Molecular Cloning, Characterization, and Function of Insulin-Related Peptide 1 (IRP1) in the Haliotis discus hanna.

Abalone is a popular mollusk in the marine aquaculture industry of China. However, existing challenges, like slow growth, individual miniaturization, and the absence of abundant abalone, have emerged as significant obstacles impeding its long-term progress in aquaculture. Studies have demonstrated that insulin-related peptide (IRP) is a crucial factor in the growth of marine organisms. However, limited studies have been conducted on IRP in abalone. This study indicated that the hdh-MIRP1 open reading frame (ORF) was composed of 456 base pairs, which encoded 151 amino acids. Based on the gene expression and immunofluorescence analyses, the cerebral ganglion of Haliotis discus hannai (H. discus hannai) was the primary site of hdh-MIRP1 mRNA expression. Moreover, hdh-MIRP1 expression was observed to be higher in the larger group than in the smaller group abalones. Only single nucleotide polymorphism (SNP) was related to their growth characteristics. However, approximately 82 proteins that may interact with hdh-MIRP1 were identified. The functional enrichment analysis of the 82 genes indicated that hdh-MIRP1 may be involved in the regulation of glucose metabolism and the process of growth. This study established a benchwork for further investigating the role of IRP in the growth of abalone.

Silk Gland Factor 1 Plays a Pivotal Role in Larval Settlement of the Fouling Mussel Mytilopsis sallei.

Most fouling organisms have planktonic larval and benthic adult stages. Larval settlement, the planktonic-benthic transition, is the critical point when biofouling begins. However, our understanding of the molecular mechanisms of larval settlement is limited. In our previous studies, we identified that the AMP-activated protein kinase-silk gland factor 1 (AMPK-SGF1) pathway was involved in triggering the larval settlement in the fouling mussel M. sallei. In this study, to further confirm the pivotal role of SGF1, multiple targeted binding compounds of SGF1 were obtained using high-throughput virtual screening. It was found that the targeted binding compounds, such as NAD+ and atorvastatin, could significantly induce and inhibit the larval settlement, respectively. Furthermore, the qRT-PCR showed that the expression of the foot proteins' genes was significantly increased after the exposure to 10 µM NAD+, while the gene expression was significantly suppressed after the exposure to 10 µM atorvastatin. Additionally, the production of the byssus threads of the adults was significantly increased after the exposure to 10-20 µM of NAD+, while the production of the byssus threads was significantly decreased after the exposure to 10-50 µM of atorvastatin. This work will deepen our understanding of SGF1 in triggering the larval settlement in mussels and will provide insights into the potential targets for developing novel antifouling agents.

miR-423-5p Regulates Skeletal Muscle Growth and Development by Negatively Inhibiting Target Gene SRF.

The process of muscle growth directly affects the yield and quality of pork food products. Muscle fibers are created during the embryonic stage, grow following birth, and regenerate during adulthood; these are all considered to be phases of muscle development. A multilevel network of transcriptional, post-transcriptional, and pathway levels controls this process. An integrated toolbox of genetics and genomics as well as the use of genomics techniques has been used in the past to attempt to understand the molecular processes behind skeletal muscle growth and development in pigs under divergent selection processes. A class of endogenous noncoding RNAs have a major regulatory function in myogenesis. But the precise function of miRNA-423-5p in muscle development and the related molecular pathways remain largely unknown. Using target prediction software, initially, the potential target genes of miR-423-5p in the Guangxi Bama miniature pig line were identified using various selection criteria for skeletal muscle growth and development. The serum response factor (SRF) was found to be one of the potential target genes, and the two are negatively correlated, suggesting that there may be targeted interactions. In addition to being strongly expressed in swine skeletal muscle, miR-423-5p was also up-regulated during C2C12 cell development. Furthermore, real-time PCR analysis showed that the overexpression of miR-423-5p significantly reduced the expression of myogenin and the myogenic differentiation antigen (p < 0.05). Moreover, the results of the enzyme-linked immunosorbent assay (ELISA) demonstrated that the overexpression of miR-423-5p led to a significant reduction in SRF expression (p < 0.05). Furthermore, miR-423-5p down-regulated the luciferase activities of report vectors carrying the 3' UTR of porcine SRF, confirming that SRF is a target gene of miR-423-5p. Taken together, miR-423-5p's involvement in skeletal muscle differentiation may be through the regulation of SRF.

