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As the most common post-transcriptional RNA modification, m6A methylation extensively regulates the structure and function of RNA. The dynamic and reversible modification of m6A is coordinated by m6A writers and erasers. m6A reader proteins recognize m6A modification on RNA, mediating different downstream biological functions. mRNA m6A modification and its corresponding regulators play an important role in cancers, but its characteristics in the precancerous stage are still unclear. In this study, we used oral precancerous DOK cells as a model to explore the characteristics of transcriptome-wide m6A modification and major m6A regulator expression in the precancerous stage compared with normal oral epithelial cell HOEC and oral cancer cell SCC-9 through MeRIP-seq and RT-PCR. Compared with HOEC cells, we found 1180 hyper-methylated and 1606 hypo-methylated m6A peaks and 354 differentially expressed mRNAs with differential m6A peaks in DOK cells. Although the change of m6A modification in DOK cells was less than that in SCC-9 cells, mRNAs with differential m6A in both cell lines were enriched into many identical GO terms and KEGG pathways. Among the 20 known m6A regulatory genes, FTO, ALKBH5, METTL3 and VIRMA were upregulated or downregulated in DOK cells, and the expression levels of 10 genes such as METTL14/16, FTO and IGF2BP2/3 were significantly changed in SCC-9 cells. Our data suggest that precancerous cells showed, to some extent, changes of m6A modification. Identifying some key m6A targets and corresponding regulators in precancerous stage may provide potential intervention targets for the prevention of cancer development through epigenetic modification in the future.

Insects and animals are attracted to, and feed on ripe fruit, thereby promoting seed dispersal. As a vital vitamin and nutrient source, fruit make up an indispensable and enjoyable component of the human diet. Fruit ripening involves a series of physiological and biochemical changes in, among others, pigmentation, chlorophyll (Chl) degradation, texture, sugar accumulation, and flavor. Growing evidence indicates that the coordinated and ordered trait changes during fruit ripening depend on a complex regulatory network consisting of transcription factors, co-regulators, hormonal signals, and epigenetic modifications. As one of the predominant transcription factor families in plants and a downstream component of ethylene signaling, more and more studies are showing that APETALA2/ethylene responsive factor (AP2/ERF) family transcription factors act as critical regulators in fruit ripening. In this review, we focus on the regulatory mechanisms of AP2/ERFs in fruit ripening, and in particular the recent results on their target genes and co-regulators. We summarize and discuss the role of AP2/ERFs in the formation of key fruit-ripening attributes, the enactment of their regulatory mechanisms by interaction with other proteins, their role in the orchestration of phytohormone-signaling networks, and the epigenetic modifications associated with their gene expression. Our aim is to provide a multidimensional perspective on the regulatory mechanisms of AP2/ERFs in fruit ripening, and a reference for understanding and furthering research on the roles of AP2/ERF in fruit ripening.

In addition to liquid-based cytology (LBC) and HR HPV testing, p16/ki-67 dual-staining is another method for cervical cancer screening. The combination of any two methods can improve the accuracy of screening, but some cervical lesions are still missed or misdiagnosed. In this retrospective study, the significance of LBC, HR HPV testing and especially p16/ki-67 dual-staining in cervical lesion screening was evaluated with reference to histological diagnosis. At the same time, we tried to explore the value of p16/ki-67 dual-staining combined with LBC and HR HPV testing (triple detection) in improving the diagnostic specificity of CIN2+ and reducing the missed diagnosis of CIN2+ lesions. We found that p16/ki-67 dual-staining was valuable in identifying cervical CIN2+ lesions and reducing the missed diagnosis of CIN2+ in HPV negative patients. More than 96% of CIN2+ patients were positive for two or three tests of triple detection. Whole positive triple detection can effectively predict high grade cervical lesions. In conclusion, the triple detection can distinguish almost all cervical CIN2+ lesions. Our data put forward and highlight the feasibility and significance of triple detection in cervical lesion screening.

The AID/APOBEC family which converts cytidine to uridine on RNA or DNA experienced dynamic expansion in primates in order to resist exogenous viruses and endogenous retrotransposons. Recently, expansion of AID/APOBEC-like homologs has also been observed in the extant jawless vertebrate lamprey. To reveal what causes such expansion and leads to the functional diversification of lamprey cytosine deaminases (CDAs), we reassessed the CDA genes in Lethenteron japonicum (Lj). We first confirmed the expansion of LjCDA1L1 (CDA1-like 1) genes and found the expression correlation of LjCDA2 and LjCDA1L2 with LjVLRs (variable lymphocyte receptors). Among up to 14 LjCDA1L1 proteins, LjCDA1L1_4a has an extremely high deamination activity on ssDNA and buDNA and, unexpectedly, on dsDNA. LjCDA1L1s can also restrict the infection of HSV-1 particles. Thus, the arms race between the host and pathogens along with the recruitment by VLR assembly may participate together to form a driving force in the expansion and diversification of the lamprey AID/APOBEC family.

