A high-throughput transient expression system for rice.

Rice is an important global crop and represents a vital source of calories for many food insecure regions. Efforts to improve this crop by improving yield, nutritional content, stress tolerance, or resilience to climate change are certain to include biotechnological approaches, which rely on the expression of transgenes in planta. The throughput and cost of currently available transgenic expression systems is frequently incompatible with modern, high-throughput molecular cloning methods. Here, we present a protocol for isolating high yields of green rice protoplasts and for PEG-mediated transformation of isolated protoplasts. Factors affecting transformation efficiency were investigated, and the resulting protocol is fast, cheap, robust, high-throughput, and does not require specialist equipment. When coupled to a high-throughput modular cloning system such as Golden Gate, this transient expression system provides a valuable resource to help break the "design-build-test" bottleneck by permitting the rapid screening of large numbers of transgenic expression cassettes prior to stable plant transformation. We used this system to rapidly assess the expression level, subcellular localisation, and protein aggregation pattern of nine single-gene expression cassettes, which represent the essential component parts of the ß-cyanobacterial carboxysome.

The iron-sulfur cluster biosynthesis protein SUFB is required for chlorophyll synthesis, but not phytochrome signaling.

Proteins that contain iron-sulfur (Fe-S) clusters play pivotal roles in various metabolic processes such as photosynthesis and redox metabolism. Among the proteins involved in the biosynthesis of Fe-S clusters in plants, the SUFB subunit of the SUFBCD complex appears to be unique because SUFB has been reported to be involved in chlorophyll metabolism and phytochrome-mediated signaling. To gain insights into the function of the SUFB protein, we analyzed the phenotypes of two SUFB mutants, laf6 and hmc1, and RNA interference (RNAi) lines with reduced SUFB expression. When grown in the light, the laf6 and hmc1 mutants and the SUFB RNAi lines accumulated higher levels of the chlorophyll biosynthesis intermediate Mg-protoporphyrin IX monomethylester (Mg-proto MME), consistent with the impairment of Mg-proto MME cyclase activity. Both SUFC- and SUFD-deficient RNAi lines accumulated the same intermediate, suggesting that inhibition of Fe-S cluster synthesis is the primary cause of this impairment. Dark-grown laf6 seedlings also showed an increase in protoporphyrin IX (Proto IX), Mg-proto, Mg-proto MME and 3,8-divinyl protochlorophyllide a (DV-Pchlide) levels, but this was not observed in hmc1 or the SUFB RNAi lines, nor was it complemented by SUFB overexpression. In addition, the long hypocotyl in far-red light phenotype of the laf6 mutant could not be rescued by SUFB overexpression and segregated from the pale-green SUFB-deficient phenotype, indicating it is not caused by mutation at the SUFB locus. These results demonstrate that biosynthesis of Fe-S clusters is important for chlorophyll biosynthesis, but that the laf6 phenotype is not due to a SUFB mutation.

Children retain implicitly learned phonological sequences better than adults: a longitudinal study.

Whereas adults often rely on explicit memory, children appear to excel in implicit memory, which plays an important role in the acquisition of various cognitive skills, such as those involved in language. The current study aimed to test the assertion of an age-dependent shift in implicit versus explicit learning within a theoretical framework that explains the link between implicit sequence memory and word-form acquisition, using the Hebb repetition paradigm. We conducted a one-year, multiple-session longitudinal study in which we presented auditory sequences of syllables, co-presented with pictures of aliens, for immediate serial recall by a group of children (8-9 years) and by an adult group. The repetition of one Hebb sequence was explicitly announced, while the repetition of another Hebb sequence was unannounced and, therefore, implicit. Despite their overall inferior recall performance, the children showed better offline retention of the implicit Hebb sequence, compared with adults who showed a significant decrement across the delays. Adults had gained more explicit knowledge of the implicit sequence than children, but this could not explain the age-dependent decline in the delayed memory for it. There was no significant age-effect for delayed memory of the explicit Hebb sequence, with both age groups showing retention. Overall performance by adults was positively correlated with measures of post-learning awareness. Performance by children was positively correlated with vocabulary knowledge. We conclude that children outperform adults in the retention over time of implicitly learned phonological sequences that will gradually consolidate into novel word-forms. The findings are discussed in the light of maturational differences for implicit versus explicit memory systems that also play a role in language acquisition. A video abstract of this article can be viewed at: https://youtu.be/G5nOfJB72t4.

