Robotic multiwell planar patch-clamp for native and primary mammalian cells

Robotic multiwell planar patch-clamp has become common in drug development and safety programs because it enables efficient and systematic testing of compounds against ion channels during voltage-clamp. It has not, however, been adopted significantly in other important areas of ion channel research, where conventional patch-clamp remains the favored method. Here, we show the wider potential of the multiwell approach with the ability for efficient intracellular solution exchange, describing protocols and success rates for recording from a range of native and primary mammalian cells derived from blood vessels, arthritic joints and the immune and central nervous systems. The protocol involves preparing a suspension of single cells to be dispensed robotically into 4-8 microfluidic chambers each containing a glass chip with a small aperture. Under automated control, giga-seals and whole-cell access are achieved followed by preprogrammed routines of voltage paradigms and fast extracellular or intracellular solution exchange. Recording from 48 chambers usually takes 1-6 h depending on the experimental design and yields 16-33 cell recordings.

Native speakers and task performance: comparing effects on complexity, fluency, and lexical diversity

This article argues that a native-speaker baseline is a neglected dimension of studies into second language (L2) performance. If we investigate how learners perform language tasks, we should distinguish what performance features are due to their processing an L2 and which are due to their performing a particular task. Having defined what we mean by “native speaker,” we present the background to a research study into task features on nonnative task performance, designed to include native-speaker data as a baseline for interpreting nonnative-speaker performance. The nonnative results, published in this journal (Tavakoli & Foster, 2008) are recapitulated and then the native-speaker results are presented and discussed in the light of them. The study is guided by the assumption that limited attentional resources impact on L2 performance and explores how narrative design features—namely complexity of storyline and tightness of narrative structure— affect complexity, fluency, accuracy, and lexical diversity in language. The results show that both native and nonnative speakers are prompted by storyline complexity to use more subordinated language, but narrative structure had different effects on native and nonnative fluency. The learners, who were based in either London or Tehran, did not differ in their performance when compared to each other, except in lexical diversity, where the learners in London were close to native-speaker levels. The implications of the results for the applicability of Levelt’s model of speaking to an L2 are discussed, as is the potential for further L2 research using native speakers as a baseline.

Pausing patterns: differences between L2 learners and native speakers

This paper reports on a comparative study of pauses made by L2 learners and native speakers of English while narrating picture stories. The comparison is based on the number of pauses and total amount of silence in themiddle and at the end of clauses in the performance of 40 native speakers and 40 L2 learners of English. The results of the quantitative analyses suggest that, although the L2 learners generally pausemore repeatedly and have longer periods of silence than the native speakers, the distinctive feature of their pausing pattern is that they pause frequently in the middle of clauses rather than at the end. The qualitative analysis of the data suggests that some of the L2 learners’mid-clause pauses are associated with processes such as replacement, reformulation, and online planning. Formulaic sequences, however, contain very few pauses and therefore appear to facilitate the learners’ fluency.

Wild pollinators enhance fruit set of crops regardless of honey bee abundance

Diversity and abundance of wild-insect pollinators have declined in many agricultural landscapes. Whether such declines reduce crop yields, or are mitigated by managed pollinators such as honey bees, is unclear. Here, we show universally positive associations of fruit set with wild-insect visits to flowers in 41 crop systems worldwide, and thus clearly demonstrate their agricultural value. In contrast, fruit set increased significantly with visitation by honey bees in only 14% of the systems surveyed. Overall, wild insects pollinated crops more effectively, because increase in their visitation enhanced fruit set by twice as much as an equivalent increase in honey bee visitation. Further, visitation by wild insects and honey bees promoted fruit set independently, so high abundance of managed honey bees supplemented, rather than substituted for, pollination by wild insects. Our results suggest that new practices for integrated management of both honey bees and diverse wild-insect assemblages will enhance global crop yields.

Effect of cultivar type and ripening on the polyphenol content of date palm fruit

Date palm (Pheonix dactylifera) fruit contains an array of polyphenols, although how these levels alter with cultivar type and fruit ripening is unclear. Utilizing HPLC and LC-ESI-MS/MS, this study define and quantify an array of hydroxybenzoic acids, hydroxycinnamic acids, and flavonoids in three common cultivars of dates (Ajwa, Barni, and Khalas) at the main ripening stages (kimri, khalal, rutab, and tamr). Polyphenols were at highest concentration at earlier stages of ripening, with concentrations reducing with ripening. The khalal stage of the Ajwa cultivar contained significantly higher (P < 0.001) levels of polyphenols than measured in the Barni and Khalas dates at the same degree of ripening. Furthermore, the Ajwa cultivar was the only one to contain significant quantities of anthocyanidins, in particular at the khalal stage. These data suggest dates are a significant source of polyphenols, especially if the earlier edible ripening stages are consumed or utilized as food ingredients.

