Transcriptomic and gene-family dynamic analyses reveal gene expression pattern and evolution in toxin-producing tissues of Asiatic toad (Bufo gargarizans)

Comprising a major clade of Anura, toads produce and secrete numerous toxins from both the parotoid glands behind their eyes and their dorsal skin. These toxins, made of various proteins and compounds, possess pharmacological potential to be repurposed to benefit human health. However, the detailed genetic regulation of toad toxin production is still poorly understood. A recent publication uncovering the genome of the representative Asiatic toad (Bufo gargarizans) provides a good reference to resolve this issue. In the present study, we sequenced the transcriptomes of parotoid gland, dorsal skin and liver from the Asiatic toad. Combining our data with 35 previously published transcriptomes across eight different tissues from the same species but from different locations, we constructed a comprehensive gene co-expression network of the Asiatic toad with the assistance of the reference genome assembly. We identified 2,701 co-expressed genes in the toxin-producing tissues (including parotoid gland and dorsal skin). By comparative genomic analysis, we identified 599 expanded gene families with 2,720 genes. Through overlapping these co-expressed genes in the toad toxin-producing tissues, we observed that three cytochrome P450 (Cyp) family members (Cyp27a1, Cyp2c29, and Cyp2c39) were significantly enriched in pathways related to cholesterol metabolism. Cholesterol is a critical precursor to steroids, and the known main steroidal toxins of bufadienolides are considered as the major bioactive components in the parotoid glands of Asiatic toad. We found 3-hydroxy-methylglutaryl CoA reductase (hmgcr), encoding the major rate-limiting enzyme for cholesterol biosynthesis, appears with multiple copies in both Asiatic toad and common toad, possibly originating from a tandem duplication event. The five copies of hmgcr genes consistently displayed higher transcription levels in the parotoid gland when compared with the abdominal skin, suggesting it as a vital candidate gene in the involvement of toad toxin production. Taken together, our current study uncovers transcriptomic and gene-family dynamic evidence to reveal the vital role of both expanded gene copies and gene expression changes for production of toad toxins

An Updated Genome Assembly Improves Understanding of the Transcriptional Regulation of Coloration in Midas Cichlid

Midas cichlid (Amphilophus citrinellus), a popular aquarium fish, attracts extensive attention from worldwide biologists mainly due to its morphological polymorphism (dark versus gold). Continuous efforts have therefore been paid to address mechanisms of its coloration variants, while it is far away from the detailed illustration of a clear regulatory network. Some limits may come from the absence of a high-quality genome assembly and a relatively accurate gene set. In this study, we sequenced about 149 Gb of nucleotide sequences of Midas cichlid, generating a genome assembly with a total size of 933.5 Mb, which exhibits a good genome continuity with a contig N50 of 10.5 Mb. A total of 25,911 protein-coding genes were annotated and about 90% completeness was achieved, which helps to build a good gene pool for understanding expressional differences of color variation. With the assistance of the final gene set, we identified a total of 277 differential expressional genes (DEGs), of which 97 up- and 180 downregulated were determined in dark-vs-gold comparisons. Two protein-protein interaction (PPI) networks were constructed from these DEGs, and three key functional modules were classified. Hub genes within each module were evaluated, and we found that the third key module contains tyrp1b, oca2, pmela, tyr, and slc24a5, which were previously proven to be associated with melanin formation. Two downregulated DEGs (myl1 and pgam2) in the first key module may be involved in muscle movement and spermatogenesis, implying that certain side effects could result from the morphological polymorphism. The first key module, consisting of proteins encoded by upregulated DEGs that were associated with MAPK signaling, Toll-like receptor signaling, and gonadotropin-releasing hormone pathways, may contribute to a negative upstream regulation or downstream influence on melanin biosynthesis. Taken together, our new genome assembly and gene annotation of Midas cichlid provide a high-quality genetic resource for biological studies on this species, and the newly identified key networks and hub genes in dark-vs-gold comparisons enhance our understanding of the transcriptional regulatory mechanisms underlying coloration changes not only in Midas cichlid but also in other fishes from freshwater to marine ecosystems

Genome Assembly for a Yunnan-Guizhou Plateau “3E” Fish, Anabarilius grahami (Regan), and Its Evolutionary and Genetic Applications

