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Galectins are proteins that are involved in the innate immune response against pathogenic microorganisms. In the present study, the gene expression pattern of galectin-1 (named as NaGal-1) and its function in mediating the defense response to bacterial attack were investigated. The tertiary structure of NaGal-1 protein consists of homodimers and each subunit has one carbohydrate recognition domain. Quantitative RT-PCR analysis indicated that NaGal-1 was ubiquitously distributed in all the detected tissues and highly expressed in the swim-bladder of Nibea albiflora, and its expression could be upregulated by the pathogenic Vibrio harveyi attack in the brain. Expression of NaGal-1 protein in HEK 293T cells was distributed in the cytoplasm as well as in the nucleus. The recombinant NaGal-1 protein by prokaryotic expression could agglutinate red blood cells from rabbit, Larimichthys crocea, and N. albiflora. The agglutination of N. albiflora red blood cells by the recombinant NaGal-1 protein was inhibited by peptidoglycan, lactose, D-galactose, and lipopolysaccharide in certain concentrations. In addition, the recombinant NaGal-1 protein agglutinated and killed some gram-negative bacteria including Edwardsiella tarda, Escherichia coli, Photobacterium phosphoreum, Aeromonas hydrophila, Pseudomonas aeruginosa, and Aeromonas veronii. These results set the stage for further studies of NaGal-1 protein in the innate immunity of N. albiflora.

Due to an increasing number of abused drugs dumped into the wastewater, more and more drugs are detected in the water environment, which may affect the survival of aquatic organisms. Lenvatinib is a multi-targeted tyrosine kinase inhibitor, and is clinically used to treat differentiated thyroid cancer, renal epithelial cell carcinoma and liver cancer. However, there are few reports on the effects of lenvatinib in embryos development. In this study, zebrafish embryos were used to evaluate the effect of lenvatinib on cardiovascular development. Well-developed zebrafish embryos were selected at 6 h post fertilization (hpf) and exposed to 0.05 mg/L, 0.1 mg/L and 0.2 mg/L lenvatinib up to 72 hpf. The processed embryos demonstrated cardiac edema, decreased heart rate, prolonged SV-BA distance, inhibited angiogenesis, and blocked blood circulation. Lenvatinib caused cardiac defects in the whole stage of cardiac development and increased the apoptosis of cardiomyocyte. Oxidative stress in the processed embryos was accumulated and inhibiting oxidative stress could rescue cardiac defects induced by lenvatinib. Additionally, we found that lenvatinib downregulated Notch signaling, and the activation of Notch signaling could rescue cardiac developmental defects and downregulate oxidative stress level induced by lenvatinib. Our results suggested that lenvatinib might induce cardiac developmental toxicity through inducing Notch mediated-oxidative stress generation, raising concerns about the harm of exposure to lenvatinib in aquatic organisms.

Phosphoribosyl pyrophosphate synthetases (EC 2.7.6.1) are key enzymes in the biological synthesis of phosphoribosyl pyrophosphate and are involved in diverse developmental processes. In our previous study, the PRPS1 gene was discovered as a key disease-resistance candidate gene in yellow drum, Nibea albiflora, in response to the infection of Vibrio harveyi, through genome-wide association analysis. This study mainly focused on the characteristics and its roles in immune responses of the PRPS1 gene in yellow drum. In the present study, the NaPRPS1 gene was cloned from yellow drum, encoding a protein of 320 amino acids. Bioinformatic analysis showed that NaPRPS1 was highly conserved during evolution. Quantitative RT-PCR demonstrated that NaPRPS1 was highly expressed in the head-kidney and brain, and its transcription and translation were significantly activated by V. harveyi infection examined by RT-qPCR and immunohistochemistry analysis, respectively. Subcellular localization revealed that NaPRPS1 was localized in cytoplasm. In addition, semi-in vivo pull-down assay coupled with mass spectrometry identified myeloid differentiation factor 88 (MyD88) as an NaPRPS1-interacting patterner, and their interaction was further supported by reciprocal pull-down assay and co-immunoprecipitation. The inducible expression of MyD88 by V. harveyi suggested that the linker molecule MyD88 in innate immune response may play together with NaPRPS1 to coordinate the immune signaling in yellow drum in response to the pathogenic infection. We provide new insights into important functions of PRPS1, especially PRPS1 in the innate immunity of teleost fishes, which will benefit the development of marine fish aquaculture.

From April to May, 2016, an archaeological survey for Paleolithic sites along the Muling River Basin was carried out jointly by a team from the School of Archaeology of Jilin University, the Institute of Cultural Relics and Archaeology of Heilongjiang Province, and local city and county Cultural Relics Administrative institutes. A total of 21 Paleolithic localities and 974 stone artifacts were found. This paper presents the results of this survey and a typological classification of the stone artifacts found in these localities, which are divided into different industry types (Flake, Blade, and Microblade) through their characteristics. Stratigraphic methods are used to speculate on the ages of the collected assemblages. Then, their functions are discussed through considerations of percussion technique, tool processing, and toolkits. Finally, they are compared with Paleolithic sites of the same age from surrounding areas.

