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Global yield gaps can be reduced through breeding and improved agronomy. However, signs of yield plateaus from wheat and rice grown in intensively farmed systems indicate a need for new strategies if output is to continue to increase. Approaches to improve photosynthesis are suggested as a solution. Empirical evidence supporting this approach comes from small-scale free-CO 2 air enrichment and transgenic studies. However, the likely achievable gains from improving photosynthesis are less understood. Models predict maximum increases in yield of 5.3-19.1% from genetic manipulation depending on crop, environment, and approach, but uncertainty remains in the presence of stress. This review seeks to provide context to the rationale for improving photosynthesis, highlight areas of uncertainty, and identify the steps required to create more accurate projections., Competing Interests: Declaration of Competing Interest The authors declare no conflict of interest., (Copyright © 2024 The Authors. Published by Elsevier Ltd.. All rights reserved.)

The Clark-type electrode can be used to assess the rates of photosynthesis by detecting changes in O 2 concentration in a culture. This chapter describes a method for a liquid phase measurement of light and dissolved inorganic carbon-dependent photosynthesis using the model green alga Chlamydomonas reinhardtii. The technique can be used to evaluate the presence or efficiency of carbon-concentrating mechanisms., (© 2024. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

Faculty at research institutions play a central role in advancing knowledge and careers, as well as promoting the well-being of students and colleagues in research environments. Mentorship from experienced peers has been touted as critical for enabling these myriad roles to allow faculty development, career progression, and satisfaction. However, there is little information available on who supports faculty and best ways to structure a faculty mentorship programme for early- and mid-career academics. In the interest of advocating for increased and enhanced faculty mentoring and mentoring programmes, we surveyed faculty around the world to gather data on whether and how they receive mentoring. We received responses from 457 early- and mid-career faculty and found that a substantial portion of respondents either reported having no mentor or a lack of a formal mentoring scheme. Qualitative responses on the quality of mentorship revealed that the most common complaints regarding mentorship included lack of mentor availability, unsatisfactory commitment to mentorship, and non-specific or non-actionable advice. On these suggestions, we identify a need for training for faculty mentors as well as strategies for individual mentors, departments, and institutions for funding and design of more intentional and supportive mentorship programmes for early- and mid-career faculty.

Protoplasts, which are plant cells with their cell walls removed, have been used for decades in plant research and have been instrumental in genetic transformation and the study of various aspects of plant physiology and genetics. With the advent of synthetic biology, these individualized plant cells are fundamental to accelerate the 'design-build-test-learn' cycle, which is relatively slow in plant research. Despite their potential, challenges remain in expanding the use of protoplasts in synthetic biology. The capacity of individual protoplasts to hybridize to form new varieties, and to regenerate from single cells, creating individuals with new features is underexplored. The main objective of this review is to discuss the use of protoplasts in plant synthetic biology and to highlight the challenges to exploiting protoplast technologies in this new 'age of synthetic biology'., (© The Author(s) 2023. Published by Oxford University Press on behalf of the Society for Experimental Biology. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

Rubisco activase (RCA) catalyzes the release of inhibitory sugar phosphates from ribulose-1,6-biphosphate carboxylase/oxygenase (Rubisco) and can play an important role in biochemical limitations of photosynthesis under dynamic light and elevated temperatures. There is interest in increasing RCA activity to improve crop productivity, but a lack of understanding about the regulation of photosynthesis complicates engineering strategies. In this review, we discuss work relevant to improving RCA with a focus on advances in understanding the structural cause of RCA instability under heat stress and the regulatory interactions between RCA and components of photosynthesis. This reveals substantial variation in RCA thermostability that can be influenced by single amino acid substitutions, and that engineered variants can perform better in vitro and in vivo under heat stress. In addition, there are indications RCA activity is controlled by transcriptional, post-transcriptional, post-translational, and spatial regulation, which may be important for balancing between carbon fixation and light capture. Finally, we provide an overview of findings from recent field experiments and consider the requirements for commercial validation as part of efforts to increase crop yields in the face of global climate change., (© 2023 The Author(s). Published by Portland Press Limited on behalf of the Biochemical Society.)