A Yunnan-Guizhou Plateau fish, the Kanglang white minnow (Anabarilius grahami), is a typical “3E” (Endangered, Endemic, and Economic) species in China. Its distribution is limited to Fuxian Lake, the nation’s second deepest lake, with a significant local economic value but a drastically declining wild population. This species has been evaluated as VU (Vulnerable) in the China Species Red List. As one of the “Four Famous Fish” in Yunnan province, the artificial breeding has been achieved since 2003. It has not only re-established its wild natural populations by reintroduction of the artificial breeding stocks, but also brought a wide and popular utilization of this species to the local fish farms. A. grahami has become one of the main native aquaculture species in Yunnan province, and the artificial production has been emerging in steady growth each year. To promote the conservation and sustainable utilization of this fish, we initiated its whole genome sequencing project using an Illumina Hiseq2500 platform. The assembled genome size of A. grahami is 1.006 Gb, accounting for 98.63% of the estimated genome size (1.020 Gb), with contig N50 and scaffold N50 values of 26.4 kb and 4.41 Mb, respectively. Approximately about 50.38% of the genome was repetitive. A total of 25,520 protein-coding genes were subsequently predicted. A phylogenetic tree based on 4,580 single-copy genes from A. grahami and 18 other cyprinids revealed three well-supported subclades within the Cyprinidae. This is the first inter-subfamily relationship of cyprinids at genome level, providing a simple yet useful framework for understanding the traditional but popular subfamily classification systems. Interestingly, a further population demography of A. grahami uncovered a historical relationship between this fish and Fuxian Lake, suggesting that range expansion or shrinkage of the habitat has had a remarkable impact on the population size of endemic plateau fishes. Additionally, a total of 33,836 simple sequence repeats (SSR) markers were identified, and 11 loci were evaluated for a preliminary genetic diversity analysis in this study, thus providing another useful genetic resource for studying this “3E” species

Extensive pyrosequencing reveals frequent intra-genomic variations of internal transcribed spacer regions of nuclear ribosomal DNA

BACKGROUND: Internal transcribed spacer of nuclear ribosomal DNA (nrDNA) is already one of the most popular phylogenetic and DNA barcoding markers. However, the existence of its multiple copies has complicated such usage and a detailed characterization of intra-genomic variations is critical to address such concerns. METHODOLOGY/PRINCIPAL FINDINGS: In this study, we used sequence-tagged pyrosequencing and genome-wide analyses to characterize intra-genomic variations of internal transcribed spacer 2 (ITS2)regions from 178 plant species. We discovered that mutation of ITS2 is frequent, with a mean of 35 variants per species. And on average, three of the most abundant variants make up 91% of all ITS2 copies. Moreover, we found different congeneric species share identical variants in 13 genera. Interestingly, different species across different genera also share identical variants. In particular, one minor variant of ITS2 in Eleutherococcus giraldii was found identical to the ITS2 major variant of Panax ginseng, both from Araliaceae family. In addition, DNA barcoding gap analysis showed that the intra-genomic distances were markedly smaller than those of the intra-specific or inter-specific variants. When each of 5543 variants were examined for its species discrimination efficiency, a 97% success rate was obtained at the species level. CONCLUSIONS: Identification of identical ITS2 variants across intra-generic or inter-generic species revealed complex species evolutionary history, possibly, horizontal gene transfer and ancestral hybridization. Although intra-genomic multiple variants are frequently found within each genome, the usage of the major variants alone is sufficient for phylogeny construction and species determination in most cases. Furthermore, the inclusion of minor variants further improves the resolution of species identification.Jingyuan Song, Linchun Shi, Dezhu Li, Yongzhen Sun, Yunyun Niu, Zhiduan Chen, Hongmei Luo, Xiaohui Pang, Zhiying Sun, Chang Liu, Aiping Lv, Youping Deng, Zachary Larson-Rabin, Mike Wilkinson and Shilin Che

KIF2A silencing inhibits the proliferation and migration of breast cancer cells and correlates with unfavorable prognosis in breast cancer

Background; Kinesin family member 2a (KIF2A), a type of motor protein found in eukaryotic cells, is associated with development and progression of various human cancers. The role of KIF2A during breast cancer tumorigenesis and progression was studied. Methods; Immunohistochemical staining, real time RT-PCR and western blot were used to examine the expression of KIF2A in cancer tissues and adjacent normal tissues from breast cancer patients. Patients’ survival in relation to KIF2A expression was estimated using the Kaplan–Meier survival and multivariate analysis. Breast cancer cell line, MDA-MB-231 was used to study the proliferation, migration and invasion of cells following KIF2A-siRNA transfection. Results; The expression of KIF2A in cancer tissues was higher than that in normal adjacent tissues from the same patient (P < 0.05). KIF2A expression in cancer tissue with lymph node metastasis and HER2 positive cancer were higher than that in cancer tissue without (P < 0.05). A negative correlation was found between KIF2A expression levels in breast cancer and the survival time of breast cancer patients (P < 0.05). In addition, multivariate analysis indicated that KIF2A was an independent prognostic for outcome in breast cancer (OR: 16.55, 95% CI: 2.216-123.631, P = 0.006). The proliferation, migration and invasion of cancer cells in vitro were suppressed by KIF2A gene silencing (P < 0.05). Conclusions; KIF2A may play an important role in breast cancer progression and is potentially a novel predictive and prognostic marker for breast cancer

