Exploring the Gastrointestinal “Nemabiome”: Deep Amplicon Sequencing to Quantify the Species Composition of Parasitic Nematode Communities

Parasitic helminth infections have a considerable impact on global human health as well as animal welfare and production. Although co-infection with multiple parasite species within a host is common, there is a dearth of tools with which to study the composition of these complex parasite communities. Helminth species vary in their pathogenicity, epidemiology and drug sensitivity and the interactions that occur between co-infecting species and their hosts are poorly understood. We describe the first application of deep amplicon sequencing to study parasitic nematode communities as well as introduce the concept of the gastro-intestinal “nemabiome”. The approach is analogous to 16S rDNA deep sequencing used to explore microbial communities, but utilizes the nematode ITS-2 rDNA locus instead. Gastro-intestinal parasites of cattle were used to develop the concept, as this host has many well-defined gastro-intestinal nematode species that commonly occur as complex co-infections. Further, the availability of pure mono-parasite populations from experimentally infected cattle allowed us to prepare mock parasite communities to determine, and correct for, species representation biases in the sequence data. We demonstrate that, once these biases have been corrected, accurate relative quantitation of gastro-intestinal parasitic nematode communities in cattle fecal samples can be achieved. We have validated the accuracy of the method applied to field-samples by comparing the results of detailed morphological examination of L3 larvae populations with those of the sequencing assay. The results illustrate the insights that can be gained into the species composition of parasite communities, using grazing cattle in the mid-west USA as an example. However, both the technical approach and the concept of the ‘nemabiome’ have a wide range of potential applications in human and veterinary medicine. These include investigations of host-parasite and parasite-parasite interactions during co-infection, parasite epidemiology, parasite ecology and the response of parasite populations to both drug treatments and control programs.     

Effect of local anesthetics on plasma protein secretion by rat hepatocytes

The effects of some local anesthetics on plasma protein secretion by rat liver slices have been studied and have been compared with those of colchicine. Rat liver slices were pulse-labelled with l-[¹⁴C]leucine for 9 min at 37°C, collected on filter paper, washed with non-radioactive leucine and reincubated in the presence or absence of the drug to be tested. The radioactive plasma proteins produced were obtained by immunoprecipitation from either the chase medium or from the washed slices. Chlorpomazine, (3 · 10^?5 M), dibucaine (10^?5 M), lidocaine (10^?3 M) and procaine (5 · 10^?5 M) inhibited both the synthesis and secretion of plasma protein but did not affect the uptake of l-leucine into the slices nor the incorporation of phosphate into intracellular nucleotide phosphates or into phopholipids. The inhibition of secretion elicited by these drugs is probably not due to the inhibition of protein synthesis since cycloheximide, when added to the chase medium at a concentration which completely inhibits protein synthesis, did not inhibit plasma protein secretion, while cycloheximide plus procaine did inhibit secretion and also caused a retention of non-secreted plasma proteins within the slices. Unlike colchicine, howover, procaine did not cause the retained plasma proteins to accumulate in Goli-derived secretory vesicles, but showed a more general effect causing a distribution among several cell fractions.     

Sensitivity to reinforcement in successive and delayed discriminations

Pigeons' responses were recorded in successive 15-s subintervals of 60-s components of several multiple variable-interval schedules of food reinforcement. In the standard multiple schedule or successive discrimination, discriminative stimuli were present throughout each component. In the delayed discrimination or memory procedure, red or green stimuli were present in the first 15 s of components and were followed by a white stimulus for the remainder of both components. Ratios of responses in the first 15 s of the two components, where discriminative stimuli were present, were sensitive to ratios of reinforcers obtained in the two components, to the same extent in both multiple and memory procedures. In both procedures, sensitivity to reinforcement decreased systematically over component subintervals, but to a greater extent in the memory procedure where discriminative stimuli were absent. The reduction in sensitivity with time since presentation of prior discriminative stimuli in the memory procedure was therefore influenced by two main factors: delayed stimulus control by the discriminative stimuli presented earlier in the component, and a decrease in sensitivity to reinforcement with increasing time since component alternation.     

Syncytiotrophoblast Extracellular Vesicles from Pre-Eclampsia Placentas Differentially Affect Platelet Function

Pre-eclampsia (PE) complicates around 3% of all pregnancies and is one of the most common causes of maternal mortality worldwide. The pathophysiology of PE remains unclear however its underlying cause originates from the placenta and manifests as raised blood pressure, proteinuria, vascular or systemic inflammation and hypercoagulation in the mother. Women who develop PE are also at significantly higher risk of subsequently developing cardiovascular (CV) disease. In PE, the failing endoplasmic reticulum, oxidative and inflammatory stressed syncytiotrophoblast layer of the placenta sheds increased numbers of syncytiotrophoblast extracellular vesicles (STBEV) into the maternal circulation. Platelet reactivity, size and concentration are also known to be altered in some women who develop PE, although the underlying reasons for this have not been determined. In this study we show that STBEV from disease free placenta isolated ex vivo by dual placental perfusion associate rapidly with platelets. We provide evidence that STBEV isolated from normal placentas cause platelet activation and that this is increased with STBEV from PE pregnancies. Furthermore, treatment of platelets with aspirin, currently prescribed for women at high risk of PE to reduce platelet aggregation, also inhibits STBEV-induced reversible aggregation of washed platelets. Increased platelet reactivity as a result of exposure to PE placenta derived STBEVs correlates with increased thrombotic risk associated with PE. These observations establish a possible direct link between the clotting disturbances of PE and dysfunction of the placenta, as well as the known increased risk of thromboembolism associated with this condition.     

