Uncovering the potential functions of lymph node metastasis-associated aberrant methylation differentially expressed genes and their association with the immune infiltration and prognosis in bladder urothelial carcinoma.

Background: Bladder urothelial carcinoma (BLCA) is a malignant tumor of the urinary system. This study aimed to explore the potential role of lymph node metastasis-associated aberrant methylation differentially expressed genes (DEGs) in BLCA. Methods: CHAMP and limma packages were used to identify lymph node metastasis-associated aberrant methylation DEGs. Univariate Cox analysis and Lasso analysis were performed to identify the signature genes, and multivariate Cox analysis was used to construct the risk score. Subsequently, the molecular characteristics of the signature genes and the relationship between risk score and prognosis, clinical characteristics and immune cell infiltration were analyzed. The signature gene AKAP7 was selected for functional verification. Results: A novel risk score model was constructed based on 12 signature genes. The risk score had a good ability to predict overall survival (OS). The nomogram constructed based on age, N stage and risk score had a higher value in predicting the prognosis of patients. It was also found that stromal activation in TIME may inhibit the antitumor effects of immune cells. Functional enrichment analysis revealed that ECM receptor interaction and focal adhesion were two important pathways involved in the regulation of BLCA. Immunohistochemistry showed that AKAP7 may be associated with the occurrence, clinical stages and grades, and lymph node metastasis of BLCA. In vitro cell experiments showed that the migration and invasion ability of EJ cells was significantly inhibited after AKAP7 overexpression, while the migration and invasion ability of T24 cells was significantly promoted after AKAP7 knockdown. Conclusion: The risk score model based on lymph node metastasis-associated aberrant methylation DEGs has a good ability to predict OS and is an independent prognostic factor for BLCA. It was also found that stromal activation in TIME may inhibit the antitumor effects of immune cells. This implicates aberrant methylation modifications as an important factor contributing to the heterogeneity and complexity of individual tumor microenvironments. Functional enrichment analysis revealed that ECM receptor interaction and focal adhesion were two important pathways involved in the regulation of BLCA, which contributed to the exploration of the pathological mechanism of BLCA. In addition, immunohistochemistry showed that AKAP7 may be associated with the occurrence, progression and lymph node metastasis of BLCA. In vitro cell experiments showed that AKAP7 could also inhibit the migration and invasion of cancer cells.

Effect of roscovitine pretreatment for increased utilization of small follicle-derived oocytes on developmental competence of somatic cell nuclear transfer embryos in pigs.

The objective of this study was to evaluate the effect of roscovitine pretreatment on the number of matured oocytes per ovary available for somatic cell nuclear transfer (SCNT) and their developmental competence. Irrespective of reproduction status (prepuberty/puberty), the average number of small follicles per ovary (19.3/17.2) was higher than that of medium follicles (1.5/2.7). In the small follicle group, the in vitro maturation rate of COCs pretreated with 50 µM roscovitine (56.1%) was significantly (P < 0.05) higher than that of the control, 25 or 75 µM treatment (15.5%, 23.7% and 35.2%, respectively), while, in the medium follicle group, there was no significant difference between the control, 25, 50 and 75 µM treatment (76.4%, 78.3%, 80.9% and 60.6%, respectively). As a result, a total number of matured oocytes per ovary was greater for 50 µM treatment (11.8) than for the control, 25 or 75 µM treatment (4.4, 5.0 and 6.3, respectively). In the small follicle group, COCs pretreated with 50 µM roscovitine showed dramatically increased blastocyst rate (16.0%) compared to the control (2.9%) (P < 0.05), whereas, in the medium follicle group, there was no significant difference between groups independent of roscovitine treatment (20.8 vs 23.0%). The cloning efficiency in the roscovitine-treated group was not significantly different from that in the control (2.6 vs 1.4%). In conclusion, the present study indicates that roscovitine treatment increased the number of matured oocytes per ovary available for SCNT and did not have any adverse effect on cloning efficiency in pigs.

Comparative transcriptomic analysis of two Cucumis melo var. saccharinus germplasms differing in fruit physical and chemical characteristics.

BACKGROUND: Hami melon (Cucumis melo var. saccharinus) is a popular fruit in China because of its excellent taste, which is largely determined by its physicochemical characteristics, including flesh texture, sugar content, aroma, and nutrient composition. However, the mechanisms by which these characteristics are regulated have not yet been determined. In this study, we monitored changes in the fruits of two germplasms that differed in physicochemical characteristics throughout the fruit development period. RESULTS: Ripe fruit of the bred variety 'Guimi' had significantly higher soluble sugar contents than the fruit of the common variety 'Yaolong.' Additionally, differences in fruit shape and color between these two germplasms were observed during development. Comparative transcriptome analysis, conducted to identify regulators and pathways underlying the observed differences at corresponding stages of development, revealed a higher number of differentially expressed genes (DEGs) in Guimi than in Yaolong. Moreover, most DEGs detected during early fruit development in Guimi were associated with cell wall biogenesis. Temporal analysis of the identified DEGs revealed similar trends in the enrichment of downregulated genes in both germplasms, although there were differences in the enrichment trends of upregulated genes. Further analyses revealed trends in differential changes in multiple genes involved in cell wall biogenesis and sugar metabolism during fruit ripening. CONCLUSIONS: We identified several genes associated with the ripening of Hami melons, which will provide novel insights into the molecular mechanisms underlying the development of fruit characteristics in these melons.