Singlet oxygen initiates a plastid signal controlling photosynthetic gene expression.

Retrograde signals from the plastid regulate photosynthesis-associated nuclear genes and are essential to successful chloroplast biogenesis. One model is that a positive haem-related signal promotes photosynthetic gene expression in a pathway that is abolished by the herbicide norflurazon. Far-red light (FR) pretreatment and transfer to white light also results in plastid damage and loss of photosynthetic gene expression. Here, we investigated whether norflurazon and FR pretreatment affect the same retrograde signal. We used transcriptome analysis and real-time reverse transcription-polymerase chain reaction (RT-PCR) to analyse the effects of these treatments on nuclear gene expression in various Arabidopsis (Arabidopsis thaliana) retrograde signalling mutants. Results showed that the two treatments inhibited largely different nuclear gene sets, suggesting that they affected different retrograde signals. Moreover, FR pretreatment resulted in singlet oxygen (1 O2 ) production and a rapid inhibition of photosynthetic gene expression. This inhibition was partially blocked in the executer1executer2 mutant, which is impaired in 1 O2 signalling. Our data support a new model in which a 1 O2 retrograde signal, generated by chlorophyll precursors, inhibits expression of key photosynthetic and chlorophyll synthesis genes to prevent photo-oxidative damage during de-etiolation. Such a signal would provide a counterbalance to the positive haem-related signal to fine tune regulation of chloroplast biogenesis.

The involvement of long-term serial-order memory in reading development: A longitudinal study.

Recent findings suggest that Hebb repetition learning-a paradigmatic example of long-term serial-order learning-is impaired in adults with dyslexia. The current study further investigated the link between serial-order learning and reading using a longitudinal developmental design. With this aim, verbal and visual Hebb repetition learning performance and reading skills were assessed in 96 Dutch-speaking children who we followed from first through second grade of primary school. We observed a positive association between order learning capacities and reading ability as well as weaker Hebb learning performance in early readers with poor reading skills even at the onset of reading instruction. Hebb learning further predicted individual differences in later (nonword) reading skills. Finally, Hebb learning was shown to explain a significant part of the variance in reading performance above and beyond phonological awareness. These findings highlight the role of serial-order memory in reading ability.

Increased susceptibility to proactive interference in adults with dyslexia?

Recent findings show that people with dyslexia have an impairment in serial-order memory. Based on these findings, the present study aimed to test the hypothesis that people with dyslexia have difficulties dealing with proactive interference (PI) in recognition memory. A group of 25 adults with dyslexia and a group of matched controls were subjected to a 2-back recognition task, which required participants to indicate whether an item (mis)matched the item that had been presented 2 trials before. PI was elicited using lure trials in which the item matched the item in the 3-back position instead of the targeted 2-back position. Our results demonstrate that the introduction of lure trials affected 2-back recognition performance more severely in the dyslexic group than in the control group, suggesting greater difficulty in resisting PI in dyslexia.

The 'Necksafe' head articulation control system: a novel cervical immobilisation device.

INTRODUCTION: The early application of a semirigid disposable cervical collar following trauma is considered a routine practice. The aim of these devices is to immobilise the cervical spine and minimise the risk of additional neurological damage. However, these collars provide only partial immobilisation, are uncomfortable and are associated with a number of complications. Our team designed and tested a novel cervical immobilisation device that aims to improve immobilisation with reduced complications: the 'Necksafe'. METHODS: Human volunteers were recruited and consented to test the novel Necksafe device in comparison with a conventional collar (the AMBU Perfit ACE) in a range of evaluations. These included assessments of the cervical range of movement (CROM) that occurred during scripted movements of the head and neck, and the effect of the new and conventional devices on jugular vein dimensions, assessed using ultrasound scanning. RESULTS: CROM analysis showed that, under standardised testing conditions, the Necksafe device offers cervical immobilisation that is at least equivalent to a conventional collar, and is superior in the planes of extension, lateral flexion and rotation. Ultrasound examination of the jugular veins was inconclusive and did not reveal any differences in jugular venous diameter or flow. Qualitative feedback from ambulance paramedics was highly supportive of the new design, suggesting that it is more comfortable, easier to fit, less confining and better tolerated than a conventional collar, with improved immobilisation effectiveness. CONCLUSIONS: The results of quantitative and qualitative testing are highly supportive of the new Necksafe design, with improved cervical immobilisation, comfort and access to the airway.

EXPRESS: Investigating a Metrical Hebb Effect for lists of words.

