A novel poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV)-PEG-melatonin composite scaffold enhances for inhibiting bone tumor recurrence and enhancing bone regeneration.

Introduction: Postoperative comprehensive treatment has become increasingly important in recent years. This study was to repair tissue defects resulting from the removal of diseased tissue and to eliminate or inhibit the recurrence and metastasis of residual tumors under the condition of reducing the systemic side effects of chemotherapeutic drugs. To address these challenges, multifunctional scaffolds based local drug delivery systems will be a promising solution. Methods: An optimal drug-loaded scaffold material PHBV-mPEG5k (PP5) was prepared, which is biocompatible, hydrophilic and biodegradable. Furthermore, this material showed to promote bone healing, and could be conveniently prepared into porous scaffold by freeze-drying the solution. By means of introducing melatonin (MT) into the porous surfaces, the MT loaded PP5 scaffold with desirable sustained release ability was successfully prepared. The effectiveness of the MT loaded PP5 scaffold in promoting bone repair and anti-tumor properties was evaluated through both in vivo and in vitro experiments. Results and Discussion: The MT loaded PP5 scaffold is able to achieve the desired outcome of bone tissue repair and anti-bone tumor properties. Furthermore, our study demonstrates that the PP5 scaffold was able to enhance the anti-tumor effect of melatonin by improving cellular autophagy, which provided a therapeutic strategy for the comprehensive postoperative treatment of osteosarcoma.

An efficient approach to synthesize sterile allopolyploids through the combined reproduction mode of ameiotic oogenesis and sperm-egg fusion in the polyploid Carassius complex.

The association between polyploidy and reproduction transition, which is an intriguing issue in evolutionary genetics, can also be exploited as an approach for genetic improvement in agriculture. Recently, we generated novel amphitriploids (NA3n) by integrating the genomes of the gynogenetic Carassius gibelio and sexual C. auratus, and found gynogenesis was recovered in most NA3n females (NA3n♀I). Here, we discovered a unique reproduction mode, termed ameio-fusiongenesis, which combines the abilities of both ameiotic oogenesis and sperm-egg fusion, in a few NA3n females (NA3n♀II). These females inherited ameiotic oogenesis to produce unreduced eggs from gynogenetic C. gibelio and sperm-egg fusion from sexual C. auratus. Subsequently, we utilized this unique reproduction mode to generate a group of synthetic alloheptaploids by crossing NA3n♀II with Megalobrama amblycephala. They contained all chromosomes of maternal NA3n♀II and a chromosomal set of paternal M. amblycephala. Intergenomic chromosome translocations between NA3n♀II and M. amblycephala were also observed in a few somatic cells. Primary oocytes of the alloheptaploid underwent severe apoptosis owing to incomplete double-strand break repair at prophase I. Although spermatocytes displayed similar chromosome behavior at prophase I, they underwent apoptosis due to chromosome separation failure at metaphase I. Therefore, the alloheptaploid females and males were all sterile. Finally, we established a sustainable clone for the large-scale production of NA3n♀II and developed an efficient approach to synthesize diverse allopolyploids containing genomes of different cyprinid species. These findings not only broaden our understanding of reproduction transition but also offer a practical strategy for polyploidy breeding and heterosis fixing.

Gonadal transcriptomes reveal sex-biased expression genes associated with sex determination and differentiation in red-tail catfish (Hemibagrus wyckioides).

