Metabolism response mechanism in the gill of Oreochromis mossambicus under salinity, alkalinity and saline-alkalinity stresses.

Saline-alkalinity is one of the important ecological parameter that has an impact function on the physiological metabolism, osmoregulation, survival, growth, development and distribution of teleost fish. Oreochromis mossambicus, a species of euryhaline that can withstand a wide variety of salinities, may be used as a research model animal in environmental studies. In order to detect the metabolism responses and mechanisms of different osmotic stresses tolerance in the gills of O. mossambicus, in present study, the metabolic responses of O. mossambicus subjected to salinity (25 g/L, S_S), alkalinity (4 g/L, A_S) and saline-alkalinity stress (salinity: 25 g/L, alkalinity: 4 g/L; SA_S) with the control environment (freshwater, C_S) were investigated by LC-MS/MS-based metabolomics. The metabolism results indicated that numerous metabolites were identified between the stress groups and the control group. In addition, under three osmotic stresses, the amino acid and carbohydrate metabolism, levels of amino acids, osmolytes and energy substances, such as L-lysine, arachidonic acid, docosahexaenoic acids, creatine and taurine, were significantly affected and changed in the metabolism of the gills of O. mossambicus. The metabolism data indicated that signal transduction and regulation pathways, including FoxO signaling pathway, mTOR signaling pathway and prolactin signaling pathway, were enriched in the gill during adaptation to high salinity, alkalinity and saline-alkalinity stress. The results of this study provide more comprehensive and reliable data for the osmotic pressure regulation mechanism and biological response of euryhaline teleost, and provide reliable scientific basis for the breeding and research of high salinity tolerance population, and further promote the development and utilization of saline-alkalinity water resources.

Metabolism responses in the intestine of Oreochromis mossambicus exposed to salinity, alkalinity and salt-alkalinity stress using LC-MS/MS-based metabolomics.

Multiple abiotic stresses are imposed on fish as a result of unprecedented changes in temperature and precipitation patterns in recent decades. It is unclear how teleosts respond to severe ambient salinity, alkalinity, and saline-alkalinity in terms of their metabolic and molecular osmoregulation processes. The metabolic reactions in the intestine of Oreochromis mossambicus under salinity (25 g/L, S_C), alkalinity (4 g/L, A_C), and saline-alkalinity (salinity: 25 g/L & alkalinity: 4 g/L, SA_C) stresses were examined in this research utilizing LC-MS/MS-based metabolomics. The findings demonstrated that the three osmotic-stressed groups' metabolic profiles were considerably different from those of the control group. Osmolytes, energy sources, free amino acids, and several intermediate metabolites were all synthetically adjusted as part of the osmoregulation associated with the salinity, alkalinity, and saline-alkalinity stress. Following osmotic stress, osmoregulation-related pathways, including the mTOR signaling pathway, TCA cycle, glycolysis/gluconeogenesis, etc., were also discovered in the intestine of O. mossambicus. Overall, our findings can assist in better comprehending the molecular regulatory mechanism in euryhaline fish under various osmotic pressures and can offer a preliminary profile of osmotic regulation.

Mitochondrial DNA and Microsatellite Analyses Showed Panmixia between Temporal Samples in Endangered Anguilla japonica in the Pearl River Basin (China).

The Japanese eel (Anguilla japonica) is a commercially important species in East Asia, the abundance of which has rapidly decreased in recent decades. The fishery resource in the Pearl River basin has mainly deteriorated due to overexploitation and habitat degradation. Knowledge on its genetic status is indispensable for resource management. In this study, we explored the temporal genetic structure of A. japonica on the basis of the concatenated sequences of two mitochondrial fragments (mtDNA) and eight microsatellite markers. A total of nine temporal samples (N = 127) were collected during 2019 and 2021 from Jiangmen City, China, which is located in the Pearl River estuary. mtDNA sequence analysis showed a high level of haplotype diversity, and yielded 124 haplotypes with ranging from 9 to 19 in temporal samples. All microsatellite loci were polymorphic among each of the nine temporal samples, with 150 alleles identified across all samples. Pairwise FST values were low and nonsignificant according to both mtDNA and microsatellite markers. STRUCTURE analysis showed that all temporal samples were not clearly differentiated from each other. The yielded outcomes supported a panmictic pattern in different temporal A. japonica samples. Therefore, our results call for the management of A. japonica as a single unit and joint conservation strategy of the species, since overexploitation in any region will decrease its global resource.

