Unlocking the vault: next‐generation museum population genomics

Natural history museum collections provide unique resources for understanding how species respond to environmental change, including the abrupt, anthropogenic climate change of the past century. Ideally, researchers would conduct genome‐scale screening of museum specimens to explore the evolutionary consequences of environmental changes, but to date such analyses have been severely limited by the numerous challenges of working with the highly degraded DNA typical of historic samples. Here, we circumvent these challenges by using custom, multiplexed, exon capture to enrich and sequence ~11 000 exons (~4 Mb) from early 20th‐century museum skins. We used this approach to test for changes in genomic diversity accompanying a climate‐related range retraction in the alpine chipmunks (Tamias alpinus) in the high Sierra Nevada area of California, USA. We developed robust bioinformatic pipelines that rigorously detect and filter out base misincorporations in DNA derived from skins, most of which likely resulted from postmortem damage. Furthermore, to accommodate genotyping uncertainties associated with low‐medium coverage data, we applied a recently developed probabilistic method to call single‐nucleotide polymorphisms and estimate allele frequencies and the joint site frequency spectrum. Our results show increased genetic subdivision following range retraction, but no change in overall genetic diversity at either nonsynonymous or synonymous sites. This case study showcases the advantages of integrating emerging genomic and statistical tools in museum collection‐based population genomic applications. Such technical advances greatly enhance the value of museum collections, even where a pre‐existing reference is lacking and points to a broad range of potential applications in evolutionary and conservation biology.     

Characterization of two distinct neuraminidases from avian-origin human-infecting H7N9 influenza viruses

An epidemic of an avian-origin H7N9 influenza virus has recently emerged in China, infecting 134 patients of which 45 have died. This is the first time that an influenza virus harboring an N9 serotype neuraminidase (NA) has been known to infect humans. H7N9 viruses are divergent and at least two distinct NAs and hemagglutinins (HAs) have been found, respectively, from clinical isolates. The prototypes of these viruses are A/Anhui/1/2013 and A/Shanghai/1/2013. NAs from these two viruses are distinct as the A/Shanghai/1/2013 NA has an R294K substitution that can confer NA inhibitor oseltamivir resistance. Oseltamivir is by far the most commonly used anti-influenza drug due to its potency and high bioavailability. In this study, we show that an R294K substitution results in multidrug resistance with extreme oseltamivir resistance (over 100 000-fold) using protein- and virus-based assays. To determine the molecular basis for the inhibitor resistance, we solved high-resolution crystal structures of NAs from A/Anhui/1/2013 N9 (R294-containing) and A/Shanghai/1/2013 N9 (K294-containing). R294K substitution results in an unfavorable E276 conformation for oseltamivir binding, and consequently loss of inhibitor carboxylate interactions, which compromises the binding of all classical NA ligands/inhibitors. Moreover, we found that R294K substitution results in reduced NA catalytic efficiency along with lower viral fitness. This helps to explain why K294 has predominantly been found in clinical cases of H7N9 infection under the selective pressure of oseltamivir treatment and not in the dominant human-infecting viruses. This implies that oseltamivir can still be efficiently used in the treatment of H7N9 infections.     

A point mutation in LTT1 enhances cold tolerance at the booting stage in rice

The cold tolerance of rice at the booting stage is a main factor determining sustainability and regional adaptability. However, relatively few cold tolerance genes have been identified that can be effectively used in breeding programmes. Here, we show that a point mutation in the low-temperature tolerance 1 (LTT1) gene improves cold tolerance by maintaining tapetum degradation and pollen development, by activation of systems that metabolize reactive oxygen species (ROS). Cold-induced ROS accumulation is therefore prevented in the anthers of the ltt1 mutants allowing correct development. In contrast, exposure to cold stress dramatically increases ROS accumulation in the wild type anthers, together with the expression of genes encoding proteins associated with programmed cell death and with the accelerated degradation of the tapetum that ultimately leads to pollen abortion. These results demonstrate that appropriate ROS management is critical for the cold tolerance of rice at the booting stage. Hence, the ltt1 mutation can significantly improve the seed setting ability of cold-sensitive rice varieties under low-temperature stress conditions, with little yield penalty under optimal temperature conditions. This study highlights the importance of a valuable genetic resource that may be applied in rice breeding programmes to enhance cold tolerance.     

