The key role of myostatin b in somatic growth in fishes derived from distant hybridization.

The basic mechanism of heterosis has not been systematically and completely characterized. In previous studies, we obtained three economically important fishes that exhibit rapid growth, WR (WCC ♀ × RCC ♂), WR-II (WR ♀ × WCC ♂), and WR-III (WR-II ♀ × 4nAU ♂), through distant hybridization. However, the mechanism underlying this rapid growth remains unclear. In this study, we found that WR, WR-II, and WR-III showed muscle hypertrophy and higher muscle protein and fat contents compared with their parent species (RCC and WCC). Candidate genes responsible for this rapid growth were then obtained through an analysis of 12 muscle transcriptomes. Notably, the mRNA level of mstnb (myostatin b), which is a negative regulator of myogenesis, was significantly reduced in WR, WR-II, and WR-III compared with the parent species. To verify the function of mstnb, a mstnb-deficient mutant RCC line was generated using the CRISPR-Cas9 technique. The average body weight of mstnb-deficient RCC at 12 months of age was significantly increased by 29.57% compared with that in wild-type siblings. Moreover, the area and number of muscle fibers were significantly increased in mstnb-deficient RCC, indicating hypertrophy and hyperplasia. Furthermore, the muscle protein and fat contents were significantly increased in mstnb-deficient RCC. The molecular regulatory mechanism of mstnb was then revealed by transcription profiling, which showed that genes related to myogenesis (myod, myog, and myf5), protein synthesis (PI3K-AKT-mTOR), and lipogenesis (pparγ and fabp3) were highly activated in hybrid fishes and mstnb-deficient RCC. This study revealed that low expression or deficiency of mstnb regulates somatic growth by promoting myogenesis, protein synthesis, and lipogenesis in hybrid fishes and mstnb-deficient RCC, which provides evidence for the molecular mechanism of heterosis via distant hybridization.

Formation of Different Polyploids Through Disrupting Meiotic Crossover Frequencies Based on cntd1 Knockout in Zebrafish.

Polyploidy, a significant catalyst for speciation and evolutionary processes in both plant and animal kingdoms, has been recognized for a long time. However, the exact molecular mechanism that leads to polyploid formation, especially in vertebrates, is not fully understood. Our study aimed to elucidate this phenomenon using the zebrafish model. We successfully achieved an effective knockout of the cyclin N-terminal domain containing 1 (cntd1) using CRISPR/Cas9 technology. This resulted in impaired formation of meiotic crossovers, leading to cell-cycle arrest during meiotic metaphase and triggering apoptosis of spermatocytes in the testes. Despite these defects, the mutant (cntd1-/-) males were still able to produce a limited amount of sperm with normal ploidy and function. Interestingly, in the mutant females, it was the ploidy not the capacity of egg production that was altered. This resulted in the production of haploid, aneuploid, and unreduced gametes. This alteration enabled us to successfully obtain triploid and tetraploid zebrafish from cntd1-/- and cntd1-/-/- females, respectively. Furthermore, the tetraploid-heterozygous zebrafish produced reduced-diploid gametes and yielded all-triploid or all-tetraploid offspring when crossed with wild-type (WT) or tetraploid zebrafish, respectively. Collectively, our findings provide direct evidence supporting the crucial role of meiotic crossover defects in the process of polyploidization. This is particularly evident in the generation of unreduced eggs in fish and, potentially, other vertebrate species.

Intratumoral and Peritumoral Multiparametric MRI-Based Radiomics Signature for Preoperative Prediction of Ki-67 Proliferation Status in Glioblastoma: A Two-Center Study.