Genome-wide identification and expression profiles of sex-related gene families in the Pacific abalone Haliotis discus hannai.

In recent years, members of the Dmrt family, TGF-ß superfamily and Sox family have been recognized as crucial genes for sex determination/differentiation across diverse animal species. Nevertheless, knowledge regarding the abundance and potential functions of these genes in abalone remains limited. In this study, a total of 5, 10, and 7 members of the Dmrt family, the TGF-ß superfamily and the Sox family, respectively, were identified in the Pacific abalone Haliotis discus hannai. Sequence characteristics, phylogenetic relationships and spatiotemporal expression profiles of these genes were investigated. Notably, HdDmrt-04 (Dmrt1/1L-like) emerged as a potential mollusc-specific gene with a preponderance for expression in the testis. Interestingly, none of the TGF-ß superfamily members exhibited specific or elevated expression in the gonads, highlighting the need for further investigation into their role in abalone sex differentiation. The Sox proteins in H. discus hannai were categorized into 7 subfamilies: B1, B2, C, D, E, F, and H. Among them, HdSox-07 (SoxH-like) was observed to play a crucial role in testis development, while HdSox-03 (SoxB1-like) and HdSox-04 (SoxC-like) probably cooperate in abalone ovary development. Taken together, the results of the present study suggested that HdDmrt-04 and HdSox-07 can be used as male-specific markers for gonad differentiation in H. discus hannai and imply conservation of their functions across invertebrates and vertebrates. Our findings provide new insights into the evolution and genetic structure of the Dmrt family, the TGF-ß superfamily and the Sox family in abalone and pave the way for a deeper understanding of sex differentiation in gastropods.

Molecular Characterization and Function of Bone Morphogenetic Protein 7 (BMP7) in the Pacific Abalone, Haliotis discus hannai.

Bone morphogenetic proteins (BMPs) play important roles in a lot of biological processes, such as bone development, cell proliferation, cell differentiation, growth, etc. However, the functions of abalone BMP genes are still unknown. This study aimed to better understand the characterization and biological function of BMP7 of Haliotis discus hannai (hdh-BMP7) via cloning and sequencing analysis. The coding sequence (CDS) length of hdh-BMP7 is 1251 bp, which encodes 416 amino acids including a signal peptide (1-28 aa), a transforming growth factor-ß (TGF-ß) propeptide (38-272 aa), and a mature TGF-ß peptide (314-416 aa). The analysis of expression showed that hdh-BMP7 mRNA was widely expressed in all the examined tissues of H. discus hannai. Four SNPs were related to growth traits. The results of RNA interference (RNAi) showed that the mRNA expression levels of hdh-BMPR I, hdh-BMPR II, hdh-smad1, and hdh-MHC declined after hdh-BMP7 was silenced. After RNAi experiment for 30 days, the shell length, shell width, and total weight were found to be reduced in H. discus hannai (p < 0.05). The results of real-time quantitative reverse transcription PCR revealed that the hdh-BMP7 mRNA was lower in abalone of the S-DD-group than in the L-DD-group. Based on these data, we hypothesized that BMP7 gene has a positive role in the growth of H. discus hannai.

Transcriptomic responses to thermal stress in hybrid abalone (Haliotis discus hannai ♀ × H. fulgens ♂).

