Search

Abstract Apoptotic protease activating factor 1 (Apaf-1) was traditionally defined as a scaffold protein in mammalian cells for assembling a caspase activation platform known as the ‘apoptosome’ after its binding to cytochrome c. Although Apaf-1 structurally resembles animal NOD-like receptor (NLR) and plant resistance (R) proteins, whether it is directly involved in innate immunity is still largely unknown. Here, we found that Apaf-1-like molecules from lancelets, fruit flies, mice, and humans have conserved DNA sensing functionality. Mechanistically, mammalian Apaf-1 recruits receptor-interacting protein 2 (RIP2, also known as RIPK2) via its WD40 repeat domain and promotes RIP2 oligomerization to initiate NF-κB-driven inflammation upon cytoplasmic DNA recognition. Furthermore, DNA binding of Apaf-1 determines cell fate by switching the cellular processes between intrinsic stimuli-activated apoptosis and inflammation. These findings suggest that Apaf-1 is an evolutionarily conserved DNA sensor and may serve as a cell fate checkpoint, which determines whether cells initiate inflammation or undergo apoptosis by distinct ligand binding.

Abstract Radiotherapy is often used to treat various types of cancers, but radioresistance greatly limits the clinical efficiency. Recent studies have shown that radiotherapy can lead to ferroptotic cancer cell deaths. Ferroptosis is a new type of programmed cell death caused by excessive lipid peroxidation. The induction of ferroptosis provides a potential therapeutic strategy for radioresistance. As the most common post-transcriptional modification of mRNA, m6A methylation is widely involved in the regulation of various physiopathological processes by regulating RNA function. Dynamic m6A modification controlled by m6A regulatory factors also affects the susceptibility of cells to ferroptosis, thereby determining the radiosensitivity of tumor cells to radiotherapy. In this review, we summarize the mechanism and significance of radiotherapy induced ferroptosis, analyze the regulatory characteristics of m6A modification on ferroptosis, and discuss the possibility of radiosensitization by enhancing m6A-mediated ferroptosis. Clarifying the regulation of m6A modification on ferroptosis and its significance in the response of tumor cells to radiotherapy will help us identify novel targets to improve the efficacy of radiotherapy and reduce or overcome radioresistance.

Glutathione peroxidase 8 (GPX8) is a key regulator of redox homoeostasis. Whether its antioxidant activity participates in the regulation of m6A modification is a crucial issue, which has important application value in cancer treatment. In this study, MeRIP-seq was used to explore the characteristics of transcriptome-wide m6A modification in GPX8-deficient oral cancer cells. Oxidative stress caused by the lack of GPX8 resulted in 1,279 hyper- and 2,287 hypo-methylated m6A peaks and 2,036 differentially expressed genes in GPX8-KO cells. Twenty-eight differentially expressed genes were related to the cell response to oxidative stress, and half of them changed their m6A modification. In GPX8-KO cells, m6A regulators IGF2BP2 and IGF2BP3 were upregulated, while FTO, RBM15, VIRMA, ZC3H13, and YTHDC2 were downregulated. After H2O2 treatment, the expression changes of RBM15, IGF2BP2, and IGF2BP3 were further enhanced. These data indicated that GPX8-mediated redox homoeostasis regulated m6A modification, thereby affecting the expression and function of downstream genes. This study highlights the possible significance of GPX8 and the corresponding m6A regulatory or regulated genes as novel targets for antioxidant intervention in cancer therapy.

Summary: Cancer cells usually exhibit shortened 3′ untranslated regions (UTRs) due to alternative polyadenylation (APA) to promote cell proliferation and migration. Upregulated CPSF6 leads to a systematic prolongation of 3′ UTRs, but CPSF6 expression in tumors is typically higher than that in healthy tissues. This contradictory observation suggests that it is necessary to investigate the underlying mechanism by which CPSF6 regulates APA switching in cancer. Here, we find that CPSF6 can undergo liquid-liquid phase separation (LLPS), and elevated LLPS is associated with the preferential usage of the distal poly(A) sites. CLK2, a kinase upregulated in cancer cells, destructs CPSF6 LLPS by phosphorylating its arginine/serine-like domain. The reduction of CPSF6 LLPS can lead to a shortened 3′ UTR of cell-cycle-related genes and accelerate cell proliferation. These results suggest that CPSF6 LLPS, rather than its expression level, may be responsible for APA regulation in cancer cells.