Non-concordance between genetic profiles of olive oil and fruit: a cautionary note to the use of DNA markers for provenance testing.

To investigate the contribution of paternal alleles to the DNA content of olive oil, genetic analyses of olive DNA samples from fruits, leaves, and oil derived from the same tree (cv. Leccino) were carried out. DNA extracted from maternal tissues--leaves and flesh--from different fruits showed identical genetic profiles using a set of DNA markers. Additional simple sequence repeat (SSR) alleles, not found in the maternal samples, were amplified in the embryos (stone), and they were also detected in DNA extracted from the paste obtained by crushing whole fruits and from the oil pressed from this material. These results demonstrate that the DNA profile obtained from olive oil is likely to represent a composite profile of the maternal alleles juxtaposed with alleles contributed by various pollen donors. Therefore, care needs to be taken in the interpretation of DNA profiles obtained from DNA extracted from oil for resolving provenance and authenticity issues.

Influence of encapsulation and coating materials on the survival of Lactobacillus plantarum and Bifidobacterium longum in fruit juices

The aim of this work was to compare alginate and pectin beads for improving the survival of Lactobacillus plantarum and Bifidobacterium longum during storage in pomegranate and cranberry juice, and to evaluate the influence of various coating materials, including chitosan, gelatin and glucomannan on cell survival and on the size and hardness of the beads. In pomegranate juice, free cells of L. plantarum died within 4 weeks of storage and those of B. longum within 1 week; in cranberry juice both types of cells died within one week. Encapsulation within either alginate or pectin beads improved cell survival considerably, but coating of the beads with chitosan or gelatin improved it even further; coating with glucomannan did not have any positive effect. The double gelatin coated pectin beads gave the highest protection among all types of beads, as a final concentration of approximately 108 CFU/mL and 106 CFU/mL for both L. plantarum and B. longum was obtained after 6 weeks of storage in pomegranate and cranberry juice, respectively. The good protection could be attributed to the very strong interaction between the two polymers, as measured by turbidity experiments, leading to the formation of a polyelectrolyte complex. It was also shown that the coating was able to inhibit the penetration of gallic acid within the beads, which was used in this study as a model phenolic compound with antimicrobial activity; this is a likely mechanism through which the beads were able to protect the cells from the antimicrobial activity of phenolic compounds present in both types of juices. Despite their good protective effect, the pectin beads were considerably softer than the alginate beads, an issue that should be addressed in order to increase their mechanical stability.

(PDF, 406 KB) On L3 acquisition and phonological permeability: a new test case for debates on the mental representation of non-native phonological systems

The goal of this article is to make an epistemological and theoretical contribution to the nascent field of third language (L3) acquisition and show how examining L3 development can offer a unique view into longstanding debates within L2 acquisition theory. We offer the Phonological Permeability Hypothesis (PPH), which maintains that examining the development of an L3/Ln phonological system and its effects on a previously acquired L2 phonological system can inform contemporary debates regarding the mental constitution of postcritical period adult phonological acquisition. We discuss the predictions and functional significance of the PPH for adult SLA and multilingualism studies, detailing a methodology that examines the effects of acquiring Brazilian Portuguese on the Spanish phonological systems learned before and after the so-called critical period (i.e., comparing simultaneous versus successive adult English-Spanish bilinguals learning Brazilian Portuguese as an L3).

Knowledge of A/A'-dependencies on subject extraction with two types of infinitives in non-native Portuguese adult bilingualism

Within generative L2 acquisition research there is a longstanding debate as to what underlies observable differences in L1/L2 knowledge/ performance. On the one hand, Full Accessibility approaches maintain that target L2 syntactic representations (new functional categories and features) are acquirable (e.g., Schwartz & Sprouse, 1996). Conversely, Partial Accessibility approaches claim that L2 variability and/or optionality, even at advanced levels, obtains as a result of inevitable deficits in L2 narrow syntax and is conditioned upon a maturational failure in adulthood to acquire (some) new functional features (e.g., Beck, 1998; Hawkins & Chan, 1997; Hawkins & Hattori, 2006; Tsimpli & Dimitrakopoulou, 2007). The present study tests the predictions of these two sets of approaches with advanced English learners of L2 Brazilian Portuguese (n = 21) in the domain of inflected infinitives. These advanced L2 learners reliably differentiate syntactically between finite verbs, uninflected and inflected infinitives, which, as argued, only supports Full Accessibility approaches. Moreover, we will discuss how testing the domain of inflected infinitives is especially interesting in light of recent proposals that Brazilian Portuguese colloquial dialects no longer actively instantiate them (Lightfoot, 1991; Pires, 2002, 2006; Pires & Rothman, 2009; Rothman, 2007).

