Through phylogenetic analysis, domain composition analysis, and conserved motifs profiling of the NBS domain, we detected shared and lineage-specific features between NLR genes in algae and land plants, supporting the common origin and continuous evolution of green plant NLR genes. Immune-activation assays revealed that both TNL and RNL proteins from green algae can elicit hypersensitive responses in Nicotiana benthamiana, indicating the molecular basis for immune activation has emerged in the early evolutionary stage of different types of NLR proteins. In summary, the results from this study suggest that NLR proteins may have taken a role as intracellular immune receptors in the common ancestor of green plants.

Structural and functional analysis of transforming growth factor beta regulator 1 (TBRG1) in the red swamp crayfish Procambarus clarkii: The initial insight into TBRG1's role in invertebrate immunity.

The transforming growth factor beta regulator 1 (TBRG1) is a growth inhibitory protein that acts as a tumor suppressor in human cancers, gaining its name for the transcriptional regulation by TGF-ß. While extensive research has been conducted on the tumor-related function of TBRG1 in mammals, its significance in invertebrates remains largely unexplored. In this study, a homolog of TBRG1 was first structurally and functionally analyzed in the red swamp crayfish Procambarus clarkii. The full-length cDNA sequence was 2143 base pairs (bp) with a 1305 bp open reading frame (ORF) encoding a deduced protein of 434 amino acids (aa). The changes of PcTBRG1 transcripts upon immune challenges indicated its involvement in innate immunity. After knocking down PcTBRG1, the decline of bacteria clearance capacity revealed the participation of PcTBRG1 in the immune response. Furthermore, the downregulation of AMPs' expression after the cotreatment of RNAi and bacteria challenge suggested that PcTBRG1 might participate in innate immunity through regulating AMPs' expression. These results provided initial insight into the immune-related function of TBRG1 in invertebrates.

Unraveling the Molecular Basis of Color Variation in Dioscorea alata Tubers: Integrated Transcriptome and Metabolomics Analysis.

Dioscorea alata L. (Dioscoreaceae) is a widely cultivated tuber crop with variations in tuber color, offering potential value as health-promoting foods. This study focused on the comparison of D. alata tubers possessing two distinct colors, white and purple, to explore the underlying mechanisms of color variation. Flavonoids, a group of polyphenols known to influence plant color and exhibit antioxidant properties, were of particular interest. The total phenol and total flavonoid analyses revealed that purple tubers (PTs) have a significantly higher content of these metabolites than white tubers (WTs) and a higher antioxidant activity than WTs, suggesting potential health benefits of PT D. alata. The transcriptome analysis identified 108 differentially expressed genes associated with the flavonoid synthesis pathway, with 57 genes up-regulated in PTs, including CHS, CHI, DFR, FLS, F3H, F3'5'H, LAR, ANS, and ANR. The metabolomics analysis demonstrated that 424 metabolites, including 104 flavonoids and 8 tannins, accumulated differentially in PTs and WTs. Notably, five of the top ten up-regulated metabolites were flavonoids, including 6-hydroxykaempferol-7-O-glucoside, pinocembrin-7-O-(6″-O-malonyl)glucoside, 6-hydroxykaempferol-3,7,6-O-triglycoside, 6-hydroxykaempferol-7-O-triglycoside, and cyanidin-3-O-(6″-O-feruloyl)sophoroside-5-O-glucoside, with the latter being a precursor to anthocyanin synthesis. Integrating transcriptome and metabolomics data revealed that the 57 genes regulated 20 metabolites within the flavonoid synthesis pathway, potentially influencing the tubers' color variation. The high polyphenol content and antioxidant activity of PTs indicate their suitability as nutritious and health-promoting food sources. Taken together, the findings of this study provide insights into the molecular basis of tuber color variation in D. alata and underscore the potential applications of purple tubers in the food industry and human health promotion. The findings contribute to the understanding of flavonoid biosynthesis and pigment accumulation in D. alata tubers, opening avenues for future research on enhancing the nutritional quality of D. alata cultivars.