Mucinous carcinoma with micropapillary features is morphologically, clinically and genetically distinct from pure mucinous carcinoma of breast.

Micropapillary features are seen in pure mucinous carcinoma of breast (PMC), which is termed mucinous carcinoma with micropapillary features (MPMC). However, whether MPMC can be identified as a morphologically, clinically or genetically distinct entity from PMC remains controversial. In this study, a retrospective review of 161 cases of breast mucinous carcinoma was conducted to assess the clinicopathologic features, prognostic implications, and genomic alterations of MPMC and PMC. MPMCs were identified in 32% of mucinous carcinomas showing an excellent interobserver agreement (ICC = 0.922). MPMCs occurred at a younger age and exhibited higher nuclear grade, more frequent lymph nodal metastasis, lymphovascular invasion, and HER2 amplification compared with PMCs. Survival analyses revealed that MPMCs show decreased progression-free survival compared with PMCs in both unmatched and matched cohorts. A similar outcome of distant disease-free survival was observed only in the unmatched cohort. However, no statistical difference in recurrence score was observed between MPMC and PMC using a 21-gene assay. Notably, both MPMCs and PMCs displayed low mutation burden, common mutations affecting TTN, GATA3, SF3B1, TP53, recurrent 6q14.1-q27 losses, and 8p11.21-q24.3 gains. GATA3, TP53, and SF3B1 were recurrently mutated in MPMCs, while PIK3CA mutations were exclusively detected in PMCs. Moreover, MPMCs harbored 17q and 20q gains as well as 17p losses, while PMCs displayed gains at 6p. PI3K-Akt, mTOR, ErbB, and focal adhesion pathways were more frequently deregulated in MPMCs than in PMCs, which may responsible for the aggressive tumor behavior of MPMCs. Our findings suggest that MPMC is morphologically, clinically, and genetically distinct from PMC.

Assessing concordance among human, in silico predictions and functional assays on genetic variant classification.

MOTIVATION: A variety of in silico tools have been developed and frequently used to aid high-throughput rapid variant classification, but their performances vary, and their ability to classify variants of uncertain significance were not systemically assessed previously due to lack of validation data. This has been changed recently by advances of functional assays, where functional impact of genetic changes can be measured in single-nucleotide resolution using saturation genome editing (SGE) assay. RESULTS: We demonstrated the neural network model AIVAR (Artificial Intelligent VARiant classifier) was highly comparable to human experts on multiple verified datasets. Although highly accurate on known variants, AIVAR together with CADD and PhyloP showed non-significant concordance with SGE function scores. Moreover, our results indicated that neural network model trained from functional assay data may not produce accurate prediction on known variants. AVAILABILITY AND IMPLEMENTATION: All source code of AIVAR is deposited and freely available at https://github.com/TopGene/AIvar. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Germline TP53 and MSH6 mutations implicated in sporadic triple-negative breast cancer (TNBC): a preliminary study.

BACKGROUND: Germline BRCA1/2 prevalence is relatively low in sporadic triple-negative breast cancer (TNBC). We hypothesized that non-BRCA genes may also have significant germline contribution to Chinese sporadic TNBC, and the somatic mutational landscape of TNBC may vary between ethnic groups. We therefore conducted this study to investigate germline and somatic mutations in 43 cancer susceptibility genes in Chinese sporadic TNBC. PATIENTS AND METHODS: Sixty-six Chinese sporadic TNBC patients were enrolled in this study. Germline and tumor DNA of each patient were subjected to capture-based next-generation sequencing using a 43-gene panel. Standard bioinformatic analysis and variant classification were performed to identify deleterious/likely deleterious germline mutations and somatic mutations. Mutational analysis was conducted to identify significantly mutated genes. RESULTS: Deleterious/likely deleterious germline mutations were identified in 27 (27/66, 40.9%) patients. Among the 27 patients, 9 (9/66, 13.6%) were TP53 carriers, 5 (5/66, 7.6%) were MSH6 carriers, and 5 (5/66, 7.6%) were BRCA1 carriers. Somatic mutations were identified in 64 (64/66, 97.0%) patients. TP53 somatic mutations occurred in most of the patients (45/66, 68.2%) and with highest mean allele frequency (28.1%), while NF1 and POLE were detected to have the highest mutation counts. CONCLUSIONS: Our results supported our hypotheses and suggested great potentials of TP53 and MSH6 as novel candidates for TNBC predisposition genes. The high frequency of somatic NF1 and POLE mutations in this study showed possibilities for clinical benefits from androgen-blockade therapies and immunotherapies in Chinese TNBC patients. Our study indicated necessity of multi-gene testing for TNBC prevention and treatment.

