The First High-Quality Genome Assembly of Freshwater Pearl Mussel Sinohyriopsis cumingii: New Insights into Pearl Biomineralization.

China leads the world in freshwater pearl production, an industry in which the triangle sail mussel (Sinohyriopsis cumingii) plays a pivotal role. In this paper, we report a high-quality chromosome-level genome assembly of S. cumingii with a size of 2.90 Gb-the largest yet reported among bivalves-and 89.92% anchorage onto 19 linkage groups. The assembled genome has 37,696 protein-coding genes and 50.86% repeat elements. A comparative genomic analysis revealed expansions of 752 gene families, mostly associated with biomineralization, and 237 genes under strong positive selection. Notably, the fibrillin gene family exhibited gene family expansion and positive selection simultaneously, and it also exhibited multiple high expressions after mantle implantation by transcriptome analysis. Furthermore, RNA silencing and an in vitro calcium carbonate crystallization assay highlighted the pivotal role played by one fibrillin gene in calcium carbonate deposition and aragonite transformation. This study provides a valuable genomic resource and offers new insights into the mechanism of pearl biomineralization.

Screening of linear B-cell epitopes and its proinflammatory activities of Haemophilus parasuis outer membrane protein P2.

Haemophilus parasuis is a commensal organism of the upper respiratory tract of pigs, but virulent strains can cause Glässer's disease, resulting in significant economic losses to the swine industry. OmpP2 is an outer membrane protein of this organism that shows considerable heterogeneity between virulent and non-virulent strains, with classification into genotypes I and II. It also acts as a dominant antigen and is involved in the inflammatory response. In this study, 32 monoclonal antibodies (mAbs) against recombinant OmpP2 (rOmpP2) of different genotypes were tested for reactivity to a panel of OmpP2 peptides. Nine linear B cell epitopes were screened, including five common genotype epitopes (Pt1a, Pt7/Pt7a, Pt9a, Pt17, and Pt19/Pt19a) and two groups of genotype-specific epitopes (Pt5 and Pt5-II, Pt11/Pt11a, and Pt11a-II). In addition, we used positive sera from mice and pigs to screen for five linear B-cell epitopes (Pt4, Pt14, Pt15, Pt21, and Pt22). After porcine alveolar macrophages (PAMs) were stimulated with overlapping OmpP2 peptides, we found that the epitope peptides Pt1 and Pt9, and the loop peptide Pt20 which was adjacent epitopes could all significantly upregulated the mRNA expression levels of IL-1α, IL-1ß, IL-6, IL-8, and TNF-α. Additionally, we identified epitope peptides Pt7, Pt11/Pt11a, Pt17, Pt19, and Pt21 and loop peptides Pt13 and Pt18 which adjacent epitopes could also upregulate the mRNA expression levels of most proinflammatory cytokines. This suggested that these peptides may be the virulence-related sites of the OmpP2 protein, with proinflammatory activity. Further study revealed differences in the mRNA expression levels of proinflammatory cytokines, including IL-1ß and IL-6, between genotype-specific epitopes, which may be responsible for pathogenic differences between different genotype strains. Here, we profiled a linear B-cell epitope map of the OmpP2 protein and preliminarily analyzed the proinflammatory activities and effects of these epitopes on bacterial virulence, providing a reliable theoretical basis for establishing a method to distinguish strain pathogenicity and to screen candidate peptides for subunit vaccines.

Transcriptome analysis of growth and shell color between two genetic variants of Corbicula fluminea with different shell colors.

To clarify the molecular mechanism of the black and yellow shell coloration, we performed a transcriptome analysis of whole tissue of Corbicula fluminea in Hongze Lake (Jiangsu Province, China). After assembly, 335,247 unigenes were obtained, and 136,804 unigenes were functionally identified using public databases (NR, GO, KEGG, eggnog, and Swissprot). 1567 differentially expressed genes (DEGs) were detected through pairwise comparisons, of which 941 DEGs were up-regulated and 626 were down-regulated in the black-shelled clam. We compared the DEGs between two clams and identified some coloration-related genes. Notably, the black-shelled clam was larger than the yellow-shelled. We speculated that higher digestion and anabolic ability of black-shelled clam might lead to this phenomenon. In contrast, the yellow-shelled clam appeared to be more sensitive to environmental stress. The metabolic energy of the yellow-shelled clam was depleted to maintain or recover from stress, and provide less energy for growth. In summary, our finding provides a theoretical basis for the molecular mechanism of pigmentation and the difference of somatotype in bivalve, as well as promotes the future breeding of more elite varieties.