Members of the gasdermin (GSDM) family are pore-forming effectors that cause membrane permeabilization and pyroptosis, a lytic proinflammatory type of cell death. To reveal the functional evolution of GSDM-mediated pyroptosis at the transition from invertebrates to vertebrates, we conducted functional characterization of amphioxus GSDME (BbGSDME) and found that it can be cleaved by distinct caspase homologs, yielding the N253 and N304 termini with distinct functions. The N253 fragment binds to cell membrane, triggers pyroptosis, and inhibits bacterial growth, while the N304 performs negative regulation of N253-mediated cell death. Moreover, BbGSDME is associated with bacteria-induced tissue necrosis and transcriptionally regulated by BbIRF1/8 in amphioxus. Interestingly, several amino acids that are evolutionarily conserved were found to be important for the function of both BbGSDME and HsGSDME, shedding new lights on the functional regulation of GSDM-mediated inflammation.

Abstract The conventional planar culture of adherent cells is inefficient for large‐scale manufacturing of cell and gene therapy products. We developed a facile and efficient bead‐to‐bead cell‐transfer method for serial subculture and large‐scale expansion of human mesenchymal stem cells (hMSCs) with microcarriers in bioreactors. We first compared culture medium with and without nucleosides and found the former maintained the expression of surface markers of hMSCs during their prolonged culture and enabled faster cell proliferation. Subsequently, we developed our bead‐to‐bead cell transfer method to subculture hMSCs and found that intermittent agitation after adding fresh microcarriers to cell‐populated microcarriers could promote spontaneous cell migration to fresh microcarriers, reduce microcarrier aggregation, and improve cell yield. This method enabled serial subculture of hMSCs in spinner flasks from passage 4 to passage 9 without using proteolytic enzymes, which showed faster cell proliferation than the serial planar cultures undergoing multiple enzyme treatment. Finally, we used the medium containing nucleosides and our bead‐to‐bead cell transfer method for cell culture scale‐up from 4‐ to 50‐L cultures in single‐use bioreactors. We achieved a 242‐fold increase in the number of cells to 1.45 × 1010 after 27‐day culture and found that the cells harvested from the bioreactors maintained proliferation ability, expression of their surface markers, tri‐lineage differentiation potential and immunomodulatory property. This study shows the promotive effect of nucleosides on hMSC expansion and the potential of using our bead‐to‐bead transfer method for larger‐scale manufacturing of hMSCs for cell therapy.

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 Background Bagging can improve the appearance of fruits and increase the food safety and commodification, it also has effects on intrinsic quality of the fruits, which was commonly reported negative changes. Fig can be regarded as a new model fruit with its relatively small genome size and long fruit season. Results In this study, widely targeted metabolomics based on HPLC MS/MS and RNA-seq of the fruit tissue of the ‘Zibao’ fig before and after bagging were analyzed to reveal the metabolites changes of the edible part of figs and the underneath gene expression network changes. A total of 771 metabolites were identified in the metabolome analysis using fig female flower tissue. Of these, 88 metabolites (including one carbohydrate, eight organic acids, seven amino acids, and two vitamins) showed significant differences in fruit tissue before and after bagging. Changes in 16 structural genes, 13 MYB transcription factors, and endogenous hormone (ABA, IAA, and GA) metabolism and signal transduction-related genes in the biosynthesis pathway of flavonoids after bagging were analyzed by transcriptome analysis. KEGG enrichment analysis also determined significant differences in flavonoid biosynthesis pathways in female flower tissue before and after bagging. Conclusions This work provided comprehensive information on the composition and abundance of metabolites in the female flower tissue of fig. The results showed that the differences in flavor components of the fruit before and after bagging could be explained by changes in the composition and abundance of carbohydrates, organic acids, amino acids, and phenolic compounds. This study provides new insights into the effects of bagging on changes in the intrinsic and appearance quality of fruits.

Bioprospecting of more novel probiotic strains has attained continuous interest. This study aimed to investigate the beneficial effects of Lactobacillus paracasei strain L14, an isolate from a traditional Chinese dairy product, on type 2 diabetes mellitus (T2DM) rats. Preventive supplementation of strain L14 showed excellent anti-diabetic effects on high-fat diet/low-dose streptozotocin (HFD/STZ)-induced T2DM rats. It significantly reduced hyperglycemia, protected pancreatic β-cell and liver function, and ameliorated oxidative stress while considerably improving dyslipidemia and inflammation. Furthermore, the strain modulated the gut microbiota to alleviate gut dysbiosis. Interestingly, most of these biochemical parameters could even restore to normal levels by the intervention of strain L14. The whole-genome sequencing of L14 was performed to provide a critical molecular basis for its probiotic activities. Genes related to antioxidant systems and other beneficial microbial metabolites like exopolysaccharides (EPS) biosynthesis were found. This study demonstrates that probiotic L. paracasei L14 has good potential for applications in functional food and pharmaceutical industries.