China is the world's largest abalone producing country. Currently, summer mortality caused by high temperature, is one of the biggest challenges for abalone aquaculture industry. The hybrid abalone (Haliotis discus hannai ♀ × H. fulgens ♂) was conferred on the "new variety". It has heterosis for thermal tolerance and has been cultured at large-scale in southern China. In this study, a transcriptome analysis was performed to identify the related genes in this hybrid abalone under thermal stress and recovery stage. Compared to control group (18°C), a total of 75, 2173, 1050, 1349, 2548, 494, and 305 differentially expressed genes (DEGs) were identified at 21°C, 24°C, 27°C, 30°C, 32°C, 29°C, and 26°C, respectively. In this study, 24°C is the critical temperature at which the abalone is subjected to thermal stress. With the temperature rising, the number of stress-responsive genes increased. During the temperature recovering to the optimum, the number of stress-responsive genes decreased gradually. Thus, this hybrid abalone has a rapid response and strong adaptability to the temperature. Under the thermal stress, the abalone triggered a complicated regulatory network including degrading the misfolded proteins, activating immune systems, negative regulation of DNA replication, and activating energy production processes. The more quickly feedback regulation, more abundant energy supply and more powerful immune system might be the underlying mechanisms to fight against thermal stress in this hybrid abalone. These findings could provide clues for exploring the thermal-response mechanisms in abalone. The key genes and pathways would facilitate biomarker identification and thermal-tolerant abalone breeding studies.

Kallistatin prevents ovarian hyperstimulation syndrome by regulating vascular leakage.

Angiogenesis and increased permeability are essential pathological basis for the development of ovarian hyperstimulation syndrome (OHSS). Kallistatin (KS) is an endogenous anti-inflammatory and anti-angiogenic factor that participates in a variety of diseases, but its role in OHSS remains unknown. In this study, treating a human ovarian granulosa-like tumour cell line KGN and human primary granulosa cells (PGCs) with human chorionic gonadotropin (hCG) reduced the expression of KS, but increased the expression of VEGF. Furthermore, we found that KS could attenuate the protein level of VEGF in both KGN cells and human PGCs. More interestingly, we observed that exogenous supplementation of KS significantly inhibited a series of signs of OHSS in mice, including weight gain, ovarian enlargement, increased vascular permeability and up-regulation of VEGF expression. In addition, KS was proved to be safe on mice ovulation, progression of normal pregnancy and fetus development. Collectively, these findings demonstrated that KS treatment prevented OHSS, at least partially, through down-regulating VEGF expression. For the first time, these results highlight the potential preventive value of KS in OHSS.

A Potential Negative Regulatory Function of Myostatin in the Growth of the Pacific Abalone, Haliotis discus hannai.

Myostatin, also known as GDF8, is a member of the transforming growth factor-ß (TGF-ß) superfamily. In vertebrates, myostatin negatively regulates the growth of skeletal muscle. In invertebrates, it has been reported to be closely related to animal growth. However, knowledge concerning the molecular mechanisms involved in the myostatin regulation of molluscan growth is limited. In this study, we found that the hdh-myostatin open reading frame (ORF) comprised 1470 base pairs that encoded 489 amino acids and contained structural characteristics typical of the TGF-ß superfamily, including a C-terminal signal peptide, a propeptide domain, and TGF-ß region. Gene expression analysis revealed that hdh-myostatin mRNA was widely expressed at different levels in all of the examined tissues of Haliotis discus hannai. Nine single nucleotide polymorphisms (SNPs) were associated with the growth traits. RNA interference (RNAi) against hdh-myostatin mRNA significantly downregulated hdh-myostatin at days 1, 15, and 30 post injection, and the pattern was correlated with downregulation of the genes TGF-ß receptor type-I (hdh-TßR I), activin receptor type-IIB (hdh-ActR IIB), and mothers against decapentaplegic 3 (hdh-Smad3). After one month of the RNAi experiment, the shell lengths and total weights increased in the abalone, Haliotis discus hannai. The results of qRT-PCR showed that the hdh-myostatin mRNA level was higher in the slow-growing group than in the fast-growing group. These results suggest that hdh-myostatin is involved in the regulation of growth, and that these SNPs would be informative for further studies on selective breeding in abalone.