[Cloning and functional analysis of flavanone synthase â ¡ from Dendrobium huoshanense].

Flavonoid C-glycosides are a class of natural products that are widely involved in plant defense responses and have diverse pharmacological activities. They are also important active ingredients of Dendrobium huoshanense. Flavanone synthase Ⅱ has been proven to be a key enzyme in the synthesis pathway of flavonoid C-glycosides in plants, and their catalytic product 2-hydroxyflavanone is the precursor compound for the synthesis of various reported flavonoid C-glycosides. In this study, based on the reported amino acid sequence of flavanone synthase Ⅱ, a flavanone synthase Ⅱ gene(DhuFNSⅡ) was screened and verified from the constructed D. huoshanense genome localization database. Functional validation of the enzyme showed that it could in vitro catalyze naringenin and pinocembrin to produce apigenin and chrysin, respectively. The open reading frame(ORF) of DhuFNSⅡ was 1 644 bp in length, encoding 547 amino acids. Subcellular localization showed that the protein was localized on the endoplasmic reticulum. RT-qPCR results showed that DhuFNSⅡ had the highest expression in stems, followed by leaves and roots. The expression levels of DhuFNSⅡ and other target genes in various tissues of D. huoshanense were significantly up-regulated after four kinds of abiotic stresses commonly encountered in the growth process, but the extent of up-regulation varied among treatment groups, with drought and cold stress having more significant effects on gene expression levels. Through the identification and functional analysis of DhuFNSⅡ, this study is expected to contribute to the elucidation of the molecular mechanism of the formation of quality metabolites of D. huoshanense, flavonoid C-glycosides, and provide a reference for its quality formation and scientific cultivation.

Boris knockout eliminates AOM/DSS-induced in situ colorectal cancer by suppressing DNA damage repair and inflammation.

The Brother of Regulator of Imprinted Sites (BORIS, gene symbol CTCFL) has previously been shown to promote colorectal cancer cell proliferation, inhibit cancer cell apoptosis, and resist chemotherapy. However, it is unknown whether Boris plays a role in the progression of in situ colorectal cancer. Here Boris knockout (KO) mice were constructed. The function loss of the cloned Boris mutation that was retained in KO mice was verified by testing its activities in colorectal cell lines compared with the Boris wild-type gene. Boris knockout reduced the incidence and severity of azoxymethane/dextran sulfate-sodium (AOM/DSS)-induced colon cancer. The importance of Boris is emphasized in the progression of in situ colorectal cancer. Boris knockout significantly promoted the phosphorylation of γH2AX and the DNA damage in colorectal cancer tissues and suppressed Wnt and MAPK pathways that are responsible for the callback of DNA damage repair. This indicates the strong inhibition of colorectal cancer in Boris KO mice. By considering that the DSS-promoted inflammation contributes to tumorigenesis, Boris KO mice were also studied in DSS-induced colitis. Our data showed that Boris knockout alleviated DSS-induced colitis and that Boris knockdown inhibited the NF-κB signaling pathway in RAW264.7 cells. Therefore Boris knockout eliminates colorectal cancer generation by inhibiting DNA damage repair in cancer cells and relieving inflammation in macrophages. Our findings demonstrate the importance of Boris in the development of in situ colorectal cancer and provide evidence for the feasibility of colorectal cancer therapy on Boris.

Identification and functional analysis of endoplasmic reticulum oxidoreductase 1 (ERO1) from the green mud crab Scylla paramamosain: The first evidence of ERO1 involved in invertebrate immune response.