In four experiments, we describe the first finding of a Metrical Hebb Effect. Participants are shown to exhibit a Hebb Repetition Effect for repeating, list-wide stress patterns across sequences of familiar words, even though the lexical items within the "repeating" lists do not themselves repeat. Experiment 1 established the presence of a Hebb effect for metrical patterns, demonstrating significant learning of list-wide metrical patterns over successive presentations. Experiment 2 investigated the effect's longevity, showing persistence of learned metrical information after a spacing of three non-repeating lists. Experiment 3 revealed that the effect did not persist over a longer spacing, of eight intervening lists. Experiment 4 investigated the learning mechanism, suggesting that chunking, rather than item-position binding, might account for the observed learning of metrical patterns. The authors propose that metrical-pattern learning represents a process of gradual integration of sequences of weak and strong stress accents, into higher-level units representing the stress patterns within, and across, words. We briefly discuss some implications of the Metrical Hebb Effect for phonological word-form learning, and for speech perception and production.

The influence of ascorbate on anthocyanin accumulation during high light acclimation in Arabidopsis thaliana: further evidence for redox control of anthocyanin synthesis.

Ascorbate and anthocyanins act as photoprotectants during exposure to high light (HL). They accumulate in Arabidopsis leaves in response to HL on a similar timescale, suggesting a potential relationship between them. Flavonoids and related metabolites were identified and profiled by liquid chromatography-tandem mass spectrometry (LC-MS/MS). The ascorbate-deficient mutants vtc1, vtc2 and vtc3 accumulated less anthocyanin than wild-type (WT) during HL acclimation. In contrast, kaempferol glycoside accumulation was less affected by light and not decreased by ascorbate deficiency, while sinapoyl malate levels decreased during HL acclimation. Comparison of six Arabidopsis ecotypes showed a positive correlation between ascorbate and anthocyanin accumulation in HL. mRNA-Seq analysis showed that all flavonoid biosynthesis transcripts were increased by HL acclimation in WT. RT-PCR analysis showed that vtc1 and vtc2 were impaired in HL induction of transcripts of anthocyanin biosynthesis enzymes, and the transcription factors PAP1, GL3 and EGL3 that activate the pathway. Abscisic acid (ABA) and jasmonic acid (JA), hormones that could affect anthocyanin accumulation, were unaffected in vtc mutants. It is concluded that HL induction of anthocyanin synthesis involves a redox-sensitive process upstream of the known transcription factors. Because anthocyanins accumulate in preference to kaempferol glycosides and sinapoyl malate in HL, they might have specific properties that make them useful in HL acclimation.

Predicting Need for Hospital Beds to Reduce Emergency Department Boarding.

Introduction Accurately determining the supply and demand of hospital beds for new admissions can help prevent adverse patient outcomes. Quantitative analysis of modern electronic medical record data can help predict supply and demand for unoccupied staffed hospital beds (SEDs) and aid in eliminating human approximations, standardizing daily work through concrete and objective data. The purpose of this study was to reduce variability and human error in predicting the number of SEDs needed. Methods In this study,the authors analyzed bed calculator data from a medium-sized, suburban medical center to evaluate the efficacy of a unique bed calculator prediction tool to determine the need for SEDs. The calculator aggregates multiple key reference factors available through the bed calculator system into a cohesive linear regression model. Results Compared with human estimation, the authors found that the bed calculator is able to predict the number of SEDs needed more effectively. That being said, there was no significant difference in the average boarding times pre- and postintervention, indicating that the bed calculator did not result in decreased boarding times for patients. Discussion These findings establish the efficacy of the bed calculator and its ability to align bed supply and demand. Because patient boarding times depend on the system's patient flow management, future studies should focus on how to improve various streams of communication and coordination.

"Nested" cryptic diversity in a widespread marine ecosystem engineer: a challenge for detecting biological invasions.

