Selection, identification and crystal structure of shark-derived single-domain antibodies against a green fluorescent protein.

Shark variable domain of new antigen receptors (VNARs) are the smallest naturally occurring binding domains with properties of low complexity, small size, cytoplasmic expression, and ease of engineering. Green fluorescent protein (GFP) molecules have been analyzed in conventional microscopy, but their spectral characteristics preclude their use in techniques offering substantially higher resolution. Besides, the GFP molecules can be quenched in acidic environment, which makes it necessary to develop anti-GFP antibody to solve these problems. In view of the diverse applications of GFP and unique physicochemical features of VNAR, the present study aims to generate VNARs against GFP. Here, we identified 36 VNARs targeting eCGP123, an extremely stable GFP, by phage display from three immunized sharks. These VNARs bound to eCGP123 with affinity constant KD values ranging from 6.76 to 605 nM. Among them, two lead VNARs named aGFP-14 and aGFP-15 with nanomolar eCGP123-binding affinity were selected for in-depth characterization. aGFP-14 and aGFP-15 recognized similar epitopes on eCGP123. X-ray crystallography studies clarified the mechanism by which aGFP14 interacts with eCGP123. aGFP-14 also showed cross-reaction with EGFP, with KD values of 47.2 nM. Finally, immunostaining analyses demonstrated that aGFP-14 was able to bind effectively to the EGFP expressed in both cultured cells and mouse brain tissues, and can be used as a fluorescence amplifier for EGFP. Our research demonstrates a feasible idea for the screening and production of shark-derived VNARs. The two high-affinity VNARs developed in the study contribute to the diversity of GFP sdAbs and may enhance the applications of GFP.

A New Concept for Primary Reconstruction of Skull Defects with 3D-Molded PEEK Patch Assisted by Navigation Based on Animal Study.

AIM: To perform an accurate primary repair of temporal bone defects. MATERIAL AND METHODS: The temporal bone defect models were performed in beagles. Extended estimated patches of the defects were predesigned by 3D reconstruction software and molded from polyether ether ketone (PEEK) using a lathe. The precise trimming of the extended PEEK patches was established via coordinate transformation of the patches between the navigation system and the reconstruction software, and in real-time tracing via intraoperative navigation. Trimmed PEEK patches were embedded onto the defects. Blood tests and image examinations were conducted postoperatively. RESULTS: The extended PEEK patches were prepared precisely according to the predesign. Real-time tracing of the actual skull defect profile was performed quickly and accurately. Trimmed skull patches perfectly matched the shape of the defects. No signs of infection, absorption, or translocation of the patches occurred postoperatively, and little epidural effusion was found. CONCLUSION: With the assistance of navigation and 3D reconstruction technology, customized molded PEEK patches can be used for accurate primary repair of temporal bone defects.

Characterization and functional study of a chimera galectin from yellow drum Nibea albiflora.

Galectins are protein that participates in a variety of immune responses in the process of pathogenic infections. In the present study, a chimera galectin gene was screened from the transcriptome database of Nibea albiflora, which was named as YdGal-3. The results of qRT-PCR showed that the mRNA transcripts of YdGal-3 were ubiquitously distributed in all the detected tissues. After infection with Vibrio harveyi, the expression of YdGal-3 in liver, spleen, and head kidney increased significantly. Immunohistochemistry showed that YdGal-3 protein was widely expressed in the head kidney. The purified YdGal-3 protein by prokaryotic expression agglutinated red blood cells. Sugar inhibition assay showed that the agglutinating activity of YdGal-3 protein was inhibited by different sugars including lactose, D-galactose, and lipopolysaccharide. In addition, we mutated YdGal-3 His 294 into proline (P), alanine (A), glycine (G), and aspartic acid (D), it was further proved that the residue plays a key role in agglutination. YdGal-3 agglutinated some gram-negative bacteria including Pseudomonas plecoglossicida, Vibrio parahemolyticus, V. harveyi, and Aeromonas hydrophila, and exhibited antibacterial activity. These results suggested that YdGal-3 protein played an important role in the innate immunity of N. albiflora.

[Rapid Reconstruction of Craniotomy Defects Based on Surgical Navigation].