Endoplasmic reticulum oxidoreductase 1 (ERO1) is an important mediator in regulating disulfide bond formation and maintaining endoplasmic reticulum homeostasis. Its activity is transcriptionally regulated by the unfolded protein response (UPR) in the endoplasmic reticulum, which is known to be essential in immunity. However, whether ERO1 is involved in innate immunity in invertebrates remains unclear. In the present study, two subtypes of ERO1 from Scylla paramamosain were first identified and characterized. Sequence analysis revealed the conserved ERO1 domain and the oxidative capacity assay verified the oxidative capacity of SpERO1 recombinant protein. Moreover, SpERO1s were found to be ubiquitously expressed in all the tested tissues, with the highest expression observed in hemocytes. Two SpERO1s exhibited distinct expression patterns in response to Vibrio alginolyticus and White Spot Syndrome Virus (WSSV). Importantly, the downregulation of the expression of immune factors upon bacterial challenge in SpERO1-silenced crabs was observed. These results provided an initial foundation for further investigations into the role of ERO1 in the innate immunity of invertebrates.

Identification and functional analysis of cytosolic phospholipase A2 (cPLA2) from the red swamp crayfish Procambarus clarkii: The first evidence of cPLA2 involved in immunity in invertebrates.

Cytosolic phospholipase A2 (cPLA2) specifically liberates the arachidonic acids from the phospholipid substrates. In mammals, cPLA2 serves as a key control point in inflammatory responses due to its diverse downstream products. However, the role of cPLA2 in animals lower than mammals largely remains unknown. In the current research, a homolog of cPLA2 was first identified and characterized in the red swamp crayfish Procambarus clarkii. The full-length cDNA of PccPLA2 was 4432 bp in length with a 3036 bp-long open reading frame, encoding a putative protein of 1011 amino acids that contained a protein kinase C conserved region 2 and a catalytic subunit of cPLA2. PccPLA2 was ubiquitously expressed in all examined tissues with the highest expression in the hepatopancreas, and the expression in hemocytes as well as hepatopancreas was induced upon the immune challenges of WSSV and Aeromonas hydrophila. After the co-treatment of RNA interference and bacterial infection, the decline of bacteria clearance capability was observed in the hemolymph, and the expression of some antimicrobial peptides (AMPs) was significantly suppressed. Additionally, the phagocytosis of A. hydrophila by primary hemocytes decreased when treated with the specific inhibitor CAY10650 of cPLA2. These results indicated the participation of PccPLA2 in both cellular and humoral immune responses in the crayfish, which provided an insight into the role that cPLA2 played in the innate immunity of crustaceans, and even in invertebrates.

The first identification and functional analysis of two drosophila mothers against decapentaplegic protein genes (SpSmad1 and SpSmad2/3) and their involvement in the innate immune response in Scylla paramamosain.

Smad，a member of the TGF-ß superfamily，controls cell proliferation，growth and guiding cell differentiation, thus playing a crucial role in diseases. However, the presence as well as specific function of Smad in crabs is still unknown. In this study, two Smads (Smad1 and Smad2/3) were identified for the first time from the mud crab Scylla paramamosain. The complete open reading frames of SpSmad1 and SpSmad2/3 were 1,497bp and 1,338bp, encoding deduced proteins of 498 and 445 amino acids respectively. Moreover, under the administration of Vibrio alginolyticus and WSSV, the relative expression levels of SpSmad1 and SpSmad2/3 were significantly increased, indicating their involvement in the innate immune response of mud crabs. Knockdown of SpSmad1 and SpSmad2/3 in vivo not only led to the increasement of the expressions of NF-κB signaling genes and antimicrobial peptides genes, but also significantly affected the bacterial clearance process of mud crabs. Additionally, overexpression of SpSmad1 and SpSmad2/3 in HEK293T cells could markedly activate NF-κB signaling. These results indicated that Smad1 and Smad2/3 participated in the innate immunity of Scylla paramamosain, and might provide a better understanding of the presence and immune regulatory functions of Smad1 and Smad2/3 in crabs and even invertebrates.

Characterization of fibroblast growth factor receptor 4 (FGFR4) from the red swamp crayfish Procambarus clarkii and its role in antiviral and antimicrobial immune responses.

