中国濒危兽类保护基因组学和宏基因组学研究进展与展望

揭示濒危兽类的演化历史、濒危机制及适应性演化策略,是保护生物学关注的重大科学问题。近十几年来,高通量测序技术的不断发展以及多学科交叉融合,为揭示濒危物种的演化历史、遗传结构、适应性演化及其与肠道微生物的协同演化的分子机制提供了重要的技术支撑,由此产生了保护基因组学和保护宏基因组学两个分支学科,为野生动物尤其是濒危动物的保护生物学研究提供了新的方向与思路。本文综述了我国在保护基因组学和保护宏基因组学领域取得的重要进展,并展望未来的发展趋势,以期进一步推动我国濒危兽类保护生物学的发展。     

Study of Oxidative Stress in Human Lens Epithelial Cells Exposed to 1.8 GHz Radiofrequency Fields

Objectives

The aims of the present study were to determine oxidative stress and to explore possible reasons of reactive oxygen species (ROS) increase in human lens epithelial (HLE) B3 cells exposed to low intensity 1.8 GHz radiofrequency fields (RF).

Methods

The HLE B3 cells were divided into RF exposure and RF sham-exposure groups. The RF exposure intensity was at specific absorption rate (SAR) of 2, 3, or 4 W/kg. The ROS levels were measured by a fluorescent probe 2′7′-dichlorofluorescin diacetate (DCFH-DA) assay in the HLE B3 cells exposed to 1.8 GHz RF for 0.5, 1, and 1.5 h. Lipid peroxidation and cellular viability were detected by an MDA test and Cell Counting Kit-8 (CCK-8) assays, respectively, in the HLE B3 cells exposed to 1.8 GHz RF for 6, 12, and 24 h, respectively. The mRNA expression of SOD1, SOD2, CAT, and GPx1 genes and the expression of SOD1, SOD2, CAT, and GPx1 proteins was measured by qRT-PCR and Western blot assays in the HLE B3 cells exposed to 1.8 GHz RF for 1 h.

Results

The ROS and MDA levels significantly increased (P<0.05) in the RF exposure group and that the cellular viability, mRNA expression of four genes, and expression of four proteins significantly decreased (P<0.05) compared with the RF sham-exposure group.

Conclusions

Oxidative stress is present in HLE B3 cells exposed to 1.8 GHz low-intensity RF and that the increased production of ROS may be related to down-regulation of four antioxidant enzyme genes induced by RF exposure.     

Identification of quantitative trait loci for grain size and the contributions of major grain-size QTLs to grain weight in rice

Grain weight, one of the three major components of rice yield, is largely determined by grain size, which is controlled by quantitative trait loci (QTLs). In a previous study, we identified qGS5 as a major QTL for grain width. Here, we report our identification of two more major grain-size QTLs (qGL3 and qGW2a) by using a recombinant inbred line (RIL) population from a cross of two indica varieties, ‘Zhenshan 97’ and ‘SLG’. To investigate the contribution of the three grain-size QTLs to final grain weight, we developed near-isogenic lines (NILs) NIL-qGL3, NIL-qGW2a, and NIL-qGS5 and used these to build the combined QTLs–NIL in the genetic background of ‘Zhenshan 97’ by marker-assisted selection and conventional backcrossing, respectively. A BCF₂ population of 957 individuals was developed from the combined QTLs-NIL for further study of the genetic control of grain size. The QTL analysis revealed that qGW2a and qGL3 played more important roles in grain weight gain than qGS5. All three QTLs showed additive effects with respect to grain weight, with no interaction. These results clearly indicate that pyramiding of major grain-size QTLs is a useful approach for improving rice yield.     

Mixed bumblebee and blowfly pollination of Cypripedium flavum (Orchidaceae) in Sichuan,China

Most Cypripedium species are specialized orchids pollinated by, in a broad sense, bees or flies. Here we present the first evidence that a slipper orchid, Cypripedium flavum, is pollinated by both bees and flies, i.e., bumblebees and blowflies. Artificial pollination experiments demonstrated that the flowers of C. flavum are self-compatible, but need pollen vectors for successful reproduction. Field observations detected 25 insects visiting the flowers, and 14 of these insects entered into the labellum of the flowers, but only female bumblebees, Bombus hypnorum, B. remotus, and the blowfly Calliphora vomitoria exited of the labellum with pollen smears of C. flavum. The floral functional morphology of C. flavum appears to be more suited to bumblebees than to blowflies. The bumblebees are more efficient pollinators of the orchid, but blowflies are more frequent visitors, so they pollinated more flowers despite being less efficient.     