In neurosurgery, skull repair caused by surgical approach is one of the important research contents. In this paper, a rapid reconstruction method of the skull defect with optical navigation system is proposed. This method can automatically reconstruct the structure of skull defect with the intraoperative defect edge points and preoperative medical image data. The head model experiment was used to evaluate the effect of the method, the average error of the reconstruction of the defect in the right orbit was 0.424 mm, while the average error of the reconstruction of the defect in the posterior skull base was 0.377 mm. The experimental results show that the structure of the defect is consistent with the actual defect, and the reconstruction accuracy satisfies the clinical requirements in neurosurgery.

Characterizations of intracellular copper/zinc superoxide dismutase from yellow drum (Nibea albiflora, Richardson 1846) and its gene expressions under the ammonia/nitrite stress.

Intracellular copper/zinc superoxide dismutase (icCuZnSOD) is a member of superoxide dismutase family that is capable of catalyzing the superoxide radicals into either hydrogen peroxide (H2O2) or ordinary molecular oxygen (O2). Unlike mammals, the study of icCuZnSOD in aquatic animals is still in the infancy stage. Here, we identified the cDNA of na-iccuznsod from yellow drum (Nibea albiflora, Richardson 1846) and obtained its fusion protein for the first time. The mRNA expressions of na-iccuznsod were investigated in different tissues, and the dominant distribution was found in head-kidney, followed by brain, liver, heart, and gill. The effects of ammonia-N/nitrite-N on the mRNA expressions of na-iccuznsod were investigated. Na-iccuznsod transcription levels showed a general tendency of an initial up-regulation followed by a down-regulation in liver, gill, and head-kidney when yellow drum were exposed to ammonia-N/nitrite-N at the lethal concentration 50 at 96 h post-treatment, suggesting the important role of Na-icCuZnSOD in eliminating reactive oxygen species (ROS) induced by ammonia-N/nitrite-N. In addition, the characteristics of Na-icCuZnSOD protein and its comparative analysis with Na-ecCuZnSOD were investigated. Na-icCuZnSOD protein showed high enzyme stabilities over a wide range of temperature (10 to 60 °C) and pH (4.9 to 11.0), indicating its broad in vitro applications in many industries. Furthermore, the comparative analysis of Na-icCuZnSOD and Na-ecCuZnSOD gives a new perspective for the study of their structure-function relationship. Collectively, the present study will advance our understanding of the toxicity of ammonia-N/nitrite-N on yellow drum through testing the mRNA expression of iccuznsod gene, and broaden our knowledge of the protein characteristics of icCuZnSOD from fish.

Near-complete genome assembly and annotation of the yellow drum (Nibea albiflora) provide insights into population and evolutionary characteristics of this species.

Yellow drum (Nibea albiflora) is an important fish species in capture fishery and aquaculture in East Asia. We herein report the first and near-complete genome assembly of an ultra-homologous gynogenic female yellow drum using Illumina short sequencing reads. In summary, a total of 154.2 Gb of raw reads were generated via whole-genome sequencing and were assembled to 565.3 Mb genome with a contig N50 size of 50.3 kb and scaffold N50 size of 2.2 Mb (BUSCO completeness of 97.7%), accounting for 97.3%-98.6% of the estimated genome size of this fish. We further identified 22,448 genes using combined methods of ab initio prediction, RNAseq annotation, and protein homology searching, of which 21,614 (96.3%) were functionally annotated in NCBI nr, trEMBL, SwissProt, and KOG databases. We also investigated the nucleotide diversity (around 1/390) of aquacultured individuals and found the genetic diversity of the aquacultured population decreased due to inbreeding. Evolutionary analyses illustrated significantly expanded and extracted gene families, such as myosin and sodium: neurotransmitter symporter (SNF), could help explain swimming motility of yellow drum. The presented genome will be an important resource for future studies on population genetics, conservation, understanding of evolutionary history and genetic breeding of the yellow drum and other Nibea species.

A high-density genetic linkage map and QTL mapping for growth and sex of yellow drum (Nibea albiflora).

A high-density genetic linkage map is essential for the studies of comparative genomics and gene mapping, and can facilitate assembly of reference genome. Herein, we constructed a high-density genetic linkage map with 8,094 SNPs selected from 113 sequenced fish of a F1 family. Ultimately, the consensus map spanned 3818.24 cM and covered nearly the whole genome (99.4%) with a resolution of 0.47 cM. 1,457 scaffolds spanning 435.15 Mb were anchored onto 24 linkage groups, accounting for 80.7% of the draft genome assembly of the yellow drum. Comparative genomic analyses with medaka and zebrafish genomes showed superb chromosome-scale synteny between yellow drum and medaka. QTL mapping and association analysis congruously revealed 22 QTLs for growth-related traits and 13 QTLs for sex dimorphism. Some important candidate genes such as PLA2G4A, BRINP3 and P2RY1 were identified from these growth-related QTL regions. A gene family including DMRT1, DMRT2 and DMRT3 was identified from these sex-related QTL regions on the linkage group LG9. We demonstrate that this linkage map can facilitate the ongoing marker-assisted selection and genomic and genetic studies for yellow drum.