The basic helix–loop–helix (bHLH) transcription factor family is the second largest transcription factor family in plants, and participates in various plant growth and development processes. A total of 118 bHLH genes were identified from fig (Ficus carica L.) by whole-genome database search. Phylogenetic analysis with Arabidopsis homologs divided them into 25 subfamilies. Most of the bHLHs in each subfamily shared a similar gene structure and conserved motifs. Seventy-two bHLHs were found expressed at fragments per kilobase per million mapped (FPKM) > 10 in the fig fruit; among them, 15 bHLHs from eight subfamilies had FPKM > 100 in at least one sample. bHLH subfamilies had different expression patterns in the female flower tissue and peel during fig fruit development. Comparing green and purple peel mutants, 13 bHLH genes had a significantly different (≥ 2-fold) expression. Light deprivation resulted in 68 significantly upregulated and 22 downregulated bHLH genes in the peel of the fruit. Sixteen bHLH genes in subfamily III were selected by three sets of transcriptomic data as candidate genes related to anthocyanin synthesis. Interaction network prediction and yeast two-hybrid screening verified the interaction between FcbHLH42 and anthocyanin synthesis-related genes. The transient expression of FcbHLH42 in tobacco led to an apparent anthocyanin accumulation. Our results confirm the first fig bHLH gene involved in fruit color development, laying the foundation for an in-depth functional study on other FcbHLH genes in fig fruit quality formation, and contributing to our understanding of the evolution of bHLH genes in other horticulturally important Ficus species.

Fig is an ancient gynodioecious fruit tree with females for commercial fruit production and hermaphrodites (males) sometimes used as pollen providers. An early sex-identification method would improve breeding efficiency. Three AGAMOUS (AG) genes were recruited from the Ficus carica genome using AG sequences from Ficus microcarpa and Ficus hispida. FcAG was 5230 bp in length, with 7 exons and 6 introns, and a 744-bp coding sequence. The gene was present in both female and male fig genomes, with a 15-bp deletion in the 7th exon. The other two AG genes (FcAG2-Gall_Stamen and FcAG3-Gall_Stamen) were male-specific, without the 15-bp deletion (759-bp coding sequence), and were only expressed in the gall and stamen of the male fig fruit. Using the deletion as the forward primer (AG-Marker), male plants were very efficiently identified by the presence of a 146-bp PCR product. The previously reported fig male and female polymorphism gene RESPONSIVE-TO-ANTAGONIST1 (RAN1) was also cloned and compared between male and female plants. Fifteen SNPs were found in the 3015-bp protein-coding sequence. Among them, 12 SNPs were identified as having sex-differentiating capacity by checking the sequences of 27 known male and 24 known female cultivars. A RAN1-Marker of 608 bp, including 6 SNPs, was designed, and a PCR and sequencing-based method was verified with 352 fig seedlings from two hybrid populations. Our results confirmed that the newly established AG-Marker is as accurate as the RAN1-Marker, and provide new clues to understanding Ficus sex determination.

The apextrin C-terminal (ApeC) domain is a class of newly discovered protein domains with an origin dating back to prokaryotes. ApeC-containing proteins (ACPs) have been found in various marine and aquatic invertebrates, but their functions and the underlying mechanisms are largely unknown. Early studies suggested that amphioxus ACP1 and ACP2 bind to bacterial cell walls and have a role in immunity. Here we identified another two amphioxus ACPs (ACP3 and ACP5), which belong to the same phylogenetic clade with ACP1/2, but show distinct expression patterns and sequence divergence (40-50% sequence identities). Both ACP3 and ACP5 were mainly expressed in the intestine and hepatic cecum, and could be up-regulated after bacterial challenge. Both prokaryotic-expressed recombinant ACP3 and ACP5 could bind with several species of bacteria and yeasts, showing agglutinating activity but no microbicidal activity. ELISA assays suggested that their ApeC domains could interact with peptidoglycan (PGN), but not with lipoteichoic acid (LTA), lipopolysaccharides (LPS) and zymosan A. Furthermore, they can only bind to Lys-type PGN from Staphylococcus aureus, but not to DAP-type PGN from Bacillus subtilis and not to moieties of PGN such as MDPs, NAMs and NAGs. This recognition spectrum is different from that of ACP1/2. We also found that when expressed in mammalian cells, ACP3 could interact with TRAF6 via a conserved non-ApeC region, which inhibited the ubiquitination of TRAF6 and hence suppressed downstream NF-κB activation. This work helped define a novel subfamily of ACPs, which have conserved structures, and have related yet diversified molecular functions. Its members have dual roles, with ApeC as a lectin and a conserved unknown region as a signal transduction regulator. These findings expand our understanding of the ACP functions and may guide future research on the role of ACPs in different animal clades.