Low-complexity domain-containing protein (LCDP), induced accumulation of calcium carbonate individual crystals to irregular polycrystals in the triangle sail mussel Hyriopsis cumingii.

The nacre layer is composed of sheet-like aragonite crystals, with often loosely arranged polycrystal aragonite sheets which may induce poor mechanical properties in shells. In this study, a full-length low-complexity domain-containing protein (LCDP) cDNA from the triangle sail mussel Hyriopsis cumingii was generated and its role in shell formation investigated. The full-length cDNA was 1058 bp; it had an open reading frame (ORF) of 714 bp encoding 237 amino acids and contained a 20-amino acid signal peptide at the N-terminus and two low-complexity domains. H. cumingii LCDP was not homologous with other species. Tissue expression analyses showed that LCDP was specifically expressed in the mantle. In shell repair assays, significantly higher LCDP expression was observed in the shell repair group from days 12-21 (p < 0.01). After LCDP silencing, aragonite flake shapes in pearl layers became irregular with disordered deposition, while calcium carbonate (CaCO3) crystal surfaces in prismatic layers became rougher and organic matrices between crystals appeared skeletonized, indicating the importance of biomineralization. Our in vitro CaCO3 crystallization assays showed that LCDP induced single crystals to polycrystals, probably via loose arrangement between aragonite flakes. These results provide new insights on freshwater mollusk biomineralization and a theoretical basis for improved pearl quality.

Prevalence and genetic diversity of porcine circovirus type 2 in northern Guangdong Province during 2016-2021.

The emergence and widespread of porcine circovirus-associated diseases (PCVADs), mainly caused by porcine circovirus type 2 (PCV2), threatens the Chinese swine industry. In this study, to investigate the recent prevalence of PCV2 in northern Guangdong Province of China, 573 tissue samples from 132 pig farms were collected during 2016-2021 and analyzed via PCR. Overall, 51.38% (297/573, 95%CI 47.74-55.92) samples were tested PCV2 positive. The detection rate of PCV2 was significantly lower in samples collected before 2016-2018 than after the outbreak of African Swine Fever (2019-2021), being 59.85% (158/264, 95%CI 53.94-65.76) and 41.47% (141/340, 95%CI 36.43-46.71), respectively. On the other end, the genetic characteristics of 26 PCV2 strains were further analyzed. These PCV2 strains belonged to three genotypes, including PCV2a, PCV2b, and PCV2d. Specifically, the predominant genotype prevalent during two periods (2016-2018 and 2019-2021) wasPCV2b (81.82%, 9/11) and PCV2d (80.0%, 12/15), respectively. The results above illustrated the high prevalence and the genetic evolution feature of PCV2 in Guangdong Province in recent years.

Molecular mutations associated with the in vitro passage of virulent porcine reproductive and respiratory syndrome virus.

Mutants of a highly pathogenic, porcine reproductive, and respiratory syndrome virus (PRRSV), JXA1 strain, were prepared by continuous in vitro passage. Genomic sequence comparisons were made between mutants obtained at different passages and the parental strain JXA1. The mutant strain obtained at passage 80 contained a 12 nucleotide insertion and 108 nucleotide mutations that resulted in 45 amino acid changes. Most of these changes (89%) occurred between passage 10 and 45 and were genetically stable for the next 35-70 passages. A comparison of the mutants, their parental strain, and several American PRRSV strains, identified 13 characteristic amino acid changes. These sites, as well as the distinct 12 nucleotide insertion, represent possible genetic markers for the evaluation of live vaccine applications, particularly for additional studies of the safety and potency of live PRRSV vaccines.

N-myc downstream-regulated gene 1 as a downregulated target gene of PTEN in the controlling of tumourigenesis in endometrioid carcinoma.