BACKGROUND: Red-tail catfish (Hemibagrus wyckioides) is an important commercially farmed catfish in southern China. Males of red-tail catfish grow faster than females, suggesting that all-male catfish will produce more significant economic benefits in aquaculture practice. However, little research has been reported on sex determination and gonadal development in red-tail catfish. RESULTS: In this study, we performed the first transcriptomic analysis of male and female gonads at four developmental stages at 10, 18, 30, and 48 days post hatching (dph) using RNA-seq technology. A total of 23,588 genes were screened in 24 sequenced samples, of which 28, 213, 636, and 1381 differentially expressed genes (DEGs) were detected at four developmental stages, respectively. Seven candidate genes of sex determination and differentiation were further identified. Real-time quantitative PCR (RT-qPCR) further confirmed that anti-Mullerian hormone (amh), growth differentiation factor 6a (gdf6a), testis-specific gene antigen 10 (tsga10), and cytochrome P450 family 17 subfamily A (cyp17a) were highly expressed mainly in the male, while cytochrome P450 family 19 subfamily A polypeptide 1b (cyp19a1b), forkhead box L2 (foxl2), and hydroxysteroid 17-beta dehydrogenase 1 (hsd17b1) were highly expressed in the female. The KEGG pathway enrichment data showed that these identified DEGs were mainly involved in steroid hormone biosynthesis and TGF-ß signaling pathways. CONCLUSIONS: Based on RNA-seq data of gonads at the early developmental stages, seven DEGs shared by the four developmental stages were identified, among which amh and gdf6a may be the male-biased expression genes, while foxl2, cyp19a1b and hsd17b1 may be the female-biased expression genes in red-tail catfish. Our study will provide crucial genetic information for the research on sex control in red-tail catfish, as well as for exploring the evolutionary processes of sex determination mechanisms in fish.

Changes in Ploidy Drive Reproduction Transition and Genomic Diversity in a Polyploid Fish Complex.

Unisexual animals are commonly found in some polyploid species complexes, and most of these species have had a long evolutionary history. However, their method for avoiding genomic decay remains unclear. The polyploid Carassius complex naturally comprises the sexual amphidiploid C. auratus (crucian carp or goldfish) (AABB) and the gynogenetic amphitriploid C. gibelio (gibel carp) (AAABBB). Recently, we developed a fertile synthetic amphitetraploid (AAAABBBB) male from C. gibelio by incorporating a C. auratus genome. In this study, we generated novel amphitriploids (AAABBB) by backcrossing the amphitetraploid male with the amphidiploid C. auratus. Whole-genome resequencing revealed the genomic changes, including recombination and independent assortment between homologs of C. gibelio and C. auratus. The fertility, sex determination system, oocyte development, and fertilization behaviors of the novel amphitriploids were investigated. Approximately 80% of the novel amphitriploid females recovered the unisexual gynogenesis ability. Intriguingly, two types of primary oocyte (with and without homolog synapsis) were discovered, and their distinct development fates were observed. Type I oocytes entered apoptosis due to improper synaptonemal complex assembly and incomplete double-strand break repair, whereas subsequent type II oocytes bypassed meiosis through an alternative ameiotic pathway to develop into mature eggs. Moreover, gynogenesis was stabilized in their offspring, and a new array of diverse gynogenetic amphitriploid clones was produced. These revealed genomic changes and detailed cytological data provide comprehensive evidence that changes in ploidy drive unisexual and sexual reproduction transition, thereby resulting in genomic diversity and allowing C. gibelio avoid genomic decay.

Comparative genome anatomy reveals evolutionary insights into a unique amphitriploid fish.

Triploids are rare in nature because of difficulties in meiotic and gametogenic processes, especially in vertebrates. The Carassius complex of cyprinid teleosts contains sexual tetraploid crucian carp/goldfish (C. auratus) and unisexual hexaploid gibel carp/Prussian carp (C. gibelio) lineages, providing a valuable model for studying the evolution and maintenance mechanism of unisexual polyploids in vertebrates. Here we sequence the genomes of the two species and assemble their haplotypes, which contain two subgenomes (A and B), to the chromosome level. Sequencing coverage analysis reveals that C. gibelio is an amphitriploid (AAABBB) with two triploid sets of chromosomes; each set is derived from a different ancestor. Resequencing data from different strains of C. gibelio show that unisexual reproduction has been maintained for over 0.82 million years. Comparative genomics show intensive expansion and alterations of meiotic cell cycle-related genes and an oocyte-specific histone variant. Cytological assays indicate that C. gibelio produces unreduced oocytes by an alternative ameiotic pathway; however, sporadic homologous recombination and a high rate of gene conversion also exist in C. gibelio. These genomic changes might have facilitated purging deleterious mutations and maintaining genome stability in this unisexual amphitriploid fish. Overall, the current results provide novel insights into the evolutionary mechanisms of the reproductive success in unisexual polyploid vertebrates.