The large yellow croaker (Larimichthys crocea) is one of the most economically valuable mariculture fish in China. Infection of Pseudomonas plecoglossicida can cause an outbreak of “internal organ white-spot disease”, which seriously affects the aquaculture of the large yellow croaker. Ubiquitylation is closely related to the post-translation modification of proteins and plays a vital role in many hosts’ immune defense pathways, while the E2-binding enzyme is a key factor in ubiquitination. Our previous genome-wide association study found that the ubiquitin-binding enzyme E2G1 (designed LcUbe2g1) was one of the candidate genes related to disease resistance in large yellow croaker. In this study, we analyzed the molecular characteristics, function, and immune mechanism of the LcUbe2g1. The full-length cDNA is 812 bp, with an open reading frame of 513 bp, encoding 170 amino acid residues. The results of the RT-qPCR and immunohistochemistry analysis revealed that its transcription and translation were significantly activated by the infection of P. plecoglossicida in large yellow croaker. Immunocytochemistry experiments verified the co-localization of LcUBE2G1 and the ubiquitin proteins in the head kidney cells of large yellow croaker. Through GST pull-down, we found that LcUBE2G1 interacted with NEDD8 to co-regulate the ubiquitination process. The above results indicate that LcUBE2G1 is essential in the regulation of ubiquitination against P. plecoglossicida infection in large yellow croaker, which lays a foundation for further study on the resistance mechanism of internal organ white-spot disease.

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.

The Rho family GTPase Rac1 acts as a molecular switch for signal transduction to regulate various cellular functions. Here, a Rac1 homolog (LcRac1) was identified in large yellow croaker (Larimichthys crocea), one of the most economically important marine fishes. The LcRac1 protein was expressed in Escherichia coli and purified. Subsequently the specific antibody was raised using the purified fusion protein (GST-LcRac1). LcRac1 was ubiquitously expressed in all 12 tissues we examined, with the highest expression in heart and blood and the weakest expression in head-kidney and spleen. Moreover, time course analysis revealed that LcRac1 expression was obviously up-regulated in liver, spleen and head-kidney after immunization with Poly I:C, LPS and Vibrio parahemolyticus. On the other hand, on the basis of protein interaction, it was found that the LcRac1 interacted with Tropomyosin, a crucial protein in the process of phagocytosis. Furthermore, RNAi assays indicated that the phagocytic percentage and phagocytic index were significantly decreased when the LcRac1 gene was silenced by sequence-specific siRNA. Fluorescence microscopy assays revealed FITC-labeled V. parahemolyticus were remarkably decreased after LcRac1 was silenced by sequence-specific siRNA at 24 h. These findings implicate the vital role of LcRac1 in innate immunity in the large yellow croaker.

Aluminum-based localized surface plasmon resonance (LSPR) holds attractive properties include low cost, high natural abundance, and ease of processing by a wide variety of methods including complementary metal oxide semiconductor process, making itself having an edge over conventional ones induced by noble metal. However, the inherent drawbacks of plasmonic mode limited on UV-green wavelength, low refractive index sensitivity, as well as heavy-shape-dependence greatly prevent aluminum plasmonics from real-life biosensing. Here, we demonstrated a uniform quasi-3-dimensional Al nanopyramid array (NPA) structure with tunable ultraviolet-visible-infrared (UV-vis-NIR) plasmon resonances for biosensing. By changing the reflection measuring angle, we could easily obtain typical peaks simultaneously exhibited on the reflectance spectrum across UV-vis-NIR wave region. The Al NPAs carried out high refractive index sensitivities which even comparable with that of noble metal, and can be used as a biosensor for directly detecting cytochrome c and carbohydrate antigen 199 in air after the sensing surface was washed cleanly and dried; the limits of detection were determined to be 800 nM and 29 ng/mL, respectively. Our proposed work therefore initiates the low-cost, high-performance biosensing using aluminum plasmonics, which would find wide applications in rapid diagnosis, mobile-healthcare and environmental monitoring.