BACKGROUND: Ecosystem engineers facilitate habitat formation and enhance biodiversity, but when they become invasive, they present a critical threat to native communities because they can drastically alter the receiving habitat. Management of such species thus needs to be a priority, but the poorly resolved taxonomy of many ecosystem engineers represents a major obstacle to correctly identifying them as being either native or introduced. We address this dilemma by studying the sea squirt Pyura stolonifera, an important ecosystem engineer that dominates coastal communities particularly in the southern hemisphere. Using DNA sequence data from four independently evolving loci, we aimed to determine levels of cryptic diversity, the invasive or native status of each regional population, and the most appropriate sampling design for identifying the geographic ranges of each evolutionary unit. RESULTS: Extensive sampling in Africa, Australasia and South America revealed the existence of "nested" levels of cryptic diversity, in which at least five distinct species can be further subdivided into smaller-scale genetic lineages. The ranges of several evolutionary units are limited by well-documented biogeographic disjunctions. Evidence for both cryptic native diversity and the existence of invasive populations allows us to considerably refine our view of the native versus introduced status of the evolutionary units within Pyura stolonifera in the different coastal communities they dominate. CONCLUSIONS: This study illustrates the degree of taxonomic complexity that can exist within widespread species for which there is little taxonomic expertise, and it highlights the challenges involved in distinguishing between indigenous and introduced populations. The fact that multiple genetic lineages can be native to a single geographic region indicates that it is imperative to obtain samples from as many different habitat types and biotic zones as possible when attempting to identify the source region of a putative invader. "Nested" cryptic diversity, and the difficulties in correctly identifying invasive species that arise from it, represent a major challenge for managing biodiversity.

Expression analysis of the VTC2 and VTC5 genes encoding GDP-L-galactose phosphorylase, an enzyme involved in ascorbate biosynthesis, in Arabidopsis thaliana.

Arabidopsis thaliana contains two GDP-L-galactose phosphorylase genes, VTC2 and VTC5, which are critical for ascorbate (AsA) biosynthesis. We investigated the expression levels of both VTC2 and VTC5 genes in wild-type A. thaliana and the AsA deficient mutants during early seedling growth. Ascorbate accumulated to an equal extent in all genotypes up to 5 d post-germination (DPG). The transcript level of VTC2 was dominant, and increased in parallel with AsA accumulation in the wild type. On the other hand, the expression of VTC5 compensated for the reduced VTC2 transcription levels in the AsA deficient mutant vtc2-1 in young seedlings. A luciferase activity assay indicated that the VTC5 promoter was more active in young (2 DPG) cotyledons and that the VTC2 and VTC5 promoters drove a day-to-night variation in expression. The present work provides clues to the precise roles of VTC2 and VTC5 in AsA biosynthesis in A. thaliana at the young seedling stage.

Overexpression of chloroplast-targeted ferrochelatase 1 results in a genomes uncoupled chloroplast-to-nucleus retrograde signalling phenotype.

Chloroplast development requires communication between the progenitor plastids and the nucleus, where most of the genes encoding chloroplast proteins reside. Retrograde signals from the chloroplast to the nucleus control the expression of many of these genes, but the signalling pathway is poorly understood. Tetrapyrroles have been strongly implicated as mediators of this signal with the current hypothesis being that haem produced by the activity of ferrochelatase 1 (FC1) is required to promote nuclear gene expression. We have tested this hypothesis by overexpressing FC1 and specifically targeting it to either chloroplasts or mitochondria, two possible locations for this enzyme. Our results show that targeting of FC1 to chloroplasts results in increased expression of the nuclear-encoded chloroplast genes GUN4, CA1, HEMA1, LHCB2.1, CHLH after treatment with Norflurazon (NF) and that this increase correlates to FC1 gene expression and haem production measured by feedback inhibition of protochlorophyllide synthesis. Targeting FC1 to mitochondria did not enhance the expression of nuclear-encoded chloroplast genes after NF treatment. The overexpression of FC1 also increased nuclear gene expression in the absence of NF treatment, demonstrating that this pathway is operational in the absence of a stress treatment. Our results therefore support the hypothesis that haem synthesis is a promotive chloroplast-to-nucleus retrograde signal. However, not all FC1 overexpression lines enhanced nuclear gene expression, suggesting there is still a lot we do not understand about the role of FC1 in this signalling pathway. This article is part of the theme issue 'Retrograde signalling from endosymbiotic organelles'.

Literacy improves short-term serial recall of spoken verbal but not visuospatial items - Evidence from illiterate and literate adults.

It is widely accepted that specific memory processes, such as serial-order memory, are involved in written language development and predictive of reading and spelling abilities. The reverse question, namely whether orthographic abilities also affect serial-order memory, has hardly been investigated. In the current study, we compared 20 illiterate people with a group of 20 literate matched controls on a verbal and a visuospatial version of the Hebb paradigm, measuring both short- and long-term serial-order memory abilities. We observed better short-term serial-recall performance for the literate compared with the illiterate people. This effect was stronger in the verbal than in the visuospatial modality, suggesting that the improved capacity of the literate group is a consequence of learning orthographic skills. The long-term consolidation of ordered information was comparable across groups, for both stimulus modalities. The implications of these findings for current views regarding the bi-directional interactions between memory and written language development are discussed.