Modifying the tumour microenvironment and reverting tumour cells: New strategies for treating malignant tumours

The tumour microenvironment (TME) plays a pivotal role in tumour fate determination. The TME acts together with the genetic material of tumour cells to determine their initiation, metastasis and drug resistance. Stromal cells in the TME promote the growth and metastasis of tumour cells by secreting soluble molecules or exosomes. The abnormal microenvironment reduces immune surveillance and tumour killing. The TME causes low anti‐tumour drug penetration and reactivity and high drug resistance. Tumour angiogenesis and microenvironmental hypoxia limit the drug concentration within the TME and enhance the stemness of tumour cells. Therefore, modifying the TME to effectively attack tumour cells could represent a comprehensive and effective anti‐tumour strategy. Normal cells, such as stem cells and immune cells, can penetrate and disrupt the abnormal TME. Reconstruction of the TME with healthy cells is an exciting new direction for tumour treatment. We will elaborate on the mechanism of the TME to support tumours and the current cell therapies for targeting tumours and the TME—such as immune cell therapies, haematopoietic stem cell (HSC) transplantation therapies, mesenchymal stem cell (MSC) transfer and embryonic stem cell‐based microenvironment therapies—to provide novel ideas for producing breakthroughs in tumour therapy strategies.     

具备定点偶联功能的HBc-VLPs的制备与性质鉴定

乙型肝炎病毒核心蛋白HBc,可在体外自组装形成二十面体对称结构的病毒样微粒VLPs。VLPs可将外源序列重复且高密度地展示在表面,VLPs进入机体后能够快速诱导机体产生针对外源性抗原的特异性体液免疫及细胞免疫应答,具有极强的免疫原性与生物活性。因此,HBc-VLPs可以作为一种安全、有效的疫苗载体。文中设计了一种能够实现与抗原定点偶联的HBc-VLPs,并开发了一套高效制备HBc-VLPs的方法。通过定点突变技术,使翻译后的多肽序列第80位氨基酸由Ala变为Cys,在HBc-VLPs的主要免疫显性区域引入一个定点交联位点,构建了原核表达载体pET28a(+)-hbc,表达、纯化获得了高纯度的HBc(A80C) 单体蛋白;在PB缓冲体系中,HBc(A80C) 蛋白自组装形成HBc-VLPs纳米粒子。粒度仪的测定结果表明,HBc-VLPs纳米微粒的平均粒径为29.8 nm,透射电子显微镜观察到HBc-VLPs形成粒径约为30 nm的球形微粒,其形态与天然的HBV微粒相似。以流感病毒M2e抗原肽为模式抗原,通过Sulfo-SMCC氨基-巯基双功能交联剂,将M2e定点连接于HBc-VLPs通过突变引入的Cys残基处,制备了M2e-HBc-VLPs模式疫苗,通过细胞荧光示踪,验证了HBc-VLPs结构的完整性与M2e的正确交联。动物免疫实验表明该疫苗能够有效刺激小鼠产生抗原特异性的IgG抗体,验证了疫苗载体HBc-VLPs的有效性。研究结果为HBc-VLPs作为疫苗载体的研究奠定了基础,能够促进HBc-VLPs载体疫苗的研发以及HBc-VLPs在其他领域的应用。     

基于成果导向教育理念的生物化学多维度教学改革体系的构建

生物化学是生物类相关专业的重要基础课,具有发展迅速、信息量大且理论性和实践性均强等特点,针对教与学过程中普遍存在的学生学习难度大、实验课缺乏整体性、综合性和设计性实验少等问题,笔者在成果导向教育 (Outcome-based education,OBE) 理念的指导下,从理论教学及实践教学入手,通过引入灵活多样的教学方法、用好在线课程、实施双语教学、加强实践教学环节、改进考核模式等方面,构建了生物化学多维度教学改革体系。实践证明,该教学改革体系能够使学生由“被动学”变“主动学”,充分调动了学生的学习积极性、培养了学生的创新能力,在提升高校人才培养质量方面起到了重要作用。     

Exosomal HOTAIR promotes proliferation,migration and invasion of lung cancer by sponging miR-203

