Implementing Patch Clamp and Live Fluorescence Microscopy to Monitor Functional Properties of Freshly Isolated PKD Epithelium

Cyst initiation and expansion during polycystic kidney disease is a complex process characterized by abnormalities in tubular cell proliferation, luminal fluid accumulation and extracellular matrix formation. Activity of ion channels and intracellular calcium signaling are key physiologic parameters which determine functions of tubular epithelium. We developed a method suitable for real-time observation of ion channels activity with patch-clamp technique and registration of intracellular Ca²⁺ level in epithelial monolayers freshly isolated from renal cysts. PCK rats, a genetic model of autosomal recessive polycystic kidney disease (ARPKD), were used here for ex vivo analysis of ion channels and calcium flux. Described here is a detailed step-by-step procedure designed to isolate cystic monolayers and non-dilated tubules from PCK or normal Sprague Dawley (SD) rats, and monitor single channel activity and intracellular Ca²⁺ dynamics. This method does not require enzymatic processing and allows analysis in a native setting of freshly isolated epithelial monolayer. Moreover, this technique is very sensitive to intracellular calcium changes and generates high resolution images for precise measurements. Finally, isolated cystic epithelium can be further used for staining with antibodies or dyes, preparation of primary cultures and purification for various biochemical assays.     

High genetic diversity and geographic subdivision of three lance nematode species (Hoplolaimus spp.) in the United States

Lance nematodes (Hoplolaimus spp.) feed on the roots of a wide range of plants, some of which are agronomic crops. Morphometric values of amphimictic lance nematode species overlap considerably, and useful morphological characters for their discrimination require high magnification and significant diagnostic time. Given their morphological similarity, these Hoplolaimus species provide an interesting model to investigate hidden diversity in crop agroecosystems. In this scenario, H. galeatus may have been over-reported and the related species that are morphologically similar could be more widespread in the United States that has been recognized thus far. The main objectives of this study were to delimit Hoplolaimus galeatus and morphologically similar species using morphology, phylogeny, and a barcoding approach, and to estimate the genetic diversity and population structure of the species found. Molecular analyses were performed using sequences of the cytochrome c oxidase subunit 1 (Cox1) and the internal transcribed spacer (ITS1) on 23 populations. Four morphospecies were identified: H. galeatus, H. magnistylus, H. concaudajuvencus, and H. stephanus, along with a currently undescribed species. Pronounced genetic structure correlated with geographic origin was found for all species, except for H. galeatus. Hoplolaimus galeatus also exhibited low genetic diversity and the shortest genetic distances among populations. In contrast, H. stephanus, the species with the fewest reports from agricultural soils, was the most common and diverse species found. Results of this project may lead to better delimitation of lance nematode species in the United States by contributing to the understanding the diversity within this group.     

Estuarine intertidal meiofauna and nematode communities as indicator of ecosystem's recovery following mitigation measures

The Mondego estuary (Portugal) has been under environmental pressure since the early 1990s due to different anthropogenic stresses. The system has been studied following benthic communities’ features from an impacted situation until the recovery phase, focusing mostly on macrobenthos. Following the application of mitigation measures in the estuary, this study is focused on the variability of the intertidal meiobenthic and nematode communities in a system that has recovered after different anthropogenic stresses. While at the spatial level (among areas along the eutrophication gradient) no significant differences were observed regarding the structure and function of the nematode communities, at the seasonal level some differences stood out. These results broadly suggest that the system has recovered from the early situations of pressures being, to the best of our knowledge, the first attempt to investigate the variability of intertidal meiobenthic and nematode communities in the scope of a system's recovery along an estuarine gradient of eutrophication. Even if performed in a short timeline, this study provides a good baseline analysis of conditions, being important for future comparisons.     

Gastrointestinal nematodes and dietary fibre: Two factors to consider when using FN for wildlife nutrition monitoring

Faecal nitrogen (FN) – the combination of metabolic nitrogen and residual food nitrogen – has been used as a proxy for diet quality in wild and domestic ruminants for over half a century. However, a common misconception in some of these studies is that FN is a direct proxy for dietary N, in spite of experimental evidence that links FN to general diet digestibility. Additionally, gastrointestinal nematodes (GIN) can alter N metabolism and increase FN by various mechanisms. To clarify the role of dietary N, fibre and GIN as a factor in FN excretion, 10 naturally parasitised sheep were fed two different isocaloric diets (LPF: low-protein, low-fibre; HPF: high-protein, high-fibre). One month after these diets began, a single anthelmintic treatment was applied to remove GIN, after which the sheep were kept on the same diet for an additional 2 weeks. Throughout the experiment, individual faecal samples were obtained to estimate both FN and GIN intensity (using faecal egg counts, FEC). In addition, two blood samples were taken before and after deworming to measure serum total protein concentrations (TP) as a proxy for protein absorption. In spite of the difference in dietary protein, FN was higher on an LPF diet, supporting the overall digestibility concept. The influence of GIN on FN was later revealed by the anthelmintic treatment, which led to a decrease of FEC and FN in both dietary groups. Serum total protein showed a slight but non-significant increase in both groups after the anthelmintic treatment. Our study supports not only the concept that FN is a proxy for diet digestibility, and not directly for dietary N, but also that gastrointestinal nematodes limit its use as a proxy for diet quality in ruminants, especially under high parasite loads (e.g., 1000 faecal eggs per gram of faeces). Such limitations should be considered before using FN for wildlife nutrition monitoring. Some recommendations are given to avoid misinterpretations.     