We recently demonstrated that accelerating the relaxation of nonphotochemical quenching leads to higher soybean photosynthetic efficiency and yield. In response, Sinclair et al . assert that improved photosynthesis cannot improve crop yields and that there is only one valid experimental design for proving a genetic improvement in yield. We explain here why neither assertion is valid.

Crop leaves in full sunlight dissipate damaging excess absorbed light energy as heat. This protective dissipation continues after the leaf transitions to shade, reducing crop photosynthesis. A bioengineered acceleration of this adjustment increased photosynthetic efficiency and biomass in tobacco in the field. But could that also translate to increased yield in a food crop? Here we bioengineered the same change into soybean. In replicated field trials, photosynthetic efficiency in fluctuating light was higher and seed yield in five independent transformation events increased by up to 33%. Despite increased seed quantity, seed protein and oil content were unaltered. This validates increasing photosynthetic efficiency as a much needed strategy toward sustainably increasing crop yield in support of future global food security.

Photosynthesis is not optimized in modern crop varieties, and therefore provides an opportunity for improvement. Speeding up the relaxation of non-photochemical quenching (NPQ) has proven to be an effective strategy to increase photosynthetic performance. However, the potential to breed for improved NPQ and a complete understanding of the genetic basis of NPQ relaxation is lacking due to limitations of oversampling and data collection from field-grown crop plants. Building on previous reports, we present a high-throughput assay for analysis of NPQ relaxation rates in Glycine max (soybean) using pulse amplitude modulated (PAM) chlorophyll fluorometry. Leaf disks are sampled from field-grown soybeans before transportation to a laboratory where NPQ relaxation is measured in a closed PAM-fluorometer. NPQ relaxation parameters are calculated by fitting a bi-exponential function to the measured NPQ values following a transition from high to low light. Using this method, it is possible to test hundreds of genotypes within a day. The procedure has the potential to screen mutant and diversity panels for variation in NPQ relaxation, and can therefore be applied to both fundamental and applied research questions.

Photosynthesis is an important remaining opportunity for further improvement in the genetic yield potential of our major crops. Measurement, analysis, and improvement of leaf CO 2 assimilation ( A ) have focused largely on photosynthetic rates under light-saturated steady-state conditions. However, in modern crop canopies of several leaf layers, light is rarely constant, and the majority of leaves experience marked light fluctuations throughout the day. It takes several minutes for photosynthesis to regain efficiency in both sun-shade and shade-sun transitions, costing a calculated 10-40% of potential crop CO 2 assimilation. Transgenic manipulations to accelerate the adjustment in sun-shade transitions have already shown a substantial productivity increase in field trials. Here, we explore means to further accelerate these adjustments and minimize these losses through transgenic manipulation, gene editing, and exploitation of natural variation. Measurement andanalysis of photosynthesis in sun-shade and shade-sun transitions are explained. Factors limiting speeds of adjustment and how they could be modified to effect improved efficiency are reviewed, specifically nonphotochemical quenching (NPQ), Rubisco activation, and stomatal responses.

Scientists routinely use images to display data. Readers often examine figures first; therefore, it is important that figures are accessible to a broad audience. Many resources discuss fraudulent image manipulation and technical specifications for image acquisition; however, data on the legibility and interpretability of images are scarce. We systematically examined these factors in non-blot images published in the top 15 journals in 3 fields; plant sciences, cell biology, and physiology (n = 580 papers). Common problems included missing scale bars, misplaced or poorly marked insets, images or labels that were not accessible to colorblind readers, and insufficient explanations of colors, labels, annotations, or the species and tissue or object depicted in the image. Papers that met all good practice criteria examined for all image-based figures were uncommon (physiology 16%, cell biology 12%, plant sciences 2%). We present detailed descriptions and visual examples to help scientists avoid common pitfalls when publishing images. Our recommendations address image magnification, scale information, insets, annotation, and color and may encourage discussion about quality standards for bioimage publishing., Competing Interests: The authors have declared that no competing interests exist.