Folate Receptor-Positive Circulating Tumor Cells as a Novel Diagnostic Biomarker in Non-Small Cell Lung Cancer

The study aims to determine the efficacy and feasibility of a novel folate receptor (FR)-based circulating tumor cell (CTC) detection method in the diagnosis of non-small cell lung cancer (NSCLC). CTCs were collected from 3 ml of blood based on negative enrichment by immunomagnetic beads and then labeled by a conjugate of a tumor-specific ligand folate and an oligonucleotide. After washing off redundant conjugates, the bound conjugates were removed and analyzed by quantitative polymerase chain reaction. The captured cells were validated as tumor cells by immunofluorescence staining. In the evaluation of clinical utility, the results showed that the CTC levels of 153 patients with NSCLC were significantly higher than the controls (49 healthy donors and 64 patients with benign lung diseases; P < .001). With a threshold of 8.64 CTC units, the method showed a sensitivity of 73.2% and a specificity of 84.1% in the diagnosis of NSCLC, especially a sensitivity of 67.2% in stage I disease. Compared with the existing clinical biomarkers such as neuron-specific enolase (NSE), carcinoembryonic antigen (CEA), cancer antigen 125 (CA125), cyfra21-1, and squamous cell carcinoma antigen (SCC Ag), the method showed the highest diagnostic efficiency (area under the curve, 0.823; 95% confidence interval, 0.773–0.874). Together, our results demonstrated that FR-positive CTCs were feasible diagnostic biomarkers in patients with NSCLC, as well as in early-stage tumors.     

METTL3/N6-methyladenosine/ miR-21-5p promotes obstructive renal fibrosis by regulating inflammation through SPRY1/ERK/NF-κB pathway activation

Renal fibrosis induced by urinary tract obstruction is a common clinical occurrence; however, effective treatment is lacking, and a deeper understanding of the mechanism of renal fibrosis is needed. Previous studies have revealed that miR-21 impacts liver and lung fibrosis progression by activating the SPRY1/ERK/NF-kB signalling pathway. However, whether miR-21 mediates obstructive renal fibrosis through the same signalling pathway has not been determined. Additionally, studies have shown that N6-methyladenosine (m⁶A) modification-dependent primary microRNA (pri-microRNA) processing is essential for maturation of microRNAs, but its role in the maturation of miR-21 in obstructive renal fibrosis has not yet been investigated in detail. To address these issues, we employed a mouse model of unilateral ureteral obstruction (UUO) in which the left ureters were ligated for 3, 7 and 14 days to simulate the fibrotic process. In vitro, human renal proximal tubular epithelial (HK-2) cells were transfected with plasmids containing the corresponding sequence of METTL3, miR-21-5p mimic or miR-21-5p inhibitor. We found that the levels of miR-21-5p and m⁶A modification in the UUO model groups increased significantly, and as predicted, the SPRY1/ERK/NF-kB pathway was activated by miR-21-5p, confirming that miR-21-5p plays an important role in obstructive renal fibrosis by enhancing inflammation. METTL3 was found to play a major catalytic role in m⁶A modification in UUO mice and drove obstructive renal fibrosis development by promoting miR-21-5p maturation. Our research is the first to demonstrate the role of the METTL3-m⁶A-miR-21-5p-SPRY1/ERK/NF-kB axis in obstructive renal fibrosis and provides a deeper understanding of renal fibrosis.     

A Survey of Chinese Citizens’ Perceptions on Farm Animal Welfare

Farm animal welfare has been gradually recognized as an important issue in most parts of the world. In China, domestic animals were traditionally raised in backyard and treated as an important component of family wealth. Industrialization of animal production brings forth the farm animal welfare concerns recently in China, yet the modern concept of animal welfare has not been publicized and a comprehensive recognition on how consumers and farmers perceive animal welfare is lacking. Therefore, we conducted a survey on public opinions toward farm animal welfare in China, based on pigs (including sows, piglets, and fattening pigs), domestic fowls (including layers and broilers) and their products. From 6,006 effective questionnaires approximately two thirds of the respondents had never heard of ‘animal welfare’; 72.9% of the respondents claimed that, for the sake of animal derived food safety, human beings should improve the rearing conditions for pigs and domestic fowls; 65.8% of the respondents totally or partly agreed on establishing laws to improve animal welfare; more than half of the respondents were willing, or to some extent willing, to pay more for high-welfare animal products, whereas 45.5% of the respondents were not willing or reluctant to pay more. In summary, farm animal welfare is still in its early stage of development and more efforts are needed to improve the public conception to animal welfare in the process of establishing farm animal welfare standards and legislations in China.     

MicroRNA-Mediated Myostatin Silencing in Caprine Fetal Fibroblasts

Myostatin functions as a negative regulator of skeletal muscle growth by suppressing proliferation and differentiation of myoblasts. Dysfunction of the myostatin gene, either due to natural mutation or genetic manipulations such as knockout or knockdown, has been reported to increase muscle mass in mammalian species. RNA interference (RNAi) mediated by microRNAs (miRNAs) is a promising method for gene knockdown studies. In the present study, transient and stable silencing of the myostatin gene in caprine fetal fibroblasts (CFF) was evaluated using the two most effective constructs selected from four different miRNA expression constructs screened in 293FT cells. Using these two miRNA constructs, we achieved up to 84% silencing of myostatin mRNA in transiently transfected CFF cells and up to 31% silencing in stably transfected CFF cells. Moreover, off-target effects due to induction of interferon (IFN) response genes, such as interferon beta (IFN-β) and 2′-5′-oligoadenylate synthetase 2 (OAS2), were markedly fewer in stably transfected CFF cells than in transiently transfected cells. Stable expression of anti-myostatin miRNA with minimal induction of interferon shows great promise for increasing muscle mass in transgenic goats.