Ancient duplications and functional divergence in the interferon regulatory factors of vertebrates provide insights into the evolution of vertebrate immune systems.

Interferon regulatory factors (IRFs) were first discovered as transcription factors that regulate the transcription of human interferon (IFN)-ß. Increasing evidence shows that they might be important players involved in Adaptive immune system (AIS) evolution. Although numbers of IRFs have been identified in chordates, the evolutionary history and functional diversity of this gene family during the early evolution of vertebrates have remained obscure. Using IRF HMM profile and HMMER searches, we identified 148 IRFs in 11 vertebrates and 4 protochordates. For them, we reconstructed the phylogenetic relationships, determined the synteny conservation, investigated the profile of natural selection, and analyzed the expression patterns in four "living fossil" vertebrates: lamprey, elephant shark, coelacanth and bichir. The results from phylogeny and synteny analysis imply that vertebrate IRFs evolved from three predecessors, instead of four as suggested in a previous study, as results from an ancient duplication followed by special expansions and lost during the vertebrate evolution. The profile of natural selection and expression reveals functional dynamics during the process. Together, they suggest that the 2nd whole-genome duplication (2WGD) provided raw materials for innovation in the IRF family, and that the birth of type-I IFN might be an important factor inducing the establishment of IRF-mediated immune networks. As a member involved in the AIS evolution, IRF provide insights into the process and mechanism involved in the complexity and novelties of vertebrate immune systems.

Phylogeographic structure, cryptic speciation and demographic history of the sharpbelly (Hemiculter leucisculus), a freshwater habitat generalist from southern China.

BACKGROUND: Species with broad distributions frequently divide into multiple genetic forms and may therefore be viewed as "cryptic species". Here, we used the mitochondrial cytochrome b (Cytb) and 12 nuclear DNA loci to investigate phylogeographic structures of the sharpbelly (Hemiculter leucisculus) in rivers in southern China and explored how the geological and climatic factors have shaped the genetic diversity and evolutionary history of this species. RESULTS: Our mitochondrial phylogenetic analysis identified three major lineages (lineages A, B, and C). Lineages B and C showed a relatively narrower geographic distribution, whereas lineage A was widely distributed in numerous drainages. Divergence dates suggested that H. leucisculus populations diverged between 1.61-2.38 Ma. Bayesian species delimitation analysis using 12 nuclear DNA loci indicated the three lineages probably represented three valid taxa. Isolation-with-migration (IM) analysis found substantial gene flow has occurred among the three lineages. Demographic analyses showed that lineages B and C have experienced rapid demographic expansion at 0.03 Ma and 0.08 Ma, respectively. CONCLUSIONS: Hemiculter leucisculus populations in drainages in southern China comprise three mtDNA lineages, and each of which may represent a separate species. Intense uplift of the Qinghai-Tibetan Plateau, evolution of Asian monsoons, changes in paleo-drainages, and poor dispersal ability may have driven the divergence of the three putative species. However, gene flow occurs among the three lineages. Climatic fluctuations have a prominent impact on the populations from the lineages B and C, but exerted little influence on the lineage A.

The first complete mitogenome of the South China deep-sea giant isopod Bathynomus sp. (Crustacea: Isopoda: Cirolanidae) allows insights into the early mitogenomic evolution of isopods.