The Cub Sushi Multiple Domains-1 (CSMD1) protein is a tumour suppressor which has been shown to play a role in regulating human mammary duct development in vitro. CSMD1 knockdown in vitro demonstrated increased cell proliferation, invasion and motility. However, the role of Csmd1 in vivo is poorly characterised when it comes to ductal development and is therefore an area which warrants further exploration. In this study a Csmd1 knockout (KO) mouse model was used to identify the role of Csmd1 in regulating mammary gland development during puberty. Changes in duct development and protein expression patterns were analysed by immunohistochemistry. This study identified increased ductal development during the early stages of puberty in the KO mice, characterised by increased ductal area and terminal end bud number at 6 weeks. Furthermore, increased expression of various proteins (Stat1, Fak, Akt, Slug/Snail and Progesterone receptor) was shown at 4 weeks in the KO mice, followed by lower expression levels from 6 weeks in the KO mice compared to the wild type mice. This study identifies a novel role for Csmd1 in mammary gland development, with Csmd1 KO causing significantly more rapid mammary gland development, suggesting an earlier adult mammary gland formation.

Background: Preprint usage is growing rapidly in the life sciences; however, questions remain on the relative quality of preprints when compared to published articles. An objective dimension of quality that is readily measurable is completeness of reporting, as transparency can improve the reader's ability to independently interpret data and reproduce findings., Methods: In this observational study, we initially compared independent samples of articles published in bioRxiv and in PubMed-indexed journals in 2016 using a quality of reporting questionnaire. After that, we performed paired comparisons between preprints from bioRxiv to their own peer-reviewed versions in journals., Results: Peer-reviewed articles had, on average, higher quality of reporting than preprints, although the difference was small, with absolute differences of 5.0% [95% CI 1.4, 8.6] and 4.7% [95% CI 2.4, 7.0] of reported items in the independent samples and paired sample comparison, respectively. There were larger differences favoring peer-reviewed articles in subjective ratings of how clearly titles and abstracts presented the main findings and how easy it was to locate relevant reporting information. Changes in reporting from preprints to peer-reviewed versions did not correlate with the impact factor of the publication venue or with the time lag from bioRxiv to journal publication., Conclusions: Our results suggest that, on average, publication in a peer-reviewed journal is associated with improvement in quality of reporting. They also show that quality of reporting in preprints in the life sciences is within a similar range as that of peer-reviewed articles, albeit slightly lower on average, supporting the idea that preprints should be considered valid scientific contributions.

C 4 photosynthesis evolved repeatedly from the ancestral C 3 state, improving photosynthetic efficiency by ~50%. In most C 4 lineages, photosynthesis is compartmented between mesophyll and bundle sheath cells, but how gene expression is restricted to these cell types is poorly understood. Using the C 3 model Arabidopsis thaliana, we identified cis-elements and transcription factors driving expression in bundle sheath strands. Upstream of the bundle sheath preferentially expressed MYB76 gene, we identified a region necessary and sufficient for expression containing two cis-elements associated with the MYC and MYB families of transcription factors. MYB76 expression is reduced in mutant alleles for these transcription factors. Moreover, downregulated genes shared by both mutants are preferentially expressed in the bundle sheath. Our findings are broadly relevant for understanding the spatial patterning of gene expression, provide specific insights into mechanisms associated with the evolution of C 4 photosynthesis and identify a short tuneable sequence for manipulating gene expression in the bundle sheath.

Quinoline-based scaffolds have been the mainstay of antimalarial drugs, including many artemisinin combination therapies (ACTs), over the history of modern drug development. Although much progress has been made in the search for novel antimalarial scaffolds, it may be that quinolines will remain useful, especially if very potent compounds from this class are discovered. We report here the results of a structure-activity relationship (SAR) study assessing potential unsymmetrical bisquinoline antiplasmodial drug candidates using in vitro activity against intact parasites in cell culture. Many unsymmetrical bisquinolines were found to be highly potent against both chloroquine-sensitive and chloroquine-resistant Plasmodium falciparum parasites. Further work to develop such compounds could focus on minimizing toxicities in order to find suitable candidates for clinical evaluation.