FGFRs involved multiple physiological processes, such as endocrine homeostasis, wound repair, and cellular behaviors including proliferation, differentiation and survival. In the present study, the homologs of fibroblast growth factor receptor 4 (FGFR4) were identified and characterized from the red swamp crayfish Procambarus clarkii for the first time. The full-length cDNAs of pcFGFR4 were 2878 bp with 2451 bp open reading frame (ORF), respectively. The deduced pcFGFR4 protein contained an immunoglobulin, two immunoglobulin C-2 Type, a transmembrane region and a catalytic domain. Real-time PCR analysis showed that pcFGFR4 were highly expressed in muscle and hemocyte. Moreover, the expression levels of pcFGFR4 in the hepatopancreas and hemocyte were positively stimulated after challenge with Aeromonas hydrophila and WSSV, implying the involvement of pcFGFR4 against bacterial and viral infections in innate immune responses. While pcFGFR4 were silenced in vivo, the expression levels of antimicrobial peptide (AMP) genes (pcALF1-5,8 and pcCrustin1-2) and NF-κB signaling components (pcDrosal and pcRelish) were significantly reduced. Additionally, NF-κB signaling could be markedly activated by overexpression of pcFGFR4 in HEK293T cells. Finally, our results indicated that pcFGFR4 regulated crayfish's innate immunity by modulating NF-κB signaling. These findings may provide new insights into pcFGFR4-mediated signaling cascades in crustaceans and provide a better understanding of crustacean innate immune system.

Genome Sequence Resource for Colletotrichum gloeosporioides, an Important Pathogenic Fungus Causing Anthracnose of Dioscorea alata.

Anthracnose disease is one of the most important diseases of Dioscorea alata and many other food yams, which is caused by Colletotrichum gloeosporioides fungus from the Glomerellaceae family of the Sordariomycetes class. In the present study, a C. gloeosporioides starin named CgDa01 was isolated from D. alata, and its genome was sequenced based on Oxford Nanopore technology (ONT) and the Illumina sequencing platform. The high-quality genome of CgDa01 was assembled with a 62.78 Mb genome size and 15,845 predicted protein-coding genes. The proteins of predicted genes were annotated using multiple public databases, including the nonredundant protein database, the InterProScan databases, and Kyoto Encyclopedia of Genes and Genomes. Among the annotated protein-coding genes, 55 were predicted as potential virulence genes by the fungal virulence factor database. The C. gloeosporioides CgDa01 genome assembly described in this study can serve as a resource for better understanding the pathogenic mechanism of C. gloeosporioides on yam hosts.

Pan-Plastome of Greater Yam (Dioscorea alata) in China: Intraspecific Genetic Variation, Comparative Genomics, and Phylogenetic Analyses.

Dioscorea alata L. (Dioscoreaceae), commonly known as greater yam, water yam, or winged yam, is a popular tuber vegetable/food crop worldwide, with nutritional, health, and economical importance. China is an important domestication center of D. alata, and hundreds of cultivars (accessions) have been established. However, genetic variations among Chinese accessions remain ambiguous, and genomic resources currently available for the molecular breeding of this species in China are very scarce. In this study, we generated the first pan-plastome of D. alata, based on 44 Chinese accessions and 8 African accessions, and investigated the genetic variations, plastome evolution, and phylogenetic relationships within D. alata and among members of the section Enantiophyllum. The D. alata pan-plastome encoded 113 unique genes and ranged in size from 153,114 to 153,161 bp. A total of four whole-plastome haplotypes (Haps I-IV) were identified in the Chinese accessions, showing no geographical differentiation, while all eight African accessions shared the same whole-plastome haplotype (Hap I). Comparative genomic analyses revealed that all four whole plastome haplotypes harbored identical GC content, gene content, gene order, and IR/SC boundary structures, which were also highly congruent with other species of Enantiophyllum. In addition, four highly divergent regions, i.e., trnC-petN, trnL-rpl32, ndhD-ccsA, and exon 3 of clpP, were identified as potential DNA barcodes. Phylogenetic analyses clearly separated all the D. alata accessions into four distinct clades corresponding to the four haplotypes, and strongly supported that D. alata was more closely related to D. brevipetiolata and D. glabra than D. cirrhosa, D. japonica, and D. polystachya. Overall, these results not only revealed the genetic variations among Chinese D. alata accessions, but also provided the necessary groundwork for molecular-assisted breeding and industrial utilization of this species.