Two duplicated gsdf homeologs cooperatively regulate male differentiation by inhibiting cyp19a1a transcription in a hexaploid fish.

Although evolutionary fates and expression patterns of duplicated genes have been extensively investigated, how duplicated genes co-regulate a biological process in polyploids remains largely unknown. Here, we identified two gsdf (gonadal somatic cell-derived factor) homeologous genes (gsdf-A and gsdf-B) in hexaploid gibel carp (Carassius gibelio), wherein each homeolog contained three highly conserved alleles. Interestingly, gsdf-A and gsdf-B transcription were mainly activated by dmrt1-A (dsx- and mab-3-related transcription factor 1) and dmrt1-B, respectively. Loss of either gsdf-A or gsdf-B alone resulted in partial male-to-female sex reversal and loss of both caused complete sex reversal, which could be rescued by a nonsteroidal aromatase inhibitor. Compensatory expression of gsdf-A and gsdf-B was observed in gsdf-B and gsdf-A mutants, respectively. Subsequently, we determined that in tissue culture cells, Gsdf-A and Gsdf-B both interacted with Ncoa5 (nuclear receptor coactivator 5) and blocked Ncoa5 interaction with Rora (retinoic acid-related orphan receptor-alpha) to repress Rora/Ncoa5-induced activation of cyp19a1a (cytochrome P450, family 19, subfamily A, polypeptide 1a). These findings illustrate that Gsdf-A and Gsdf-B can regulate male differentiation by inhibiting cyp19a1a transcription in hexaploid gibel carp and also reveal that Gsdf-A and Gsdf-B can interact with Ncoa5 to suppress cyp19a1a transcription in vitro. This study provides a typical case of cooperative mechanism of duplicated genes in polyploids and also sheds light on the conserved evolution of sex differentiation.

Two Duplicated Ptpn6 Homeologs Cooperatively and Negatively Regulate RLR-Mediated IFN Response in Hexaploid Gibel Carp.

Src homology region 2 domain-containing phosphatase 1 (SHP1), encoded by the protein tyrosine phosphatase nonreceptor type 6 (ptpn6) gene, belongs to the family of protein tyrosine phosphatases (PTPs) and participates in multiple signaling pathways of immune cells. However, the mechanism of SHP1 in regulating fish immunity is largely unknown. In this study, we first identified two gibel carp (Carassius gibelio) ptpn6 homeologs (Cgptpn6-A and Cgptpn6-B), each of which had three alleles with high identities. Then, relative to Cgptpn6-B, dominant expression in adult tissues and higher upregulated expression of Cgptpn6-A induced by polyinosinic-polycytidylic acid (poly I:C), poly deoxyadenylic-deoxythymidylic (dA:dT) acid and spring viremia of carp virus (SVCV) were uncovered. Finally, we demonstrated that CgSHP1-A (encoded by the Cgptpn6-A gene) and CgSHP1-B (encoded by the Cgptpn6-B gene) act as negative regulators of the RIG-I-like receptor (RLR)-mediated interferon (IFN) response via two mechanisms: the inhibition of CaTBK1-induced phosphorylation of CaMITA shared by CgSHP1-A and CgSHP1-B, and the autophagic degradation of CaMITA exclusively by CgSHP1-A. Meanwhile, the data support that CgSHP1-A and CgSHP1-B have sub-functionalized and that CgSHP1-A overwhelmingly dominates CgSHP1-B in the process of RLR-mediated IFN response. The current study not only sheds light on the regulative mechanism of SHP1 in fish immunity, but also provides a typical case of duplicated gene evolutionary fates.

Identification of Novel Biomarkers for Predicting Prognosis and Immunotherapy Response in Head and Neck Squamous Cell Carcinoma Based on ceRNA Network and Immune Infiltration Analysis.