Photosynthesis measurements are traditionally taken under steady-state conditions; however, leaves in crop fields experience frequent fluctuations in light and take time to respond. This slow response reduces the efficiency of carbon assimilation. Transitions from low to high light require photosynthetic induction, including the activation of Rubisco and the opening of stomata, whereas transitions from high to low light require the relaxation of dissipative energy processes, collectively known as non-photochemical quenching (NPQ). Previous attempts to assess the impact of these delays on net carbon assimilation have used simplified models of crop canopies, limiting the accuracy of predictions. Here, we use ray tracing to predict the spatial and temporal dynamics of lighting for a rendered mature Glycine max (soybean) canopy to review the relative importance of these delays on net cumulative assimilation over the course of both a sunny and a cloudy summer day. Combined limitations result in a 13% reduction in crop carbon assimilation on both sunny and cloudy days, with induction being more important on cloudy than on sunny days. Genetic variation in NPQ relaxation rates and photosynthetic induction in parental lines of a soybean nested association mapping (NAM) population was assessed. Short-term NPQ relaxation (<30 min) showed little variation across the NAM lines, but substantial variation was found in the speeds of photosynthetic induction, attributable to Rubisco activation. Over the course of a sunny and an intermittently cloudy day these would translate to substantial differences in total crop carbon assimilation. These findings suggest an unexplored potential for breeding improved photosynthetic potential in our major crops., (© 2020 The Authors. The Plant Journal published by Society for Experimental Biology and John Wiley & Sons Ltd.)

The majority of plants use C 3 photosynthesis, but over 60 independent lineages of angiosperms have evolved the C 4 pathway. In most C 4 species, photosynthesis gene expression is compartmented between mesophyll and bundle-sheath cells. We performed DNaseI sequencing to identify genome-wide profiles of transcription factor binding in leaves of the C 4 grasses Zea mays , Sorghum bicolor , and Setaria italica as well as C 3 Brachypodium distachyon In C 4 species, while bundle-sheath strands and whole leaves shared similarity in the broad regions of DNA accessible to transcription factors, the short sequences bound varied. Transcription factor binding was prevalent in gene bodies as well as promoters, and many of these sites could represent duons that influence gene regulation in addition to amino acid sequence. Although globally there was little correlation between any individual DNaseI footprint and cell-specific gene expression, within individual species transcription factor binding to the same motifs in multiple genes provided evidence for shared mechanisms governing C 4 photosynthesis gene expression. Furthermore, interspecific comparisons identified a small number of highly conserved transcription factor binding sites associated with leaves from species that diverged around 60 million years ago. These data therefore provide insight into the architecture associated with C 4 photosynthesis gene expression in particular and characteristics of transcription factor binding in cereal crops in general., (© 2019 The author(s). All rights reserved.)

Peer-reviewed journal publication is the main means for academic researchers in the life sciences to create a permanent public record of their work. These publications are also the de facto currency for career progress, with a strong link between journal brand recognition and perceived value. The current peer-review process can lead to long delays between submission and publication, with cycles of rejection, revision, and resubmission causing redundant peer review. This situation creates unique challenges for early career researchers (ECRs), who rely heavily on timely publication of their work to gain recognition for their efforts. Today, ECRs face a changing academic landscape, including the increased interdisciplinarity of life sciences research, expansion of the researcher population, and consequent shifts in employer and funding demands. The publication of preprints, publicly available scientific manuscripts posted on dedicated preprint servers prior to journal-managed peer review, can play a key role in addressing these ECR challenges. Preprinting benefits include rapid dissemination of academic work, open access, establishing priority or concurrence, receiving feedback, and facilitating collaborations. Although there is a growing appreciation for and adoption of preprints, a minority of all articles in life sciences and medicine are preprinted. The current low rate of preprint submissions in life sciences and ECR concerns regarding preprinting need to be addressed. We provide a perspective from an interdisciplinary group of ECRs on the value of preprints and advocate their wide adoption to advance knowledge and facilitate career development., Competing Interests: I have read the journal’s policy and the authors of this manuscript declare no financial competing interests. Non-financial competing interests: All authors are members of the eLife Ambassadors program to promote use of preprints. Benjamin Schwessinger is a member of eLife early career advisory group. Steven J. Burgess, Edward Emmott, Humberto J Debat and Zach Hensel are members of the ASAPBio Ambassador program to promote the use of preprints.

