The function of the cytoplasmic dynein light chain PTKM23 in the transport of PTSMAD2 during spermatogenesis in Portunus trituberculatus.

Cytoplasmic dynein participates in transport functions and is essential in spermatogenesis. KM23 belongs to the dynein light chain family. The TGFß signaling pathway is indispensable in spermatogenesis, and Smad2 is an important member of this pathway. We cloned PTKM23 and PTSMAD2 from Portunus trituberculatus and measured their expression during spermatogenesis. PTKM23 may be related to cell division, acrosome formation and nuclear remodeling, and PTSMAD2 may participate in regulating the expression of genes related to spermatogenesis. We assessed the localization of PTKM23 with PTDHC and α-Tubulin, and the results suggested that PTKM23 functions in intracellular transport during spermatogenesis. We knocked down PTKM23 in vivo, and the expression of p53, B-CATAENIN and CYCLIN B decreased significantly, further suggesting a role of PTKM23 in transport and cell division. The localization of PTDIC with α-Tubulin and that of PTSMAD2 with PTDHC changed after PTKM23 knockdown. We transfected PTKM23 and PTSMAD2 into HEK-293 T cells and verified their colocalization. These results indicate that PTKM23 is involved in the assembly of cytoplasmic dynein and microtubules during spermatogenesis and that PTKM23 mediates the participation of cytoplasmic dynein in the transport of PTSMAD2 during spermatogenesis. This study provides a theoretical molecular biological basis for the breeding of P. trituberculatus.

Cellular localization and potential ligands of a novel scavenger receptor class B/CD36 protein homolog (Pt-SRB2) identified in the marine crab, Portunustrituberculatus.

The scavenger receptor class B family proteins (SRB) are multiligand membrane receptor proteins. Herein, a novel SRB homolog (Pt-SRB2) was identified in Portunus trituberculatus. The open reading frame of Pt-SRB2 was predicted to encode 520 amino acid residues comprising a typical CD36 domain. Phylogenetic analysis showed that Pt-SRB2 distinctly clustered with the SRB homologs of most crustaceans and Drosophila but was separate from all vertebrate CD36/SRB. Semi-quantitative and Real-time quantitative PCR revealed that the abundance of Pt-SRB2 transcripts was the highest in hepatopancreas than in other tested tissues. Overexpressed Pt-SRB2 was distributed primarily in the cell membrane and cytoplasm of HEK293T or Drosophila Schneider 2 cells. In crab hemocytes, Pt-SRB2 was distributed primarily in the cell membrane by immunofluorescence staining. In addition, the immunofluorescence staining showed that green fluorescence signals were mainly located in the inner lumen membrane of the hepatopancreatic tubules. Moreover, solid-phase enzyme-linked immunosorbent assay revealed that rPt-SRB2-L exhibited relative high affinity with lipopolysaccharides, and relative moderate binding affinity with lipoteichoic acid or peptidoglycan. Of note, rPt-SRB2-L showed high binding affinity with eicosapentaenoic acid among a series of long-chain polyunsaturated fatty acids. Taken together, this study provided valuable data for understanding the functions of the crab CD36/SRB.

The Functions of Pt-DIC and Pt-Lamin B in Spermatogenesis of Portunus trituberculatus.

Cytoplasmic Dynein is a multiple-subunit macromolecular motor protein involved in the transport process of cells. The Dynein intermediate chain (DIC) is one of the subunits of Dynein-1. In our previous studies, we showed that Pt-DIC may play an important role in the nuclear deformation of spermiogenesis in Portunus trituberculatus. Lamin B is essential for maintaining nuclear structure and functions. Surprisingly, Pt-Lamin B was expressed not only in the perinucleus but also in the pro-acrosome during spermiogenesis in P. trituberculatus. Studies have also shown that Dynein-1 can mediate the transport of Lamin B in mammals. Thus, to study the relationship of Pt-DIC and Pt-Lamin B in the spermatogenesis of P. trituberculatus, we knocked down the Pt-DIC gene in P. trituberculatus by RNAi. The results showed that the distribution of Pt-DIC and Pt-Lamin B in spermiogenesis was abnormal, and the colocalization was weakened. Moreover, we verified the interaction of Pt-DIC and Pt-Lamin B via coimmunoprecipitation. Therefore, our results suggested that both Pt-DIC and Pt-Lamin B were involved in the spermatogenesis of P. trituberculatus, and one of the functions of Dynein-1 is to mediate the transport of Lamin B in the spermiogenesis of P. trituberculatus.