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.

Abstract Pyruvate kinase is a terminal enzyme in the glycolytic pathway, where it catalyzes the conversion of phosphoenolpyruvate to pyruvate and production of ATP via substrate level phosphorylation. PKM2 is one of four isoforms of pyruvate kinase and is widely expressed in many types of tumors and associated with tumorigenesis. In addition to pyruvate kinase activity involving the metabolic pathway, increasing evidence demonstrates that PKM2 exerts a non-metabolic function in cancers. PKM2 has been shown to be translocated into nucleus, where it serves as a protein kinase to phosphorylate various protein targets and contribute to multiple physiopathological processes. We discuss the nuclear localization of PKM2, its protein kinase function and association with cancers, and regulation of PKM2 activity.

Lancelet expresses an ancestral RAG transposon, ProtoRAG, which predates human RAGgenes that are responsible for V(D)J recombination and adaptive immunity repertoire generation. Here the authors show that ProtoRAG is functionally regulated by a tran s-acting factor, bbYY1, for tuning transposon activity and maintaining genome stability.

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.

Abstract Cervical cancer is the fourth most common malignant tumor in women worldwide. The persistent infection of high-risk Human Papillomavirus (hrHPV) is considered to be the primary cause of this disease. As an innate immune receptor, the nucleotide-binding oligomerization domain protein-1 (NOD1) recognizes the pathogen-associated molecular pattern (PAMP), subsequently initiating immune responses. NOD1 is also involved in the apoptotic signaling pathway and mutates in many cancer cells. In the study, we revealed that NOD1 expression decreased during the progression of cervical intraepithelial neoplasia to cervical cancer and that HPV16 E6/E7 oncoproteins induced down-regulation of NOD1. Moreover, the activation of NOD1 promoted the apoptosis of HPV16-positive cervical cancer cells. The data indicated that the dysregulation of NOD1-mediated inflammation and apoptosis may contribute to cervical intraepithelial neoplasia progression and cervical cancer.

Abstract Background Color directly affects fruit quality and consumer preference. In fig syconia, the female flower tissue is contained in a receptacle. Anthocyanin pigmentation of this tissue and the peel differs temporally and spatially. A transcriptome study was carried out to elucidate key genes and transcription factors regulating differences in fig coloring. Results Anthocyanins in the female flower tissue were identified mainly as pelargonidin-3-glucoside and cyanidin-3-rutinoside; in the peel, the major anthocyanins were cyanidin 3-O-glucoside and cyanidin-3-rutinoside. Anthocyanin content was significantly higher in the female flower tissue vs. peel before fig ripening, whereas at ripening, the anthocyanin content in the peel was 5.39 times higher than that in the female flower tissue. Light-deprivation treatment strongly inhibited peel, but not female flower tissue, anthocyanin pigmentation. RNA-Seq revealed 522 differentially expressed genes (recruited with criteria log2 ≥ 2 and P

Abstract Background Fig fruit are highly perishable at the tree-ripe (TR) stage. Commercial-ripe (CR) fruit, which are harvested before the TR stage for their postharvest transportability and shelf-life advantage, are inferior to TR fruit in size, color and sugar content. The succulent urn-shaped receptacle, serving as the protective structure and edible part of the fruit, determines fruit quality. Quantitative iTRAQ and RNA-Seq were performed to reveal the differential proteomic and transcriptomic traits of the receptacle at the two harvest stages. Results We identified 1226 proteins, of which 84 differentially abundant proteins (DAPs) were recruited by criteria of abundance fold-change (FC) ≥1.3 and p

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.

