Effects of high hydrostatic pressure (HHP) and storage temperature on bacterial counts, color change, fatty acids and non-volatile taste active compounds of oysters (Crassostrea ariakensis).

The effects of HHP and storage temperature on bacterial counts, color, fatty acids and flavor compounds of oysters Crassostrea ariakensis were investigated. Counts of Vibrio vulnificus and Vibrio parahaemolyticus decreased to undetectable levels in ≥ 400 MPa-treated oysters. Storage at -20 °C significantly restrained microbial growth compared to 4 °C (P < 0.05). L* values of HHP-treated oysters significantly increased compared to raw oysters (P < 0.05). Storage slightly affected the color according to total color difference (ΔE*) values. Fatty acid profiles and betaine contents in 400 and 600 MPa-treated oysters at 0 and 15 d were almost the same as raw samples. Contents of total free amino acids (FAAs), Na+ and Ca2+ were significantly higher in 400 and 600 MPa-treated oysters than those in raw oysters at 0 d (P < 0.05), while the opposite results were observed in 5'-adenosine monophosphate (AMP), 5'-guanosine monophosphate (GMP), citric acid, succinic acid, K+ and PO43- (P < 0.05). At 400 and 600 MPa, FAAs significantly decreased after 15-d storage at 4 °C and -20 °C (P < 0.05), while no significant changes were observed in nucleotides, organic acids and inorganic ions.

FGD5-AS1 facilitates the osteogenic differentiation of human bone marrow-derived mesenchymal stem cells via targeting the miR-506-3p/BMP7 axis.

BACKGROUND: Osteoporosis is a systemic disease characterized by impaired bone formation, increased bone resorption, and brittle bone fractures. The osteogenic differentiation of human bone marrow-derived mesenchymal stem cells (hBMSCs) is considered to be a vital process for bone formation. Numerous studies have reported that long non-coding RNAs (lncRNAs) are involved in the osteogenic differentiation of hBMSCs. The present study aimed to investigate the effect of FGD5 antisense RNA 1 (FGD5-AS1) on osteogenic differentiation. METHODS: RT-qPCR was performed to detect the expression of FGD5-AS1, miR-506-3p, and osteogenesis-related genes OCN, OPN, OSX, and RUNX2. Western blotting was carried out to detect the protein levels of osteogenesis-related markers. In addition, the regulatory effect of FGD5-AS1 on osteogenic differentiation was detected through alkaline phosphatase (ALP) activity, Alizarin Red S (ARS) staining, and Cell Counting Kit-8 (CCK-8). Bioinformatics analysis and luciferase reporter assay were used to predict and validate the interaction between FGD5-AS1 and miR-506-3p as well as miR-506-3p and bone morphogenetic protein 7 (BMP7). RESULTS: The RT-qPCR analysis revealed that FGD5-AS1 was upregulated in hBMSCs following induction of osteogenic differentiation. In addition, FGD5-AS1 knockdown attenuated hBMSC viability and osteogenic differentiation. Bioinformatics analysis and luciferase reporter assays verified that FGD5-AS1 could directly interact with microRNA (miR)-506-3p. Furthermore, miR-506-3p could directly target the 3'-untranslated region (3'-UTR) of BMP7. Additionally, functional assays demonstrated that miR-506-3p silencing could restore the suppressive effect of FGD5-AS1 knockdown on osteogenic differentiation and viability of hBMSCs, and miR-506-3p could attenuate osteogenic differentiation via targeting BMP7. CONCLUSIONS: Taken together, the results of the present study suggested that FGD5-AS1 could positively regulate the osteogenic differentiation of hBMSCs via targeting the miR-506-3p/BMP7 axis.

Comparison of biochemical composition and non-volatile taste active compounds in raw, high hydrostatic pressure-treated and steamed oysters Crassostrea hongkongensis.