OBJECTIVES: Patients with head and neck squamous cell carcinoma (HNSCC) have poor prognosis and show poor responses to immune checkpoint (IC) inhibitor (ICI) therapy. Competing endogenous RNA (ceRNA) networks, tumor-infiltrating immune cells (TIICs), and ICIs may influence tumor prognosis and response rates to ICI therapy. This study is aimed at identifying prognostic and IC-related biomarkers and key TIIC signatures to improve prognosis and ICI therapy response in HNSCC patients. METHODS AND RESULTS: Ninety-five long noncoding RNAs (lncRNAs), microRNAs (miRNAs), and 1746 mRNAs were identified using three independent methods. We constructed a ceRNA network and estimated the proportions of 22 immune cell subtypes. Ten ceRNAs were related to prognosis according to Kaplan-Meier analysis. Two risk signatures based, respectively, on nine ceRNAs (ANLN, CFL2, ITGA5, KDELC1, KIF23, NFIA, PTX3, RELT, and TMC7) and three immune cell types (naïve B cells, neutrophils, and regulatory T cells) via univariate Cox regression, least absolute shrinkage and selection operator, and multivariate Cox regression analyses could accurately and independently predict the prognosis of HNSCC patients. Key mRNAs in the ceRNA network were significantly correlated with naïve B cells and regulatory T cells and with stage, grade, and immune and molecular subtype. Eight IC genes exhibited higher expression in tumor tissues and were correlated with eight key mRNAs in the ceRNA network in HNSCC patients with different HPV statuses according to coexpression and TIMER 2.0 analyses. Most drugs were effective in association with expression of these key signatures (ANLN, CFL2, ITGA5, KIF23, NFIA, PTX3, RELT, and TMC7) based on GSCALite analysis. The prognostic value of key biomarkers and associations between key ceRNAs and IC genes were validated using online databases. Eight key ceRNAs were confirmed to predict response to ICI in other cancers based on TIDE analysis. CONCLUSIONS: We constructed two risk signatures to accurately predict prognosis in HNSCC. Key IC-related signatures may be associated with response to ICI therapy. Combinations of ICIs with inhibitors of eight key mRNAs may improve survival outcomes of HNSCC patients.

Divergent Antiviral Mechanisms of Two Viperin Homeologs in a Recurrent Polyploid Fish.

Polyploidy and subsequent diploidization provide genomic opportunities for evolutionary innovations and adaptation. The researches on duplicated gene evolutionary fates in recurrent polyploids have seriously lagged behind that in paleopolyploids with diploidized genomes. Moreover, the antiviral mechanisms of Viperin remain largely unclear in fish. Here, we elaborate the distinct antiviral mechanisms of two viperin homeologs (Cgviperin-A and Cgviperin-B) in auto-allo-hexaploid gibel carp (Carassius gibelio). First, Cgviperin-A and Cgviperin-B showed differential and biased expression patterns in gibel carp adult tissues. Subsequently, using co-immunoprecipitation (Co-IP) screening analysis, both CgViperin-A and CgViperin-B were found to interact with crucian carp (C. auratus) herpesvirus (CaHV) open reading frame 46 right (ORF46R) protein, a negative herpesvirus regulator of host interferon (IFN) production, and to promote the proteasomal degradation of ORF46R via decreasing K63-linked ubiquitination. Additionally, CgViperin-B also mediated ORF46R degradation through autophagosome pathway, which was absent in CgViperin-A. Moreover, we found that the N-terminal α-helix domain was necessary for the localization of CgViperin-A and CgViperin-B at the endoplasmic reticulum (ER), and the C-terminal domain of CgViperin-A and CgViperin-B was indispensable for the interaction with degradation of ORF46R. Therefore, the current findings clarify the divergent antiviral mechanisms of the duplicated viperin homeologs in a recurrent polyploid fish, which will shed light on the evolution of teleost duplicated genes.

Functional Divergence of Multiple Duplicated Foxl2 Homeologs and Alleles in a Recurrent Polyploid Fish.

