Transcriptome Analysis Reveals Molecular Underpinnings of Common Carp (Cyprinus carpio) Under Hypoxia Stress.

As an essential environmental factor that affects the economic benefits of aquaculture, hypoxia is one of the urgent problems to be solved in the aquaculture fish breeding industry. Common carp (Cyprinus carpio) is a critical economic fish in China, and at present, there are many breeding strains of common carp with different character advantages in China, including Hebao red carp (C. carpio var wuyuanesis) and Songpu mirror carp (C. carpio var specularis). Even if the environmental adaptation of common carp is generally strong, the genetic background of hypoxia tolerance in different strains of common carp is unclear yet. This study tested the hypoxia tolerance of Songpu minor carp, Hebao red carp, and their hybrid F1 population by an acute hypoxia treatment. Muscle and liver tissues were used for transcriptome sequencing analysis to identify the key factors for hypoxia tolerance and explore the potential genetic mechanism for breeding high hypoxia tolerance in common carp. The comparative transcriptomic analysis revealed abundant hypoxia response-related genes and their differential regulation mechanism in these two tissues of different common carp strains under acute hypoxia, including immune response, cellular stress response, HIFs (hypoxia-inducible factors), MAP kinase, iron ion binding, and heme binding. Our findings will facilitate future investigation on the hypoxia response mechanism and provide a solid theoretical basis for breeding projects in common carp.

Carotid intima-media thickness in patients with hyperuricemia: a systematic review and meta-analysis.

OBJECTIVE: Despite the high incidence and mortality of cardiovascular events in hyperuricemia patients, the role of serum uric acid in cardiovascular diseases is still controversial. The aim of this meta-analysis was to explore the difference of carotid intima-media thickness in hyperuricemia and control groups. METHODS: We performed this meta-analysis by searching the PubMed, Cochrane Library, Embase and Web of Science databases up to July 2020. The 95% confidence intervals and standard mean differences were calculated to analyze the differences in carotid intima-media thickness in hyperuricemia groups and control groups. Sensitivity analysis, subgroup analysis and meta-regression were used to explore the sources of heterogeneity. Publication bias was evaluated by funnel plot and Begg's regression test. We used Stata 14.0 software to complete our analyses. RESULTS: A total of 8 articles were included. The results showed that there was a significant increase in carotid intima-media thickness in the hyperuricemia groups compared with the control groups [SMD = 0.264, 95% CI (0.161-0.366), P < 0.001]. Subgroup analyses showed that age, sample size, blood pressure and body mass index were not the source of heterogeneity. Meta-regression enrolled the method of CIMT measurement, location, age, smoking and diabetes mellitus as categorical variables, but none of these factors was found to be significant in the model. The Begg's test value (P = 0.174) was greater than 0.05, indicating there was no publication bias. CONCLUSION: The results showed that carotid intima-media thickness was increased in hyperuricemia patients compared with controls, which indicated that hyperuricemia patients may have a higher risk of cardiovascular diseases.

Does vitamin D improve symptomatic and structural outcomes in knee osteoarthritis? A systematic review and meta-analysis.

OBJECTIVE: To provide evidence on the effects of vitamin D supplementation on knee osteoarthritis (KOA) and new targets for clinical prevention and treatment of KOA. METHOD: The PubMed, Embase, Web of science, Wanfang, CNKI and SinoMed databases were retrieved to investigate the effects of vitamin D supplementation on patients with KOA. The search time was from databases establishment to 15 November 2020. RevMan5.3 software was used for meta-analysis. The results were expressed as standardized mean difference (SMD) with 95% confidence interval (CI) or weighted mean difference (WMD) with 95% confidence interval (CI). RESULTS: A total of 1599 patients with osteoarthritis of the knee were included in the study, which involved six articles. The results of the meta-analysis showed that vitamin D supplementation is statistically significant for WOMAC score (SMD = - 0.67, 95% CI - 1.23 to - 0.12) in patients with KOA, including WOMAC pain score (SMD = - 0.32, 95% CI - 0.63 to - 0.02), function score (SMD = - 0.34, 95% CI - 0.60 to - 0.08) and stiffness score (SMD = - 0.13, 95% CI - 0.26 to - 0.01). In subgroup analysis, vitamin D supplementation less than 2000 IU was statistically significant for the reduction of stiffness score (SMD = - 0.22, 95% CI - 0.40 to - 0.04). Vitamin D supplements can reduce synovial fluid volume progression in patients with KOA (SMD = - 0.20, 95% CI - 0.39 to - 0.02). There was no statistical significance in improving tibia cartilage volume (SMD = 0.12, 95% CI - 0.05 to 0.29), joint space width (SMD = - 0.10, 95% CI - 0.26 to 0.05) and bone marrow lesions (SMD = 0.03, 95% CI - 0.26 to 0.31). CONCLUSION: Vitamin D supplements can improve WOMAC pain and function in patients with KOA. But there is a lack of strong evidence that vitamin D supplementation can prevent structural progression in patients with KOA.