In this study, the complete mitochondrial (mt) genome sequence of the South China deep-sea giant isopod Bathynomus sp. was determined, and this study is the first to explore in detail the mt genome of a deep-sea member of the order Isopoda. This species belongs to the genus Bathynomus, the members of which are saprophagous residents of the deep-sea benthic environment; based on their large size, Bathynomus is included in the "supergiant group" of isopods. The mt genome of Bathynomus sp. is 14,965 bp in length and consists of 13 protein-coding genes, two ribosomal RNA genes, only 18 transfer RNA genes, and a noncoding control region 362 bp in length, which is the smallest control region discovered in Isopoda to date. Although the overall genome organization is typical for metazoans, the mt genome of Bathynomus sp. shows a number of derived characters, such as an inversion of 10 genes when compared to the pancrustacean ground pattern. Rearrangements in some genes (e.g., cob, trnT, nad5, and trnF) are shared by nearly all isopod mt genomes analyzed thus far, and when compared to the putative isopod ground pattern, five rearrangements were found in Bathynomus sp. Two tRNAs exhibit modified secondary structures: The TΨC arm is absent from trnQ, and trnC lacks the DHU. Within the class Malacostraca, trnC arm loss is only found in other isopods. Phylogenetic analysis revealed that Bathynomus sp. (Cymothoida) and Sphaeroma serratum (Sphaeromatidea) form a single clade, although it is unclear whether Cymothoida is monophyletic or paraphyletic. Moreover, the evolutionary rate of Bathynomus sp. (dN/dS [nonsynonymous mutational rate/synonymous mutational rate] = 0.0705) is the slowest measured to date among Cymothoida, which may be associated with its relatively constant deep-sea environment. Overall, our results may provide useful information for understanding the evolution of deep-sea Isopoda species.

Divergent DNA Methylation Provides Insights into the Evolution of Duplicate Genes in Zebrafish.

The evolutionary mechanism, fate and function of duplicate genes in various taxa have been widely studied; however, the mechanism underlying the maintenance and divergence of duplicate genes in Danio rerio remains largely unexplored. Whether and how the divergence of DNA methylation between duplicate pairs is associated with gene expression and evolutionary time are poorly understood. In this study, by analyzing bisulfite sequencing (BS-seq) and RNA-seq datasets from public data, we demonstrated that DNA methylation played a critical role in duplicate gene evolution in zebrafish. Initially, we found promoter methylation of duplicate genes generally decreased with evolutionary time as measured by synonymous substitution rate between paralogous duplicates (Ks). Importantly, promoter methylation of duplicate genes was negatively correlated with gene expression. Interestingly, for 665 duplicate gene pairs, one gene was consistently promoter methylated, while the other was unmethylated across nine different datasets we studied. Moreover, one motif enriched in promoter methylated duplicate genes tended to be bound by the transcription repression factor FOXD3, whereas a motif enriched in the promoter unmethylated sequences interacted with the transcription activator Sp1, indicating a complex interaction between the genomic environment and epigenome. Besides, body-methylated genes showed longer length than body-unmethylated genes. Overall, our results suggest that DNA methylation is highly important in the differential expression and evolution of duplicate genes in zebrafish.

Correlated expression of retrocopies and parental genes in zebrafish.

Previous studies of the function and evolution of retrocopies in plants, Drosophila and non-mammalian chordates provided new insights into the origin of novel genes. However, little is known about retrocopies and their parental genes in teleosts, and it remains obscure whether there is any correlation between them. The present study aimed to characterize the spatial and temporal expression profiles of retrogenes and their parental genes based on RNA-Seq data from Danio rerio embryos and tissues from adult. Using a modified pipeline, 306 retrocopies were identified in the zebrafish genome, most of which exhibited ancient retroposition, and 76 of these showed a Ks < 2.0. Expression of a retrocopy is generally expected to present no correlation with its parental gene, as regulatory regions are not part of the retroposition event. Here, this assumption was tested based on RNA-Seq data from eight stages and thirteen tissue types of zebrafish. However, the result suggested that retrocopies displayed correlated expression with their parental genes. The level of correlation was found to decrease during embryogenesis, but to increase slightly within a tissue using Ks as the proxy for the divergence time. Tissue specificity was also observed: retrocopies were found to be expressed at a more specific level compared with their parental genes. Unlike Drosophila, which has sex chromosomes, zebrafish do not show testis-biased expression. Our study elaborated temporal and spatial patterns of expression of retrocopies in zebrafish, examined the correlation between retrocopies and parental genes and analyzed potential source of regulated elements of retrocopies, which lay a foundation for further functional study of retrocopies.

[Activity Study of Icarlin and Epimedin C Monomer under the Same Molarity in Osteoporosis Ze- brafish Model].