RNA m6A reader YTHDF2 facilitates lung adenocarcinoma cell proliferation and metastasis by targeting the AXIN1/Wnt/ß-catenin signaling.

Lung adenocarcinoma (LUAD) remains a leading cause of cancer-related deaths worldwide. YTHDF2 is a reader of N6-methyladenosine (m6A) on RNA and plays a critical role in the initiation and propagation of myeloid leukemia; however, whether YTHDF2 controls the development of LUAD remains to be explored. Here, we found that YTHDF2 was significantly upregulated in LUAD compared with paracancerous normal tissues, and YTHDF2 knockdown drastically inhibited, while its overexpression promoted, cell growth, colony formation and migration of LUAD cells in vitro. In addition, YTHDF2 knockdown significantly inhibited tumorigenesis in a murine tumor xenograft model. Through the integrative analysis of RNA-seq, m6A-seq, CLIP-seq, and RIP-seq datasets, we identified a set of potential direct targets of YTHDF2 in LUAD, among which we confirmed AXIN1, which encodes a negative regulator of the Wnt/ß-catenin signaling, as a direct target of YTHDF2. YTHDF2 promoted AXIN1 mRNA decay and subsequently activated the Wnt/ß-catenin signaling. Knockout of AXIN1 sufficiently rescued the inhibitory effect of YTHDF2 depletion on lung cancer cell proliferation, colony-formation, and migration. These results revealed YTHDF2 to be a contributor of LUAD development acting through the upregulation of the AXIN1/Wnt/ß-catenin signaling, which can be a potential therapeutic target for LUAD.

Platelet-rich plasma protects human melanocytes from oxidative stress and ameliorates melanogenesis induced by UVB irradiation.

To investigate the role of platelet-rich plasma (PRP) from different sources in alleviating oxidative stress and ameliorating melanogenesis in UVB-irradiated PIG1 cells, PIG1 cells were irradiated with 80 mJ/cm2 UVB prior to 1% PRP application and the following experiments were taken: the viability of UVB-irradiated PIG1 cells, cellular malondialdehyde (MDA) and reactive oxygen species (ROS) content, and activities of antioxidant enzymes. Western blotting was utilized to detect the expression level of proteins associated with melanin synthesis, apoptosis, and DNA lesions. We found that PRP intervention promoted cell proliferation, reduced MDA and ROS content, increased the activities of series of antioxidant enzymes, and alleviated DNA damages in UVB-damaged PIG1 cells. It is important to note that PRP treatment inhibited UVB-induced melanogenesis via the PI3K/Akt/GSK3ß signal pathway. Therefore, we suppose PRP treatment exerts a protective role through their antioxidation effect on UVB-damaged PIG1 cells and hinders melanogenesis induced by UVB irradiation.

The Anti-photoaging Effects of Pre- and Post-treatment of Platelet-rich Plasma on UVB-damaged HaCaT Keratinocytes.

Platelet-rich plasma (PRP) has seen wide clinical use owing to its regenerative and repair abilities. OBJECTIVE: To investigate the anti-photoaging effects of pre- and post-treatment of PRP on UVB-damaged HaCaT cells. METHODS: HaCaT cells were irradiated with 80 mJ/cm2 UVB, before or after PRP treatment (1000 × 107 /L), and following measurements were taken: survival rate of UVB-irradiated HaCaT cells, malondialdehyde (MDA) content and activities of glutathione peroxidase (GSH-Px), superoxide dismutase (SOD) and catalase (CAT). Western blot was used to determine the effect of different PRP intervention on the expression of PI3K, AKT, ERK, MMP-1, MMP-9, TIMP-1 and γ-H2AX in the UVB-irradiated HaCaT cells. RESULTS: pre- and post-PRP treatment reduced MDA content and increased the activities of GSH-Px, SOD and CAT in photoaged HaCaT cells. These changes resulted in reduced cytotoxic effects. Besides, different PRP intervention promoted cell proliferation via PI3K/AKT pathway. Furthermore, PRP application suppressed the expression of γ-H2AX. Also, PRP intervention alleviated photoaging effects by upregulating the expression level of tissue inhibitor of metalloproteinases-1 (TIMP-1) while downregulating matrix metalloproteinase (MMP) expression level in photoaged HaCaT cells. CONCLUSION: pre- and post-PRP treatment play anti-photoaging role through strengthening cellular oxidative defense capacity, mitigating MMP expression, alleviating DNA damages and promoting proliferation of UVB-irradiated HaCaT cells.