Population genetic analysis of aquaculture salmonid populations in China using a 57K rainbow trout SNP array.

Various salmonid species are cultivated in cold water aquaculture. However, due to limited genomic data resources, specific high-throughput genotyping tools are not available to many of the salmonid species. In this study, a 57K single nucleotide polymorphism (SNP) array for rainbow trout (Oncorhynchus mykiss) was utilized to detect polymorphisms in seven salmonid species, including Hucho taimen, Oncorhynchus masou, Salvelinus fontinalis, Brachymystax lenok, Salvelinus leucomaenis, O. kisutch, and O. mykiss. The number of polymorphic markers per population ranged from 3,844 (O. kisutch) to 53,734 (O. mykiss), indicating that the rainbow trout SNP array was applicable as a universal genotyping tool for other salmonid species. Among the six other salmonid populations from four genera, 28,882 SNPs were shared, whereas 525 SNPs were polymorphic in all four genera. The genetic diversity and population relationships of the seven salmonid species were studied by principal component analysis (PCA). The phylogenetic relationships among populations were analyzed using the maximum likelihood method, which indicated that the shared SNP markers provide reliable genomic information for population genetic analyses in common aquaculture salmonid fishes. Furthermore, this obtained genomic information may be applicable for population genetic evaluation, marker-assisted breeding, and propagative parent selection in fry production.

Genomic Basis of Adaptive Evolution: The Survival of Amur Ide (Leuciscus waleckii) in an Extremely Alkaline Environment.

The Amur ide (Leuciscus waleckii) is a cyprinid fish that is widely distributed in Northeast Asia. The Lake Dali Nur population inhabits one of the most extreme aquatic environments on Earth, with an alkalinity up to 50 mmol/L (pH 9.6), thus providing an exceptional model with which to characterize the mechanisms of genomic evolution underlying adaptation to extreme environments. Here, we developed the reference genome assembly for L. waleckii from Lake Dali Nur. Intriguingly, we identified unusual expanded long terminal repeats (LTRs) with higher nucleotide substitution rates than in many other teleosts, suggesting their more recent insertion into the L. waleckii genome. We also identified expansions in genes encoding egg coat proteins and natriuretic peptide receptors, possibly underlying the adaptation to extreme environmental stress. We further sequenced the genomes of 10 additional individuals from freshwater and 18 from Lake Dali Nur populations, and we detected a total of 7.6 million SNPs from both populations. In a genome scan and comparison of these two populations, we identified a set of genomic regions under selective sweeps that harbor genes involved in ion homoeostasis, acid-base regulation, unfolded protein response, reactive oxygen species elimination, and urea excretion. Our findings provide comprehensive insight into the genomic mechanisms of teleost fish that underlie their adaptation to extreme alkaline environments.

Complete mitochondrial genome of burbot (Lota lota: family Gadidae) from Amur basin lineage, Northeastern China.

The mitochondrial genome was amplified in two large overlapping fragments by long PCR and one gap PCR. The complete Northeastern China burbot mitogenome sequence (KC844053) is 16,575 bp in length. Similar to most vertebrata, the mitogenome has a set of 37 genes: including 13 protein-coding, 22 tRNA and 2 rRNA genes. More than 98% nucleotide sequence was simil to the reported individual.

Diversification of the duplicated Rab1a genes in a hypoxia-tolerant fish, common carp (Cyprinus carpio).