RATIONALE AND OBJECTIVES: To assess the predictive ability of intratumoral and peritumoral multiparametric magnetic resonance imaging (MRI)-based radiomics signature (RS) for preoperative prediction of Ki-67 proliferation status in glioblastoma. MATERIALS AND METHODS: A total of 205 patients with glioblastoma at two institutions were retrospectively analyzed. Data from institution 1 (n = 158) were used to develop the predictive model, and as an internal test dataset, data from institution 2 (n = 47) constitute the external test dataset. Feature selection was performed using spearman correlation coefficient, univariate ranking method, and the least absolute shrinkage and selection operator algorithm. RSs were established using a logistic regression algorithm. The predictive performance of the RSs was assessed using calibration curve, decision curve analysis (DCA), and area under the curve (AUC). RESULTS: In the RSs based on single-parametric (contrast-enhanced T1-weighted image, T2-weighted image, or apparent diffusion coefficient maps), the AUCs of intratumoral, peritumoral, and combined area (intratumoral and peritumoral) were 0.60-0.67, with no significant difference among them. The RSs that using multiparametric features (integrating the previously mentioned three sequences) showed improved AUC compared to the single-parametric RSs; AUC reached 0.75-0.89. Among them, the multiparametric RS based on radiomics features of the combined area (Multi-Com) exhibited the highest performance, with an internal test dataset AUC of 0.89 (95% confidence interval (CI) 0.75-1.00) and an external test dataset AUC of 0.88 (95% CI 0.78-0.97). The calibration curve and DCA display RS (Multi-Com) have good calibration ability and clinical applicability. CONCLUSION: The multiparametric MRI-based RS combining intratumoral and peritumoral features can serve as a noninvasive and effective tool for preoperative assessment of Ki-67 proliferation status in glioblastoma.

Ccdc57 is required for straightening the body axis by regulating ciliary motility in the brain ventricle of zebrafish.

Recently, cilia defects have been proposed to contribute to scoliosis. Here, we demonstrate that coiled-coil domain-containing 57 (Ccdc57) plays an essential role in straightening the body axis of zebrafish by regulating ciliary beating in the brain ventricle (BV). Zygotic ccdc57 (Zccdc57) mutant zebrafish developes scoliosis without significant changes in their bone density and calcification, and the maternal-zygotic ccdc57 (MZccdc57) mutant embryos display curved bodies since the long-pec stage. The expression of ccdc57 is enriched in ciliated tissues and immunofluorescence analysis reveals colocalization of Ccdc57-HA with acetylated α-tubulin, implicating it in having a role in ciliary function. Further examination reveals that it is the coordinated cilia beating of multiple cilia bundles (MCB) in the MZccdc57 mutant embryos that is affected at 48 hours post fertilization, when the compromised cerebrospinal fluid flow and curved body axis have already occurred. Either ccdc57 mRNA injection or epinephrine treatment reverses the spinal curvature in MZccdc57 mutant larvae from ventrally curly to straight or even dorsally curly and significantly upregulates urotensin signaling. This study reveals the role of ccdc57 in maintaining coordinated cilia beating of MCB in the BV.

Spatiotemporal characteristics of the pharyngeal teeth in interspecific distant hybrids of cyprinid fish: Phylogeny and expression of the initiation marker genes.

As an important feeding organ and taxonomical characteristic, the pharyngeal teeth of cyprinid fish have very high morphological diversity and exhibit species-specific numbers and arrangements. Many genes have been verified to regulate the pharyngeal teeth development and act as the initiation marker for teeth. Six initiation marker genes for pharyngeal teeth were used as RNA probes to investigate the expression pattern, and these genes were further used to construct a phylogenetic tree for cyprinid fish including some distant hybrids. The results from in situ hybridization showed that similarities and differences existed in the expression of dlx2b, dlx4b, dlx5a, pitx2, fth1b, and scpp5 in the pharyngeal region of the hybrids (BT) by the crosses of blunt snout bream (BSB, ♀) × topmouth culter (TC, ♂). Particularly, we found a high specificity marker gene scpp5 for the early development of pharyngeal teeth. The Scpp5 expression pattern established a clear graphic representation on the spatiotemporal characteristics of the early morphogenesis of pharyngeal teeth in BT and BSB. Our results suggested that the scpp5 expression in 4V1, 3V1, and 5V1 in BT occurred earlier than that in BSB, while the replacement rate of pharyngeal teeth (4V2, 3V2, and 5V2) was faster in BSB. Phylogenetic analysis revealed that the six marker genes were highly conserved and could be used as the molecular marker for identifying the parents of the distant hybrids in cyprinid fish. The expression patterns of the scpp5 gene was examined in various tissues, including the brain, gill, heart, liver, muscle, skin, fins, gonad, eye, and kidney, showing that the scpp5 gene was ubiquitously expressed, indicating its important role in cyprinid fish.

An Optimized Weighted Naïve Bayes Method for Flood Risk Assessment.