Somatic mutation is associated with the occurrence, development and recurrence of oral cancer. Oral squamous cell carcinoma cell lines have been widely used in oral cancer research. So it's important to understand their genetic background, especially the mutation profile. Using the data of The Catalogue Of Somatic Mutations In Cancer (COSMIC), we analyzed the mutation characteristics of six tongue squamous cell carcinoma cell lines, identified the most common mutation cancer genes in oral squamous cell carcinoma, and compared these genes with those in colon cancer and lung cancer. Six tongue cancer cells have similar mutation spectrum in the coding region, of which C > T transition is the most common mutation, mainly located in the context of CpG dinucleotides. Profiles of mutation genes vary in different tongue cancer cell lines and TP53 is the most common mutation gene, followed by CDKN2A, KMT2D, and LRP1B. The 20 most common mutation genes in oral cancer are different from those in colon cancer and lung cancer, although TP53 is the most common mutation gene in these three cancers. The mutation characteristics of tongue cancer cell lines and mutation gene profiles of oral cancer provide valuable information for the study of oral cancers.

Metabolic reprogramming is one of the hallmarks of a tumor. It not only promotes the development and progression of tumor but also contributes to the resistance of tumor cells to chemotherapeutics. The difference in the metabolism between drug-resistant and sensitive tumor cells indicates that drug-resistant tumor cells have experienced metabolic adaptation. The metabolic response induced by chemotherapy is dynamic, but the early metabolic response of tumor cells to anticancer drugs and the effect of an initial response on the development of drug resistance have not been well studied. Early metabolic intervention may prevent or slow down the development of drug resistance. The differential metabolic responses of normal cells and tumor cells to drugs are unclear. The specific metabolites or metabolic pathways of tumor cells to chemotherapeutic drugs can be used as the target of metabolic intervention in tumor therapy. In this study, we used comparative metabolomics to analyze the differential metabolic responses of oral cancer cells and normal oral epithelial cells to short-term cisplatin exposure, and to identify the marker metabolites of early response in oral cancer cells. Oral cancer cells showed a dynamic metabolic response to cisplatin. Seven and five metabolites were identified as specific response markers to cisplatin exposure in oral cancer cell SCC-9 and normal oral epithelial cell HOEC, respectively. Glyoxylate and dicarboxylate metabolism and fructose, malate, serine, alanine, sorbose and glutamate were considered as specific enriched metabolic pathways and biomarkers of SCC-9 cells in response to cisplatin, respectively. The existence of differential metabolic responses lays a foundation for tumor chemotherapy combined with metabolic intervention.

Abstract Background Gibberellin (GA) treatments can induce parthenocarpy in the main crop of San Pedro-type figs, the native non-parthenocarpic fruit, however, the underlying mechanism is still largely unclear. Results In our study, GA3 was applied to San Pedro-type fig main crop at anthesis. Sharply increased GA3 content was detected in both female flowers and receptacle, along with significantly decreased indole-3-acetic acid (IAA), zeatin and abscisic acid (ABA) levels in female flowers, and increased zeatin peak intensity and earlier ABA peak in receptacles. Transcriptome comparison between control and treatment groups identified more differentially expressed genes (DEGs) in receptacles than in female flowers 2 and 4 days after treatment (DAT); 10 DAT, the number of DEGs became similar in the two tissues. Synchronized changing trends of phytohormone-associated DEGs were observed in female flowers and receptacles with fruit development. Modulation of ethylene and GA signaling and auxin metabolism by exogenous GA3 occurred mainly 2 DAT, whereas changes in auxin, cytokinin and ABA signaling occurred mainly 10 DAT. Auxin-, ethylene- and ABA-metabolism and response pathways were largely regulated in the two tissues, mostly 2 and 10 DAT. The major components altering fig phytohormone metabolic and response patterns included downregulated GA2ox, BAS1, NCED and ACO, and upregulated ABA 8′-h and AUX/IAA. Conclusions Thus GA-induced parthenocarpy in fig is co-modulated by the female flowers and receptacle, and repression of ABA and ethylene biosynthesis and GA catabolism might be the main forces deflecting abscission and producing fig parthenocarpy.

Abstract Global shortening of 3′UTRs by alternative polyadenylation (APA) has been observed in cancer cells. However, the role of APA in cancer remains unknown. CCND1 is a proto-oncogene that regulates progression through the G1-S phase of the cell cycle; moreover, it has been observed to be switching to proximal APA sites in cancer cells. To investigate the biological function of the APA of CCND1, we edited the weak poly(A) signal (PAS) of the proximal APA site to a canonical PAS using the CRISPR/Cas9 method, which can force the cells to use a proximal APA site. Cell cycle profiling and proliferation assays revealed that the proximal APA sites of CCND1 accelerated the cell cycle and promoted cell proliferation, but UTR-APA and CR-APA act via different molecular mechanisms. These results indicate that PAS editing with CRISPR/Cas9 provides a good method by which to study the biological function of APA.