Hypodermin A improves survival of skin allografts.

BACKGROUND: Hypodermin A (HA) is a serine esterase that degrades complement, a key element of the innate immune system. Immunosuppressive properties of HA have previously been studied in vitro. However, such properties have not been fully demonstrated in vivo. The aim of this study was to evaluate the effect of HA in inhibiting allograft rejection in an HA transgenic mouse model. METHODS: FVB (HA transgenic mice or wild-type mice) to BALB/c mice skin transplantation model were used. Skin grafts were analyzed by histology, immunohistochemistry, and Western blotting. RESULTS: HA overexpression resulted in significantly prolonged skin allograft survival. Histologic changes in the skin allografts paralleled the gross appearance of rejection. ELISA and Western blotting showed that HA significantly reduced the content of complement C3 and C9 in HA skin allografts. The expressions of CD4, B7-2, and MHC class II were all significantly suppressed in HA skin allografts compared with the control group. CONCLUSIONS: These findings suggest that HA effectively prolongs skin allograft survival. The study results provide insight into a promising strategy to improve the survival of grafts in humans.

Generation of GGTA1 biallelic knockout pigs via zinc-finger nucleases and somatic cell nuclear transfer.

Genetically modified pigs are valuable models of human disease and donors of xenotransplanted organs. Conventional gene targeting in pig somatic cells is extremely inefficient. Zinc-finger nuclease (ZFN) technology has been shown to be a powerful tool for efficiently inducing mutations in the genome. However, ZFN-mediated targeting in pigs has rarely been achieved. Here, we used ZFNs to knock out the porcine α-1, 3-galactosyl-transferase (GGTA1) gene, which generates Gal epitopes that trigger hyperacute immune rejection in pig-to-human transplantation. Primary pig fibroblasts were transfected with ZFNs targeting the coding region of GGTA1. Eighteen mono-allelic and four biallelic knockout cell clones were obtained after drug selection with efficiencies of 23.4% and 5.2%, respectively. The biallelic cells were used to produce cloned pigs via somatic cell nuclear transfer (SCNT). Three GGTA1 null piglets were born, and one knockout primary fibroblast cell line was established from a cloned fetus. Gal epitopes on GGTA1 null pig cells were completely eliminated from the cell membrane. Functionally, GGTA1 knockout cells were protected from complement-mediated immune attacks when incubated with human serum. This study demonstrated that ZFN is an efficient tool in creating gene-modified pigs. GGTA1 null pigs and GGTA1 null fetal fibroblasts would benefit research and pig-to-human transplantation.

The effect of the number of transferred embryos, the interval between nuclear transfer and embryo transfer, and the transfer pattern on pig cloning efficiency.

To improve the efficiency of producing cloned pigs, we investigated the influence of the number of transferred embryos, the culturing interval between nuclear transfer (NT) and embryo transfer, and the transfer pattern (single oviduct or double oviduct) on cloning efficiency. The results demonstrated that transfer of either 150-200 or more than 200NT embryos compared to transfer of 100-150 embryos resulted in a significantly higher pregnancy rate (48 ± 16, 50 ± 16 vs. 29 ± 5%, p<0.05) and average litter size (4.1 ± 2.3, 7 ± 3.6 vs. 2.5 ± 0.5). In vitro culture of reconstructed embryos for a longer time (40 h vs. 20 h) resulted in higher (p<0.05) pregnancy rate (44 ± 9 vs. 31 ± 3%) and delivery rate (44 ± 9 vs. 25 ± 9%). Furthermore, double oviductal transfer dramatically increased pregnancy rate (83 ± 6 vs. 27+8%, p<0.05), delivery rate (75 ± 2 vs. 27+8%, p<0.05) and average litter size (6.5 ± 2.8 vs. 2.6 ± 1.2) compared to single oviductal transfer. Our study demonstrated that an improvement in pig cloning efficiency is achieved by adjusting the number and in vitro culture time of reconstructed embryos as well as the embryo transfer pattern.

Detection of spotted fever group Rickettsia in Haemaphysalis longicornis from Hebei Province, China.

DNA samples from 737 tick pools, representing 6,850 Haemaphysalis longicornis and 51 Dermacentor nuttalli collected from Hebei Province, China, were analyzed by polymerase chain reaction (PCR) for the presence of spotted fever group Rickettsia. Fifty (6.9%) of 724 H. longicornis in the tick pool were positive, but no positive samples were found in 13 D. nuttalli. Sequence analysis of the partial outer membrane protein A (ompA) genes from the 10 positive samples showed 97.4-99.8% identity, but were different from the homologous sequence of Rickettsia previously deposited in GenBank. Phylogenetic analysis of ompA genes indicated that the Rickettsia detected in this study belonged to a novel haplotype, and formed a clade distinct from Rickettsia heilongjiangii, Rickettsia sibirica, and Rickettsia hulinii in China. The new strain, named Candidatus Rickettsia hebeiii, appears to represent a distinct lineage and could constitute a new species with a minimum prevalence of about 0.7% in H. longicornis from Hebei Province, China.