Floods occur frequently and cause considerable damage to local environments. Effectively assessing the flood risk contributes to reducing loss caused by such disasters. In this study, the weighted naïve Bayes (WNB) method was selected to evaluate flood risk, and the entropy weight method was employed to compute the weights. A sampling and verifying model was employed to generate the most accurate conditional probability table (MACPT) to calculate the probability of flooding. When using the framework integrating WNB with the sampling and verifying model, previous studies could not obtain a WNB-based MACPT and the WNB classification accuracy, for lacking WNB functions that could be called directly. Facing this issue, in this study we developed WNB functions with the MATLAB platform to directly integrate with the sampling and verifying model to generate a WNB-based MACPT, contributing to the greater interpretability and extensibility of the model. Shantou and Jieyang cities in China were selected as the study area. The results demonstrate that: (1) a WNB-based MACPT can reflect the real spatial distribution of flood risk and (2) the WNB outperform the NB when integrated with the sampling and verifying model. The resulting gridded estimation reveal a detailed spatial pattern of flood risk, which can serve as a realistic reference for decision making related to floods. Furthermore, the proposed method uses less data, which would be helpful in developing countries where long-term intensive hydrologic monitoring is limited.

Evidence for the paternal mitochondrial DNA in the crucian carp-like fish lineage with hybrid origin.

In terms of taxonomic status, common carp (Cyprinus carpio, Cyprininae) and crucian carp (Carassius auratus, Cyprininae) are different species; however, in this study, a newborn homodiploid crucian carp-like fish (2n=100) (2nNCRC) lineage (F1-F3) was established from the interspecific hybridization of female common carp (2n=100)×male blunt snout bream (Megalobrama amblycephala, Cultrinae, 2n=48). The phenotypes and genotypes of 2nNCRC differed from those of its parents but were closely related to those of the existing diploid crucian carp. We further sequenced the whole mitochondrial (mt) genomes of the 2nNCRC lineage from F1 to F3. The paternal mtDNA fragments were stably embedded in the mt-genomes of F1-F3 generations of 2nNCRC to form chimeric DNA fragments. Along with this chimeric process, numerous base sites of F1-F3 generations of 2nNCRC underwent mutations. Most of these mutation sites were consistent with the existing diploid crucian carp. Moreover, the mtDNA organization and nucleotide composition of 2nNCRC were more similar to those of the existing diploid crucian carp than those of the parents. The inheritable chimeric DNA fragments and mutant loci in the mt-genomes of different generations of 2nNCRC provided important evidence of the mtDNA change process in the newborn lineage derived from hybridization of different species. Our findings demonstrated for the first time that the paternal mtDNA were transmitted into the mt-genomes of homodiploid lineage, which provided new insights into the existence of paternal mtDNA in the mtDNA inheritance.

The subgenomes show asymmetric expression of alleles in hybrid lineages of Megalobrama amblycephala × Culter alburnus.

Hybridization drives rapid speciation by shaping novel genotypic and phenotypic profiles. Genomic incompatibility and transcriptome shock have been observed in hybrids, although this is rarer in animals than in plants. Using the newly sequenced genomes of the blunt snout bream (Megalobrama amblycephala [BSB]) and the topmouth culter (Culter alburnus [TC]), we focused on the sequence variation and gene expression changes in the reciprocal intergeneric hybrid lineages (F1-F3) of BSB × TC. A genome-wide transcriptional analysis identified 145-974 expressed recombinant genes in the successive generations of hybrid fish, suggesting the rapid emergence of allelic variation following hybridization. Some gradual changes of gene expression with additive and dominance effects and various cis and trans regulations were observed from F1 to F3 in the two hybrid lineages. These asymmetric patterns of gene expression represent the alternative strategies for counteracting deleterious effects of the subgenomes and improving adaptability of novel hybrids. Furthermore, we identified positive selection and additive expression patterns in transforming growth factor, beta 1b (tgfb1b), which may account for the morphological variations of the pharyngeal jaw in the two hybrid lineages. Our current findings provide insights into the evolution of vertebrate genomes immediately following hybridization.

Targeted disruption of tyrosinase causes melanin reduction in Carassius auratus cuvieri and its hybrid progeny.

