Sequencing of the Complete Mitochondrial Genome of the Big Brown Mactra Clam, Mactra grandis (Venerida: Mactridae).

Mitochondrial genomes are playing an increasingly important role in molluscan taxonomy, germplasm, and evolution studies. The first complete mitochondrial genome of the commercial big brown mactra clam, Mactra grandis, was characterized using Illumina next-generation sequencing in this study. The 17,289 bp circular genome has a typical gene organization of 13 protein-coding genes (PCGs), 2 rRNAs, and 22 tRNAs, with an obvious (A + T)-bias of 64.54%. All PCGs exhibited a homogeneous bias in nucleotide composition with a (A + T)-bias, a positive GC skew, and a negative AT skew. Results of phylogenetic analysis showed that Mactra grandis was most closely related to Mactra cygnus. The functional gene arrangement of the two species was identical but different from other Mactra species. The congeneric relationships among Mactra species were demonstrated by genetic distance analysis. Additionally, the selective pressure analysis suggested that cox1 was highly efficient for discriminating closely related species in genus Mactra, while nad2 was the most appropriate marker for population genetic analysis.

Effects of pneumatic compression therapy on wound healing in patients with venous ulcers: A meta-analysis.

This meta-analysis assessed the effect of pneumatic compression therapy on the wound healing of venous ulcers, with the aim of providing a basis for the selection of clinical treatment. Randomised controlled trials (RCTs) on the application of pneumatic compression therapy to venous ulcers were collected by searching PubMed, Embase, Cochrane Library, China National Knowledge Infrastructure, VIP, and Wanfang databases, with a timeframe from database inception to August 2023. After two researchers independently screened the literature, extracted information, and evaluated the quality of the included studies, a meta-analysis was performed using RevMan 5.4 software. Six RCTs with 367 patients were included, with 172 patients in the intervention group and 195 in the control group. The results showed that pneumatic and bandage compression therapies had a similar impact on wound healing rates of venous ulcers (54.65% vs. 53.84%, odds ratio [OR]: 1.02, 95% confidence interval [CI]: 0.49-2.12, p = 0.96), changes in wound area (standardised mean difference: -0.16, 95% CIs: -0.45 to 0.12, p = 0.26), adverse event rates (76.56% vs. 67.07%, OR: 1.62, 95% CI: 0.77-3.39, p = 0.20), and the differences were not statistically significant. Thus, current evidence suggests that the effects of pneumatic compression therapy on wound healing rates, changes in wound area, and the incidence of adverse events in patients with venous ulcers are similar to those of bandage pressure therapy. However, owing to the limitations in the number and quality of studies, more high-quality RCTs are needed to clarify the feasibility and economics of pneumatic compression therapy in patients with venous ulcers.

Parallel evolution in Crassostrea oysters along the latitudinal gradient is associated with variation in multiple genes involved in adipogenesis.

Parallel diversification provides a proper framework for studying the role of natural selection in evolution. Yet, empirical studies from ecological 'non-model' species of invertebrates are limited at the whole genome level. Here, we presented a chromosome-scale genome assembly for Crassostrea angulata and investigated the parallel genomic evolution in oysters. Specifically, we used population genomics approaches to compare two southern-northern oyster species pairs (C. angulata-C. gigas and southern-northern C. ariakensis) along the coast of China. The estimated divergence time of C. angulata and C. gigas is earlier than that of southern and northern C. ariakensis, which aligns with the overall elevated genome-wide divergence. However, the southern-northern C. ariakensis FST profile represented more extremely divergent "islands". Combined with recent reciprocal hybridization studies, we proposed that they are currently at an early stage of speciation. These two southern-northern oyster species pairs exhibited significant repeatability in patterns of genome-wide differentiation, especially in genomic regions with extremely high and low divergence. This suggested that divergent and purifying selection has contributed to the genomic parallelism between southern and northern latitudes. Top differentiated genomic regions shared in these two oyster species pairs contained candidate genes enriched for functions in energy metabolism, especially adipogenesis, which are closely related to reproductive behaviours. These genes might be good candidates for further investigation in vivo. In conclusion, our results suggest that similar divergent selection and shared genomic features could predictably transform standing genetic variation within one species pair into differences in another.

Effects of Dietary Cystine and Tyrosine Supplementation on Melanin Synthesis in the Pacific Oyster (Crassostrea gigas).