Quantitative secretome analysis of polymyxin B resistance in Escherichia coli.

Bacterial resistance has become a serious threat to human health. In particular, the gradual development of resistance to polymyxins, the last line of defense for human infections, is a major issue. Secreted proteins contribute to the interactions between bacteria and the environment. In this study, we compared the secretomes of polymyxin B-sensitive and -resistant Escherichia coli strains by data-independent acquisition mass spectrometry. In total, 87 differentially expressed secreted proteins were identified in polymyxin B-resistant E. coli compared to the sensitive strain. A GO enrichment analysis indicated that the differentially expressed proteins were involved in biological processes, including bacterial-type flagellum-dependent cell motility, ion transport, carbohydrate derivative biosynthetic process, cellular response to stimulus, organelle organization, and cell wall organization or biogenesis. The differentially expressed secreted proteins in polymyxin B-resistant bacteria were enriched for multiple pathways, suggesting that the resistance phenotype depends on complex regulatory mechanisms. A potential biomarker or drug target (YebV) was found in polymyxin B-resistant E. coli. This work clarifies the secretome changes associated with the acquisition of polymyxin resistance and may contribute to drug development.

An Effective Microinjection Method and TALEN-Mediated Genome Editing in Pacific Abalone.

Pacific abalone, Haliotis discus hannai, is an economically important marine mollusk species and an important model animal for studies on ecological, fertilization and developmental biology. While embryonic injection and genome editing have been wildly used in gene function study and trait improvement in many species, they have not been developed in abalones. In this study, we reported an effective method to inject exogenous materials in H. discus hannai unfertilized eggs. The injected eggs could be fertilized at a ratio of 52.6% ± 5.9% and hatch at a ratio of 14.6% ± 1.6%. On the base of this, we further developed an efficient genome editing approach in this species with the transcription activator-like effector nuclease (TALEN) technique. Two TALEN pairs targeting the coding sequence of the abalone nodal gene were assembled and tested. While one of the TALEN pairs showed no detectable mutation efficacy, the other one generated mutations in 50% of the targeted loci. The mutation includes small insertions and deletions and base pair replacements like that reported in other species when the TALEN method was applied. Overall, this is the first study to demonstrate site-specific genome editing in abalone. This work can serve as a reference for future studies focusing on the functional genomics in mollusks.

Identification and characteristics of muscle growth-related microRNA in the Pacific abalone, Haliotis discus hannai.

BACKGROUND: The Pacific abalone, Haliotis discus hannai, is the most important cultivated abalone in China. Improving abalone muscle growth and increasing the rate of growth are important genetic improvement programs in this industry. MicroRNAs are important small noncoding RNA molecules that regulate post-transcription gene expression. However, no miRNAs have been reported to regulate muscle growth in H. discus hannai. RESULTS: we profiled six small RNA libraries for three large abalone individuals (L_HD group) and three small individuals (S_HD group) using RNA sequencing technology. A total of 205 miRNAs, including 200 novel and 5 known miRNAs, were identified. In the L_HD group, 3 miRNAs were up-regulated and 7 were down-regulated compared to the S_HD specimens. Bioinformatics analysis of miRNA target genes revealed that miRNAs participated in the regulation of cellular metabolic processes, the regulation of biological processes, the Wnt signaling pathway, ECM-receptor interaction, and the MAPK signaling pathway, which are associated with regulating growth. Bone morphogenetic protein 7 (BMP7) was verified as a target gene of hdh-miR-1984 by a luciferase reporter assay and we examined the expression pattern in different developmental stages. CONCLUSION: This is the first study to demonstrate that miRNAs are related to the muscle growth of H. discus hannai. This information could be used to study the mechanisms of abalone muscle growth. These DE-miRNAs may be useful as molecular markers for functional genomics and breeding research in abalone and closely related species.