Common carp is a widely cultivated fish with longer than 2,000 years domestication history, due to its strong environmental adaptabilities, especially hypoxia tolerance. The common carp genome has experienced a very recent whole genome duplication (WGD) event. Among a large number of highly similar duplicated genes, a pair of Ras-associated binding-GTPase 1a (Rab1a) genes were found fast diverging. Four analogous Rab1a genes were identified in the common carp genome. Comparisons of gene structures and sequences indicated Rab1a-1 and Rab1a-2 was a pair of fast diverging duplicates, while Rab1a-3 and Rab1a-4 was a pair of less diverged duplicates. All putative Rab1a proteins shared conserved GTPase domain, which enabled the proteins serve as molecular switches for vesicular trafficking. Rab1a-1 and Rab1a-2 proteins varied in their C-terminal sequences, which were generally considered to encode the membrane localization signals. Differential expression patterns were observed between Rab1a-1 and Rab1a-2 genes. In blood, muscle, spleen, and heart, the mRNA level of Rab1a-1 was higher than that of Rab1a-2. In liver and intestine, the mRNA level of Rab1a-2 was higher. Expression of Rab1a-1 and Rab1a-2 showed distinct hypoxia responses. Under severe hypoxia, Rab1a-1 expression was down-regulated in blood, while Rab1a-2 expression was up-regulated in liver. Compared with the less diverged Rab1a-3/4 gene pair, common carp Rab1a-1/2 gene pair exhibited strong characteristics of sub-functionalization, which might contribute to a sophisticated and efficient Ras-dependent regulating network for the hypoxia-tolerant fish.

The complete mitochondrial genome of the silvertip tetra, Hasemania nana (Characiformes: Characidae).

We first sequenced the complete mitochondrial genome of silvertip tetra (Hasemania nana). The mitogenome was determined to be 16,581 bp long circular molecule with a typical gene arrangement of vertebrate mitochondrial DNA. All genes were encoded on the heavy strand with the exception of ND6 and eight tRNA genes. Mitochondrial DNA information provided the basis for the studies in species identification and conservation of the species' natural resources.

Complete mitochondrial genome of rosy barb, Puntius conchonius.

In this study, we sequenced and determined the complete mitochondrial genome of rosy barb (Puntius conchonius). The circular mitochondrial genome (17,082 bp) consisted of 13 protein-coding genes, 22 tRNA genes, 2 rRNA genes and 1 control region. This is the first report on the complete mitogenome sequence of rosy barb (P. conchonius).

Duplication and differentiation of common carp (Cyprinus carpio) myoglobin genes revealed by BAC analysis.

Two distinct myoglobin (mb) transcripts have been reported in common carp, Cyprinus carpio, which is a hypoxia-tolerant fish living in habitats with greatly fluctuant dissolved oxygen levels. Recombinant protein analysis has shown functional specialization of the two mb transcripts. In this work, analysis for mb-containing bacterial artificial chromosome (BAC) clones indicated different genome loci for common carp myoglobin-1 (mb-1) and myoglobin-2 (mb-2) genes. Fluorescence in situ hybridization (FISH) revealed that mb-1 and mb-2 are located on separate chromosomes. In both of the mb-1 and mb-2 containing BAC clones, gene synteny was well conserved with the homologous region on zebrafish chromosome 1, supporting that the common carp specific mb-2 gene originated from the recent whole genome duplication event in cyprinid lineage. Transcription factor binding sites search indicated that both common carp mb genes lacked specificity Protein 1 (Sp1) and myocyte enhancer factor-2 (MEF2) binding sites, which mediated muscle-specific and calcium-dependent expression in the well-studied mb promoters. Potential hypoxia response elements (HREs) were predicted in the regulatory region of common carp mb genes. These characteristics of common carp mb gene regulatory region well interpreted the hypoxia-inducible, non-muscle expression pattern of mb-1. In the case of mb-2, a 10 bp insertion to the binding site of upstream stimulatory factor (USF), which was a co-factor of hypoxia inducible factor (HIF), might cause the non-response to hypoxia treatment of mb-2. The case of common carp mb gene duplication and subsequent differentiation in expression pattern and protein function provided an example for adaptive evolution toward aquatic hypoxia tolerance.