Melanogenesis is a multistep process to produce melanin for dark pigmentation in skin coloration. Previous studies in vertebrates demonstrated that cystine and tyrosine amino acids are involved in the melanin synthesis. However, very little is known about the melanogenesis in bivalve. In this study, cystine supplementation for 30 days significantly upregulated the expression of CgB-aat1, CgCbs and CgTyr and pheomelanin content in the Pacific oyster Crassostrea gigas. Transmission electron microscope (TEM) results revealed more melanosomes in the connective tissue and melanin granules were secreted in epithelium of mantle. In contrast, tyrosine supplementation had no clear effect on melanogenesis except the gene expression changes of CgB-aat1 and CgCbs. In addition, prolonged supplementation of cystine or tyrosine for 60 days had a negative impact on melanogenesis. Indeed, after 60 days, expression of most of the melanin synthesis-related genes under study was decreased, and melanin content was significantly reduced, indicating that cystine and tyrosine might inhibit production of eumelanin and pheomelanin, respectively. In addition, in vitro analysis using primary cell culture from mantle tissue indicated that incubation with cystine, tyrosine, or B-AAT1 polypeptide, CBS/TYR recombinant proteins induced the increase of CgB-aat1 and CgCbs expression in a dose-dependent manner, suggesting the presence of a regulatory network in response to cystine and tyrosine amino acids intakes in pheomelanin synthesis-related gene expression. Taken together, these data indicate that cystine-CgB-aat1-CgCbs-CgTyr axis is a potential regulator of the pheomelanin biosynthesis pathway, and thus plays an important role in the mantle pigmentation in C. gigas. This work provides a new clue for selective cultivation of oyster strains with specific shell colors in bivalve breeding.

Transcription factor CgPOU3F4-like regulates expression of pheomelanin synthesis related gene CgB-aat1 in the Pacific oyster (Crassostrea gigas).

Previous study has found that b (0, +) -type amino acid transporter 1 (CgB-aat1) plays an essential role on mantle pigmentation in the Pacific oyster Crassostrea gigas. However, the molecular regulation of CgB-aat1 gene expression remains unclear. Herein, three POU domain family members, CgPOU2F1, CgPOU3F4-like and CgPOU4F3-X1 were characterized and they all had POUs and HOX domains, respectively, which were important in transcriptional regulation. CgPOU3F4-like gene expression was the highest in mantle edge. Subsequently, the dual-luciferase reporter result showed that the core regulatory region of CgB-aat1 gene was from -632 to -350 bp of promoter. In transient co-transfection assays, the strongest activity was activated only by CgPOU3F4-like, suggesting CgPOU3F4-like was a valid transcriptional activator of CgB-aat1 gene promoter. And the structural integrity of CgPOU3F4-like was essential for its activation function. In addition, site directed mutagenesis assay was applied to detect three key binding sites between CgPOU3F4-like and core region of CgB-aat1 gene promoter, and this interaction was verified by ChIP test. Furthermore, CgPOU3F4-like knockdown by RNA interference led to obvious decreases in CgB-aat1 and cystathionine beta-synthase (CgCbs) expressions at both mRNA and protein levels. Collectively, these results indicate that CgPOU3F4-like positively regulate CgB-aat1 gene expression and it may be a critical upstream transcriptional regulation factor in pheomelanin synthesis in C. gigas.

Involvement of B-aat1 and Cbs in regulating mantle pigmentation in the Pacific oyster (Crassostrea gigas).

BACKGROUND: Shell color formation is an important physiological process in bivalves, the molecular genetic basis has potential application in bivalve aquaculture, but there is still remaining unclear about this issue. The cystine/glutamate transporter (Slc7a11) and cystathionine beta-synthase (Cbs) are integral genes in pheomelanin synthesis pathway, which is vital to skin pigmentation. METHODS AND RESULTS: Here, the sequences of b (0, +) -type amino acid transporter 1 (B-aat1) and Cbs in Pacific oyster (Crassostrea gigas) (CgB-aat1, CgCbs) were characterized. Phylogenetically, the deduced amino acid sequences of CgB-aat1 and CgCbs both possessed conserved features. Genes were both ubiquitously expressed in six tested tissues with more abundant expression level in central mantle. Besides, the polyclonal antibodies of CgB-aat1, CgCbs, CgTyr, and CgTyrp2 were successfully prepared. Immunofluorescence analysis revealed that CgB-aat1 and CgCbs proteins were both expressed in gill rudiments of eyed-larvae and concentrated mainly in cytoplasm of epithelial cell and nerve axons in mantle. Additionally, after CgB-aat1 or CgCbs silencing, expressions at mRNA and protein levels of CgB-aat1 and CgCbs involved in pheomelanin synthesis were significantly suppressed, and CgTyr, CgTyrp1 and CgTyrp2 related to eumelanin synthesis were also down-regulated but no apparent differences, respectively. Moreover, micrographic examination found less brown-granules at mantle edge in CgB-aat1 interference group. CONCLUSION: These results implied that pheomelanin synthesis was possible induced by CgB-aat1-CgTyr-CgCbs axis, and it played an essential role on mantle pigmentation in the oysters. These findings provide the useful genetic knowledge and enrich the physiological information for the shell color formation in bivalve aquaculture.