Expression profiling of lncRNAs and mRNAs reveals regulation of muscle growth in the Pacific abalone, Haliotis discus hannai.

Long non-coding RNAs (lncRNAs) are known to play a major role in the epigenetic regulation of muscle development. Unfortunately there is little understanding of the mechanisms with which they regulate muscle growth in abalone. Therefore, we used RNA-seq to study the muscle transcriptomes of six Haliotis discus hannai specimens: three large (L_HD group) and three small (S_HD group). We identified 2463 lncRNAs in abalone muscle belonging to two subtypes: 160 anti-sense lncRNAs and 2303 intergenic lncRNAs (lincRNAs). In the L_HD group, we identified 204 significantly differentially expressed lncRNAs (55 upregulated and 149 downregulated), and 2268 significantly differentially expressed mRNAs (994 upregulated and 1274 downregulated), as compared to the S_HD group. The bioinformatics analysis indicated that lncRNAs were relate to cell growth, regulation of growth, MAPK signaling pathway, TGF-ß signaling pathway, PI3K-Akt and insulin signaling pathway, which involved in regulating muscle growth. These findings contribute to understanding the possible regulatory mechanisms of muscle growth in Pacific abalone.

Tumor suppressor miR-449a inhibits the development of gastric cancer via down-regulation of SGPL1.

MicroRNAs (miRNAs) are small noncoding RNAs that are known to participate in the regulation of many physiological and pathological processes, which can indirectly influence the development of malignant behaviors. Numerous studies have demonstrated that miR-449a plays important roles in human carcinogenesis. However, its precise functional and regulatory roles remain unclear. In this study, we mainly explored the functional role of miR-449a in gastric cancer (GC). The expression levels of miR-449a in 98 cases of GC tissues and cell lines were determined by qRT-PCR. The possible mechanisms of miR-449a in GC cells were explored by fluorescence reporter assay. miR-449a expression was significantly lower in GC tissues compared to matched para-carcinoma tissues and was associated with tumor differentiation. Furthermore, in vitro knockdown of miR-449a by siRNA significantly inhibited MKN-28 cell proliferation, migration and invasion as well as tumorigenesis via inducing G0/G1 arrest of GC cells. In addition, we identified SGPL1 as a target of miR-449a and demonstrated that miR-449a regulated SGPL1 expression via binding its 3'-UTR region. The experiments indicated that miR-449a functions as a novel tumor suppressor in GC and its anti-oncogenic activity may involve its inhibition of the target gene SGPL1. These findings suggested that miR-449a may be a promising candidate for the development of antitumor drugs targeting GC.

iTRAQ-Based Identification of Proteins Related to Muscle Growth in the Pacific Abalone, Haliotis discus hannai.

The abalone Haliotis discus hannai is an important aquaculture species that is grown for human consumption. However, little is known of the genetic mechanisms governing muscle growth in this species, particularly with respect to proteomics. The isobaric tag for relative and absolute quantitation (iTRAQ) method allows for sensitive and accurate protein quantification. Our study was the first to use iTRAQ-based quantitative proteomics to investigate muscle growth regulation in H. discus hannai. Among the 1904 proteins identified from six samples, 125 proteins were differentially expressed in large specimens of H. discus hannai as compared to small specimens. In the large specimens, 47 proteins were upregulated and 78 were downregulated. Many of the significant Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways, including these differentially expressed proteins, were closely related to muscle growth, including apoptosis, thyroid hormone signaling, regulation of the actin cytoskeleton, and viral myocarditis (p < 0.05). Our quantitative real-time polymerase chain reaction (qRT-PCR) analyses suggested that the alterations in expression levels observed in the differentially expressed proteins were consistent with the alterations observed in the encoding mRNAs, indicating the repeatability of our proteomic approach. Our findings contribute to the knowledge of the molecular mechanisms of muscle growth in H. discus hannai.