In this study, the effects of high hydrostatic pressure (HHP) and steam on biochemical composition and non-volatile taste active compounds of oysters Crassostrea hongkongensis were investigated. The moisture content in steamed oysters significantly decreased when compared to raw samples, subsequently their crude protein, crude lipid, glycogen and ash contents (% wet weight) were all increased (P < 0.05). In addition, though the moisture content in HHP oysters decreased, no significant differences were observed in proximate compositions compared to raw oysters, except crude protein. There were no significant differences in saturated fatty acids (SFA), monounsaturated fatty acids (MUFA) and polyunsaturated fatty acids (PUFA) profiles between raw and HHP oysters, however, C20:3n6 content in HHP oysters was significantly higher than that in raw samples (P < 0.05). The PUFA profile of steamed oysters, mostly contributed by n-3 PUFA, was significantly higher than that of both raw and HHP samples (P < 0.05). Major free amino acids (FAA) (taste activity value, TAV > 1) in oysters with three treatments were alanine, glycine, glutamic acid and histidine, and their contents were significantly higher in raw and HHP groups than that in steamed group. The 5'-inosine monophosphate (IMP) and 5'-guanosine monophosphate (GMP) in HHP and steamed oysters decreased compared to raw samples, while AMP content in steam oysters were significantly increased (P < 0.05). The equivalent umami concentration (EUC) of oysters of raw, HHP and steamed groups were 8.80, 3.66 and 1.44 g MSG/100 g, respectively, with significant differences observed among different treatments (P < 0.05). Succinic acid was the major organic acid in raw and HHP oysters, while lactic acid was the major organic acid in steamed groups. Further, Na+, K+, PO43- and Cl- were the main inorganic ions (TAV > 1), and their contents were significantly higher in raw and HHP groups than that in steamed group (P < 0.05). This study demonstrated that HHP treatment slightly influenced the changes in the biochemical composition and non-volatile taste active compounds to raw oysters, compared to steamed process.

The effects of cryoprotectants on sperm motility of the Chinese pearl oyster, Pinctada fucata martensii.

Cryopreservation has been widely employed to preserve genetic material of aquatic animals. Although of common use in bivalves, resulting effects due to the toxicity of the cryoprotectants dimethyl sulfoxide (DMSO), propanediol (PG), methanol (MET) and ethylene glycol (EG), upon sperm motility in the Chinese pearl oyster, Pinctada fucata martensii, has remained undocumented. This study endeavors to identify the least toxic among the effective cryoprotectant agents by observing and comparing their toxic effects on sperm motility under varying concentrations and duration of exposure. Sperm samples were exposed during controlled experiments, for 1, 3, 6, 9, 12 and 15 min durations, to each of the listed cryoprotectants at 5, 10, 15, and 20% (volume:volume) concentrations. Sperm motility was observed to diminish when exposed to all cryoprotectant solutions, and observations demonstrated that toxicity increased relative to both concentration and equilibration time. After 6 min of exposure to the cryoprotectants, sperm motility was seen to have diminished significantly in DMSO at just 5% concentration, and in MET, PG and EG at 10% concentrations, respectively (the values of the lowest observed effect concentrations). The relationship between the quantity of immotile sperm and the cryoprotectant concentration was described using the logarithmic regression equation. MET exhibited the lowest effective concentration required to inhibit sperm motility by 50% (EC50), followed by EG, PG and DMSO, in order. Therefore, MET proved most toxic under the test conditions for sperm of P. fucata martensii, whereas DMSO, PG and EG were observed as comparatively safer, suggesting that DMSO, PG and EG warrant further study in the application of cryopreservation of Chinese P. fucata martensii sperm.

Comparison of Surgical Outcomes Between Short-Segment Open and Percutaneous Pedicle Screw Fixation Techniques for Thoracolumbar Fractures.