Histological, elemental, and ultrastructural analysis of melanin in mantle of Pacific oyster (Crassostrea gigas).

Colorful shell of bivalve is mainly because of the biological pigments, of which melanin plays an important role in shell color formation. More and more studies focus on the genes function involved in melanin synthesis, but relatively few studies address the biochemical character and ultrastructure of melanin in bivalve from microscopic perspective. Here, we investigated the histological structure of mantle of Crassostrea gigas with orange shell color. Distribution of melanin in mantle was verified with histochemical staining. In addition, immunofluorescence technique showed that strongly positive signal of CgTYR was specific to the mantle margin, which is consistence with the location of brown granules in H&E staining. The further result of elementary composition of melanin displayed that metal Ca, Fe, and Zn were detected using scanning transmission electron microscope and energy dispersive spectroscopy mapping methods. Next, based on TEM observations, it was speculated that the series of cellular events leading to the formation and release of melanin. Melanocyte in the primary stage showed many mitochondria and rough endoplasmic reticulum as well as an extensive Golgi complex with numerous vesicles intermingled with melanosome. Subsequently, melanosome was expended and their hue gradually intensified, and Golgi complex and mitochondria were still observed in the cytoplasm. Finally, after melanosome was discharged into intercellular spaces, the disintegration of membranes in some cells, and severe cellular vacuolization. These data enrich the understanding of ultrastructural characteristic and formation of melanin in mantle of bivalve and pave the way for further investigating shell coloration at the cellular level.

Molecular characterization of Pax7 and its role in melanin synthesis in Crassostrea gigas.

The paired-box 7 (Pax7) is a transcription factor crucial for skin color polymorphism. However, the mechanism underlying the pigmentation associated with Pax7 in mollusks have yet to be elucidated. In this study, the cDNA sequence of Pax7 in the Pacific oyster Crassostrea gigas (CgPax7) was characterized. Phylogenetically, the identity of deduced amino acid sequence was similar to that of other mollusks and contained 463 amino acids, with conserved features of paired domain (PRD), homeobox domain (HD) and octapeptide. Gene expression analysis revealed that CgPax7 was markedly increased at D-shaped larvae stage and ubiquitously expressed in six examined tissues in adult oyster. The result of whole-mount in situ hybridization (WMISH) showed a restricted pattern of CgPax7 expression on margins of shell valves at D-shaped and umbo larvae stages. Additionally, although CgPax7 silencing had no significant effect on CgMitf expression, it significantly inhibited the expressions of CgPax7, CgTyr, CgTyrp1, CgTyrp2 and CgCdk2, genes involved in Tyr-mediated melanin synthesis. Furthermore, CgPax7 knockdown obviously decreased the tyrosinase activity. Less brown-granules at mantle edge was detected by micrographic examination and melanosomes defect was observed by transmission electron microscopy. It was demonstrated that CgPax7 play a key role in melanin synthesis by regulating Tyr-pathway in C. gigas. These findings indicated the potential framework by which mollusks pigmentation.

Integrated Analysis of Coding Genes and Non-coding RNAs Associated with Shell Color in the Pacific Oyster (Crassostrea gigas).