The white crucian carp (Carassius auratus cuvieri, WCC) not only is one of the most economically important fish in Asia, characterized by strong reproductive ability and rapid growth rates, but also represents a good germplasm to produce hybrid progenies with heterosis. Gene knockout technique provides a safe and acceptant way for fish breeding. Achieving gene knockout in WCC and its hybrid progeny will be of great importance for both genetic studies and hybridization breeding. Tyrosinase (TYR) is a key enzyme in melanin synthesis. Depletion of tyr in zebrafish and mice results in mosaic pigmentation or total albinism. Here, we successfully used CRISPR-Cas9 to target tyr in WCC and its hybrid progeny (WR) derived from the cross of WCC (♀) and red crucian carp (Carassius auratus red var., RCC, ♂). The level of TYR protein was significantly reduced in mutant WCC. Both the mutant WCC and the mutant WR showed different degrees of melanin reduction compared with the wild-type sibling control fish, resulting from different mutation efficiency ranging from 60% to 90%. In addition, the transcriptional expression profiles of a series of pivotal pigment synthesis genes, i.e. tyrp1, mitfa, mitfb, dct and sox10, were down-regulated in tyr-CRISPR WCC, which ultimately caused a reduction in melanin synthesis. These results demonstrated that tyr plays a key role in melanin synthesis in WCC and WR, and CRISPR-Cas9 is an effective tool for modifying the genome of economical fish. Furthermore, the tyr-CRISPR models could be valuable in understanding fundamental mechanisms of pigment formation in non-model fish.

Depletion of Tissue-Specific Ion Transporters Causes Differential Expression of PRL Targets in Response to Increased Levels of Endogenous PRL.

Prolactin (PRL) has been considered a key regulator of ion uptake in zebrafish. The genes slc12a10.2 and slc12a3, which are Na+ and chloride Cl- co-transporters, have been reported to be regulated by PRL in freshwater fish. The integrative network of PRL signaling dissected from the knockout of tissue-specific downstream PRL ion transporters remains poor. In the present study, zebrafish models with increased endogenous levels of PRL were generated through the knockout of slc12a10.2 or slc12a3, and the developmental consequences were analyzed. The increased levels of pituitary PRL were observed in both slc12a10.2- and slc12a3-deficient fish. Unlike the slc12a3-deficient fish, which could survive to adulthood, the slc12a10.2-deficient fish began to die at 9 days post-fertilization (dpf) and did not survive beyond 17 dpf. This survival defect is a result of defective Cl- uptake in this mutant, indicating that Slc12a10.2 plays an essential role in Cl- uptake. Intriguingly, compared to the levels in control fish, no significant differences in the levels of Na+ in the body were observed in slc12a10.2- or slc12a3-deficient zebrafish. The upregulations of the PRL downstream transporters, slc9a3.2, slc12a10.2, and atp1a1a.5 were observed in slc12a3-deficient fish in both the gills/skin and the pronephric duct. However, this type of response was not observed in the pronephric duct of slc12a10.2-deficient fish, except under Na+-deprived conditions. Our results show that PRL is susceptible to deficiencies in downstream ion transporters. Moreover, both the gills/skin and pronephric duct show differential expression of downstream PRL targets in response to increased levels of pituitary PRL caused by the depletion of tissue-specific ion transporters.

A spatial assessment of urban waterlogging risk based on a Weighted Naïve Bayes classifier.

Urban waterlogging occurs frequently and often causes considerable damage that seriously affects the natural environment, human life, and the social economy. The spatial evaluation of urban waterlogging risk represents an essential analytic step that can be used to prevent urban waterlogging and minimize related losses. The Weighted Naïve Bayes (WNB) classifier is a powerful method for knowledge discovery and probability inference under conditions of uncertainty; a WNB classifier can be applied to estimate the likelihood of hazards. Six spatial factors were considered to be added to the WNB, which may improve the efficiency in predicting urban waterlogging risk during analysis. As such, a spatial framework integrating WNB with GIS was developed to assess the risk of urban waterlogging using the primary urban area of Guangzhou in China as an example. The results show that 1) the rationality of six spatial factors was determined according to the Conditional Probability Tables and weights; 2) the Most Accurate Sampling Table has objectivity; and 3) the areas with a high likelihood of waterlogging risk were mainly located in the southwestern part of the study area. The northeastern zones are relatively free of waterlogging risk. The results reveal a more accurate spatial pattern of urban waterlogging risk that can be used to identify risk "hot spots". The resulting gridded estimates provide a realistic reference for decision making related to urban waterlogging.