Droplet-based microfluidics has been used to facilitate high-throughput analysis of individual prokaryote and mammalian cells. However, there is a scarcity of similar workflows applicable to rapid phenotyping of plant systems where phenotyping analyses typically are time-consuming and low-throughput. We report on-chip encapsulation and analysis of protoplasts isolated from the emergent plant model Marchantia polymorpha at processing rates of >100,000 cells per hour. We use our microfluidic system to quantify the stochastic properties of a heat-inducible promoter across a population of transgenic protoplasts to demonstrate its potential for assessing gene expression activity in response to environmental conditions. We further demonstrate on-chip sorting of droplets containing YFP-expressing protoplasts from wild type cells using dielectrophoresis force. This work opens the door to droplet-based microfluidic analysis of plant cells for applications ranging from high-throughput characterisation of DNA parts to single-cell genomics to selection of rare plant phenotypes.

If the highly efficient C 4 photosynthesis pathway could be transferred to crops with the C 3 pathway there could be yield gains of up to 50%. It has been proposed that the multiple metabolic and developmental modifications associated with C 4 photosynthesis are underpinned by relatively few master regulators that have allowed the evolution of C 4 photosynthesis more than 60 times in flowering plants. Here we identify a component of one such regulator that consists of a pair of cis -elements located in coding sequence of multiple genes that are preferentially expressed in bundle sheath cells of C 4 leaves. These motifs represent duons as they play a dual role in coding for amino acids as well as controlling the spatial patterning of gene expression associated with the C 4 leaf. They act to repress transcription of C 4 photosynthesis genes in mesophyll cells. These duons are also present in the C 3 model Arabidopsis thaliana , and, in fact, are conserved in all land plants and even some algae that use C 3 photosynthesis. C 4 photosynthesis therefore appears to have coopted an ancient regulatory code to generate the spatial patterning of gene expression that is a hallmark of C 4 photosynthesis. This intragenic transcriptional regulatory sequence could be exploited in the engineering of efficient photosynthesis of crops., Competing Interests: The authors declare no conflict of interest.

Building on our earlier work of attaching a chemosensitizer (reversal agent) to a known drug pharmacophore, we have now expanded the structure-activity relationship study to include simplified versions of the chemosensitizer. The change from two aromatic rings in this head group to a single ring does not appear to detrimentally affect the antimalarial activity of the compounds. Data from in vitro heme binding and β -hematin inhibition assays suggest that the single aromatic RCQ compounds retain activities against Plasmodium falciparum similar to those of CQ, although other mechanisms of action may be relevant to their activities., (Copyright © 2017 Gunsaru et al.)

Most terrestrial plants use C 3 photosynthesis to fix carbon. In multiple plant lineages a modified system known as C 4 photosynthesis has evolved. To better understand the molecular patterns associated with induction of C 4 photosynthesis, the genus Flaveria that contains C 3 and C 4 species was used. A base to tip maturation gradient of leaf anatomy was defined, and RNA sequencing was undertaken along this gradient for two C 3 and two C 4 Flaveria species. Key C 4 traits including vein density, mesophyll and bundle sheath cross-sectional area, chloroplast ultrastructure, and abundance of transcripts encoding proteins of C 4 photosynthesis were quantified. Candidate genes underlying each of these C 4 characteristics were identified. Principal components analysis indicated that leaf maturation and the photosynthetic pathway were responsible for the greatest amount of variation in transcript abundance. Photosynthesis genes were over-represented for a prolonged period in the C 4 species. Through comparison with publicly available data sets, we identify a small number of transcriptional regulators that have been up-regulated in diverse C 4 species. The analysis identifies similar patterns of expression in independent C 4 lineages and so indicates that the complex C 4 pathway is associated with parallel as well as convergent evolution., (© The Author 2017. Published by Oxford University Press on behalf of the Society for Experimental Biology.)

C 4 photosynthesis acts as a carbon concentrating mechanism that leads to large increases in photosynthetic efficiency. The C 4 pathway is found in more than 60 plant lineages 1 but the molecular enablers of this evolution are poorly understood. In particular, it is unclear how non-photosynthetic proteins in the ancestral C 3 system have repeatedly become strongly expressed and integrated into photosynthesis gene regulatory networks in C 4 leaves. Here, we provide clear evidence that in C 3 leaves, genes encoding key enzymes of the C 4 pathway are already co-regulated with photosynthesis genes and are controlled by both light and chloroplast-to-nucleus signalling. In C 4 leaves this regulation becomes increasingly dependent on the chloroplast. We propose that regulation of C 4 cycle genes by light and the chloroplast in the ancestral C 3 state has facilitated the repeated evolution of the complex and convergent C 4 trait.