In the course of a reverse genetic screen in the Belgian Blue cattle breed, we uncovered a 10-bp deletion (c.87_96del) in the first coding exon of the melanophilin gene (MLPH), which introduces a premature stop codon (p.Glu32Aspfs*1) in the same exon, truncating 94% of the protein. Recessive damaging mutations in the MLPH gene are well known to cause skin, hair, coat or plumage color dilution phenotypes in numerous species, including human, mice, dog, cat, mink, rabbit, chicken and quail. Large-scale array genotyping undertaken to identify p.Glu32Aspfs*1 homozygous mutant animals revealed a mutation frequency of 5% in the breed and allowed for the identification of 10 homozygous mutants. As expression of a colored coat requires at least one wild-type allele at the co-dominant Roan locus encoded by the KIT ligand gene (KITLG), homozygous mutants for p.Ala227Asp corresponding with the missense mutation were excluded. The six remaining colored calves displayed a distinctive dilution phenotype as anticipated. This new coat color was named 'cool gray'. It is the first damaging mutation in the MLPH gene described in cattle and extends the already long list of species with diluted color due to recessive mutations in MLPH and broadens the color palette of gray in this breed.

DNA methylation is an essential epigenetic modification that significantly regulates gene expression during development and differentiation. In this study, genome-wide methylation analysis of different gonads of the large yellow croaker was performed using whole-genome bisulfite sequencing (WGBS), which has characterized DNA methylation patterns in gonad tissue and identified candidate regions for future studies. Clustering analysis revealed that male and neomale methylation patterns were close compared to female. Based on KEGG pathway analysis of differentially methylated genes, we obtained signaling pathways related to gonadal development. We further investigated the methylation status of previously reported sex determination genes, and found that these genes showed different methylation status in three types of gonads, which may provide important clues to reveal the sex determination genes in the large yellow croaker. Furthermore, combined with transcriptome analysis, we found 7 sex-related genes in three comparison groups where expression negatively correlated with methylation.

Bamaxiang pig is from Guangxi province in China, characterized by its small body size and two-end black coat colour. It is an important indigenous breed for local pork market and excellent animal model for biomedical research. In this study, we performed genomewide association studies (GWAS) on 43 growth and carcass traits in 315 purebred Bamaxiang pigs based on a 1.4 million SNP array. We observed considerable phenotypic variability in the growth and carcass traits in the Bamaxiang pigs. The corresponding SNP based heritability varied greatly across the 43 traits and ranged from 9.0% to 88%. Through a conditional GWAS, we identified 53 significant associations for 35 traits at p value threshold of 10-6 . Among which, 26 associations on chromosome 3, 7, 14 and X passed a genomewide significance threshold of 5 × 10-8 . The most remarkable loci were at around 30.6 Mb on chromosome 7, which had growth stage-dependent effects on body lengths and cannon circumferences and showed large effects on multiple carcass traits. We discussed HMGA1 NUDT3, EIF2AK1, TMEM132C and AFF2 that near the lead SNP of significant loci as plausible candidate genes for corresponding traits. We also showed that including phenotypic covariate in GWAS can help to reveal additional significant loci for the target traits. The results provide insight into the genetic architecture of growth and carcass traits in Bamaxiang pigs.

Four newborn purebred Belgian Blue calves presenting a severe form of epidermolysis bullosa were recently referred to our heredo-surveillance platform. SNP array genotyping followed by autozygosity mapping located the causative gene in a 8.3-Mb interval on bovine chromosome 24. Combining information from (i) whole-genome sequencing of an affected calf, (ii) transcriptomic data from a panel of tissues and (iii) a list of functionally ranked positional candidates pinpointed a private G to A nucleotide substitution in the LAMA3 gene that creates a premature stop codon (p.Arg2609*) in exon 60, truncating 22% of the corresponding protein. The LAMA3 gene encodes the alpha 3 subunit of the heterotrimeric laminin-332, a key constituent of the lamina lucida that is part of the skin basement membrane connecting epidermis and dermis layers. Homozygous loss-of-function mutations in this gene are known to cause severe junctional epidermolysis bullosa in human, mice, horse, sheep and dog. Overall, our data strongly support the causality of the identified gene and mutation.

Localized surface plasmon resonance (LSPR) combined with immunoassay shows greatly potential in fast detection of tumor markers. In this paper, a highly sensitive LSPR substrate has been fabricated and modified for direct detection of alpha-fetoprotein (AFP). The biosensor was prepared by interference lithography, and modified by covalently immobilizing anti-AFP on the surface of gold nano-mushroom arrays (GNMA). The modification process was investigated by Vis-NIR reflectance spectra and cyclic voltammogram measurements. We revealed the optical properties of the modified GNMA by measuring the Vis-NIR reflectance spectra and simulating its electric intensity field distribution under light illumination. The GNMA substrate was highly sensitive, with a refractive index sensitivity of ~465 nm/RIU. The substrate can be applied to label-free detection of AFP, with the linear range and the limit of detection determined to be 20-200 ng/mL and 24 ng/mL (S/N=3), respectively. We also demonstrated its clinical application by directly detecting AFP in human serum samples. It is expected that our biosensor could be integrated on microfluidic chips for high-throughput detection in portable early diagnosis, post-operative and point-of-care (POC) in clinical applications.