E-box元件修饰端粒酶核心启动子序列及体外转录活性分析

人类端粒酶启动子(hTERT启动子)在肿瘤基因治疗中的有效性已经得到了证实. 然而,hTERT启动子有限的肿瘤靶向转录活性困扰着它的临床应用.早期研究已经揭示,核心hTERT启动子上的-34位E-box元件与该启动子的肿瘤靶向转录活性有关.为进一步探索核心hTERT启动子序列3′端富余E-box元件是否能提高启动子的肿瘤靶向转录能力,用化学合成方法在野生型hTERT(WT-hTERT)核心启动子片段(编码蛋白起始子ATG上游-268 bp～-10 bp)的3′端接入3个E-box序列, 构建成修饰型hTERT(Mod-hTERT)启动子. 然后,分别用WT-hTERT和Mod-hTERT启动子去调控增强型绿色荧光蛋白(EGFP)及荧光素酶报告基因在293FT、HepGⅡ、SGC7901、U2OS、以及原代培养人成纤维细胞(PHF)中表达. 结果表明, 在Mod-hTERT启动子的各实验组细胞中,能够在端粒酶阳性的293FT、HepGⅡ及 SGC7901细胞组中观测到EGFP的表达,而在端粒酶阴性的U2OS及PHF细胞组中没有观测到EGFP的表达;在端粒酶阳性的293FT、HepGⅡ和SGC7901细胞株中,Mod-hTERT启动子调控下的荧光素酶活性要高于WT-hTERT启动子组（P＜0.01）; 而在端粒酶阴性的U2OS细胞组中,Mod-hTERT启动子调控下的荧光素酶活性则低于WT-hTERT启动子组（P＜0.01); 在PHF细胞组中,Mod-hTERT启动子组与WT-hTERT启动子组的荧光素酶活性差异不显著(P＞0.05).研究提示,在3′端增加E-box元件可以提高核心hTERT启动子序列的肿瘤靶向转录活性.     

Trade-offs between activity and thermoregulation in a small carnivore,the least weasel Mustela nivalis

We studied factors influencing daily energy expenditures (DEE) of male least weasels (Mustela nivalis) using the doubly labelled water technique. The relationship between ambient temperature and DEE formed a triangular pattern, characterized by invariance of the maximum DEE and an inverse relationship between minimum DEE and temperature. A simple energetic model relating the DEE of male weasels to activity time (AT) and ambient temperature predicted that, across seasons, less than 10 per cent of measurements approach the upper bound of observed DEE. Male weasels were able to maintain a relatively constant maximum energy output across varying temperatures by adjusting their AT to changes in temperature. They achieved maximum energy expenditures in winter due to high thermoregulatory costs, and in spring and summer due to high levels of physical activity. This pattern exemplifies a ‘metabolic niche’ of a small mammal having extremely high energy expenditures primarily driven by ambient temperature.     

The impacts of flow on chemical communication strategies and fight dynamics of crayfish

Signal transmission is influenced by the physics of an environment. Consequently, a physical effect on sensory signals can influence how animals send or sample sensory information. Habitat-specific physics may constrain or enhance signal transmission (e.g. sound transmission in a flowing river versus a still pond) and provide a mechanism for the evolution of sensory biases. This study investigated how the transmission of chemically mediated social signals in crayfish is influenced by two different aquatic environments. Agonistic bouts between crayfish were performed under lotic (flowing water) and lentic (nonflowing, still water) conditions. When crayfish (Orconectes rusticus) collected from a lotic system (river) interacted under lotic conditions, we noted that dominant O. rusticus spent more time upstream than subordinate O. rusticus. Orconectes rusticus positioned themselves randomly and spent equal amounts of time with respect to upstream and downstream in the nonflowing environment. We tested another species, Orconectes virilis, collected from a nonflowing environment (lake) and they showed no positional preference when tested in flow. Additionally, both O. rusticus and O. virilis took longer to reach high fight intensities under flow conditions. It was possible to visualize O. rusticus urine release, and they released urine more often when upstream of an opponent in a flow environment during these agonistic bouts. These results suggest that O. rusticus collected from lotic environments release urine to maximize the transmission of chemical cues to a fight opponent. It appears that crayfish may adapt their signalling processes based upon their long-term ambient environments.