Tdrd12 Is Essential for Germ Cell Development and Maintenance in Zebrafish.

The regularity of Piwi-interacting RNA (piRNA) biogenesis is crucial to germline development. Functioning as Piwi-interacting proteins, Tudor domain-related proteins (Tdrds) have been demonstrated to be involved in spermatogenesis and the piRNA pathway. In this study, zebrafish tdrd12 was identified, and the maternal and germ cell-specific expression patterns of zebrafish tdrd12 were observed. Utilizing TALEN (transcription activator-like effector nuclease) techniques, two independent tdrd12 mutant zebrafish lines were generated. Although no defects were found during the generation of the primordial germ cells (PGCs) in the tdrd12-null fish progenies obtained from the heterozygous tdrd12 mutant parents, all Tdrd12-deficient fish developed into infertile males. The reduced numbers and eventually loss of the germ cells by 35 days post fertilization (dpf) led to masculinization and infertility of the Tdrd12-deficient fish. Meiosis defects of the germ cells in the tdrd12 mutants during the gonad-transitioning period were observed, revealing the indispensable functions of Tdrd12 in gametogenesis. Our studies demonstrated that zebrafish Tdrd12 is essential for germ cell development and maintenance.

The basal function of teleost prolactin as a key regulator on ion uptake identified with zebrafish knockout models.

Prolactin (PRL) is an anterior pituitary hormone with a broad range of functions. Its ability to stimulate lactogenesis, maternal behavior, growth and development, osmoregulation, and epithelial ion transport has been reported in many vertebrates. In our present study, we have targeted the zebrafish prl locus via transcription activator-like effector nucleases (TALENs). Two independent targeted mutant lines with premature termination of the putative sequence of PRL peptides were generated. All prl-deficient zebrafish progeny died at 6-16 days post-fertilization stage (dpf) in egg water. However, the prl-deficient larvae thrived and survived through adulthood in brackish water (5175 mg/L ocean salts), without obvious defects in somatic growth or reproduction. When raised in egg water, the expression levels of certain key Na(+)/Cl(-) cotransporters in the gills and Na(+)/K(+)-ATPase subunits, Na(+)/H(+) exchangers and Na(+)/Cl(-) transporters in the pronephros of prl-deficient larvae were down-regulated at 5 dpf, which caused Na(+)/K(+)/Cl(-) uptake defects in the mutant fish at 6 dpf. Our present results demonstrate that the primary function of zebrafish prl is osmoregulation via governing the uptake and homeostasis of Na(+), K(+) and Cl(-). Our study provides valuable evidence to understand the mechanisms of PRL function better through both phylogenetic and physiological perspectives.

[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.

A new method for estimating carbon dioxide emissions from transportation at fine spatial scales.

Detailed estimates of carbon dioxide (CO2) emissions at fine spatial scales are useful to both modelers and decision makers who are faced with the problem of global warming and climate change. Globally, transport related emissions of carbon dioxide are growing. This letter presents a new method based on the volume-preserving principle in the areal interpolation literature to disaggregate transportation-related CO2 emission estimates from the county-level scale to a 1 km2 grid scale. The proposed volume-preserving interpolation (VPI) method, together with the distance-decay principle, were used to derive emission weights for each grid based on its proximity to highways, roads, railroads, waterways, and airports. The total CO2 emission value summed from the grids within a county is made to be equal to the original county-level estimate, thus enforcing the volume-preserving property. The method was applied to downscale the transportation-related CO2 emission values by county (i.e. parish) for the state of Louisiana into 1 km2 grids. The results reveal a more realistic spatial pattern of CO2 emission from transportation, which can be used to identify the emission 'hot spots'. Of the four highest transportation-related CO2 emission hotspots in Louisiana, high-emission grids literally covered the entire East Baton Rouge Parish and Orleans Parish, whereas CO2 emission in Jefferson Parish (New Orleans suburb) and Caddo Parish (city of Shreveport) were more unevenly distributed. We argue that the new method is sound in principle, flexible in practice, and the resultant estimates are more accurate than previous gridding approaches.