BACKGROUND This study aimed to compare the surgical outcomes between open pedicle screw fixation (OPSF) and percutaneous pedicle screw fixation (PPSF) for the treatment of thoracolumbar fractures, which has received scant research attention to date. MATERIAL AND METHODS Eight-four patients with acute and subacute thoracolumbar fractures who were treated with SSPSF from January 2013 to June 2014 at the Changzhou Hospital of Traditional Chinese Medicine (Changzhou, China) were retrospectively reviewed. The patients were divided into 4 groups: the OPSF with 4 basic screws (OPSF-4) group, the OPSF with 4 basic and 2 additional screws (OPSF-6) group, the PPSF with 4 basic screws (PPSF-4) group, and the PPSF with 4 basic and 2 additional screws (PPSF-6) group. The intraoperative, immediate postoperative, and over 1-year follow-up outcomes were evaluated and compared among these groups. RESULTS Blood loss in the PPSF-4 group and the PPSF-6 group was significantly less than in the OPSF-4 group and the OPSF-6 group (P<0.05). The OPSF-6 group exhibited significantly higher immediate postoperative correction percentage of anterior column height of fractured vertebra than the other 3 groups (P<0.05), and higher correction of sagittal regional Cobb angle and kyphotic angle of injured vertebra than in the PPSF-4 and -6 groups (P<0.05). In addition, there was no significant difference in the correction loss of percentage of anterior column height, and loss of sagittal Cobb angle and kyphotic angle of fractured vertebrae at final follow-up among the 4 groups (P>0.05). CONCLUSIONS OPSF with 6 screws had an advantage in the correction of injured vertebral height and kyphosis, and PPSF reduced the intraoperative blood loss of patients.

cDNA Microarray Analysis Revealing Candidate Biomineralization Genes of the Pearl Oyster, Pinctada fucata martensii.

Biomineralization is a common biological phenomenon resulting in strong tissue, such as bone, tooth, and shell. Pinctada fucata martensii is an ideal animal for the study of biomineralization. Here, microarray technique was used to identify biomineralization gene in mantle edge (ME), mantle center (MC), and both ME and MC (ME-MC) for this pearl oyster. Results revealed that 804, 306, and 1127 contigs expressed at least three times higher in ME, MC, and ME-MC as those in other tissues. Blast against non-redundant database showed that 130 contigs (16.17 %), 53 contigs (17.32 %), and 248 contigs (22.01 %) hit reference genes (E ≤ -10), among which 91 contigs, 48 contigs, and 168 contigs could be assigned to 32, 26, and 63 biomineralization genes in tissue of ME, MC, and ME-MC at a threshold of 3 times upregulated expression level. The ratios of biomineralization contigs to homologous contigs were similar at 3 times, 10 times, and 100 times of upregulated expression level in either ME, MC, or ME-MC. Moreover, the ratio of biomineralization contigs was highest in MC. Although mRNA distribution characters were similar to those in other studies for eight biomineralization genes of PFMG3, Pif, nacrein, MSI7, mantle gene 6, Pfty1, prismin, and the shematrin, most biomineralization genes presented different expression profiles from existing reports. These results provided massive fundamental information for further study of biomineralization gene function, and it may be helpful for revealing gene nets of biomineralization and the molecular mechanisms underlining formation of shell and pearl for the oyster.

Characterization of the pearl oyster (Pinctada martensii) mantle transcriptome unravels biomineralization genes.

Pearl oyster, Pinctada martensii, is a marine bivalve species widely distributed in tropic and subtropic marine coasts. Mantle is the special tissue of P. martensii that secretes biomineralization proteins inducing shell deposition as well as iridescent nacre both in the inner shell and artificial nucleus. The pearl oyster is very efficient for artificial pearl production and is therefore an ideal organism for studies into the processes of biomineralization. However, deficiency of transcriptome information limits the insight into biomineralization mechanisms and pearl formation. In this study, we sequenced and characterized the P. martensii mantle transcriptome using 454 pyrosequencing. A total of 25,723 unique transcripts were assembled from 220,824 quality reads, followed by annotation and Gene Ontology classification analysis. A total of 146 unique transcript segments homologous to 49 reference biomineralization genes were identified, including calcineurin-binding protein, amorphous calcium carbonate binding protein 1, calmodulin, calponin-like protein, carbonic anhydrase 1, glycine-rich shell matrix protein, lysine-rich matrix protein, mantle gene or protein, nacrein, pearlin, PIF, regucalcin, and shematrin. The sequence data enabled the identification of 10,285 potential single nucleotide polymorphism loci and 7,836 putative indels, providing a resource for molecular biomarker, population genetics, and functional genomic studies. A large number of candidate genes for biomineralization were identified, considerably enriching resources for the study of shell formation. These sequence data will notably advance biomineralization and transcriptome study in pearl oyster and other Pinctada species.