The transcriptomes of the crucian carp complex (Carassius auratus) provide insights into the distinction between unisexual triploids and sexual diploids.

Both sexual reproduction and unisexual reproduction are adaptive strategies for species survival and evolution. Unisexual animals have originated largely by hybridization, which tends to elevate their heterozygosity. However, the extent of genetic diversity resulting from hybridization and the genomic differences that determine the type of reproduction are poorly understood. In Carassius auratus, sexual diploids and unisexual triploids coexist. These two forms are similar morphologically but differ markedly in their modes of reproduction. Investigation of their genomic differences will be useful to study genome diversity and the development of reproductive mode. We generated transcriptomes for the unisexual and sexual populations. Genes were identified using homology searches and an ab initio method. Estimation of the synonymous substitution rate in the orthologous pairs indicated that the hybridization of gibel carp occurred 2.2 million years ago. Microsatellite genotyping in each individual from the gibel carp population indicated that most gibel carp genes were not tri-allelic. Molecular function and pathway comparisons suggested few gene expansions between them, except for the progesterone-mediated oocyte maturation pathway, which is enriched in gibel carp. Differential expression analysis identified highly expressed genes in gibel carp. The transcriptomes provide information on genetic diversity and genomic differences, which should assist future studies in functional genomics.

Rates and patterns of microsatellite mutations in common carp (Cyprinus carpio L.).

During genotyping 150 microsatellites in a F1 family of common carp, six mutations were found at five microsatellite loci. The overall mutation rate of common carp microsatellites was 2.53 X 10(-4) per locus per generation. At five loci, mutations increased the length of alleles by at least one repeat unit, suggesting mutations at microsatellite loci in common carp do not follow strict stepwise mutation model. The data on mutation rates and patterns can facilitate population genetics studies, and provide useful parameters for estimating a long-term effective population size of common carp.

Self-assembled film of hydrophobins on gold surfaces and its application to electrochemical biosensing.

Hydrophobins are small fungal proteins which self-assemble on interfaces and significantly change the surface wettability. The self-assembled film of hydrophobin HFBI on a gold surface improved the surface hydrophilicity with water contact angle changing from 73.8+/-1.8 degrees to 45.3+/-1.4 degrees . A quartz crystal microbalance (QCM) analysis indicated that the HFBI coverage density on a gold surface was 588 ng cm(-2), and the self-assembled film remained stable under different pH values ranging from 1 to 13. A hydrophilic protein such as choline oxidase (ChOx) was then successfully immobilized on the HFBI modified gold surface. To evaluate the bioactivity of immobilized enzyme, an amperometric choline biosensor was constructed based on the Gold/HFBI/ChOx electrode, which produced as large as 4578.27 nA response current by 0.238 microg immobilized ChOx, when saturated by choline substrate. Comparing with our choline biosensors previously reported, the HFBI self-assembled film exhibited excellent capability to preserve the bioactivity of ChOx, hence a great potential in electrochemical biosensing is suggested.

Amperometric glucose biosensor based on self-assembly hydrophobin with high efficiency of enzyme utilization.

Hydrophobins are a family of natural self-assembling proteins with high biocompability, which are apt to form strong and ordered assembly onto many kinds of surfaces. These physical-chemical and biological properties make hydrophobins suitable for surface modification and biomolecule immobilization purposes. A class II hydrophobin HFBI was used as enzyme immobilization matrix on platinum electrode to construct amperometric glucose biosensor. Permeability of HFBI self-assembling film was optimized by selecting the proper HFBI concentration for electrode modification, in order to allow H(2)O(2) permeating while prevent interfering compounds accessing. HFBI self-assembly and glucose oxidase (GOx) immobilization was monitored by quartz crystal microbalance (QCM), and characterization of the modified electrode surface was obtained by scanning electron microscope (SEM). The resulting glucose biosensors showed rapid response time within 6s, limits of detection of 0.09 mM glucose (signal-to-noise ratio=3), wide linear range from 0.5 to 20mM, high sensitivity of 4.214 x 10(-3)AM(-1)cm(-2), also well selectivity, reproducibility and lifetime. The all-protein modified biosensor exhibited especially high efficiency of enzyme utilization, producing at most 712 microA responsive current for per unit activity of GOx. This work provided a promising new immobilization matrix with high biocompatibility and adequate electroactivity for further research in biosensing and other surface functionalizing.