Androstenedione and 17α-methyltestosterone induce early ovary development of Anguilla japonica.

Endocrine effects as 11-ketotestosterone (11-KT), an unaromatizable androgen, regulating the follicles growth in the previtellogenic stage of eel reproduction have been widely elucidated. However, the influence of aromatizable androgens on the brain-pituitary-gonad axis during oogenesis in A. japonica has not been clearly elaborated. In the study, androstenedione (AD) and 17α-methyltestosterone (MT) were employed together to induce ovary development of seven-year-old female Anguilla japonica through feeding or exposure in the migration season. After female A. japonica had been fed with commercial diet containing 5 mg AD and MT kg d-1 body weight respectively for 45 d in fresh water (Trial I), the development of oocytes still remained at the oil droplet stage, but the GSI and follicle diameter increased significantly. The serum 11-KT level and expression of liver vitellogenin mRNA were significantly elevated. After female fish had been exposed to seawater containing 50 µg L-1 AD and MT respectively for 45 d (Trial II), the ovaries of A. japonica almost reached midvitellogenic stage and the GSI and follicle diameter increased significantly. Yolk granular layer was observed in the peripheral ooplasm. The serum 11-KT level maintained consistently low, and the serum E2 level declined significantly to a relatively low level. The expression levels of ovarian arα and cyp19a1, brain (with pituitary together) mGnRH and lhß increased significantly. The results showed that A. japonica in Trial II appeared a higher ovarian development than those in Trial I. These findings indicated that AD and MT increased the oil droplet and enlarged follicle diameter in previtellogenic stage, while the vitellogenesis and gonadotropin release did not occur in Trial I. In Trial II, AD and MT promoted vitellogenesis by stimulating the ovary expression of arα and by up-regulating brain mGnRH and pituitary lhß expression.

[Automatic Robotic Puncture System for Accurate Liver Cancer Ablation Based on Optical Surgical Navigation].

This paper designed an automatic robotic puncture system for accurate liver cancer ablation based on optical surgical navigation. The near-infrared optical surgical navigation system we constructed for liver ablation was applied to carry out surgical planning and simulation, the near-infrared cameras dynamically tracked the current position of puncture needle relative to the location of the patient's anatomy, then guided the surgery robot to position precisely in three-dimensional space and performed the surgery.

The channel catfish genome sequence provides insights into the evolution of scale formation in teleosts.

Catfish represent 12% of teleost or 6.3% of all vertebrate species, and are of enormous economic value. Here we report a high-quality reference genome sequence of channel catfish (Ictalurus punctatus), the major aquaculture species in the US. The reference genome sequence was validated by genetic mapping of 54,000 SNPs, and annotated with 26,661 predicted protein-coding genes. Through comparative analysis of genomes and transcriptomes of scaled and scaleless fish and scale regeneration experiments, we address the genomic basis for the most striking physical characteristic of catfish, the evolutionary loss of scales and provide evidence that lack of secretory calcium-binding phosphoproteins accounts for the evolutionary loss of scales in catfish. The channel catfish reference genome sequence, along with two additional genome sequences and transcriptomes of scaled catfishes, provide crucial resources for evolutionary and biological studies. This work also demonstrates the power of comparative subtraction of candidate genes for traits of structural significance.

Phosphoinositide 3-kinase family in channel catfish and their regulated expression after bacterial infection.

The phosphoinositide-3-kinase (PI3Ks) family of lipid kinases is widely conserved from yeast to mammals. In this work, we identified a total of 14 members of the PI3Ks from the channel catfish genome and transcriptome and conducted phylogenetic and syntenic analyses of these genes. The expression profiles after infection with Edwardsiella ictaluri and Flavobacterium columnare were examined to determine the involvement of PI3Ks in immune responses after bacterial infection in catfish. The results indicated that PI3Ks genes including all of the catalytic subunit and several regulatory subunits genes were widely regulated after bacterial infection. The expression patterns were quite different when challenged with different bacteria. The PI3Ks were up-regulated rapidly at the early stage after ESC infection, but their induced expression was much slower, at the middle stage after columnaris infection. RNA-Seq datasets indicated that PI3K genes may be expressed at different levels in different catfish differing in their resistance levels against columnaris. Future studies are required to confirm and validate these observations. Taken together, this study indicated that PI3K genes may be involved as a part of the defense responses of catfish after infections, and they could be one of the determinants for disease resistance.