Rise and metabolic roles of Vibrio during the fermentation of crab paste.

Microbial community may systematically promote the development of fermentation process of foods. Traditional fermentation is a spontaneous natural process that determines a unique nutritional characteristic of crab paste of Portunus trituberculatus, However, rare information is available regarding the development pattern and metabolic role of bacterial community during the fermentation of crab paste. Here, using a 16S rRNA gene amplicon sequencing technology, we investigated dynamics of bacterial community and its relationship with metabolites during the fermentation of crab paste. The results showed that bacterial community changed dynamically with the fermentation of crab paste which highlighted by consistently decreased α-diversity and overwhelming dominance of Vibrio at the later days of fermentation. Vibrio had a positive correlation with trimethylamine, hypoxanthine, formate, and alanine while a negative correlation with inosine and adenosine diphosphate. In contrast, most of other bacterial indicators had a reverse correlation with these metabolites. Moreover, Vibrio presented an improved function potential in the formation of the significantly increased metabolites. These findings demonstrate that the inexorable rise of Vibrio not only drives the indicator OTUs turnover in the bacterial community but also has incriminated the quality of crab paste from fresh to perished.

ATP catabolism and bacterial succession in postmortem tissues of mud crab (Scylla paramamosain) and their roles in freshness.

The seafood microbiome is highly diverse and plays an essential role in the spoilage of seafood. Nevertheless, how such a diverse microbiome influences freshness of mud crab (Scylla paramamosain) remains unclear. Here we investigated the postmortem ATP catabolism and succession of the bacterial community in the hepatopancreas and muscle of S. paramamosain using a high-performance liquid chromatography method and 16S rRNA gene amplicon sequencing. Our results showed a tissue-dependent change in ATP catabolism determinized the differences in the changes of nucleotide freshness indices of hepatopancreas and muscle over postmortem time of mud crab. The muscle K value could be used as an optimal nucleotide freshness indicator for the freshness of mud crab, with a proposed threshold of 20%. From a microbiota perspective, a more significant bacterial community change was observed in the muscle than in the hepatopancreas. These changes could result in a close relationship between ATP and its catabolites and microbial taxa in the muscle. Moreover, Photobacterium, Peptostreptococcaceae, average path distance, OTU richness, and Shannon index of muscle bacterial community markedly contributed to K value. These findings suggest that the mud crab of 4 h postmortem at room temperature is still edible. Notably, the importance of microbial community composition and interaction for the spoilage of mud crab should be carefully considered.

Gene Transcriptional and Metabolic Profile Changes in Mimetic Aging Mice Induced by D-Galactose.

D-galactose injection has been shown to induce many changes in mice that represent accelerated aging. This mouse model has been widely used for pharmacological studies of anti-aging agents. The underlying mechanism of D-galactose induced aging remains unclear, however, it appears to relate to glucose and 1ipid metabolic disorders. Currently, there has yet to be a study that focuses on investigating gene expression changes in D-galactose aging mice. In this study, integrated analysis of gas chromatography/mass spectrometry-based metabonomics and gene expression profiles was used to investigate the changes in transcriptional and metabolic profiles in mimetic aging mice injected with D-galactose. Our findings demonstrated that 48 mRNAs were differentially expressed between control and D-galactose mice, and 51 potential biomarkers were identified at the metabolic level. The effects of D-galactose on aging could be attributed to glucose and 1ipid metabolic disorders, oxidative damage, accumulation of advanced glycation end products (AGEs), reduction in abnormal substance elimination, cell apoptosis, and insulin resistance.

PCR-RFLP identification of four Chinese soft-shelled turtle Pelodiscus sinensis strains using mitochondrial genes.

A PCR-RFLP method to confirm the identification of four Pelodiscus sinensis strains (Taihu Lake strain, Taiwan strain, Yellow River strain and Japanese strain) was developed and evaluated. In this study, we sequenced and analyzed the partial sequences of mitochondrial NADH4, COX I and NADH5-NADH6 genes of 60 individuals from the four P. sinensis strains. Bgl I, Hpa II and Cla I were selected for cutting NADH4, COX I and NADH5-NADH6 PCR products, respectively, and each strain has its unique restriction band patterns. The result showed that all the 140 samples tested can be correctly identified based on the combination of the three digested fragments pattern. This study provides an effective method to distinguish the four main strains of P. sinensis.