BACKGROUND & OBJECTIVE: Mutation/deletion of PTEN has been known to be involved in the development of many cancers including endometrial carcinoma. NDRG1 (N-myc downstream-regulated gene 1) is reported to be associated with tumourigenesis. PTEN expression has been shown to be correlated with NDRG1 in both prostate and breast cancer. In this study, we explored the possibility that PTEN alteration may cause carcinogenesis of endometrioid carcinoma by regulating the expression of the NDRG1 gene. METHODS: Tissue blocks of 103 patients with pathologically confirmed endometrioid carcinoma were included. All the carcinoma tissues were accompanied with varied degree of necrosis. Using two-step method and avidin-biotin peroxidase complex immunohistochemistry method, the correlation of the two genes expression in ischaemic area and the relationship of NDRG1 expression between ischaemic and non-ischaemic area in endometrioid carcinomas was evaluated. RESULTS: PTEN alteration and NDRG1 expressions were significantly increased in the ischaemic area of endometrioid carcinoma compared with their expressions in the normal endometrium respectively (P<0.001, P<0.001). A positive correlation was found between PTEN alteration and NDRG1 expression in the ischaemic area of endometrioid carcinoma. INTERPRETATION & CONCLUSION: We suggest that NDRG1 may be an important candidate gene in facilitating endometrium carcinogenesis in the adaptation of hypoxia for survival. Alteration of PTEN may upregulate NDRG1 expression, which plays an important role in the process leading to endometrial carcinogenesis.

Correlation between NDRG1 and PTEN expression in endometrial carcinoma.

N-myc Downstream-Regulated Gene 1 (NDRG1) is known as a differentiation-related gene that plays important roles in cell differentiation, organ formation, and embryonic development. NDRG1 has recently been shown to be associated with carcinogenesis and tumor progression in a wide variety of tumors. Phosphatase and tensin homolog deleted from chromosome (PTEN), a phosphatase and tensin homolog located on chromosome 10, is shown to be a tumor suppressor and is often mutated or deleted in various tumor cells, particularly in endometrial carcinoma. Using an immunohistochemical approach, we investigated the expression of NDRG1 and PTEN in normal endometrium, atypical hyperplasia, and endometrial carcinoma. All tumor tissues harvested in this study were derived from endometrioid carcinoma Type I, that were estrogen-related. Our results demonstrate that the expression of NDRG1 was up-regulated in 5/40 (12.5%), 18/34 (52.94%), and 86/103 (83.5%) normal endometrium, atypical hyperplasia, and endometrial carcinoma cases, respectively (P < 0.01), while in 6/40 (15%), 20/34 (58.82%), and 89/103 (86.41%) normal endometrium, atypical hyperplasia, and endometrial carcinoma cases, respectively. PTEN expression was significantly decreased (P < 0.01). Statistical analyzes demonstrated a positive correlation between NDRG1 up-regulation and PTEN down-regulation (P < 0.01). The expression of NDRG1 had no correlation with the differentiation degree of the tumor cells, lymph-node metastasis, and/or abdominal cavity implantation (P > 0.05). Our results indicated that development of endometrial carcinoma is associated with an overexpression of NDRG1 and the loss of PTEN expression. Identification of changes in the NDRG1 and PTEN expression may be a significant diagnostic tool for the early detection of endometrial carcinoma.

[A synthetic peptide selected by bioinformatics inhibits mouse corneal neovascularization].

OBJECTIVE: To identify the active anti-angiogenic region in the amino acid sequence of human apolipoprotein (a) [apo (a)] kringle V (KV), and to evaluate the role of this synthetic peptide on VEGF-induced angiogenesis of mouse cornea in vivo. METHODS: The characterization of the structure and biological activity of the amino acid sequence of apo (a) KV was analyzed using the bioinformatic methods which included sequence alignment, analysis of antigenicity, surface accessibility and hydrophilicity, and then a peptides was selected. The peptide was synthesized with a high efficiency solid-phase method. Corneal neovascularization was induced with a pellet containing 160 ng vascular endothelial growth factor (VEGF) in a mouse corneal micropocket model. 40 C57BL/6 mice (40 eyes) were divided randomly into 4 groups (10 eyes per group). Four kinds of pellets were made containing 160 ng VEGF plus the dose range of 0.0, 0.5, 1.0 and 1.5 microg synthetic peptide for control group, group A, group B and group C, respectively. Neovascularization was observed biomicroscopically on day 7 after the operation, and the corneas were then examined histologically. RESULTS: The result of bioinformatic analysis showed that the peptide contained a majority of conservative residues and possessed fine properties of antigenicity, surface accessibility and hydrophilicity. The synthetic peptide at the doses of 1.0 microg and 1.5 microg showed significant inhibition of mouse corneal neovascularization induced by VEGF in the parameters of vessel length, clock hours and area compared with the control group on day 7 after the operation (P < 0.01). There was no difference in the two doses (1.0 microg and 1.5 microg peptide) in the inhibition of the neovascularization. The dose of 0.5 microg peptide did not show any significant inhibition of the neovascularization compared with the control group (P > 0.05). CONCLUSIONS: The peptide, selected from the amino acid sequence of apo (a) KV by bioinformatics, appears to inhibit VEGF-induced angiogenesis in a mouse corneal micropocket assay in vivo, therefore, the study suggest that this amino acid sequence may locate at the active anti-angiogenic region of apo (a) KV.