C4 photosynthesis is a complex phenotype that allows more efficient carbon capture than the ancestral C3 pathway. In leaves of C4 species, hundreds of transcripts increase in abundance compared with C3 relatives and become restricted to mesophyll (M) or bundle sheath (BS) cells. However, no mechanism has been reported that regulates the compartmentation of multiple enzymes in M or BS cells. We examined mechanisms regulating CARBONIC ANHYDRASE4 (CA4) in C4 Gynandropsis gynandra. Increased abundance is directed by both the promoter region and introns of the G. gynandra gene. A nine-nucleotide motif located in the 5' untranslated region (UTR) is required for preferential accumulation of GUS in M cells. This element is present and functional in three additional 5' UTRs and six 3' UTRs where it determines accumulation of two isoforms of CA and pyruvate,orthophosphate dikinase in M cells. Although the GgCA4 5' UTR is sufficient to direct GUS accumulation in M cells, transcripts encoding GUS are abundant in both M and BS. Mutating the GgCA4 5' UTR abolishes enrichment of protein in M cells without affecting transcript abundance. The work identifies a mechanism that directs cell-preferential accumulation of multiple enzymes required for C4 photosynthesis., (© 2016 American Society of Plant Biologists. All rights reserved.)

Under anoxic conditions the green alga Chlamydomonas reinhardtii activates various fermentation pathways leading to the creation of formate, acetate, ethanol and small amounts of other metabolites including d-lactate and hydrogen. Progress has been made in identifying the enzymes involved in these pathways and their subcellular locations; however, the identity of the enzyme involved in reducing pyruvate to d-lactate has remained unclear. Based on sequence comparisons, enzyme activity measurements, X-ray crystallography, biochemical fractionation and analysis of knock-down mutants, we conclude that pyruvate reduction in the chloroplast is catalyzed by a tetrameric NAD(+)-dependent d-lactate dehydrogenase encoded by Cre07.g324550. Its expression during aerobic growth supports a possible function as a 'lactate valve' for the export of lactate to the mitochondrion for oxidation by cytochrome-dependent d-lactate dehydrogenases and by glycolate dehydrogenase. We also present a revised spatial model of fermentation based on our immunochemical detection of the likely pyruvate decarboxylase, PDC3, in the cytoplasm., (© The Author 2015. Published by Oxford University Press on behalf of Japanese Society of Plant Physiologists.)

Chloroquine (CQ) has been the mainstay of malaria treatment for more than 60 years. However, the emergence and spread of CQ resistance now restrict its use to only a few areas where malaria is endemic. The aim of the present study was to investigate whether a novel combination of a CQ-like moiety and an imipramine-like pharmacophore can reverse CQ resistance ex vivo. Between March to October 2011 and January to September 2013, two "reversed chloroquine" (RCQ) compounds (PL69 and PL106) were tested against multidrug-resistant field isolates of Plasmodium falciparum (n = 41) and Plasmodium vivax (n = 45) in Papua, Indonesia, using a modified ex vivo schizont maturation assay. The RCQ compounds showed high efficacy against both CQ-resistant P. falciparum and P. vivax field isolates. For P. falciparum, the median 50% inhibitory concentrations (IC50s) were 23.2 nM for PL69 and 26.6 nM for PL106, compared to 79.4 nM for unmodified CQ (P < 0.001 and P = 0.036, respectively). The corresponding values for P. vivax were 19.0, 60.0, and 60.9 nM (P < 0.001 and P = 0.018, respectively). There was a significant correlation between IC50s of CQ and PL69 (Spearman's rank correlation coefficient [r s] = 0.727, P < 0.001) and PL106 (rs = 0.830, P < 0.001) in P. vivax but not in P. falciparum. Both RCQs were equally active against the ring and trophozoite stages of P. falciparum, but in P. vivax, PL69 and PL106 showed less potent activity against trophozoite stages (median IC50s, 130.2 and 172.5 nM) compared to ring stages (median IC50s, 17.6 and 91.3 nM). RCQ compounds have enhanced ex vivo activity against CQ-resistant clinical isolates of P. falciparum and P. vivax, suggesting the potential use of reversal agents in antimalarial drug development. Interspecies differences in RCQ compound activity may indicate differences in CQ pharmacokinetics between the two Plasmodium species., (Copyright © 2015, American Society for Microbiology. All Rights Reserved.)

