Sodium molybdate inhibits the growth of ovarian cancer cells via inducing both ferroptosis and apoptosis.

Ovarian cancer has the most mortality of all gynecologic malignancies. High-grade serous ovarian carcinoma (HGSOC) is the most common and deadly type of ovarian cancer. Tumor recurrence occurs due to the emergence of chemotherapy resistance. Thus, searching for new therapeutic strategies is essential for the management of ovarian cancer. Deregulation of iron metabolism can be used by ovarian cancer cells to survive, proliferate and metastasize. Here we report that sodium molybdate, a soluble molybdenum (Mo) compound, induces the elevation of the labile iron pool (LIP) in ovarian cancer cells, correlated with the down-regulation of genes involved in extracellular matrix organization. Sodium molybdate also induces depletion of glutathione (GSH) through mediating the production of nitric oxide (NO). Elevation of LIP and depletion of GSH promote the ferroptosis of ovarian cancer cells. Meanwhile, nitric oxide induces mitochondrial damage through inhibiting mitochondrial aconitase activity, ATP production, and mitochondrial membrane potential, leading to apoptosis of ovarian cancer cells. In vivo study shows that sodium molybdate reduces tumor burden in nude mice. Xenografts treated with sodium molybdate are characterized by obvious iron accumulation, increased expression of the iron storage protein ferritin, and lipid peroxide product 4-hydroxynonenal. In addition, an elevated percentage of apoptotic cells is observed in xenografts treated with sodium molybdate. Taken together, these results demonstrate that sodium molybdate can induce both ferroptosis and apoptosis of ovarian cancer cells, making it a potential therapeutic candidate for ovarian cancer.

Transcriptome analysis of Xanthomonas oryzae pv. oryzicola exposed to H2O2 reveals horizontal gene transfer contributes to its oxidative stress response.

Xanthomonas oryzae pv. oryzicola (Xoc), the causal agent of bacterial leaf streak, is one of the most severe seed-borne bacterial diseases of rice. However, the molecular mechanisms underlying Xoc in response to oxidative stress are still unknown. In this study, we performed a time-course RNA-seq analysis on the Xoc in response to H2O2, aiming to reveal its oxidative response network. Overall, our RNA sequence analysis of Xoc revealed a significant global gene expression profile when it was exposed to H2O2. There were 7, 177, and 246 genes that were differentially regulated at the early, middle, and late stages after exposure, respectively. Three genes (xoc_1643, xoc_1946, xoc_3249) showing significantly different expression levels had proven relationships with oxidative stress response and pathogenesis. Moreover, a hypothetical protein (XOC_2868) showed significantly differential expression, and the xoc_2868 mutants clearly displayed a greater H2O2 sensitivity and decreased pathogenicity than those of the wild-type. Gene localization and phylogeny analysis strongly suggests that this gene may have been horizontally transferred from a Burkholderiaceae ancestor. Our study not only provides a first glance of Xoc's global response against oxidative stress, but also reveals the impact of horizontal gene transfer in the evolutionary history of Xoc.

Autophagy Is Indispensable for the Self-Renewal and Quiescence of Ovarian Cancer Spheroid Cells with Stem Cell-Like Properties.

Epithelial ovarian cancer has the highest mortality rate of all gynecologic cancers. Cancer stem cells are considered to be the initiating cells of tumors. It is known that spheroid culture promotes ovarian cancer cells to acquire stem cell characteristics and to become stem cell-like. But the mechanisms remain largely unclear. Our data show that autophagy is sustainably activated in ovarian cancer spheroid cells. Inhibition of autophagy by knockdown of ATG5 abolishes the self-renewal ability of ovarian cancer spheroid cells. Knockdown of ATG5 prevents ovarian cancer spheroid cells to enter quiescent state. Autophagy is critical for quiescent ovarian cancer spheroid cells to reenter the cell cycle because rapamycin can promote quiescent ovarian cancer spheroid cells to form colonies on soft agar and knockdown of ATG5 can arrest ovarian cancer cells in G0/G1. Autophagy and NRF2 form a positive feedback regulation loop to regulate reactive oxygen species (ROS) levels in ovarian cancer spheroid cells. The optimal ROS level, neither too high nor too low, facilitates the self-renewal marker, NOTCH1, to reach to the highest level. Bafilomycin A1 can impair the self-renewal of ovarian cancer spheroid cells by disturbing ROS levels.