[Subretinal transplantation of human fetal lung fibroblasts expressed ciliary neurotrophic factor gene prevent photoreceptor degeneration in RCS rats].

OBJECTIVE: To investigate the efficacy of subretinal transplantation of CNTF gene transfected fibroblasts for preventing photoreceptor degeneration in RCS. METHODS: The human fetal lung fibroblasts with high level expression of CNTF were established by liposome mediated gene transfer and MTX selection. A 5 microl of cell suspension, containing 1 x 10(5) cells, was injected through pars plana of ciliary body into the subretinal space of the right eye at postnatal 4-5 weeks, the left eye was left without injection or injected with PBS as controls. The both eyes were enucleated for histopathological examinations at 2, 4, 6, 8, 10, 12 and 15 weeks following transplantation. RESULTS: The level of CNTF protein (91,046.15 pg/ml) expressed in the transfected cells was determined by sandwich enzyme-linked immunosorbent assay (ELISA). The four of seven eyes examined by light microscopy and the ten of 14 eyes examined by electro microscopy showed rescue effect. The prolonged photoreceptor survival, reduction of apoptotic cells and debris were observed in transplanted eyes in comparison with untreated or sham-injected eyes. CONCLUSION: This study provides the first indication that transplanted human fibroblasts with high level expression of CNTF are able to rescue photoreceptor degeneration in RCS dystrophic rat retina.

[Exogenous advanced glycosylation end products induce diabetes-like vascular dysfunction in normal rats: a factor for occurrence of diabetic retinopathy].

OBJECTIVE: To disclose the relationship between the deposition of advanced glycosylation end products (AGE) in the retinal vascular tissues and damage of retinal vessels in diabetic retinopathy. METHODS: Sixteen SD rats aged 2 months were divided into 4 groups, with 4 rats in each group. Rats in normal group received no treatment. Diabetes was induced by AGE in the diabetes group. Rat serum albumin (RSA, 40 mg/kg weight) was administered daily to healthy non-diabetic rats through tail veins for 2 weeks (RSA group). AGE-modified RSA was injected to rats in another group at the same route and dosage (AGE-RSA group). The number of pericytes in retinal capillary vessels was counted 2 weeks later. RESULTS: After two weeks continuous AGE treatment, the average amount of pericytes of capillary vessel per 10 microscope visual field (x 100 magnification) in AGE group (4.31 +/- 0.34) was significantly less than that of RSA group (5.80 +/- 0.48) (P < 0.01). Meanwhile, in the AGE-RSA group, AGE were identified in the retinal vascular tissues by immunohistochemical staining. CONCLUSION: Injection of exogenous AGE into healthy rats induces vascular changes resembling those find in the diabetic retinopathy. AGE might be one of the independent pathogenic factors in the occurrence of diabetic retinopathy.

Exogenous advanced glycosylation end products induce diabetes-like vascular dysfunction in normal rats: a factor in diabetic retinopathy.

BACKGROUND: Diabetic retinopathy has been shown to be directly associated with the degree and duration of hyperglycemia, and advanced glycosylation end products (AGEs) have been implicated in this pathological process. The purpose of the experiments reported here was to study the effect of AGE deposition on retinal vascular damage which leads to diabetic retinopathy. METHODS: Intravenous injection of exogenous AGEs was used to treat wild-type non-diabetic Sprague-Dawley rats. One of the two retinal slides from each animal was treated using immunohistochemical staining to label retinal vascular AGE deposition, the other H&E staining for counting of capillary pericytes. The results were compared with the findings in untreated wild-type and diabetic controls and in rats treated with unmodified rat serum albumin (RSA). RESULTS: After 2 weeks of continuous treatment, AGEs were identified in the retinal vascular tissue of the AGE-RSA-injected group. The average number of retinal capillary pericytes per 10x100 microscope power field was 4.313+/-0.34 (mean +/- SD) in the AGE-RSA-injected group, compared with 5.798+/-0.481 in the control group ( P<0.01). CONCLUSION: These experiments demonstrate that AGEs, independent of other metabolic factors, can induce vascular change resembling that of diabetic retinopathy.

[Liposome-mediated gene transfer into retina].