We investigated bioactive peptides obtained from muscle protein hydrolysate of bighead carp by evaluating in vitro dipeptidyl peptidase IV (DPP-IV) and angiotensin-converting enzyme (ACE) inhibitory capacities and antioxidant activity. Peptide sequences were identified from pepsin hydrolysates. Met-Lys-Ala-Val-Cys-Phe-Ser-Leu was one of the most effective sequences with 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical scavenging activity (EC50 = 4.58 ± 0.15 μM) and relatively high ACE inhibitory capacity (IC50 = 3.68 ± 0.13 μM). Tyr-Asn-Leu-Lys-Glu-Arg-Tyr-Ala-Ala-Trp (IC50 = 1.35 ± 0.23 μM) and Tyr-Asn-Arg-Leu-Pro-Glu-Leu (IC50 = 3.42 ± 0.39 μM) exhibited the most potent ACE inhibitory activity. Ile-Ala-Asp-His-Phe-Leu showed the highest DPP-IV inhibitory activity with an IC50 value of 610.1 ± 82.6 μM. All selected peptides were non-toxic, and most were non-allergenic according to in silico predictions. These results indicate the promising potential of bighead carp muscle hydrolysates as functional additives in foods and pharmaceuticals.

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.

RNA processing by alternative polyadenylation (APA) can result in 3′UTR diversity and regulation of target RNA function. Here the authors survey APA in viral infection of macrophages.

Metabolic reprogramming is one of the hallmarks of tumors. Alterations of cellular metabolism not only contribute to tumor development, but also mediate the resistance of tumor cells to antitumor drugs. The metabolic response of tumor cells to various chemotherapy drugs can be analyzed by metabolomics. Although cancer cells have experienced metabolic reprogramming, the metabolism of drug resistant cancer cells has been further modified. Metabolic adaptations of drug resistant cells to chemotherapeutics involve redox, lipid metabolism, bioenergetics, glycolysis, polyamine synthesis and so on. The proposed metabolic mechanisms of drug resistance include the increase of glucose and glutamine demand, active pathways of glutaminolysis and glycolysis, promotion of NADPH from the pentose phosphate pathway, adaptive mitochondrial reprogramming, activation of fatty acid oxidation, and up-regulation of ornithine decarboxylase for polyamine production. Several genes are associated with metabolic reprogramming and drug resistance. Intervening regulatory points described above or targeting key genes in several important metabolic pathways may restore cell sensitivity to chemotherapy. This paper reviews the metabolic changes of tumor cells during the development of chemoresistance and discusses the potential of reversing chemoresistance by metabolic regulation.

Lactobacillus strains were screened for inhibition of dipeptidyl peptidase IV (DPP-IV) and α-glucosidase. Cell-free excretory supernatants (CFS) and cell-free extracts (CFE) of 21 strains were prepared, and most of them showed DPP-IV inhibition in a concentration-dependent manner, which started at the exponential phase and peaked at the stationary phase of the Lactobacillus. The CFS bioactivity was heat-resistant, stable to acid–alkali and glucosidase treatment. Trypsin treatment specifically elevated CFS's DPP-IV inhibition. Lactobacillus plantarum strains CFS displayed different protein patterns and trypsin sensitive bands on SDS-PAGE, demonstrating specific peptide-cleavage contributes to higher inhibition. Seven strains with highest DPP-IV inhibition also displayed α-glucosidase inhibitory activity, of which, three strains of L. plantarum and one of Lactobacillus brevis showed tolerance to simulated gastrointestinal tract conditions and high HT-29 cell adhesion. This initial report of lactobacilli secreting strong DPP-IV and α-glucosidase inhibition components will be beneficial for selection and application of anti-diabetic probiotics.

