Mouse Sertoli cells isolation by lineage tracing and sorting

Sertoli cells play a key role in spermatogenesis by supporting the germ cells throughout differentiation. The isolation of Sertoli cells is essential to study their functions. However, the close contact of Sertoli cells with other testicular cell types and the high proliferation of contaminating cells are obstacles to obtain pure primary cultures. Current rodent Sertoli cell isolation protocols result in enriched, rather than pure Sertoli cells. Therefore, novel approaches are necessary to improve the purity of Sertoli cell primary cultures. The goal of this study is to obtain pure mouse Sertoli cells using lineage tracing and fluorescence‐activated cell sorting (FACS). We bred the Amh‐Cre mouse line with tdTomato line to generate mice constitutively expressing red fluorescence specifically in Sertoli cells. Primary cultures of Sertoli cells isolated from prepubertal mice showed that 79% of cells expressed tdTomato, as evaluated by fluorescence microscopy and flow cytometry; however, nearly all adherent cells were positive for vimentin. Most of the tomato‐negative cells expressed α‐smooth muscle actin (α‐SMA), a peritubular myoid cell marker, but double‐negative populations were also present. These findings suggest that vimentin lacks Sertoli cell‐specificity and that α‐SMA is not adequate to identify all of the contaminating cells. Upon FACS sorting; however, virtually 100% of the cells were tdTomato positive, expressed vimentin, but not α‐SMA. Prepubertal mice yielded a higher number of Sertoli cells compared to adults, but both could be adequately sorted. In conclusion, our study shows that lineage tracing and sorting is an efficient strategy for acquiring pure populations of murine Sertoli cells.     

Genetic diversity of Puccinia kuehnii,the causal agent of orange rust of sugarcane,from Brazil

The use of resistant genotypes is the preferred method to control orange rust of sugarcane (Saccharum spp) caused by Puccinia kuehnii. This approach has been adopted in Brazil but outbreaks of the disease on previously resistant varieties showed that the efficacy of this method is limited and requires a better understanding of pathogen diversity. Nevertheless, adequate molecular markers for examining pathogen diversity at population level are not available, which limits the success of orange rust control by genetic resistance. Therefore, two independent investigations were conducted to examine genetic diversity of P. kuehnii from São Paulo state, the most important sugarcane growing state of Brazil. First, simple-sequence repeat (SSR) markers were developed in the present work and genotypic diversity of orange rust isolates from different locations investigated. Second, phenotypic diversity was examined by the single-pustule inoculation technique on P. kuehnii isolates retrieved from three susceptible commercial sugarcane cultivars. A total of 96 SSR markers were generated and tested for this species. Subsequently, 29 isolates of P. kuehnii were fingerprinted with nine SSR markers to estimate the genotypic diversity by neighbour-joining and 3D principal coordinates. The 29 isolates of the pathogen clustered into four main groups, which were identified by three SSR markers (NPRL_PK_108a, NPRL_PK_162_spka and NPRL_PK_221_spka). Phenotypic data at 21 days after the single-pustule inoculation showed that P. kuehnii from highly susceptible commercial cultivars harboured a small proportion of variants capable of causing disease on resistant cultivars. A differential reaction was demonstrated for the most virulent variant in a repeated experiment confirming the existence of races within P. kuehnii in Brazil.     

分子标记在濒危物种保护中的应用

分子标记可揭示种群遗传和进化信息, 为制定濒危物种保护措施、指导恢复实践提供重要依据。本文主要介绍了分子标记在濒危物种保护过程不同环节中的应用, 包括: (1)正确识别保护单元, 如排除隐存种和杂交种的影响; (2)确定优先保护单元, 包括优先保护区域、优先保护物种、优先保护种群等; (3)指导迁地保护; (4)对保护工作的动态监测和评估。文章最后探讨了分子标记应用于保护的发展方向, 如开展长期的种群遗传组成监测、切实应用于保护管理实践、将基因组学等遗传信息用于全球变化背景下保护策略的制定等, 期望为分子标记技术在生物多样性保护的研究和实践中提供参考。     

Studies on reproductive strategies of Vitex negundo L. var. heterophylla (Franch.) Rehder (Lamiaceae) based on morphological characteristics and SSR markers

Vitex negundo L. var. heterophylla (Franch.) Rehder (Lamiaceae) is an important tree species for soil and water conservation, yet the reproductive ecology of this species remains to be elucidated. To investigate the reproductive traits of V. negundo var. heterophylla, the phenology, morphological characteristics (a suite of characters was assessed: floral morphology, nectar production, pollen viability, and stigma receptivity) and mating system of this species were systematically revealed for the first time in this study. Phenological observations, morphological measurements, and nectar production analysis were conducted during anthesis. Pollen viability and stigma receptivity at different flowering stages were measured by biochemical methods. Finally, genetic analysis based on SSR markers was used to reveal the mating system; outcrossing index and pollen‐ovule ratio were also calculated to help analysis. V. negundo var. heterophylla showed several obvious characteristics of outcrossing, such as abundant and attractive flowers, secreting nectar, and emitting scent. In addition, mechanisms such as homogamy and a short anther‐stigma distance that can promote self‐fertilization were also identified in this species. The coexistence of selfing and outcrossing characteristics demonstrates a predominantly outcrossed mixed mating system (outcrossing rate, t = 95%). The scientific information provided by this study may contribute to conservation of V. negundo var. heterophylla from a reproductive perspective.     