The papain-like cysteine proteases (PLCPs) are the most abundant family of cysteine proteases in plants, with essential roles in biotic/abiotic stress responses, growth and senescence. Papain, bromelain and ficin are widely used in food, medicine and other industries. In this study, 31 PLCP genes (FcPCLPs) were identified in the fig (Ficus carica L.) genome by HMM search and manual screening, and assigned to one of nine subfamilies based on gene structure and conserved motifs. SAG12 and RD21 were the largest subfamilies with 10 and 7 members, respectively. The FcPCLPs ranged from 1,128 to 5,075 bp in length, containing 1–10 introns, and the coding sequence ranged from 624 to 1,518 bp, encoding 207–505 amino acids. Subcellular localization analysis indicated that 24, 2, and 5 PLCP proteins were targeted to the lysosome/vacuole, cytoplasm and extracellular matrix, respectively. Promoter (2,000 bp upstream) analysis of FcPLCPs revealed a high number of plant hormone and low temperature response elements. RNA-seq revealed differential expression of 17 FcPLCPs in the inflorescence and receptacle, and RD21 subfamily members were the major PLCPs expressed in the fruit; 16 and 5 FcPLCPs responded significantly to ethylene and light, respectively. Proteome analyses revealed 18 and 5 PLCPs in the fruit cell soluble proteome and fruit latex, respectively. Ficins were the major PLCP in fig fruit, with decreased abundance in inflorescences, but increased abundance in receptacles of commercial-ripe fruit. FcRD21B/C and FcALP1 were aligned as the genes encoding the main ficin isoforms. Our study provides valuable multi-omics information on the FcPLCP family and lays the foundation for further functional studies.

DExD/H-box helicases play essential roles in RNA metabolism, and emerging data suggests that they have additional functions in antiviral immunity across species. However, little is known about this evolutionarily conserved family in antiviral responses in lower species. Here, through isolation of poly(I:C)-binding proteins in amphioxus, an extant basal chordate, we found that DExD/H-box helicases DHX9, DHX15, and DDX23 are responsible for cytoplasmic dsRNA detection in amphioxus. Since the antiviral roles of DDX23 have not been characterized in mammals, we performed further poly(I:C) pull-down assays and found that human DDX23 binds to LMW poly(I:C) through its N-terminal region, suggesting that DDX23 is an evolutionarily conserved dsRNA sensor. Knockdown of human DDX23 enhanced the replication of VSV and reduced the activation of the NF-κB and IRF3. Moreover, when stimulated with poly(I:C) or VSV, human DDX23 translocated from the nucleus to the cytoplasm and formed complexes with TRIF or MAVS to initiate downstream signaling. Collectively, this comparative immunological study not only defined DDX23 as an emerging nuclear pattern recognition receptor (PRR) for the innate sensing of an RNA virus, but also extended the essential role of the DExD/H helicase family in viral RNA sensing from mammals to basal chordates.