Chloramphenicol (Chl) is an effective antimicrobial agent widely used in veterinary medicine and commonly used in fish. Its use is restricted in the clinic because of adverse effects on the immune system and oxidative stress in mammals. However, the effects of Chl treatment on invertebrates remain unclear. Amphioxus, a basal chordate, is an ideal model to study the origin and evolution of the vertebrate immune system as it has a primary vertebrate‐like arachidonic acid (AA) metabolic system. Here, we combined transcriptomic and lipidomic approaches to investigate the immune system and observe the oxygenated metabolites of AA to address the antibiotic effects on amphioxus. Tissue necrosis of the gill slits occurred in the Chl‐treated amphioxus, but fewer epithelial cells were lost when treated with both Chl and ampicillin (Amp). The immune related pathways were dysregulated in both of the antibiotic treatment groups. The Chl alone treatment resulted in immunosuppression with down‐regulation of the innate immune genes. In contrast, the Chl + Amp treatment resulted in immunostimulation to some extent, as shown by KEGG clustering. Furthermore, Chl induced a 3‐fold reduction in the level of the eicosanoids, while the Chl + Amp treatment resulted in 1.7‐fold increase of eicosanoid level. Thus in amphioxus, Amp might relieve the effects of the Chl‐induced immune suppression and increase the level of eicosanoids from AA. Finally, the oxygenated metabolites from AA might be crucial to evaluate the effects of Chl treatment in animals.

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.

We investigated the nuclear DNA content and genetic diversity of a river lamprey, the Korean lamprey Lampetra morii, which is distributed in the northeast of China. L. morii spends its whole life cycle in fresh water, and its adult size is relatively small (~160 mm long) compared with that of other lampreys. The haploid nuclear DNA content of L. morii is 1.618 pg (approximately 1.582 Gb) in germline cells, and there is ~15% germline DNA loss in somatic cells. These values are significantly smaller than those of Petromyzon marinus, a lamprey with a published draft genome. The chromosomes of L. morii are small and acrocentric, with a diploid modal number of 2n = 132, lower than some other lampreys. Sequence and AFLP analyses suggest that the allelic polymorphism rate (~0.14% based on examined nuclear and mitochondrial DNA sequences) of L. morii is much lower than that (~2%) of P. marinus. Phylogenetic analysis based on a mitochondrial DNA fragment confirms that L. morii belongs to the genus Lampetra, which, together with the genus Lethenteron, forms a sister group to P. marinus. These genetic background data are valuable for subsequent genetic and genomic research on L. morii.

Exendin-4, a glucagon-like protein-1 (GLP-1) receptor agonist, is an excellent therapeutic peptide drug for type 2 diabetes due to longer lasting biological activity compared to GLP-1. This study explored the feasibility of using probiotic Lactobacillus paracasei as an oral vector for recombinant exendin-4 peptide delivery, an alternative to costly chemical synthesis and inconvenient administration by injection. L. paracasei transformed with a plasmid encoding the exendin-4 gene (L. paracasei L14/pMG76e-exendin-4) with a constitutive promotor was successfully constructed and showed efficient secretion of exendin-4. The secreted exendin-4 significantly enhanced insulin secretion of INS-1 β-cells, along with an increment in their proliferation and inhibition of their apoptosis, corresponding to the effect of GLP-1 on these cells. The transcription level of the pancreatic duodenal homeobox-1 gene (PDX-1), a key transcription factor for cellular insulin synthesis and secretion, was upregulated by the treatment with secreted exendin-4, paralleling the upregulation of insulin gene expression. Caco-2 cell monolayer permeability assay showed a 34-fold increase in the transport of exendin-4 delivered by L. paracasei vs. that of free exendin-4 (control), suggesting effective facilitation of exendin-4 transport across the intestinal barrier by this delivery system. This study demonstrates that the probiotic Lactobacillus can be engineered to secrete bioactive exendin-4 and facilitate its transport through the intestinal barrier, providing a novel strategy for oral exendin-4 delivery using this lactic acid bacterium.

To seek evidence of a primitive adaptive immune system (AIS) before vertebrate, we examined whether lymphocytes or lymphocyte-like cells and the related molecules participating in the lymphocyte function existed in amphioxus. Anatomical analysis by electron microscopy revealed the presence of lymphocyte-like cells in gills, and these cells underwent morphological changes in response to microbial pathogens that are reminiscent of those of mammalian lymphocytes executing immune response to microbial challenge. In addition, a systematic comparative analysis of our cDNA database of amphioxus identified a large number of genes whose vertebrate counterparts are involved in lymphocyte function. Among these genes, several genes were found to be expressed in the vicinity of the lymphocyte-like cells by in situ hybridization and up-regulated after exposure to microbial pathogens. Our findings in the amphioxus indicate the twilight for the emergence of AIS before the invertebrate-vertebrate transition during evolution.