Pyrosequencing of the Camptotheca acuminata transcriptome reveals putative genes involved in camptothecin biosynthesis and transport

Background: Camptotheca acuminata is a Nyssaceae plant, often called the "happy tree", which is indigenous in Southern China. C. acuminata produces the terpenoid indole alkaloid, camptothecin (CPT), which exhibits clinical effects in various cancer treatments. Despite its importance, little is known about the transcriptome of C. acuminata and the mechanism of CPT biosynthesis, as only few nucleotide sequences are included in the GenBank database.Results: From a constructed cDNA library of young C. acuminata leaves, a total of 30,358 unigenes, with an average length of 403 bp, were obtained after assembly of 74,858 high quality reads using GS De Novo assembler software. Through functional annotation, a total of 21,213 unigenes were annotated at least once against the NCBI nucleotide (Nt), non-redundant protein (Nr), Uniprot/SwissProt, Kyoto Encyclopedia of Genes and Genomes (KEGG), and Arabidopsis thaliana proteome (TAIR) databases. Further analysis identified 521 ESTs representing 20 enzyme genes that are involved in the backbone of the CPT biosynthetic pathway in the library. Three putative genes in the upstream pathway, including genes for geraniol-10-hydroxylase (CaPG10H), secologanin synthase (CaPSCS), and strictosidine synthase (CaPSTR) were cloned and analyzed. The expression level of the three genes was also detected using qRT-PCR in C. acuminata. With respect to the branch pathway of CPT synthesis, six cytochrome P450s transcripts were selected as candidate transcripts by detection of transcript expression in different tissues using qRT-PCR. In addition, one glucosidase gene was identified that might participate in CPT biosynthesis. For CPT transport, three of 21 transcripts for multidrug resistance protein (MDR) transporters were also screened from the dataset by their annotation result and gene expression analysis.Conclusion: This study produced a large amount of transcriptome data from C. acuminata by 454 pyrosequencing. According to EST annotation, catalytic features prediction, and expression analysis, novel putative transcripts involved in CPT biosynthesis and transport were discovered in C. acuminata. This study will facilitate further identification of key enzymes and transporter genes in C. acuminata

Essays on Political Economy

This dissertation is a collection of essays focused on understanding: (1) the strategic interactions related to censorship; and (2) the force behind changes across time in public policies, including emigration policies and governmental structures, in autocracies. Essay 1 theoretically and empirically analyzes censorship on social media platforms in China. If the government sets a higher tax rate for the next tax year, it will benefit more from the profit made by the company. Hence the government has weaker incentives to punish the company in case of disobedience. Therefore, the company has weaker incentives to comply with the government's order to censor sensitive content. The effort of censorship may be all in vain due to the paradox of censorship: The more the censor suppresses whatever the censor dislikes, the more attention that disliked subject receives. Essay 2 explains how the censor strategically times censorship when considering that it may backfire. In equilibrium, if the censor stops the discussion about a piece of news and the learning process sooner, the receiver believes he is more likely a bad type. Essay 3 uses an infinite-horizon principal-agent model to explore the interaction between the hierarchical structures of governments and the career concerns of local officials in China. This paper shows that there exists a vertical structure that dominates all horizontal structures. Vertical structures generate less uncertainty in the promotion process and hence a clearer career path than horizontal structures. Therefore, vertical structures are more efficient in incentivizing local officials to work. Essay 4 explains why autocrats and dictators draft the emigration policy so that emigration is sometimes easy for citizens, sometimes almost impossible. There are clear gains in opening the floodgates: If more citizens leave the country, fewer citizens will participate in revolutionary activities, making it more likely for the government to remain in power. Under certain conditions, this improvement in stability dominates the loss in GDP caused by emigration

The paddy frog genome provides insight into the molecular adaptations and regulation of hibernation in ectotherms

Summary: Amphibians, like the paddy frog (Fejervarya multistriata), have played a critical role in the transition from water to land. Hibernation is a vital survival adaptation in cold environments with limited food resources. We decoded the paddy frog genome to reveal the molecular adaptations linked to hibernation in ectotherms. The genome contained 13 chromosomes, with a significant proportion of repetitive sequences. We identified the key genes encoding the proteins of AANAT, TRPM8, EGLN1, and VEGFA essential for circadian rhythms, thermosensation, and hypoxia during hibernation by comparing the hibernator and non-hibernator genomes. Examining organ changes during hibernation revealed the central regulatory role of the brain. We identified 21 factors contributing to hibernation, involving hormone biosynthesis, protein digestion, DNA replication, and the cell cycle. These findings provide deeper insight into the complex mechanisms of ectothermic hibernation and contribute to our understanding of the broader significance of this evolutionary adaptation