Development of expressed sequence tags from the pearl oyster, Pinctada martensii Dunker.

The pearl oyster, Pinctada martensii, is the primary species used for the aquaculture production of marine pearls in China and Japan. Genetic tools and resources are needed to study the genome of this species and to understand the molecular basis of development, growth, host defense, pearl formation, and other important traits. In this study, we developed a set of expressed sequence tags (ESTs) for P. martensii. We constructed cDNA libraries from adult tissues and sequenced 7,128 ESTs. Clustering analysis identified 788 contigs (covering 5,769 ESTs) and 1,351 singletons, yielding a total of 2,139 unique genes. Of these unique genes, only 935 had significant (E-value ≤ 0.005) hits in GenBank, and the remaining 1,204 (56.3%) were novel. Most of the known genes are related to cellular structure, protein binding, and metabolic processes. Putative host-defense genes (86) were identified including C-type lectin, ferritin, polyubiquitin, proteases, protease inhibitors, scavenger receptors, heat shock proteins, and RAS oncogenes. The EST sequences developed in this study provide a valuable resource for future efforts on gene identification, marker development, and studies on molecular mechanism of host defense in pearl oysters.

A Dicer-1 gene from white shrimp Litopenaeus vannamei: expression pattern in the processes of immune response and larval development.

Dicer is a member of the RNAase III family which catalyzes the cleavage of double-stranded RNA to small interfering RNAs and micro RNAs, and then directs sequence-specific gene silencing. In this paper, the full-length cDNA of Dicer-1 was cloned from white shrimp Litopenaeus vannamei (designated as LvDcr1). It was of 7636 bp, including a poly A tail, a 5' UTR of 136 bp, a 3' UTR of 78 bp, and an open reading frame (ORF) of 7422 bp encoding a putative protein of 2473 amino acids. The predicted amino acid sequence comprised all recognized functional domains found in other Dicer-1 homologues and showed the highest (97.7%) similarity to the Dicer-1 from tiger shrimp Penaeus mondon. Quantitative real-time PCR was employed to investigate the tissue distribution of LvDcr1 mRNA, and its expression in shrimps under virus challenge and larvae at different developmental stages. The LvDcr1 mRNA could be detected in all examined tissues with the highest expression level in hemocyte, and was up-regulated in hemocytes and gills after virus injection. These results indicated that LvDcr1 was involved in antiviral defense in adult shrimp. During the developmental stages from fertilized egg to postlarva VII, LvDcr1 was constitutively expressed at all examined development stages, but the expression level varied significantly. The highest expression level was observed in fertilized eggs and followed a decrease from fertilized egg to nauplius I stage. Then, the higher levels of expression were detected at nauplius V and postlarva stages. LvDcr1 expression regularly increased at the upper phase of nauplius, zoea and mysis stages than their prophase. The different expression of LvDcr1 in the larval stages could provide clues for understanding the early innate immunity in the process of shrimp larval development.

Predominant expression and cellular distribution of fish Agr2 in renal collecting system.