Identification and functional analysis of two drosophila mothers against decapentaplegic protein(Smad)genes and their involvement in immune responses in the red swamp crayfish Procambarus clarkii.

Drosophila mothers against decapentaplegic proteins (Smads), the crucial signal transducers in activating downstream gene transcription through transforming growth factor beta (TGF-ß) receptors, are the pleiotropic factors with important role in mediating cell proliferation, homeostasis, differentiation, apoptosis and immune response. However, whether Smads are involved in immune response in crustaceans remains unexplored. In the present study, the Smad3 and Smad4 were firstly identified and functionally characterized from the Red Swamp Crayfish Procambarus clarkii. The full-length cDNAs of pcSmad3 and pcSmad4 were 1, 670 bp and 3, 060 bp with 1, 326 bp and 1, 875 bp open reading frame (ORF), respectively. Real-time PCR analysis of the expression profiles demonstrated that pcSmad3 and pcSmad4 were predominantly expressed at in stomach, heart, and hemocytes. Notably, the expression levels of pcSmad3 and pcSmad4 both Aeromonas hydrophila and WSSV challenges were significantly altered, suggesting the involvement of pcSmad3 and pcSmad4 in innate immune responses. Knockdown of pcSmad3 and pcSmad4 in vivo dramatically activated the transcriptions of NF-κB signaling genes and anti-lipopolysaccharide factor genes. The overexpression of pcSmad3 and pcSmad4 could significantly activate NF-κB signaling in HEK293T cells. Meanwhile, the clearance of bacteria was significantly reduced with knockdown of pcSmad3 and pcSmad4 in vivo. Results indicated that pcSmad3 and pcSmad4 played an immune-regulatory role in crayfish's innate immunity, which might pave the for a better understanding of the TGF-ß superfamily members in crustacean.

Identification and function analysis of two fibroblast growth factor receptor (FGFR) from Scylla paramamosain: The evidence of FGFR involved in innate immunity in crustacean.

The fibroblast growth factor receptor (FGFR) belongs to the tyrosine kinase family consisting of four members (FGFR1-4). This study involved identification and characterization of FGFR1 and FGFR3 from mud crab Scylla paramamosain for the first time. The obtained cDNAs of SpFGFR1 and SpFGFR3 were 2,380 bp and 2,982 bp in length with a 1,503 bp and 2,310 bp open reading frame, respectively. The predicted SpFGFR1 protein included three immunoglobulin domains and a transmembrane region, while SpFGFR3 protein possessed a typical TyrKc (Tyrosine kinase, catalytic) domain. Real-time PCR analysis showed that SpFGFR1 and SpFGFR3 were highly expressed in the hepatopancreas. Furthermore, the expression levels of SpFGFR1 and SpFGFR3 in the hepatopancreas were enhanced following challenges with Vibro alginolyticus, Staphylococcus aureus, Poly (I:C) and White spot syndrome virus, which shows the involvement of SpFGFR1 and SpFGFR3 in innate immune response to infections from bacteria and virus. There was significant suppression of six antimicrobial peptide genes (SpALF1-5 and SpCrustin) and three NF-κB members (SpDorsal, SpIKK and SpRelish) when SpFGFR1 and SpFGFR3 was interfered in vivo. Also, treatment of the hemocytes with specific inhibitor of SpFGFR for 24 h consistently down-regulated SpDorsal, SpRelish and AMPs. These results suggested that SpFGFR1 and SpFGFR3 played important roles in regulating the Toll signaling pathway and immune deficiency (IMD) pathway through NF-κB signaling pathway. These findings may provide new insights into the role of FGFRs in the innate immune function of crustaceans.

Identification and functional analysis of drosophila mothers against decapentaplegic protein gene 1 (Smad1) from the red swamp crayfish Procambarus clarkii: The first evidence of Smad1 involved in immunity in invertebrates.