正Dear Editor,Lung cancer is one of the most common causes of cancerrelated deaths worldwide. Despite great progress in the development of lung cancer treatments, the prognoses of patients with advanced stage cancer remains poor (Torre et al.,2015). Growing evidence has demonstrated that long non-     

Effects of seasonal precipitation change on soil respiration processes in a seasonally dry tropical forest

Precipitation is projected to change intensity and seasonal regime under current global projections. However, little is known about how seasonal precipitation changes will affect soil respiration, especially in seasonally dry tropical forests. In a seasonally dry tropical forest in South China, we conducted a precipitation manipulation experiment to simulate a delayed wet season (DW) and a wetter wet season (WW) over a three‐year period. In DW, we reduced 60% throughfall in April and May to delay the onset of the wet season and irrigated the same amount water into the plots in October and November to extend the end of the wet season. In WW, we irrigated 25% annual precipitation into plots in July and August. A control treatment (CT) receiving ambient precipitation was also established. Compared with CT, DW significantly increased soil moisture by 54% during October to November, and by 30% during December to April. The treatment of WW did not significantly affect monthly measured soil moisture. In 2015, DW significantly increased leaf area index and soil microbial biomass but decreased fine root biomass. In contrast, WW significantly decreased fine root biomass and forest floor litter stocks. Soil respiration was not affected by DW, which could be attributed to the increased microbial biomass offsetting the decrease in fine root biomass. In contrast, WW significantly increased soil respiration from 3.40 to 3.90 μmol m^?2 s^?1 in the third year, mainly due to the increased litter decomposition and soil pH (from 4.48 to 4.68). The present study suggests that both a delayed wet season and a wetter wet season will have significant impacts on soil respiration‐associated ecosystem components. However, the ecosystem components can respond in different directions to the same change in precipitation, which ultimately affected soil respiration.     

Characterization of the plasma membrane proteins and receptor-like kinases associated with secondary vascular differentiation in poplar

The constituents of plasma membrane proteins, particularly the integral membrane proteins, are closely associated with the differentiation of plant cells. Secondary vascular differentiation, which gives rise to the increase in plant stem diameter, is the key process by which the volume of the plant body grows. However, little is known about the plasma membrane proteins that specifically function in the vascular differentiation process. Proteomic analysis of the membrane proteins in poplar differentiating secondary vascular tissues led to the identification 226 integral proteins in differentiating xylem and phloem tissues. A majority of the integral proteins identified were receptors (55 proteins), transporters (34 proteins), cell wall formation related (27 proteins) or intracellular trafficking (17 proteins) proteins. Gene expression analysis in developing vascular cells further demonstrated that cambium differentiation involves the expression of a group of receptor kinases which mediate an array of signaling pathways during secondary vascular differentiation. This paper provides an outline of the protein composition of the plasma membrane in differentiating secondary vascular tissues and sheds light on the role of receptor kinases during secondary vascular development.     

Response of in vitro strawberry to antibiotics

By identifying antibiotics that had the least phytotoxic effects on explants during genetic transformation, we evaluated the effect of various antibiotics on callus induction and morphogenesis from leaf explants and in vitro growth of Fragaria × ananassa Duch. cv. Toyonaka. Results showed that kanamycin (Kan) significantly inhibited callus induction, bud differentiation and root morphogenesis while carbenicillin (Carb), cefotaxime (Cef) and an equal concentration of Cef and Carb up to 500 mg L⁻¹ had no significant effects on callus induction and shoot growth. Kan, even at 2.5 mg L⁻¹, significantly inhibited callus induction, shoot regeneration and root formation, while no shoots regenerated at concentrations above 15 mg L⁻¹. Rooting was completely inhibited in the presence of 50 mg L⁻¹ Kan. Cef had negative effects on shoot regeneration from leaf explants and in vitro growth of strawberry. Compared to Cef, Carb at ≤300 mg L⁻¹ significantly promoted shoot and root organogenesis. However, an equal concentration of Carb plus Cef could alleviate the negative effect of Cef on strawberry. Results from relative electrolyte leakage, root and antioxidant activities, O₂^·− production rate, H₂O₂, proline and MDA contents showed that Kan, Cef and Carb caused electrolyte leakage and triggered active enzymatic processes and metabolism. This offers a possible mechanism for the inhibition or stimulation of strawberry growth caused by these antibiotics.