Evolutionary fates of duplicated genes have been widely investigated in many polyploid plants and animals, but research is scarce in recurrent polyploids. In this study, we focused on foxl2, a central player in ovary, and elaborated the functional divergence in gibel carp (Carassius gibelio), a recurrent auto-allo-hexaploid fish. First, we identified three divergent foxl2 homeologs (Cgfoxl2a-B, Cgfoxl2b-A, and Cgfoxl2b-B), each of them possessing three highly conserved alleles and revealed their biased retention/loss. Then, their abundant sexual dimorphism and biased expression were uncovered in hypothalamic-pituitary-gonadal axis. Significantly, granulosa cells and three subpopulations of thecal cells were distinguished by cellular localization of CgFoxl2a and CgFoxl2b, and the functional roles and the involved process were traced in folliculogenesis. Finally, we successfully edited multiple foxl2 homeologs and/or alleles by using CRISPR/Cas9. Cgfoxl2a-B deficiency led to ovary development arrest or complete sex reversal, whereas complete disruption of Cgfoxl2b-A and Cgfoxl2b-B resulted in the depletion of germ cells. Taken together, the detailed cellular localization and functional differences indicate that Cgfoxl2a and Cgfoxl2b have subfunctionalized and cooperated to regulate folliculogenesis and gonad differentiation, and Cgfoxl2b has evolved a new function in oogenesis. Therefore, the current study provides a typical case of homeolog/allele diversification, retention/loss, biased expression, and sub-/neofunctionalization in the evolution of duplicated genes driven by polyploidy and subsequent diploidization from the recurrent polyploid fish.

Regain of sex determination system and sexual reproduction ability in a synthetic octoploid male fish.

Polyploids in vertebrates are generally associated with unisexual reproduction, but the direct consequences of polyploidy on sex determination system and reproduction mode remain unknown. Here, we synthesized a group of artificial octoploids between unisexual gynogenetic hexaploid Carassius gibelio and sexual tetraploid Carassius auratus. The synthetic octoploids were revealed to have more than 200 chromosomes, in which 50 chromosomes including the X/Y sex determination system were identified to transfer from sexual tetraploid C. auratus into the unisexual gynogenetic hexaploid C. gibelio. Significantly, a few synthetic octoploid males were found to be fertile, and one octoploid male was confirmed to regain sexual reproduction ability, which exhibits characteristics that are the same to sexual reproduction tetraploid males, such as 1:1 sex ratio occurrence, meiosis completion and euploid sperm formation in spermatogenesis, as well as normal embryo development and gene expression pattern during embryogenesis. Therefore, the current finding provides a unique case to explore the effect of sex determination system incorporation on reproduction mode transition from unisexual gynogenesis to sexual reproduction along with genome synthesis of recurrent polyploidy in vertebrates.

Prognostic Value and Immune Infiltrates of ABCA8 and FABP4 in Stomach Adenocarcinoma.

BACKGROUND: Stomach adenocarcinoma (STAD) is a common malignancy worldwide with poor prognosis. Therefore, it is important to identify a valuable prognostic biomarker for STAD. The aim of present study was to identify novel prognostic biomarkers for STAD and evaluate the potential role of hub genes in STAD. METHODS: Gene Expression Profiling Interactive Analysis (GEPIA) and Cancer RNA-Seq Nexus were performed to identify differentially expressed genes (DEGs). Subsequently, hub genes were selected by a Venn diagram, and the expression of key genes was confirmed by UALCAN database. Furthermore, survival analysis of these hub genes was performed using Oncolnc and Human Protein Atlas (HPA) database. Gene alteration status of hub genes was assessed by cBioPortal. Finally, we investigated the association between hub genes and immune cell infiltration in STAD through the Tumor Immune Estimation Resource (TIMER) and GEPIA database. RESULTS: Three common hub genes were obtained, including 2 downregulated DEGs (ABCA8 and FABP4) and one upregulated DEG (SLC52A3). Furthermore, increased expression of ABCA8 and FABP4 and decreased expression of SLC52A3 were correlated with poor prognosis. Meanwhile, three hub genes showed genetic alterations in various datasets of STAD. Finally, our results showed that ABCA8 and FABP4 displayed a positive correlation with immune infiltration, especially in M2 macrophages. CONCLUSIONS: The results of this study suggest that ABCA8 and FABP4 may be used as prognostic biomarkers and correlated with immune infiltration in STAD.