Amperometric glucose biosensor based on multilayer films via layer-by-layer self-assembly of multi-wall carbon nanotubes, gold nanoparticles and glucose oxidase on the Pt electrode.

A novel amperometric glucose biosensor based on the nine layers of multilayer films composed of multi-wall carbon nanotubes (MWCNTs), gold nanoparticles (GNp) and glucose oxidase (GOD) was developed for the specific detection of glucose. MWCNTs were chemically modified with the H(2)SO(4)-HNO(3) pretreatment to introduce carboxyl groups which were used to interact with the amino groups of poly(allylamine) (PAA) and cysteamine via 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide/N-hydroxysuccinimide cross-linking reaction, respectively. A cleaned Pt electrode was immersed in PAA, MWCNTs, cysteamine and GNp, respectively, followed by the adsorption of GOD, assembling the one layer of multilayer films on the surface of Pt electrode (GOD/GNp/MWCNTs/Pt electrode). Repeating the above process could assemble different layers of multilayer films on the Pt electrode. PBS washing was applied at the end of each assembly deposition for dissociating the weak adsorption. Film assembling and characterization were studied by transmission electron microscopy and quartz crystal microbalance, and properties of the resulting glucose biosensors were measured by electrochemical measurements. The marked electrocatalytic activity of Pt electrode based on multilayer films toward H(2)O(2) produced during GOD enzymatic reactions with glucose permitted effective low-potential amperometric measurement of glucose. Taking the sensitivity and selectivity into consideration, the applied potential of 0.35 V versus Ag/AgCl was chosen for the oxidation detection of H(2)O(2) in this work. Among the resulting glucose biosensors, the biosensor based on nine layers of multilayer films was best. It showed a wide linear range of 0.1-10mM glucose, with a remarkable sensitivity of 2.527 microA/mM, a detection limit of 6.7 microM estimated at a signal-to-noise ratio of 3 and fast response time (within 7s). Moreover, it exhibited good reproducibility, long-term stability and the negligible interferences of ascorbic acid, uric acid and acetaminophen. The study can provide a feasible approach on developing new kinds of oxidase-based amperometric biosensors, and can be used as an illustration for constructing various hybrid structures.

Amperometric glucose biosensor based on layer-by-layer assembly of multilayer films composed of chitosan, gold nanoparticles and glucose oxidase modified Pt electrode.

A new strategy for fabricating glucose biosensor was presented by layer-by-layer assembled chitosan (CS)/gold nanoparticles (GNp)/glucose oxidase (GOD) multilayer films modified Pt electrode. First, a cleaned Pt electrode was immersed in poly(allylamine) (PAA), and then transferred to GNp, followed by the adsorption of GOD (GOD/GNp/PAA/Pt). Second, the GOD/GNp/PAA/Pt electrode was immersed in CS, and then transferred to GNp, followed by the adsorption of GOD (GOD/GNp/CS/GOD/GNp/PAA/Pt). Third, different layers of multilayer films modified Pt electrodes were assembled by repeating the second process. Film assembling and characterization were studied by quart crystal microbalance, and properties of the resulting glucose biosensors were measured by electrochemical measurements. The results confirmed that the assembling process of multilayer films was simple to operate, the immobilized GOD displayed an excellent catalytic property to glucose, and GNp in the biosensing interface efficiently improved the electron transfer between analyte and electrode surface. The amperometric response of the biosensors uniformly increased from one to six layers of multilayer films, and then reached saturation after the seven layers. Among the resulting biosensors, the biosensor based on the six layers of multilayer films was best. It showed a wide linear range of 0.5-16 mM, with a detection limit of 7.0 microM estimated at a signal-to-noise ratio of 3, fast response time (within 8s). Moreover, it exhibited good reproducibility, long-term stability and interference free. This method can be used for constructing other thin films, which is a universal immobilization method for biosensor fabrication.