Smads, part of signaling cascades that represent downstream pathways of the TGF-ß super family proteins, are pleiotropic cytokines with important role in mediating cell proliferation, homeostasis, differentiation, apoptosis and immune response. However, the specific functions of Smads remain unknown in crustaceans. In the present study, the drosophila mothers against decapentaplegic protein gene 1 (Smad1) was firstly identified and characterized from the Red Swamp Crayfish Procambarus clarkii. The obtained cDNA sequence of pcSmad1was 2, 503 bp long with a 1, 488 bp open reading fame, which encoded a putative protein of 496 amino acids. Furthermore, pcSmad1 responded to both Aeromonas hydrophila and WSSV challenge, suggesting the involvement of pcSmad1 in innate immune responses. Knockdown of pcSmad1 in vivo dramatically increased the expressions of NF-κB signaling genes and anti-lipopolysaccharide factor genes. Additionally, overexpression of pcSmad1 in HEK293T cells could markedly activate NF-κB signaling. Taken together, these results indicated that pcSmad1 played an immune-regulatory role in crayfish's innate immunity, which may provide a better understanding of TGF-ß superfamily members in crustacean.

N-n-Butyl haloperidol iodide ameliorates liver fibrosis and hepatic stellate cell activation in mice.

N-n-Butyl haloperidol iodide (F2) is a novel compound that has antiproliferative and antifibrogenic activities. In this study we investigated the therapeutic potential of F2 against liver fibrosis in mice and the underlying mechanisms. Two widely used mouse models of fibrosis was established in mice by injection of either carbon tetrachloride (CCl4) or thioacetamide (TAA). The mice received F2 (0.75, 1.5 or 3 mg·kg-1·d-1, ip) for 4 weeks of fibrosis induction. We showed that F2 administration dose-dependently ameliorated CCl4- or TAA-induced liver fibrosis, evidenced by significant decreases in collagen deposition and c-Jun, TGF-ß receptor II (TGFBR2), α-smooth muscle actin (α-SMA), and collagen I expression in the liver. In transforming growth factor beta 1 (TGF-ß1)-stimulated LX-2 cells (a human hepatic stellate cell line) and primary mouse hepatic stellate cells, treatment with F2 (0.1, 1, 10 µM) concentration-dependently inhibited the expression of α-SMA, and collagen I. In LX-2 cells, F2 inhibited TGF-ß/Smad signaling through reducing the levels of TGFBR2; pretreatment with LY2109761 (TGF-ß signaling inhibitor) or SP600125 (c-Jun signaling inhibitor) markedly inhibited TGF-ß1-induced induction of α-SMA and collagen I. Knockdown of c-Jun decreased TGF-ß signaling genes, including TGFBR2 levels. We revealed that c-Jun was bound to the TGFBR2 promoter, whereas F2 suppressed the binding of c-Jun to the TGFBR2 promoter to restrain TGF-ß signaling and inhibit α-SMA and collagen I upregulation. In conclusion, the therapeutic benefit of F2 against liver fibrosis results from inhibition of c-Jun expression to reduce TGFBR2 and concomitant reduction of the responsiveness of hepatic stellate cells to TGF-ß1. F2 may thus be a potentially new effective pharmacotherapy for human liver fibrosis.

Relationships between rumen microbes, short-chain fatty acids, and markers of white adipose tissue browning during the cold season in grazing Mongolian sheep (Ovisaries).