Characterization of Peptacetobacter hominis gen. nov., sp. nov., isolated from human faeces, and proposal for the reclassification of Clostridium hiranonis within the genus Peptacetobacter.

A novel, Gram-stain-positive, rod-shaped, non-motile, non-spore-forming, obligately anaerobic bacterium, designated strain ZHW00191T, was isolated from human faeces and characterized by using a polyphasic taxonomic approach. Growth occurred at 25-45 °C (optimum, 37-42 °C), at pH 5.5-10.0 (optimum, pH 6.5-7.0) and with 0-2 % (w/v) NaCl (optimum, 0 %). The end products of glucose fermentation were acetic acid, isobutyric acid and isovaleric acid and a small amount of propionic acid. The dominant cellular fatty acids (>10 %) of strain ZHW00191T were C16 : 0, C18 : 1 ω9с and C18 : 2ω6,9с. Its polar lipid profile comprised diphosphatidylglycerol, phosphatidylglycerol, three unidentified phospholipids and ten unidentified glycolipids. Respiratory quinones were not detected. The cell-wall peptidoglycan contained meso-2,6-diaminopimelic acid, and the whole-cell sugars were ribose and glucose. The genomic DNA G+C content was 32.8 mol%. Analysis of the 16S rRNA gene sequence indicated that ZHW00191T was most closely related to Clostridium hiranonis TO-931T (95.3 % similarity). Average nucleotide identity (ANI) and digital DNA-DNA hybridization (dDDH) analyses with closely related reference strains indicated that reassociation values were both well below the thresholds of 95-96% and 70 % for species delineation, respectively. Based on phenotypic, chemotaxonomic and genetic studies, a novel genus, Peptacetobacter gen. nov., is proposed. The novel isolate ZHW00191T (=JCM 33482T=GDMCC 1.1530T) is proposed as the type strain of the type species Peptacetobacter hominis gen. nov., sp. nov. of the proposed new genus. Furthermore, it is proposed that Clostridium hiranonis be transferred to this novel genus, as Peptacetobacter hiranonis comb. nov.

Identification of Three Autophagy-Related Long Non-Coding RNAs as a Novel Head and Neck Squamous Cell Carcinoma Prognostic Signature.

Head and neck squamous cell carcinoma (HNSCC) has a poor prognosis. Considerable evidence indicates that autophagy and non-coding RNA play essential roles in the biological processes involved in cancers, but associations between autophagy-related long non-coding RNAs (lncRNAs) and HNSCC remain unclear. In the present study, HNSCC RNA sequences and autophagy-related gene data were extracted from The Cancer Genome Atlas database and the Human Autophagy Database. A total of 1,153 autophagy-related lncRNAs were selected via calculating Pearson's correlation coefficient. Three prognosis-related autophagy lncRNAs were identified via univariate Cox regression, least absolute shrinkage and selection operator analysis, and multivariate Cox regression analysis. We also constructed a prognostic model based on these autophagy-related lncRNAs and evaluated its ability to accurately and independently predict the prognosis of HNSCC patients. The area under the curve (AUC) was 0.864 (3-year) and 0.836 (5-year), and our model can independently predict the prognosis of HNSCC. The prognostic value of the three autophagy lncRNAs was confirmed via analysis of samples from five databases. To further identify the functions of the three lncRNAs, a co-expression network was constructed and pathway analysis was performed. In that analysis the lncRNAs were correlated with 189 related genes and 20 autophagy-related genes, and these lncRNAs mainly involved homologous recombination, the Fanconi anemia pathway, the autophagy-related pathway, and immune-related pathways. In addition, we validated the expression levels of three lncRNAs and autophagy markers (ATG12, BECN1, and MAP1LC3B) based on TIMER, Oncomine, and HPA database analysis. Our results indicated that TTTY15 was increased in HPV positive and HPV negative HNSCC patients, and three autophagy markers were up-regulated in all HNSCCC patients. Lastly, association between three lncRNAs and autophagy markers was performed, and our results showed that TTTY15 and MIF-AS1 were associated with autophagy markers. Collectively, these results suggested that three autophagy-related lncRNAs have prognostic value in HNSCC patients.