Chemical components from Sambucus adnata wall

Sambucus adnata Wall. belongs to Caprifoliaceae family. Phytochemical investigation on the aerial parts of S. adnata led to the isolation and identification of 17 known compounds (1–17) including triterpenoids and sterols. Compounds 1 and 8 are isolated from Sambucus adnata for the first time, and it is the first report that compounds 2, 5–6, 9–10, 12–15, and 17 are isolated from the plants of genus Sambucus. Importantly, three types of abundant components: (3β)-urs-12-en-3-yl stearate (0.33 g/kg, ursane type), (3β)-olean-12-en-3-yl stearate (0.17 g/kg, oleanane type), and lupenyl palmitate (0.52 g/kg, lupinane type) could be used as potential chemotaxonomic markers to distinguish among species of Sambucus.     

Flavonoids from tribe Delphineae (Ranunculaceae): Phytochemical review and chemotaxonomic value

This review summarizes the flavonoids isolated from three genera, namely, Aconitum, Delphinium, and Consolida, belonging to tribe Delphineae in the Ranunculaceae family for the first time. A total of 104 distinct flavonoid components, including 85 flavonols, 13 anthocyanins, four flavones, and two neoflavones, have been isolated from 44 members of tribe Delphineae. Flavonols account for the largest proportion and can be regarded as the dominant group of flavonoids in this tribe. Of the 104 isolated flavonoids, 55 are novel, indicating the high chemical diversity among the flavonoid constituents of Delphineae plants. Flavonoids in Delphineae plants exhibit chemotaxonomic significance, characterizing certain Delphineae species well. Flavonol glycosides, as the major flavonoid constituents in the investigated Delphineae species, could also serve as valuable chemotaxonomic markers in addition to diterpenoid alkaloids for the identification of Delphineae species.     

Genome-wide scan reveals important additive and non-additive genetic effects associated with resistance to Haemonchus contortus in Florida Native sheep

Florida Native sheep is among the sheep breeds best adapted to humid and hot climatic conditions such as those of Florida, USA, and have shown a superior ability to regulate nematode burdens. This is one of the oldest sheep breeds in North America and is an endangered species. To ensure genetic diversity and long-term survival of the breed, protection of the current genetic stock is critical and conservation efforts are required to promote its breeding and production. The objective of the present study was to investigate the importance of additive and non-additive genetic effects on resistance to natural Haemonchus contortus infections in Florida Native sheep using a whole genome scan. A total of 200 sheep were evaluated in the present study. Phenotypic records included faecal egg count (FEC, eggs/gram), FAMACHA® score, packed cell volume (PCV, %), body condition score and average daily gain (ADG, kg). Sheep were genotyped using the GGP Ovine 50K SNP chip and 45.2 k single nucleotide polymorphism (SNP) markers spanning the entire genome were available for quality control procedures. Mixed models were used to analyse the response variables and included the identity by state matrix to control for population structure. Bonferroni correction was used to control for multiple testing and a second arbitrary threshold (0.1 × 10^?3) was used. Fifteen SNPs with additive and non-additive genetic effects and located in Ovis aries chromosome OAR1, 2, 3, 6, 8, 10, 11, 12, 13 and 21 were associated with FEC, FAMACHA® score, PCV and ADG. These SNPs could be potential genetic markers for resistance to natural H. contortus exposure in Florida Native sheep.     

消癌平注射液联合表柔比星新辅助化疗对三阴性乳腺癌患者免疫功能、生活质量及血清肿瘤标志物的影响

摘要 目的：探讨消癌平注射液联合表柔比星新辅助化疗对三阴性乳腺癌（TNBC）患者免疫功能、生活质量及血清肿瘤标志物的影响。方法：选取TNBC患者89例,按照随机数字表法分为对照组和研究组,对照组（n=44）患者给予表柔比星新辅助化疗治疗,研究组（n=45）患者给予消癌平注射液联合表柔比星新辅助化疗。对比两组疗效、免疫功能、生活质量、血清肿瘤标志物及不良反应。结果：研究组治疗12周后的临床总有效率为91.11%（41/45）,高于对照组的63.64%（28/44）（P<0.05）。两组治疗12周后健康调查简表（SF-36）量表各维度评分升高,且研究组较对照组高（P<0.05）。两组治疗12周后CD4+CD25+Treg、Th17/ Treg均降低,且研究组较对照组低（P<0.05）,Th17升高,且研究组较对照组高（P<0.05）。两组治疗12周后癌胚抗原（CEA）、糖类抗原199（CA199）、糖类抗原125（CA125）均降低,且研究组较对照组低（P<0.05）。两组不良反应发生率对比未见差异（P>0.05）。结论：消癌平注射液联合表柔比星新辅助化疗治疗TNBC,具有确切的治疗效果,可降低血清肿瘤标志物水平,改善患者免疫功能和生活质量。