Production of bFGF monoclonal antibody and its inhibition of metastasis in Lewis lung carcinoma.

Basic fibroblast growth factor (bFGF) and fibroblast growth factor receptor 1 (FGFR1) are associated with drug resistance in lung cancer. In the present study, mouse monoclonal antibodies (mAb) against human bFGF, targeting the binding site of bFGF with FGFR1 were produced, and the antitumor activity and inhibition of metastasis was studied in Lewis lung carcinoma (LLC). A total of four hybridoma cell strains that stably secreted bFGF mAb were obtained. mAbE12 was selected as the most effective for use in the following studies, with a relative affinity constant of 5.66x108 l/mol. mAbE12 was demonstrated to inhibit cell proliferation and tumor growth in vitro and in vivo. Furthermore, mAbE12 blocked migration and metastasis of LLC cells in vitro and in vivo. This occurred due to a mAbE12­induced upregulation of E­cadherin expression through the protein kinase B­glycogen synthase kinase 3 ß­Snail pathway. These results suggested that mAbE12 may be a potential antibody for the treatment of lung cancer.

Chitosan-chitin nanocrystal composite scaffolds for tissue engineering.

Chitin nanocrystals (CNCs) with length and width of 300 and 20nm were uniformly dispersed in chitosan (CS) solution. The CS/CNCs composite scaffolds prepared utilizing a dispersion-based freeze dry approach exhibit significant enhancement in compressive strength and modulus compared with pure CS scaffold both in dry and wet state. A well-interconnected porous structure with size in the range of 100-200µm and over 80% porosity are found in the composite scaffolds. The crystal structure of CNCs is retained in the composite scaffolds. The incorporation of CNCs leads to increase in the scaffold density and decrease in the water swelling ratio. Moreover, the composite scaffolds are successfully applied as scaffolds for MC3T3-E1 osteoblast cells, showing their excellent biocompatibility and low cytotoxicity. The results of fluorescent micrographs images reveal that CNCs can markedly promote the cell adhesion and proliferation of the osteoblast on CS. The biocompatible composite scaffolds with enhanced mechanical properties have potential application in bone tissue engineering.

[Preparation and application of monoclonal antibody against human myoglobin].

OBJECTIVE: To prepare the monoclonal antibody (mAb) against human myoglobin (MYO) of high titer and specificity and develop double-antibody sandwich ELISA for detecting MYO in human serum samples. METHODS: The BALB/c mice were immunized with natural human MYO, and the hybridoma cell lines secreting anti-MYO mAb were established using cell fusion and hybridoma screening techniques. The characteristics of the mAb were identified after affinity purification from ascites. Then the best antibody pair was selected from mAb to establish a one-step sandwich ELISA method. Sixty human serum samples were detected by the homemade ELISA kit and the imported one, respectively. RESULTS: Nine strains of hybridoma cell lines stably secreted anti-MYO mAb. Four strains named 2M1, 3M4, 5M7 and 10M4 could secrete high-quality mAb and the titers of them were in the range of 1.0×10(6) to 2.6×10(6) (A450 value was about 1.0). Three antibody pairs (2M1/HRP-3M4, 5M7/HRP-3M4, 10M4/HRP-5M7) were selected by double-antibody sandwich ELISA. Among them, the 5M7/HRP-3M4 had higher sensitivity and larger linear range. The homemade ELISA kit had a larger linear range (25-1000 ng/mL) than the imported one (25-500 ng/mL) and showed high accuracies in detecting human serum samples, being 95% (19/20) in positive samples and 100% (40/40) in negative samples. CONCLUSION: With the anti-human MYO mAbs of high specificity and affinity, a one-step sandwich ELISA for detecting human MYO has been established successfully, which provides a basis for the development of domestic ELISA kit.