In this paper, we fabricated uniform polydimethylsiloxane (PDMS) magnetic microcapsules with eccentric internal structures and employed them as a novel delivery system for orientation-specific and dual stimuli-responsive controlled drug release. These eccentric microcapsules contained Fe3O4 nanoparticles in their inner cores. Because of the paramagnetic Fe3O4 nanoparticles, the eccentric microcapsules could be accurately moved by a magnetic field, leading to the precise control of the microcapsule locations. Also, due to the eccentric structures of the magnetic microcapsules, the capsules exhibited a non-uniform magnetic property; the capsules could be aligned by magnetic fields with their thin walls facing the magnet, resulting in a precise orientation-specific control of the microcapsules. More interestingly, the eccentric magnetic microcapsules demonstrated a dual stimuli-responsive controlled release of inclusions involving a sustained release under ultrasound and an intense release under laser stimulation. Furthermore, we studied the efficacy of doxorubicin (DOX) release from the microcapsules regulated by laser stimulation by performing in vitro cell tests with and without an applied magnetic field. The cell tests showed that the orientation-specific control of the microcapsules under a magnetic field (when the thin walls of the eccentric microcapsules were oriented towards the cell) improved the efficacy of the drug released from the microcapsules. The results suggested that our eccentric magnetic microcapsules hold all the properties needed for a site-specific, orientation-specific and dual stimuli-responsive delivery system, demonstrating a great potential application for multifunctional controlled drug release.

A paper-based microfluidic device based on unconventional principle was developed and used to detect lead ions through a two-step process including heated incubation and subsequent mixing. The device was made by generating a superhydrophobic pattern, which defines channel and reservoir barriers, on a water-impermeable paper substrate, followed by loading and drying the reagents in the defined reservoirs. Different from the conventional paper-based devices that are made of water-permeable paper, the as-prepared device holds water drops in discrete reservoirs, and the water drops will not move unless the device is titled along the direction of the predefined channels. In this way, the liquid samples applied onto the device are handled as individual drops and could be stored, transported, and mixed on demand. Different from the conventional paper-based devices that use capillary force to drive liquid, our new device uses wetting and gravity as driving force. We name this operation principle suspending-droplet mode paper-based device (SD-μPAD). The use of a Teflon contact-printing stamp makes the production of such devices rapid, cost efficient, and mass productive. Utilizing a G-quadruplex-based luminescence switch-on assay, we demonstrated rapid, convenient, highly sensitive, and low cost detection of lead(II) ions in water samples, using a custom made battery-powered portable device, and a smart phone as the detector.

In this paper, a strategy was developed for fabricating uniform polydimethylsiloxane (PDMS) microcapsules with eccentric and core-centered internal hollow structures, which can be employed as a novel controlled-release system for site-specific drug delivery under ultrasound regulation. This strategy involves the use of a microfluidic device, through which three phases (i.e., an inner water phase containing drug molecules, a middle oil phase of PDMS solution, and an outer water phase) were delivered at independently adjustable flow rates, allowing the formation of water-in-oil-in-water (W/O/W) emulsion droplets in a microfluidic device. After baking the as-prepared microcapsules, microcapsules with different inner hollow cores were obtained. The sizes of the inner hollow structures could be tuned, leading to a series of microcapsules with different densities. The densities of these microcapsules were all lower than that of water, which showed a long gastric residence time. Most interestingly, eccentric hollow microcapsules with well-controlled sizes and shapes were also prepared using this method. The eccentric and core-centered hollow microcapsules demonstrated triggered and controlled the release of encapsulation under ultrasound, for which the release profiles were consistent with the theoretical simulation. The results showed that the microcapsules had all the properties of a floating drug delivery system and controlled release system, and demonstrated great potential to be used for controlled release, in particular, for the delivery of drugs that are absorbed primarily in the upper segments of the gastrointestinal tract.

Summary Muscle fibre traits are related with meat quality in meat animals. In this study, a whole-genome scan with 183 microsatellite markers covering the pig genome was performed to identify quantitative trait loci (QTL) for cross-sectional area, numerical percentage and relative area of type I, IIA and IIB myofibres, fibre number per square centimetre and total fibre number in the longissimus muscle by using 120 F2 animals in a White Duroc × Erhualian intercross. In total, 20 QTL were mapped on pig chromosomes (SSC) 1, 2, 7, 8, 9, 11, 15, 16 and X, of which eight reached genome-wide significance levels and explained large proportions (6.53–34.63%) of phenotypic variance. Five QTL detected in this study confirmed the previous QTL reports and the others were detected for the first time. Chinese Erhualian alleles are generally associated with muscle fibre traits favourable for meat quality.