Genome-wide identification of Hsp70 genes in channel catfish and their regulated expression after bacterial infection.

Heat shock proteins 70/110 (Hsp70/110) are a family of conserved ubiquitously expressed heat shock proteins which are produced by cells in response to exposure to stressful conditions. Besides the chaperone and housekeeping functions, they are also known to be involved in immune response during infection. In this study, we identified 16 Hsp70/110 geness in channel catfish (Ictalurus punctatus) through in silico analysis using RNA-Seq and genome databases. Among them 12 members of Hsp70 (Hspa) family and 4 members of Hsp110 (Hsph) family were identified. Phylogenetic and syntenic analyses provided strong evidence in supporting the orthologies of these HSPs. In addition, we also determined the expression patterns of Hsp70/110 genes after Flavobacterium columnare and Edwardsiella ictaluri infections by meta-analyses, for the first time in channel catfish. Ten out of sixteen genes were significantly up/down-regulated after bacterial challenges. Specifically, nine genes were found significantly expressed in gill after F. columnare infection. Two genes were found significantly expressed in intestine after E. ictaluri infection. Pathogen-specific pattern and tissue-specific pattern were found in the two infections. The significantly regulated expressions of catfish Hsp70 genes after bacterial infections suggested their involvement in immune response in catfish.

Hsp90, Hsp60 and sHsp families of heat shock protein genes in channel catfish and their expression after bacterial infections.

Heat shock proteins (Hsps) are a suite of highly conserved proteins whose expressions are generally induced by elevated temperature. However, many Hsps play important roles in both innate and adaptive immunity. On the basis of our previous work on Hsp40 and Hsp70 gene families in channel catfish (Ictalurus punctatus), the objective of this study was to characterize Hsp90, Hsp60, Hsp10, and small Hsp genes, and to investigate their expression profiles after bacterial infections. A total of 20 Hsp genes were identified and annotated in the channel catfish genome, including five Hsp90 genes, one Hsp60 gene, one Hsp10 gene, and 13 sHsp genes. Six Hsp genes were differentially expressed after Edwardsiella ictaluri infection, and 12 were differentially expressed after Flavobacterium columnare infection. Although expression of these genes exhibited both temporal and spatial regulation, the induction of Hsp genes was observed soon after bacterial infection, while the suppression of Hsp genes was observed at later time-points, suggesting their distinct roles in immune responses and disease defenses. A pathogen-specific expression pattern of Hsp90 was observed. After F. columnare infection, all Hsp90 genes were found up-regulated except Hsp90ab1, which was not significantly regulated. However, after E. ictaluri infection, only one Hsp90 gene was found significantly down-regulated. Both pathogen-specific and tissue-specific pattern of expression were observed with small Hsps after E. ictaluri and F. columnare bacterial infections. These results suggested that most of Hsp genes may play important roles in immune response and/or disease defense in channel catfish.

[Research progress on techniques for artificial propagation of corals].

The natural coral reef resources degrade rapidly because of climate change, environmental pollution and exploitation of aquarium species. Artificial propagation is an effective way to facilitate the reduction of wild harvesting, reef restoration, preservation of biodiversity. This paper reviewed the technique and research progresses focused on coral artificial propagation. We compared the advantages and disadvantages of sexual reproduction and asexual reproduction as well as in situ and ex situ propagation. Moreover, we summarized the important roles of irradiation, flow rate, nutrients, feed and other factors in coral propagation within recirculating aquaculture system (RAS). Irradiation is the key to successful ex situ coral culture and different species show different needs of radiation intensity and light spectrum. Therefore, artificial lighting in RAS, as well as. power and maintenance costs, are very important for ex situ coral aquaculture. In addition, corals are very sensitive to NH4+, NO3-, NO2- as well as phosphate in RAS, and many physical, chemical and biological methods are acquired to maintain low nutrients condition. Although RAS has progressed a lot in terms of irradiation, flow rate and nutrient control, future studies also should focus on sexual reproduction, genetic modification and disease control.