Proteolysis of milk heated at high temperatures by native enzymes analysed by trinitrobenzene sulphonic acid (TNBS) method

Native enzymes play a significant role in proteolysis of milk during storage. This is significant for heat resistant native enzymes. Plasmin is one of the most heat resistant enzymes found in milk. It has been reported to survive several heat treatments, causing spoilage during storage. The aim of this study was to assess susceptibility of high temperature heated milk to proteolysis by native enzymes. The trinitrobenzene sulphonic acid (TNBS) method was used for this purpose. Raw milk was heated at 110, 120, 130,142°C for 2 s and 85°C for 15 s and milk processed at low temperature (85°C /15s) was selected to mimic pasteurisation. TNBS method confirmed that raw milk and milk processed at 85°C /15s were the most proteolysed, whereas treatment of milk at high temperatures (110, 120, 130 and 142°C for 2 s) inactivated the native enzymes. It may thus be concluded that high temperature processing positively affects proteolysis by lowering its susceptibility to spoilage during storage.

A SEPALLATA gene is involved in the development and ripening of strawberry (Fragaria X ananassa Duch.) fruit, a non-climacteric tissue

Climacteric and non-climacteric fruits have traditionally been viewed as representing two distinct programmes of ripening associated with differential respiration and ethylene hormone effects. In climacteric fruits, such as tomato and banana, the ripening process is marked by increased respiration and is induced and co-ordinated by ethylene, while in non-climacteric fruits, such as strawberry and grape, it is controlled by an ethylene-independent process with little change in respiration rate. The two contrasting mechanisms, however, both lead to texture, colour, and flavour changes that probably reflect some common programmes of regulatory control. It has been shown that a SEPALLATA(SEP)4-like gene is necessary for normal ripening in tomato. It has been demonstrated here that silencing a fruit-related SEP1/2-like (FaMADS9) gene in strawberry leads to the inhibition of normal development and ripening in the petal, achene, and receptacle tissues. In addition, analysis of transcriptome profiles reveals pleiotropic effects of FaMADS9 on fruit development and ripening-related gene expression. It is concluded that SEP genes play a central role in the developmental regulation of ripening in both climacteric and non-climacteric fruits. These findings provide important information to extend the molecular control of ripening in a non-climacteric fruit beyond the limited genetic and cultural options currently available.

Grouping of 24 apple cultivars on the basis of starch degradation rate and their fruit pattern

The ripening processes of 24 apple cultivars were examined in the United Kingdom National Fruit Collection in 2010. Basically the starch content, and additionally ground colour, water-soluble solids content and flesh firmness were studied during ripening. The degradation of the starch content was evaluated using a 0–10 scale. A starch degradation value of 50% was taken to be the optimum harvest date, with harvest beginning at a value of 40% and finishing at 60%. Depending on the cultivar, this represented a harvest window of 9 to 21 days. Later ripening cultivars matured more slowly, leading to a longer harvesting period, with the exception of cv. Feuillemorte. Pronounced differences were observed among the cultivars on the basis of the starch degradation pattern, allowing them to be divided into four groups. Separate charts were elaborated for each group that are recommended for use in practice.

Avoiding a bad apple: insect pollination enhances fruit quality and economic value

Insect pollination is important for food production globally and apples are one of the major fruit crops which are reliant on this ecosystem service. It is fundamentally important that the full range of benefits of insect pollination to crop production are understood, if the costs of interventions aiming to enhance pollination are to be compared against the costs of the interventions themselves. Most previous studies have simply assessed the benefits of pollination to crop yield and ignored quality benefits and how these translate through to economic values. In the present study we examine the influence of insect pollination services on farmgate output of two important UK apple varieties; Gala and Cox. Using field experiments, we quantify the influence of insect pollination on yield and importantly quality and whether either may be limited by sub-optimal insect pollination. Using an expanded bioeconomic model we value insect pollination to UK apple production and establish the potential for improvement through pollination service management. We show that insects are essential in the production of both varieties of apple in the UK and contribute a total of £36.7 million per annum, over £6 million more than the value calculated using more conventional dependence ratio methods. Insect pollination not only affects the quantity of production but can also have marked impacts on the quality of apples, influencing size, shape and effecting their classification for market. These effects are variety specific however. Due to the influence of pollination on both yield and quality in Gala, there is potential for insect pollination services to improve UK output by up to £5.7 million per annum. Our research shows that continued pollinator decline could have serious financial implications for the apple industry but there is considerable scope through management of wild pollinators or using managed pollinator augmentation, to improve the quality of production. Furthermore, we show that it is critically important to consider all production parameters including quality, varietal differences and management costs when valuing the pollination service of any crop so investment in pollinator management can be proportional to its contribution.