RNA-Seq-based transcriptome analysis of dormant flower buds of Chinese cherry (Prunus pseudocerasus).

Bud dormancy is a critical biological process allowing Chinese cherry (Prunus pseudocerasus) to survive in winter. Due to the lake of genomic information, molecular mechanisms triggering endodormancy release in flower buds have remained unclear. Hence, we used Illumina RNA-Seq technology to carry out de novo transcriptome assembly and digital gene expression profiling of flower buds. Approximately 47million clean reads were assembled into 50,604 sequences with an average length of 837bp. A total of 37,650 unigene sequences were successfully annotated. 128 pathways were annotated by Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis, and metabolic, biosynthesis of second metabolite and plant hormone signal transduction accounted for higher percentage in flower bud. In critical period of endodormancy release, 1644, significantly differentially expressed genes (DEGs) were identified from expression profile. DEGs related to oxidoreductase activity were especially abundant in Gene Ontology (GO) molecular function category. Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis demonstrated that DEGs were involved in various metabolic processes, including phytohormone metabolism. Quantitative real-time PCR (qRT-PCR) analysis indicated that levels of DEGs for abscisic acid and gibberellin biosynthesis decreased while the abundance of DEGs encoding their degradation enzymes increased and GID1 was down-regulated. Concomitant with endodormancy release, MADS-box transcription factors including P. pseudocerasus dormancy-associated MADS-box (PpcDAM), Agamous-like2, and APETALA3-like genes, shown remarkably epigenetic roles. The newly generated transcriptome and gene expression profiling data provide valuable genetic information for revealing transcriptomic variation during bud dormancy in Chinese cherry. The uncovered data should be useful for future studies of bud dormancy in Prunus fruit trees lacking genomic information.

Comparative analysis of the 5S rRNA and its associated proteins reveals unique primitive rather than parasitic features in Giardia lamblia.

BACKGROUND: 5S rRNA is a highly conserved ribosomal component. Eukaryotic 5S rRNA and its associated proteins (5S rRNA system) have become very well understood. Giardia lamblia was thought by some researchers to be the most primitive extant eukaryote while others considered it a highly evolved parasite. Previous reports have indicated that some aspects of its 5S rRNA system are simpler than that of common eukaryotes. We here explore whether this is true to its entire system, and whether this simplicity is a primitive or parasitic feature. METHODOLOGY/PRINCIPAL FINDINGS: By collecting and confirming pre-existing data and identifying new data, we obtained almost complete datasets of the system of three isolates of G. lamblia, two other parasitic excavates (Trichomonas vaginalis, Trypanosoma cruzi), and one free-living one (Naegleria gruberi). After comprehensively comparing each aspect of the system among these excavates and also with those of archaea and common eukaryotes, we found all the three Giardia isolates to harbor a same simplified 5S rRNA system, which is not only much simpler than that of common eukaryotes but also the simplest one among those of these excavates, and is surprisingly very similar to that of archaea; we also found among these excavates the system in parasitic species is not necessarily simpler than that in free-living species, conversely, the system of free-living species is even simpler in some respects than those of parasitic ones. CONCLUSION/SIGNIFICANCE: The simplicity of Giardia 5S rRNA system should be considered a primitive rather than parasitically-degenerated feature. Therefore, Giardia 5S rRNA system might be a primitive system that is intermediate between that of archaea and the common eukaryotic model system, and it may reflect the evolutionary history of the eukaryotic 5S rRNA system from the archaeal form. Our results also imply G. lamblia might be a primitive eukaryote with secondary parasitically-degenerated features.

Identification and characterization of putative CIPK genes in maize.