Objective To evaluate anti-osteoporotic activity of icariin and Epimedin C monomer under the same molarity in predinsolone-induced osteoporosis zebrafish. Methods Zebrafish larvae after 4-day fertilization were divided into group S [0. 5% dimethyl sulfoxide (DMSO) , A (25 µmol/L prednisolone, 0. 5% DMSO), B (2 IU/L salmon calcitonin, 25 µmol/L prednisolone,0. 5% DMSO), C (1. 5 1,mol/L icariin, 25 µmol/L prednisolone, 0. 5% DMSO) , D (15 µLmol/L icariin,25 µmol/L prednisolone, 0. 5% DM- SO), E (150 µmol/L icariin, 25 µmol/L prednisolone, 0. 5% DMSO), F (1. 5 µmol/L Epimediri C, 25 µmol/L prednisolone, 0. 5% DMSO) , G (15 µmol/L Epimedin C, 25 µmol/L prednisolone, 0.5% DM- SO) , H (150 µmol/L Epimedin C, 25 µmol/L prednisolone, 0. 5% DMSO). All culture solution contained 0. 5% DMSO. All the young fishes were grown in a 24-well plate. The culture medium was changed every day. They were cultured in a incubator box at 28. 5 °C and killed at day 9. Zebrafish skeleton was stained with alizarin red. The stained Zebrafish ventral skull was observed using microscope, and mineralized area was quantitatively analyzed. Results Compared with group S, accumulative integrated optical densi- ty(IOD)of the mineralized area significantly decreased in group A (P <0. 01) ; accumulative IOD of the mineralized area significantly increased in group B (P <0. 01). The accumulative IOD of the mineralized area showed weakly increasing tendency in group C, D, and E along with increased concentration (P < 0. 05). Compared with group A, accumulative IOD obviously increased in group B with statistical difference (P <0. 01) , but with no statistical difference as compared with group C or group D (P >0. 05). Statistical difference existed in accumulative IOD between group A and group E (P <0. 05). The mineralized area showed increasing tendency in group F and group G along with increased concentration (P <0. 05), and accumulative IOD obviously increased as well (P <0. 05). No Zebrafish embryo survived in group H. There was no statistical difference in Zebrafish embryo survival among group E, F, or G (P >0. 05). The staining of Zebrafish skull was clearly seen in group S, with vertebrae and bilateral branchial skeleton clearly seen. The intensity of staining in the same area was obviously attenuated in group A. The osteo- genesis was speeded up under the same condition in group B, with obviously enlarged mineralized area and more darkly stained bone tissue. The mineralization of skull was gradually increasing during the stai- ning process in group C, D, E, F, and G. The mineralized area and the intensity of staining were gradually enhanced, and changes of vertebrae were most obviously seen in group C, D, E, F, and G, but they were not arrived at the stained intensity level in group B. Conclusions Osteoporosis Zebrafish model is a simple and efficient model for screening bioactive ingredients of Chinese herbs. The activity of Epimedin C at low concentration was better than icariin in this model. But possible toxicity of Epimedin C at high concentration needs to be further studied.

Analysis of the nicotinamide phosphoribosyltransferase family provides insight into vertebrate adaptation to different oxygen levels during the water-to-land transition.

One of the most important events in vertebrate evolutionary history is the water-to-land transition, during which some morphological and physiological changes occurred in concert with the loss of specific genes in tetrapods. However, the molecular mechanisms underlying this transition have not been well explored. To explore vertebrate adaptation to different oxygen levels during the water-to-land transition, we performed comprehensive bioinformatics and experimental analysis aiming to investigate the NAMPT family in vertebrates. NAMPT, a rate-limiting enzyme in the salvage pathway of NAD+ biosynthesis, is critical for cell survival in a hypoxic environment, and a high level of NAMPT significantly augments oxidative stress in normoxic environments. Phylogenetic analysis showed that NAMPT duplicates arose from a second round whole-genome duplication event. NAMPTA existed in all classes of vertebrates, whereas NAMPTB was only found in fishes and not tetrapods. Asymmetric evolutionary rates and purifying selection were the main evolutionary forces involved. Although functional analysis identified several functionally divergent sites during NAMPT family evolution, in vitro experimental data demonstrated that NAMPTA and NAMPTB were functionally conserved for NAMPT enzymatic function in the NAD+ salvage pathway. In situ hybridization revealed broad NAMPTA and NAMPTB expression patterns, implying regulatory functions over a wide range of developmental processes. The morpholino-mediated knockdown data demonstrated that NAMPTA was more essential than NAMPTB for vertebrate embryo development. We propose that the retention of NAMPTB in water-breathing fishes and its loss in air-breathing tetrapods resulted from vertebrate adaptation to different oxygen levels during the water-to-land transition.