Anterior gradient 2 (Agr2) genes encode secretory proteins, and play significant roles in anterior-posterior patterning and tumor metastasis. Agr2 transcripts were shown to display quite diverse tissue distribution in different species, and little was known about the cellular localization of Agr2 proteins. In this study, we identified an Agr2 homologue from gibel carp (Carassius auratus gibelio), and revealed the expression patterns and cellular localization during embryogenesis and in adult tissues. The full-length cDNA of CagAgr2 is 803 nucleotides (nt) with an open reading frame of 510 nt encoding 169 amino acids. The Agr2 C-terminus matches to the class I PDZ-interacting motif, suggesting that it might be a PDZ-binding protein. During embryogenesis, CagAgr2 was found to be transcribed in the mucus-secreting hatching gland from tailbud stage and later in the pharynx region, swim bladder and pronephric duct as revealed by RT-PCR and whole mount in situ hybridization. In the adult fish, its transcription was predominantly confined to the kidney, and lower transcription levels were also found in the intestine, ovary and gills. To further localize the Agr2 protein, the anti-CagAgr2 polyclonal antibody was produced and used for immunofluorescence observation. In agreement with mRNA expression data, the Agr2 protein was localized in the pronephric duct of 3dph larvae. In adult fish, Agr2 protein expression is confined to the renal collecting system with asymmetric distribution along the apical-basolateral axis. The data provided suggestive evidence that fish Agr2 might be involved in differentiation and secretory functions of kidney epithelium.

Characterization of 31 EST-derived microsatellite markers for the pearl oyster Pinctada martensii (Dunker).

We developed and characterized 31 microsatellite markers from expressed sequence tags of Pinctada martensii (Dunker). The number of alleles per locus ranged from 4 to 18 as determined in 44 individuals from a wild population. The expected heterozygosity ranged from 0.4121 to 0.9436, while the observed heterozygosity ranged from 0.4054 to 0.7273. Most of the loci are in Hardy-Weinberg equilibrium. These markers should be useful for population genetics studies, parentage and genome mapping in this species.

Generation and analysis of ESTs from the eastern oyster, Crassostrea virginica Gmelin and identification of microsatellite and SNP markers.

BACKGROUND: The eastern oyster, Crassostrea virginica (Gmelin 1791), is an economically important species cultured in many areas in North America. It is also ecologically important because of the impact of its filter feeding behaviour on water quality. Populations of C. virginica have been threatened by overfishing, habitat degradation, and diseases. Through genome research, strategies are being developed to reverse its population decline. However, large-scale expressed sequence tag (EST) resources have been lacking for this species. Efficient generation of EST resources from this species has been hindered by a high redundancy of transcripts. The objectives of this study were to construct a normalized cDNA library for efficient EST analysis, to generate thousands of ESTs, and to analyze the ESTs for microsatellites and potential single nucleotide polymorphisms (SNPs). RESULTS: A normalized and subtracted C. virginica cDNA library was constructed from pooled RNA isolated from hemocytes, mantle, gill, gonad and digestive tract, muscle, and a whole juvenile oyster. A total of 6,528 clones were sequenced from this library generating 5,542 high-quality EST sequences. Cluster analysis indicated the presence of 635 contigs and 4,053 singletons, generating a total of 4,688 unique sequences. About 46% (2,174) of the unique ESTs had significant hits (E-value

[Screen of differentially expressed genes between gastrula embryos and tail bud embryos in gynogenetic gibel carp (Carassius auratus gibelio)].

Suppression subtractive hybridization (SSH) cDNA plasmid libraries were constructed between gastrula embryos and tail bud embryos in gynogenetic gibel carp (Carassius auratus gibelio). 739 and 816 PCR positive clones were respectively selected to perform dot blot, and 72 dot blot positive clones and 98 dot blot positive clones were obtained from the SSH plasmid libraries specific for gastrula embryos and tail bud embryos. Sequencing analysis and database searches indicated that there were 19 known genes and 31 unknown/cDNA fragments in the sequenced 72 dot blot positive clones specific for gastrula embryos, and 52 known genes and 37 unknown cDNA fragments in the sequenced 98 dot blot positive clones specific for tail bud embryos. Moreover,specific expressions of partial genes were further confirmed by virtual Northern blots and RT-PCR. The screen of these differentially expressed genes will help us to understand the molecular mechanism in gibel carp embryogenesis.