Efficacy of two entomopathogenic nematodes Heterorhabditis bacteriophora and Steinernema carpocapsae for control of the leopard moth borer Zeuzera pyrina (Lepidoptera: Cossidae) larvae under laboratory conditions

The biological traits of the entomopathogenic nematodes (EPNs), Steinernema carpocapsae and Heterorhabditis bacteriophora, against the larvae of the leopard moth, Zeuzera pyrina were evaluated in the laboratory. The traits included pathogenicity, penetration potential as well as foraging behaviour. Plate assays were performed using a range of EPN concentrations (5, 10, 20, 50 and 100 infective juveniles (IJs) per larva). The LC₅₀ values for S. carpocapsae and H. bacteriophora were 6.4 and 8.4 IJs larva^?1 after 72 h. Both EPN species caused high mortality in branch experiments. Significantly higher mortality rates occurred in the larger larvae after exposure to S. carpocapsae. Both EPN species successfully penetrated the Z. pyrina larvae as well as larvae of Galleria mellonella L. (Lepidoptera: Galleridae).The proportional response of H. bacteriophora to host-associated cues was strongly higher than S. carpocapsae in Petri dishes containing agar 1, 12 and 24 h after EPN application. These results highlight the efficiency of EPNs for the control of Z. pyrina larvae. However, due to the cryptic habitat of Z. pyrina larvae in their galleries in the trees, field trails need to be conducted to further evaluate this potential.     

拟南芥AtCDPK1基因克隆与转化马铃薯的研究

以野生拟南芥生态型(Col-0)为材料,利用PCR和DNA重组技术,克隆了拟南芥钙依赖蛋白激酶基因(AtCDPK1)。AtCDPK1基因全长为2 269bp,碱基序列与已知基因At1g18890完全一致。构建AtCDPK1基因的植物表达载体pCHF3-CDPK1,并利用农杆菌介导法将pCHF3-CDPK1转入马铃薯品种‘费乌瑞它’的脱毒试管微型薯中,经筛选与植株再生,共获得50个抗性再生植株。PCR检测显示,有36个植株基因组中含有AtCDPK1基因。RT-PCR分析证实,AtCDPK1基因在这些马铃薯植株的基因组中均能正常转录表达。这一结果为进一步开展AtCDPK1基因功能分析及转基因抗旱马铃薯新品种选育研究奠定了基础。     

马铃薯WRKY2基因的克隆和非生物逆境下的表达模式

马铃薯WRKY2基因的功能尚未见有报道,WRKY蛋白是近年来发现的一类重要转录因子家族,它们在植物应对生物胁迫、非生物胁迫和生长发育过程中起到关键的调控作用。该研究采用电子克隆法获得马铃薯WRKY2基因,该基因的编码区长度1 065 bp,编码355氨基酸,序列分析表明,该蛋白属于WRKY家族第二组成员,锌指结构为C-X5-C-X23H-X-H。构建系统发育树结果表明它与番茄WRKY7亲缘关系较近,氨基酸序列相似性达96%,与烟草中WRKY蛋白的相似性为86%,利用原核表达法在大肠杆菌中获得该蛋白。通过凝胶阻滞实验证明,该蛋白在体外能结合W-box元件,而且这种结合能被冷探针所竞争,同时也表明St WRKY2不能结合含有突变W-box DNA片段,证明St WRKY2与W-box结合具有特异性。通过实时荧光定量PCR技术分析该基因在根、茎和叶中的表达量,结果表明该基因主要在根中表达,其次是叶和茎。为进一步研究该基因可能参与的生理功能,对马铃薯组培苗进行10μmol/L低磷、10μmol/L低钾、200 mmol/L Na Cl、400 mmol/L PEG溶液和4℃低温处理,处理时间6 h,实时荧光定量PCR的结果表明该基因在低磷处理后表达量明显下降,在200 mmol/L Na Cl和400 mmol/L PEG处理6 h后表达量明显升高,但在10μmol/L低钾和4℃低温处理后表达量与对照相比无明显变化。说明St WRKY2能响应低磷、Na Cl和PEG这三种非生物逆境胁迫。研究结果可为进一步深入研究马铃薯WRKY2基因的功能奠定基础。     

Understanding Reaction of Potato (Solanum tuberosum) to Ralstonia solanacearum and Relationship of Wilt Incidence to Latent Infection

Bacterial wilt caused by Ralstonia solanacearum is one of the most important diseases affecting more than 200 plant species, including solanaceous crops. The pathogen is known to cause complicated symptoms ranging from visible to latent ones. Understanding crop's reaction to the pathogen and the underlying relatedness of latent infection to wilt incidence is of paramount importance. Thus, a number of potato cultivars including improved and otherwise were evaluated under greenhouse and field conditions. Accordingly, twenty‐eight of the cultivars tested under greenhouse conditions were resistant to the pathogen with scores ranging from 0.77 to 1.17 of 5. Nonetheless, under field conditions, only 2 of 28 cultivars found to be ‘resistant’ under greenhouse conditions, showed adequate resistance to the pathogen, indicating the significant impact of environment on the activity of the pathogen and reaction of the crop. Percentage wilt incidence and latent infection showed significant (P < 0.05) positive correlation, with r = 0.9438. Thus, evaluation of crop's performance based on the combination of the parameters like field wilt incidence and proportion of latent infection gave us better picture of the overall crop feat, than using wilt incidence as a sole parameter of evaluation as has been the case in most studies. Moreover, the established correlation of latent infection with field wilt incidence will also help us understand the disease epidemiology and design effective management measures, accordingly.