Mongolian sheep are characteristically cold-tolerant and they partially depend on seasonal browning of white adipose tissue (WAT) to acclimate to cold environments. The present work aimed to examine the rumen microbes, rumen fermentation profile, and relationships between the rumen microbiota, short-chain fatty acids (SCFAs), and markers of WAT browning and are thus conducive to exploring the plateau environment adaptability of Mongolian sheep in the cold season. A comparative analysis of the rumen microbes and SCFAs in the cold and warm seasons was conducted. Rumen microbes were analyzed using Illumina sequencing of the 16S rRNA gene. Ruminal SCFAs were determined by gas chromatography. Spearman's correlation test was used to determine the relationships between the rumen microbiota, SCFAs, and markers of WAT browning. Microbial 16S rRNA sequencing revealed a marked shift in rumen microbiota composition between the two seasons, and the bacteria were characterized by increased levels of the Actinobacteria and genera Christensenellaceae R-7 group, Ruminococcaceae UCG-011, Rikenellaceae RC9 gut group, Papillibacter, and Butyrivibrio 2 and reduced levels of Prevotella 1 and Ruminococcaceae UCG-014 in the cold season (P<0.05). Furthermore, the concentrations of SCFAs such as acetate and butyrate were significantly increased in the cold season (P<0.001 and P<0.05, respectively). Correlation analysis demonstrated that the relative abundances of the Actinobacteria and the genera Christensenellaceae R-7 group, Butyrivibrio 2, Ruminococcaceae UCG-002, and Ruminococcaceae UCG-011, identified as members of the Christensenellaceae, Lachnospiraceae, and Ruminococcaceae families (all within Firmicutes), were positively correlated with markers of browning in either retroperitoneal WAT or perirenal WAT, and acetate was positively correlated with Ruminococcaceae UCG-011 and Butyrivibrio 2 and markers of browning in either retroperitoneal WAT or perirenal WAT. Overall, there are distinct relationships between the rumen microbiota, ruminal SCFAs and markers of WAT browning during the cold season in grazing Mongolian sheep.

Role of white adipose tissue browning in cold seasonal acclimation in grazing Mongolian sheep (Ovisaries).

Mongolian sheep are characteristically cold-tolerant and thus can survive well and maintain genetic stability in the extremely cold environment of the Mongolian Plateau. However, the adaptive mechanism of Mongolian sheep during the cold season in the plateau environment remains unknown. Browning of white adipose tissues (WAT) can trigger nonshivering thermogenesis as a potential strategy to promote an animal's tolerance to cold environments. Thus, a comparative analysis of the genes and proteins of uncoupling protein 1 (UCP1)-dependent and UCP1-independent browning pathways, mitochondrial biogenesis, lipogenic and lipolytic processes of WAT from grazing Mongolian sheep in the cold and warm seasons was conducted. We found seasonal browning of both retroperitoneal WAT and perirenal WAT, and the signalling of the process was mainly transduced by the UCP1- dependent pathway, primarily reflected in the upregulated gene levels of UCP1 and peroxisome proliferative activated receptor gamma coactivator 1 alpha (PGC-1α). In addition, the mean adipocyte diameter and mRNA expression of lipogenic genes in both interscapular WAT and subcutaneous WAT were significantly elevated during the cold season. The findings of this study demonstrate that grazing Mongolian sheep could depend on seasonal browning of both retroperitoneal WAT and perirenal WAT together with the expansion of both interscapular WAT and subcutaneous WAT to acclimate to cold environments of the Mongolian Plateau.

[Progress in Research on the Novel Tumor Marker circRNA].

Circular RNA(circRNA)is a novel type of endogenous non-coding RNA.Most circRNAs act as microRNA(miRNA)sponges to regulate the expression of functional genes.In addition,some circRNAs can be translated and interact with RNA-binding proteins to perform biological functions.The expression of circRNAs is prevalent in tissues and body fluids,and their abnormal expression is related to tumor progression.circRNAs are stable even under the treatment of RNase R because of their circular conformation.As circRNAs have construct stability,wide variety,specific regulation of tumor progression and high expression in body fluids,it is potential for circRNAs to serve as candidate diagnostic,prognostic and therapeutic targets.However,the knowledge about circRNAs remains poor.In addition to the not completely resolved functions and generation mechanisms of circRNAs,the annotations of circRNAs are also waiting for expanding.Here,we review the generation mechanisms,biological functions,and application of circRNAs in tumor research,aiming to provide integrated information for the future research.