The vacuole plays a central role in fruit growth and quality formation, yet its proteomic landscape is largely unknown. In the present study, a protocol for isolating intact vacuoles from grape flesh tissue was successfully established. Quantitative proteome analysis identified 2533 proteins from five sampling dates along Cabernet Sauvignon berry development from stage I to III; among them, 1443 proteins were identified on all five sampling dates in at least two biological replicates per sample and were designated core proteome, and 1820 were recruited as differentially abundant proteins (DAPs) by sequential pairwise comparisons using arbitrary fold change of >1.5 and P < 0.05. Metabolism consistently constituted the largest category of identified proteins for both core proteome and DAPs, together with a consistently high proportion of protein-fate category proteins, indicating that the classic lytic functions of vegetative cell vacuoles are maintained throughout berry development; accumulation of metabolites involved in high sugar and other berry qualities in the late developmental stage added to the conventional lytic role of the flesh cell vacuoles. Overall increases in abundance of the DAPs were seen in the transporter proteins, membrane fusion/vesicle trafficking, and protein-fate categories, and decreased abundance was seen for DAPs in the stress, energy and cytoskeleton categories as berry development progressed. A very pronounced proteomic change was revealed between late stage I and mid stage II, with 915 increased and 114 decreased DAPs, demonstrating a significant surge of the vacuolar proteome underlying the rather static phenotypical and physiological phase. We identified 161 transport proteins with differential abundance, including proton pumps, aquaporins, sugar transporters, ATP-binding cassette transporters and ion transport proteins, together with organic compound transport proteins, the highest number and variety of berry tonoplast transporters found in grape proteome efforts to date. We further found a pre-positive increment of 96 transport proteins from the middle of stage II, before the berry undergoes its dramatic physiological changes at and following véraison. Our results are the first to describe the proteome of a vacuole-enriched preparation, toward understanding the functions of the largest compartment in berry cells during grape growth and ripening.

Combined metabolomic and transcriptomic analyses were carried out with fig cultivar Green Peel and its color mutant “Purple Peel.” Five and twenty-two metabolites were identified as having significantly different contents between fruit peels of the two cultivars at young and mature stages, respectively. Cyanidin O-malonylhexoside demonstrated a 3,992-fold increase in the mature purple peel, the first identification of a major cyanidin in fig fruit; cyanidin 3-O-glucoside, cyanidin O-malonylhexoside O-hexoside and cyanidin-3,5-O-diglucoside were upregulated 100-fold, revealing the anthocyanins underlying the purple mutation. Beyond the visible differences, there was very significant accumulation of the colorless flavonoids procyanidin B1, luteolin-3′,7-di-O-glucoside, epicatechin and quercetin-3-O-rhamnoside in the mature “Purple Peel” compared to “Green Peel.” At the young stage, only cyanidin O-malonylhexoside, cyanidin O-malonylhexoside O-hexoside and esculetin were upregulated a few fold in the mutant. Transcriptome analysis revealed a downregulated expression trend of genes encoding phenylpropanoid and flavonoid biosynthetic pathway enzyme in the young “Purple Peel” compared to the young “Green Peel,” whereas significant and simultaneous upregulation was revealed in almost all of the flavonoid and anthocyanin pathway components and relevant transcription factors in the mature-stage mutant. The role of R2R3-MYB transcription factors in the color morph mutation and its possible relation to the activity of retrotransposons are discussed. Moreover, large-scale upregulation of small heat-shock protein genes was found in the mature mutant. This is the first work to reveal comprehensive metabolome and transcriptome network changes underlying a fig mutation in a single horticultural attribute, and its profound effects on fruit nutrition and quality.

As the most common post-transcriptional RNA modification, m6A methylation extensively regulates the structure and function of RNA. The dynamic and reversible modification of m6A is coordinated by m6A writers and erasers. m6A reader proteins recognize m6A modification on RNA, mediating different downstream biological functions. mRNA m6A modification and its corresponding regulators play an important role in cancers, but its characteristics in the precancerous stage are still unclear. In this study, we used oral precancerous DOK cells as a model to explore the characteristics of transcriptome-wide m6A modification and major m6A regulator expression in the precancerous stage compared with normal oral epithelial cell HOEC and oral cancer cell SCC-9 through MeRIP-seq and RT-PCR. Compared with HOEC cells, we found 1180 hyper-methylated and 1606 hypo-methylated m6A peaks and 354 differentially expressed mRNAs with differential m6A peaks in DOK cells. Although the change of m6A modification in DOK cells was less than that in SCC-9 cells, mRNAs with differential m6A in both cell lines were enriched into many identical GO terms and KEGG pathways. Among the 20 known m6A regulatory genes, FTO, ALKBH5, METTL3 and VIRMA were upregulated or downregulated in DOK cells, and the expression levels of 10 genes such as METTL14/16, FTO and IGF2BP2/3 were significantly changed in SCC-9 cells. Our data suggest that precancerous cells showed, to some extent, changes of m6A modification. Identifying some key m6A targets and corresponding regulators in precancerous stage may provide potential intervention targets for the prevention of cancer development through epigenetic modification in the future. [ABSTRACT FROM AUTHOR]