Molluscan shell color polymorphism is important in genetic breeding, while the molecular information mechanism for shell coloring is unclear. Here, high-throughput RNA sequencing was used to compare expression profiles of coding and non-coding RNAs (ncRNAs) from Pacific oyster Crassostrea gigas with orange and black shell, which were from an F2 family constructed by crossing an orange shell male with a black shell female. First, 458, 13, and 8 differentially expressed genes (DEGs), lncRNAs (DELs), and miRNAs (DEMs) were identified, respectively. Functional analysis suggested that the DEGs were significantly enriched in 9 pathways including tyrosine metabolism and oxidative phosphorylation pathways. Several genes related to melanin synthesis and biomineralization expressed higher whereas genes associated with carotenoid pigmentation or metabolism expressed lower in orange shell oyster. Then, based on the ncRNA analysis, 163 and 20 genes were targeted by 13 and 8 differentially expressed lncRNAs (DELs) and miRNAs (DEMs), severally. Potential DELs-DEMs-DEGs interactions were also examined. Seven DEMs-DEGs pairs were detected, in which tyrosinase-like protein 1 was targeted by lgi-miR-133-3p and lgi-miR-252a and cytochrome P450 was targeted by dme-miRNA-1-3p. These results revealed that melanin synthesis-related genes and miRNAs-mRNA interactions functioned on orange shell coloration, which shed light on the molecular regulation of shell coloration in marine shellfish.

Transcriptome profiles in the spleen of African catfish (Clarias gariepinus) challenged with Aeromonas veronii.

The African catfish, Clarias gariepinus, an important cultured freshwater species in many countries, possess the characteristic of high disease resistance. However, little genomic information for this character of the fish is available up to now. To address the shortfall and to better understand C. gariepinus immune response to pathogen infection at molecular level, C. gariepinus were challenged with potent A. veronii and the high-throughput RNA sequencing (RNA-seq) technology were employed to produce transcriptomes from spleen. In total, an average of 46,073,372 clean reads obtained were de novo assembled into 156,955 unigenes with an average length of 1082 bp. All of unigenes were annotated to seven public databases. Three comparisons were separately conducted between the infected groups at 3 h, 24 h, 48 h post-challenge and control group. A total of 2482 differentially expressed unigenes (DEGs) were identified. Among these, 114 immune-related DEGs were captured, including 88, 42, and 31 genes at 3 h, 24 h and 48 h after infection respectively, for analysis of expression pattern and enrichment. The 114 DEGs displayed four expression patterns by cluster analysis and they were significantly enriched in 38 pathways (q < 0.01) related to the immune or disease, five of which were NF-kappa B, TNF, NLR, TLR and RLR pathways. Finally, the expression levels of twelve selected immune-related DEGs involved in above five pathways were scrutinized. Seven of which were up-regulated at 3 h after infection, afterward, their expression dropped to control level. In summary, this study provides valuable transcriptome resource for understanding the defense mechanisms of C. gariepinus in resistance to pathogens from the gene expression viewpoint, which also open up the possibility to study the immune complexity and to better comprehend the interrelationships between some immune pathways in C. gariepinus.

[Polymorphisms of methylenetetrahydrofolate reductase gene among women of childbearing age from Shiyan area].

OBJECTIVE: To assess the association of methylenetetrahydrofolate reductase (MTHFR) gene 677C to T polymorphism with blood homocysteine (Hcy) level among women of childbearing age from Shiyan area. METHODS: PCR-chip hybridization was used to determine the genotype of MTHFR 677C to T, and a biochemical assay was used to determine the total Hcy level among 428 healthy women of childbearing age. Association of MTHFR 677C to T with total Hcy level was assessed. RESULTS: Heterozygous CT mutation was most common form for the MTHFR 677C to T polymorphisms and amounted for 49.77% among the group, while the CC wild type and homozygous TT mutation respectively accounted for 30.61% and 19.63%. These gave a frequency of 44.51% for the 677T allele. The dominant genotype among different age groups were the CT type. Of note, the proportion of MTHFR 677CC is higher in women above 30 years of age. The distribution of MTHFR 677C to T genotypes has differed significantly among different age groups (P<0.05). Compared with those with wild type alleles, carriers of MTHFR mutations had a higher plasma Hcy level. The genotypic frequencies of MTHFR C677T in Shiyan region differed significantly from those of Sichuan, Hebei, Henan and Shandong (P<0.05) but were similar to those of Jiangsu, Guangdong, Ningxia and Xinjiang. CONCLUSION: The distribution of MTHFR C677T polymorphism among women of childbearing age in Shiyan area is influenced by age and is geographically specific and associated with plasma Hcy level. Nearly 50% of women have carried the high risk alleles, for whom folic acid supplementation is crucial for the reduction of birth defect rate.