Comparative Transcriptome Analysis Reveals Differentially Expressed Genes and Signaling Pathways Between Male and Female Red-Tail Catfish (Mystus wyckioides).

Sexual dimorphism is widespread in fish species. The red-tail catfish (Mystus wyckioides) is a commercially important catfish in the lower reaches of the Lancang River and the Mekong basin, and it shows a growth advantage in males. Here, RNA-seq was for the first time used to explore the gene expression difference between the sexes in the hypothalamus and pituitary of red-tail catfish, respectively. In the hypothalamus, 5732 and 271 unigenes have significantly higher and lower expressions, respectively, in males compared with females. KEGG analysis showed that 212 DEGs were annotated to 216 signaling pathways, and enrichment analysis suggested different levels of cAMP and glutamatergic synapse signaling between male and female hypothalami and some of the DEGs appear involved in gonad development and growth. In the pituitary, we found only 19 differentially expressed unigenes, which were annotated to 32 signaling pathways, most of which play important roles in gonad development.

Whole Genome Incorporation and Epigenetic Stability in a Newly Synthetic Allopolyploid of Gynogenetic Gibel Carp.

Allopolyploidization plays an important role in speciation, and some natural or synthetic allopolyploid fishes have been extensively applied to aquaculture. Although genetic and epigenetic inheritance and variation associated with plant allopolyploids have been well documented, the relative research in allopolyploid animals is scarce. In this study, the genome constitution and DNA methylation inheritance in a newly synthetic allopolyploid of gynogenetic gibel carp were analyzed. The incorporation of a whole genome of paternal common carp sperm in the allopolyploid was confirmed by genomic in situ hybridization, chromosome localization of 45S rDNAs, and sequence comparison. Pooled sample-based methylation sensitive amplified polymorphism (MSAP) revealed that an overwhelming majority (98.82%) of cytosine methylation patterns in the allopolyploid were inherited from its parents of hexaploid gibel carp clone D and common carp. Compared to its parents, 11 DNA fragments in the allopolyploid were proved to be caused by interindividual variation, recombination, deletion, and mutation through individual sample-based MSAP and sequencing. Contrast to the rapid and remarkable epigenetic changes in most of analyzed neopolyploids, no cytosine methylation variation was detected in the gynogenetic allopolyploid. Therefore, the newly synthetic allopolyploid of gynogenetic gibel carp combined genomes from its parents and maintained genetic and epigenetic stability after its formation and subsequently seven successive gynogenetic generations. Our current results provide a paradigm for recurrent polyploidy consequences in the gynogenetic allopolyploid animals.

Origin and transition of sex determination mechanisms in a gynogenetic hexaploid fish.

Most vertebrates reproduce sexually, and plastic sex determination mechanisms including genotypic sex determination (GSD) and environmental sex determination (ESD) have been extensively revealed. However, why sex determination mechanisms evolve diversely and how they correlate with diverse reproduction strategies remain largely unclear. Here, we utilize the superiority of a hexaploid gibel carp (Carassius gibelio) that is able to reproduce by unisexual gynogenesis and contains a rare but diverse proportion of males to investigate these puzzles. A total of 2248 hexaploid specimens were collected from 34 geographic wild populations throughout mainland China, in which 24 populations were revealed to contain 186 males with various incidences ranging from 1.2 to 26.5%. Subsequently, the proportion of temperature-dependent sex determination (TSD) was revealed to be positively correlated to average annual temperature in wild populations, and male incidence in lab gynogenetic progenies was demonstrated to increase with the increasing of larval rearing temperature. Meanwhile, extra microchromosomes were confirmed to play genotypic male determination role as previously reported. Thereby, GSD and TSD were found to coexist in gibel carp, and the proportions of GSD were observed to be much higher than that of TSD in sympatric wild populations. Our findings uncover a potential new mechanism in the evolution of sex determination system in polyploid vertebrates with unisexual gynogenesis ability, and also reveal a possible association of sex determination mechanism transition between TSD and GSD and reproduction mode transition between unisexual gynogenesis and bisexual reproduction.