What is the locus of the errorless-learning advantage?

In two experiments involving word-stem completion, an advantage was found for errorless over errorful-learning conditions, for both severely and moderately memory-impaired participants. This advantage did not depend on the implicit/explicit nature of the question asked. Additional tests showed that subsequent recognition of target items was good for both groups, but only in the absence of lures derived from participants' prior errors. Source-memory was shown to be virtually absent in the severely impaired group and only weakly present in the moderately impaired group. This combination of results suggests that preserved implicit memory, in the absence of explicit memory, is sufficient for an errorless-learning advantage to accrue.

What can't functional neuroimaging tell the cognitive psychologist?

In this paper, I critically review the usefulness of functional neuroimaging to the cognitive psychologist. All serious cognitive theories acknowledge that cognition is implemented somewhere in the brain. Finding that the brain "activates" differentially while performing different tasks is therefore gratifying but not surprising. The key problem is that the additional dependent variable that imaging data represents, is often one about which cognitive theories make no necessary predictions. It is, therefore, inappropriate to use such data to choose between such theories. Even supposing that fMRI were able to tell us where a particular cognitive process was performed, that would likely tell us little of relevance about how it was performed. The how-question is the crucial question for theorists investigating the functional architecture of the human mind. The argument is illustrated with particular reference to Henson (2005) and Shallice (2003), who make the opposing case.

Repetition learning in the immediate serial recall of visual and auditory materials.

In 5 experiments, a Hebb repetition effect, that is, improved immediate serial recall of an (unannounced) repeating list, was demonstrated in the immediate serial recall of visual materials, even when use of phonological short-term memory was blocked by concurrent articulation. The learning of a repeatedly presented letter list in one modality (auditory or visual) did not transfer to give improved performance on the same list in the other modality. This result was not replicated for word lists, however, for which asymmetric transfer was observed. Inferences are made about the structure of short-term memory and about the nature of the Hebb repetition effect.

Linking memory and language: Evidence for a serial-order learning impairment in dyslexia.

The present study investigated long-term serial-order learning impairments, operationalized as reduced Hebb repetition learning (HRL), in people with dyslexia. In a first multi-session experiment, we investigated both the persistence of a serial-order learning impairment as well as the long-term retention of serial-order representations, both in a group of Dutch-speaking adults with developmental dyslexia and in a matched control group. In a second experiment, we relied on the assumption that HRL mimics naturalistic word-form acquisition and we investigated the lexicalization of novel word-forms acquired through HRL. First, our results demonstrate that adults with dyslexia are fundamentally impaired in the long-term acquisition of serial-order information. Second, dyslexic and control participants show comparable retention of the long-term serial-order representations in memory over a period of 1 month. Third, the data suggest weaker lexicalization of newly acquired word-forms in the dyslexic group. We discuss the integration of these findings into current theoretical views of dyslexia.

Climate change: time to Do Something Different.

There is now very little, if any, doubt that the global climate is changing and that this is in some way related to human behavior through unsustainable preferences in lifestyle and organizational practices. Despite the near conclusive evidence of the positive relationship between greenhouse gas emissions and global warming, a small proportion of people remain unconvinced. More importantly, even among the much larger number of people who accept a link between human behavior and climate change, many are inactive, or insufficiently active, in attempting to remedy the situation. We suggest this is partly because people are unaware both of how their day-to-day behaviors connect with energy consumption and carbon emissions, and of the behavioral alternatives that are available to them. This, we believe, is a key reason why individual lifestyles and organizational practices continue in an unsustainable way. We also suggest that the psychologists and behavioral researchers who seek to develop a better understanding of people's relationship with, and reaction to, environmental issues, might also be on track to suffer a similar blindness. They risk becoming fixed on investigating a limited range of established variables, perhaps to the detriment of alternative approaches that are more practically oriented though, so far, less well explored empirically. In this article, we present the Framework for Internal Transformation as an alternative perspective on the variables that might underpin pro-environmental activity and behavior change. After briefly reviewing the related literature, we outline that framework. Then we present some early empirical data to show its relationship to a range of pro-environmental indices. We follow with a discussion of the framework's relevance in relation to pro-environmental behavior change and make proposals for future research.