Tripartite motif protein 52 (TRIM52) promoted fibrosis in LX-2 cells through PPM1A-mediated Smad2/3 pathway.

To investigate the roles of tripartite motif containing 52 (TRIM52) in human hepatic fibrosis in vitro, human hepatic stellate cell line LX-2 cells were transfected with hepatitis B virus (HBV) replicon to establish HBV-induced fibrosis in LX-2 cells, and then treated with small interfering RNA-mediated knockdown of TRIM52 (siTRIM52). LX-2 cells without HBV replicon transfection were treated with lentiviruses-mediated overexpression of TRIM52 and phosphatase magnesium dependent 1A (PPM1A). Fibrosis response of LX-2 cells were assessed by the production of hydroxyproline (Hyp) and collagen I/III, as well as protein levels of α-smooth muscle actin (α-SMA). PPM1A and phosphorylated (p)-Smad2/3 were measured to assess the mechanism. The correlation between TRIM52 and PPM1A was determined using co-immunoprecipitation, and whether and how TRIM52 regulated the degradation of PPM1A were determined by ubiquitination assay. Our data confirmed HBV-induced fibrogenesis of LX-2 cells, as evidenced by significant increase in Hyp and collagen I/III and α-SMA, which was associated with reduction of PPM1A and elevation of transforming growth factor-ß (TGF-ß), p-Smad2/3, and p-Smad3L. However, those changes induced by HBV were significantly attenuated with additional siTRIM52 treatment. Similar to HBV, overexpression of TRIM52 exerted promoted effect in the fibrosis of LX-2 cells. Interestingly, TRIM52 induced the fibrogenesis of LX-2 cells and the activation of TGF-ß/Smad pathway were significantly reversed by PPM1A overexpression. Furthermore, our data confirmed TRIM52 as a deubiquitinase that influenced the accumulation of PPM1A protein, and subsequently regulated the fibrogenesis of LX-2 cells. TRIM52 was a fibrosis promoter in hepatic fibrosis in vitro, likely through PPM1A-mediated TGF-ß/Smad pathway.

Cancer-derived exosomal miR-221-3p promotes angiogenesis by targeting THBS2 in cervical squamous cell carcinoma.

AIMS: Recently, cancer-derived exosomes were shown to have pro-metastasis function in cancer, but the mechanism remains unclear. Angiogenesis is essential for tumor progression and is a great promising therapeutic target for advanced cervical cancer. Here, we investigated the role of cervical cancer cell-secreted exosomal miR-221-3p in tumor angiogenesis. METHODS AND RESULTS: miR-221-3p was found to be closely correlated with microvascular density in cervical squamous cell carcinoma (CSCC) by evaluating the microvascular density with immunohistochemistry and miR-221-3p expression with in situ hybridization in clinical specimens. Using the groups of CSCC cell lines (SiHa and C33A) with miR-221-3p overexpression and silencing, the CSCC exosomes were characterized by electron microscopy, western blotting, and fluorescence microscopy. The enrichment of miR-221-3p in CSCC exosomes and its transfer into human umbilical vein endothelial cells (HUVECs) were confirmed by qRT-PCR. CSCC exosomal miR-221-3p promoted angiogenesis in vitro in Matrigel tube formation assay, spheroid sprouting assay, migration assay, and wound healing assay. Then, exosome intratumoral injection indicated that CSCC exosomal miR-221-3p promoted tumor growth in vivo. Thrombospondin-2 (THBS2) was bioinformatically predicted to be a direct target of miR-221-3p, and this was verified by using the in vitro and in vivo experiments described above. Additionally, overexpression of THBS2 in HUVECs rescued the angiogenic function of miR-221-3p. CONCLUSIONS: Our results suggest that CSCC exosomes transport miR-221-3p from cancer cells to vessel endothelial cells and promote angiogenesis by downregulating THBS2. Therefore, CSCC-derived exosomal miR-221-3p could be a possible novel diagnostic biomarker and therapeutic target for CSCC progression.