Wider geographic distribution and higher diversity of hexaploids than tetraploids in Carassius species complex reveal recurrent polyploidy effects on adaptive evolution.

Polyploidy roles on adaptive evolution and ecological novelty have been extensively studied in plants but remained unclear in vertebrates owing to the rare polyploidy incidences. Here, a huge number of 3105 specimens in Carassius species complex including 2211 hexaploids and 894 tetraploids were sampled from 34 locations through mainland China. And hexaploids had wider geographic distribution than tetraploids especially in the areas with high altitude, high latitude and low annual precipitation. Then, an approximate 1050 bp transferrin (tf) fragments were amplified from all the samples, and 526 tf alleles were identified from a total of 37260 sequences at last. Intriguingly, higher nucleotide diversity of tf alleles in hexaploids than in tetraploids was revealed. Moreover, via phylogenetic analysis of tf alleles, potential origin center of Carassius species complex was deduced to be Yangtze River basin and hexaploids should undergo multiple independent polyploidy origins from sympatric tetraploids. These findings indicate that the hexaploids might possess stronger environmental adaptation and ecological novelty than the tetraploids, which provide an association paradigm of recurrent polyploidy and ecological context in polyploid vertebrates.

Numerous mitochondrial DNA haplotypes reveal multiple independent polyploidy origins of hexaploids in Carassius species complex.

Evolutionary trajectory and occurrence history of polyploidy have been extensively studied in plants, but they remain quite elusive in vertebrates. Here, we sampled and gathered 4,159 specimens of polyploid Carassius species complex including 1,336 tetraploids and 2,823 hexaploids from a large geographic scale (49 localities) across East Asia, and identified a huge number of 427 diverse haplotypes of mitochondrial control region, in which 74 haplotypes with total occurrence frequency up to 75.498% were shared by hexaploids and tetraploids. Significantly, these diverse haplotypes were clustered into four major lineages, and many haplotypes of hexaploids and tetraploids were intermixed in every lineage. Moreover, the evolutionary trajectory and occurrence history of four different lineages were revealed by a simplified time-calibrated phylogenetic tree, and their geographic distribution frequencies and haplotype diversity were also analyzed. Furthermore, lineage C and D were revealed to undergo population expansion throughout mainland China. Therefore, our current data indicate that hexaploids should undergo multiple independent polyploidy origins from sympatric tetraploids in the polyploid Carassius species complex across East Asia.

Upregulation of microRNA-98 increases radiosensitivity in esophageal squamous cell carcinoma.

Although radiation resistance is a common challenge in the clinical treatment of esophageal squamous cell carcinoma (ESCC), an effective treatment strategy has yet to be developed. Aberrant expression of microRNAs (miRNAs) is responsible for cancer sensitivity to radiation. In this study, we aimed to identify the miRNAs that are associated with radioresistance in ESCC. We used a miRNA microarray to perform a comparison of miRNA expression in both ESCC parental and acquired radioresistance cell lines. qRT-PCR was used to confirm the alterations. Cell radiosensitivity was determined with a survival fraction assay. Functional analyses of the identified miRNA in ESCC cells with regard to metastasis and apoptosis were performed by transwell assays and flow cytometry. The miRNA targets were identified with pathway analysis and confirmed with a luciferase assay. miR-98 was recognized as the most downregulated miRNA in established radioresistant cell line. AmiR-98 mimic enforced the expression of miRNA-98 and made ESCC cells sensitive to radiotherapy, while anti-miR-98 reversed this process. Optimal results were achieved by decreasing cellular proliferation, decreasing cell migration and inducing apoptosis. The luciferase target gene analysis results showed that the overexpression of miRNA-98 inhibited tumor growth and resistance tolerance by directly binding to the BCL-2 gene. Our study indicated that increasing miRNA-98 expression can be used as a potential radiosensitive therapeutic strategy for treating esophageal cancer cells.