OBJECTIVE: To evaluate the efficiency of liposome mediated gene transfer to retina, the time of gene expression in retinal cells, retinal damage caused by liposome and the possibility of using liposome mediated gene transfer to retina for treating fundus diseases. METHODS: The ratio of cationic liposome to green fluorescein protein (GFP) expression plasmid for high efficacy of transfection was tested in vitro. Then optimal liposome-plasmid mixture was injected into the subretinal space of Sprague-Dawley (SD) or Royal college of surgeon (RCS) rats. The GFP expression was observed by using fluorescence microscopy, and morphological changes were examined by using paraffin embedded sections, HE staining and transmission electron microscopy. RESULTS: The GFP expression in retinal cells was observed lasting more than 4 weeks. However, upon 4 weeks of transfection the swelling and degeneration in outer segment of photoreceptors could be observed. The aggravated degeneration with disappearance of outer segments, only one layer of photoreceptor nucleus surviving, apoptotic inner granular layer cells and neovascularization were observed 8 weeks after transfection. But no inflammatory cells were observed. Three of 5 RCS rats that received plasmid pCNTF (ciliary neurotrophic factor) delivery at 4 weeks old showed more surviving nuclei of photoreceptors after 3 to 5 weeks of transfection. CONCLUSION: Liposome is able to deliver genes to retinal cells with efficacy, and the expression of therapeutic gene such as CNTF in retinal cells can rescue photoreceptors from degeneration. But liposome used here is to some extent harmful to photoreceptors.

[Inhibitation of tumor angiogenesis, growth and metastasis by blocking VEGF paracrine pathway].

Solid tumors require an adequate vascular supply to grow beyond a certain dimension. It is known that formation of new blood vessels in tumor is mediated by unbalanced expression of angiogenic factors and their inhibitors. Among the former, the vascular endothelial growth factor (VEGF) has been assumed prime candidacy as a major positive physiological effector. To investigate the role of VEGF in angiogenesis associated with development of breast cancer, a sense VEGF and an anti-sense VEGF expression plasmids were constructed, and then were introduced into a human breast carcinoma cell line, MCF-7, expressing middle level of endogenous VEGF. Anti-sense VEGF(121) transfected MCF-7 cells that expressed reduced constitutive levels of VEGF and showed the same growing potential as untransfected MCF-7 cells in vitro, but it showed longer latency, smaller tumor, slower growth and prolonged survival time compared to parental or sense VEGF(165) transfected MCF-7 cells in vivo. Moreover, the tumors derived from anti-sense VEGF(121) transfected MCF-7 cells characterized by minimal vascularization and extensive necrosis. Finally, mice with primary subcutaneous tumors treated with intratumoral administration of anti-sense VEGF, or the plasmid expressing extracellular domain of the Flk-1 VEGF receptor (sFlk-1) followed by electroporation, showed significant tumor suppression. These results suggest that VEGF plays a major angiogenic role in breast cancer and a strategy, which blocks the VEGF paracrine pathway, may provide a means to control tumor growth topically without the risk of systemic antiangiogenesis.

[A novel model for visualization of tumor cell-induced angiogenesis in vivo].

The bladder transitional cell carcinoma cell line, BTT739 from the T739 mouse, was transfected with a plasmid that encoded an enhanced green fluorescence protein (GFP) and the cells stably expressing GFP were selected and subcloned. 1 x 10(3)-1 x 10(4) GFP-labeled BTT739 cells were injected under the skin of ear of T739 mice. On day 2-5 post injection, the most interesting manifestations observed were the chemotaxis-like movement of the tumor cells toward the pre-existing host vasculature, host vessel dilation and tortuosity and increased extravasation. On day 10 or later, the sprout from pre-existing host vasculature was observed. Once angiogenesis was triggered on, the tumor cells grew more rapidly and exhibited a specific growth pattern where tumor cells always associated with or surrounded the vessels. The newly formed microvessels always showed heavy extravasation. Immunohistochemistry staining revealed strong VEGF and VEGFR2 (Flk-1) expression in tumor cells. Angiography using Rhodamin-labeled dextran showed neovascularization with unprecedented clarity. However, the tumor mass, even bigger than 2 mm and being neovascularized, shrunk and then disappear in 3-5 days and left only delicated host vessels and recovered extravasation. The evidence from this observation indicated that angiogenesis induced by tumor cells after implantation into the host begins at very early stage. The micrometastases foci could not form or survive without vigorous and continuous angiogenesis. Furthermore, there was active VEGF paracrine and autocrine expression in tumor and high level VEGF secretion by tumor cells plays an important role in initiating angiogenesis and supporting micrometastases.