Calcium (Ca) plays a crucial role as a second messenger in intracellular signaling elicited by developmental and environmental cues. Calcineurin B-like proteins (CBLs) and their target proteins, CBL-interacting protein kinases (CIPKs) have emerged as a key Ca(2+)-mediated signaling network in response to stresses in plants. Bioinformatic analysis was used to identify 43 putative ZmCIPK (Zea mays CIPK) genes in the genome of maize inbred line B73. Based on gene structures, these ZmCIPKs were divided into intron-rich and intron-poor groups. Phylogenetic analysis indicated that the ZmCIPK family had a high evolutionary relationship with the rice CIPK family of 30 members. Microarray data and RT-PCR assay showed that ZmCIPK genes transcriptionally responded to abiotic stresses, and that 24, 31, 20 and 19 ZmCIPK genes were up-regulated by salt, drought, heat and cold stresses, respectively. There were different expression patterns of ZmCIPKs between cold-tolerant inbred line B73 and cold-sensitive inbred line Mo17 under cold stress. Our findings will aid further molecular dissection of biological functions of the CIPKs in maize, and provide new insight into the CBL-CIPK signaling network in plants.

Noisy splicing, more than expression regulation, explains why some exons are subject to nonsense-mediated mRNA decay.

BACKGROUND: Nonsense-mediated decay is a mechanism that degrades mRNAs with a premature termination codon. That some exons have premature termination codons at fixation is paradoxical: why make a transcript if it is only to be destroyed? One model supposes that splicing is inherently noisy and spurious transcripts are common. The evolution of a premature termination codon in a regularly made unwanted transcript can be a means to prevent costly translation. Alternatively, nonsense-mediated decay can be regulated under certain conditions so the presence of a premature termination codon can be a means to up-regulate transcripts needed when nonsense-mediated decay is suppressed. RESULTS: To resolve this issue we examined the properties of putative nonsense-mediated decay targets in humans and mice. We started with a well-annotated set of protein coding genes and found that 2 to 4% of genes are probably subject to nonsense-mediated decay, and that the premature termination codon reflects neither rare mutations nor sequencing artefacts. Several lines of evidence suggested that the noisy splicing model has considerable relevance: 1) exons that are uniquely found in nonsense-mediated decay transcripts (nonsense-mediated decay-specific exons) tend to be newly created; 2) have low-inclusion level; 3) tend not to be a multiple of three long; 4) belong to genes with multiple splice isoforms more often than expected; and 5) these genes are not obviously enriched for any functional class nor conserved as nonsense-mediated decay candidates in other species. However, nonsense-mediated decay-specific exons for which distant orthologous exons can be found tend to have been under purifying selection, consistent with the regulation model. CONCLUSION: We conclude that for recently evolved exons the noisy splicing model is the better explanation of their properties, while for ancient exons the nonsense-mediated decay regulated gene expression is a viable explanation.

Evidence for common short natural trans sense-antisense pairing between transcripts from protein coding genes.

BACKGROUND: There is increasing realization that regulation of genes is done partly at the RNA level by sense-antisense binding. Studies typically concentrate on the role of non-coding RNAs in regulating coding RNA. But the majority of transcripts in a cell are likely to be coding. Is it possible that coding RNA might regulate other coding RNA by short perfect sense-antisense binding? Here we compare all well-described human protein coding mRNAs against all others to identify sites 15-25 bp long that could potentially perfectly match sense-antisense. RESULTS: From 24,968 protein coding mRNA RefSeq sequences, none failed to find at least one match in the transcriptome. By randomizations generating artificial transcripts matched for G+C content and length, we found that there are more such trans short sense-antisense pairs than expected. Several further features are consistent with functionality of some of the putative matches. First, transcripts with more potential partners have lower expression levels, and the pair density of tissue specific genes is significantly higher than that of housekeeping genes. Further, the single nucleotide polymorphism density is lower in short pairing regions than it is in flanking regions. We found no evidence that the sense-antisense pairing regions are associated with small RNAs derived from the protein coding genes. CONCLUSIONS: Our results are consistent with the possibility of common short perfect sense-antisense pairing between transcripts of protein coding genes.

Alternative promoters influence alternative splicing at the genomic level.

BACKGROUND: More and more experiments have shown that transcription and mRNA processing are not two independent events but are tightly coupled to each other. Both promoter and transcription rate were found to influence alternative splicing. More than half of human genes have alternative promoters, but it is still not clear why there are so many alternative promoters and what their biological roles are. METHODOLOGY/PRINCIPAL FINDINGS: In this study, we explored whether there is a functional correlation between alternative promoters and alternative splicing by a genome-wide analysis of human and mouse genes. We constructed a large data set of genes with alternative promoter and alternative splicing annotations. By analyzing these genes, we showed that genes with alternative promoters tended to demonstrate alternative splicing compare to genes with single promoter, and, genes with more alternative promoters tend to have more alternative splicing variants. Furthermore, transcripts from different alternative promoters tended to splice differently. CONCLUSIONS/SIGNIFICANCE: Thus at the genomic level, alternative promoters are positively correlated with alternative splicing.

Phylogenetic positions of several amitochondriate protozoa--evidence from phylogenetic analysis of DNA topoisomerase II.

Several groups of parasitic protozoa, as represented by Giardia, Trichomonas, Entamoeba and Microsporida, were once widely considered to be the most primitive extant eukaryotic group--Archezoa. The main evidence for this is their 'lacking mitochondria' and possessing some other primitive features between prokaryotes and eukaryotes, and being basal to all eukaryotes with mitochondria in phylogenies inferred from many molecules. Some authors even proposed that these organisms diverged before the endosymbiotic origin of mitochondria within eukaryotes. This view was once considered to be very significant to the study of origin and evolution of eukaryotic cells (eukaryotes). However, in recent years this has been challenged by accumulating evidence from new studies. Here the sequences of DNA topoisomerase II in G lamblia, T. vaginalis and E. histolytica were identified first by PCR and sequencing, then combining with the sequence data of the microsporidia Encephalitozoon cunicul and other eukaryotic groups of different evolutionary positions from GenBank, phylogenetic trees were constructed by various methods to investigate the evolutionary positions of these amitochondriate protozoa. Our results showed that since the characteristics of DNA topoisomerase II make it avoid the defect of 'long-branch attraction' appearing in the previous phylogenetic analyses, our trees can not only reflect effectively the relationship of different major eukaryotic groups, which is widely accepted, but also reveal phylogenetic positions for these amitochondriate protozoa, which is different from the previous phylogenetic trees. They are not the earliest-branching eukaryotes, but diverged after some mitochondriate organisms such as kinetoplastids and mycetozoan; they are not a united group but occupy different phylogenetic positions. Combining with the recent cytological findings of mitochondria-like organelles in them, we think that though some of them (e.g. diplomonads, as represented by Giardia) may occupy a very low evolutionary position, generally these organisms are not as extremely primitive as was thought before; they should be polyphyletic groups diverging after the endosymbiotic origin of mitochondrion to adapt themselves to anaerobic parasitic life.

Identification of a Giardia krr1 homolog gene and the secondarily anucleolate condition of Giaridia lamblia.

Giaridia lamblia was long considered to be one of the most primitive eukaryotes and to lie close to the transition between prokaryotes and eukaryotes, but several supporting features, such as lack of mitochondrion and Golgi, have been challenged recently. It was also reported previously that G. lamblia lacked nucleolus, which is the site of pre-rRNA processing and ribosomal assembling in the other eukaryotic cells. Here, we report the identification of the yeast homolog gene, krr1, in the anucleolate eukaryote, G. lamblia. The krr1 gene, encoding one of the pre-rRNA processing proteins in yeast, is actively transcribed in G. lamblia. The deduced protein sequence of G. lamblia krr1 is highly similar to yeast KRR1p that contains a single-KH domain. Our database searches indicated that krr1 genes actually present in diverse eukaryotes and also seem to present in Archaea. However, only the eukaryotic homologs, including that of G. lamblia, have the single-KH domain, which contains the conserved motif KR(K)R. Fibrillarin, another important pre-rRNA processing protein has also been identified previously in G. lamblia. Moreover, our database search shows that nearly half of the other nucleolus-localized protein genes of eukaryotic cells also have their homologs in Giardia. Therefore, we suggest that a common mechanism of pre-RNA processing may operate in the anucleolate eukaryote G. lamblia and in the other eukaryotes and that like the case of "lack of mitochondrion," "lack of nucleolus" may not be a primitive feature, but a secondarily evolutionary condition of the parasite.