C4 photosynthesis suppresses the oxygenation activity of Ribulose Bisphosphate Carboxylase Oxygenase and so limits photorespiration. Although highly complex, it is estimated to have evolved in 66 plant lineages, with the vast majority lacking sequenced genomes. Transcriptomics has recently initiated assessments of the degree to which transcript abundance differs between C3 and C4 leaves, identified novel components of C4 metabolism, and also led to mathematical models explaining the repeated evolution of this complex phenotype. Evidence is accumulating that this complex and convergent phenotype is partly underpinned by parallel evolution of structural genes, but also regulatory elements in both cis and trans. Furthermore, it appears that initial events associated with acquisition of C4 traits likely represent evolutionary exaptations related to non-photosynthetic processes., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

Large-scale research programmes seeking to characterize the C4 pathway have a requirement for a simple, high throughput screen that quantifies photorespiratory activity in C3 and C4 model systems. At present, approaches rely on model-fitting to assimilatory responses (A/C i curves, PSII quantum yield) or real-time carbon isotope discrimination, which are complicated and time-consuming. Here we present a method, and the associated theory, to determine the effectiveness of the C4 carboxylation, carbon concentration mechanism (CCM) by assessing the responsiveness of V O/V C, the ratio of RuBisCO oxygenase to carboxylase activity, upon transfer to low O2. This determination compares concurrent gas exchange and pulse-modulated chlorophyll fluorescence under ambient and low O2, using widely available equipment. Run time for the procedure can take as little as 6 minutes if plants are pre-adapted. The responsiveness of V O/V C is derived for typical C3 (tobacco, rice, wheat) and C4 (maize, Miscanthus, cleome) plants, and compared with full C3 and C4 model systems. We also undertake sensitivity analyses to determine the impact of R LIGHT (respiration in the light) and the effectiveness of the light saturating pulse used by fluorescence systems. The results show that the method can readily resolve variations in photorespiratory activity between C3 and C4 plants and could be used to rapidly screen large numbers of mutants or transformants in high throughput studies., (© The Author 2014. Published by Oxford University Press on behalf of the Society for Experimental Biology.)

C4 photosynthesis is a complex trait that has a high degree of natural variation, involving anatomical and biochemical changes relative to the ancestral C3 state. It has evolved at least 66 times across a variety of lineages and the evolutionary route from C3 to C4 is likely conserved but not necessarily genetically identical. As such, a variety of C4 species are needed to identify what is fundamental to the C4 evolutionary process in a global context. In order to identify the genetic components of C4 form and function, a number of species are used as genetic models. These include Zea mays (maize), Sorghum bicolor (sorghum), Setaria viridis (Setaria), Flaveria bidentis, and Cleome gynandra. Each of these species has different benefits and challenges associated with its use as a model organism. Here, we propose that RNA profiling of a large sampling of C4, C3-C4, and C3 species, from as many lineages as possible, will allow identification of candidate genes necessary and sufficient to confer C4 anatomy and/or biochemistry. Furthermore, C4 model species will play a critical role in the functional characterization of these candidate genes and identification of their regulatory elements, by providing a platform for transformation and through the use of gene expression profiles in mesophyll and bundle sheath cells and along the leaf developmental gradient. Efforts should be made to sequence the genomes of F. bidentis and C. gynandra and to develop congeneric C3 species as genetic models for comparative studies. In combination, such resources would facilitate discovery of common and unique C4 regulatory mechanisms across genera.

Artificial microRNA technology was investigated as a means of down regulating metabolic pathways in the green alga Chlamydomonas reinhardtii, targeting pyruvate formate lyase (PFL1), which catalyzes the conversion of pyruvate to acetyl-CoA and formate during anoxic conditions. Two transformants with an 80-90% reduction in target protein and mRNA levels were identified. Nuclear magnetic resonance spectroscopy confirmed a substantial decrease in the production of formate in the knockdown lines during dark anoxic conditions and a re-routing of metabolism leading to enhanced production of ethanol and lactate. Under microaerobic conditions in the light, induced by sulphur-deprivation, knock-down of PFL1 resulted in reduced formate and ethanol production, increased net consumption of acetate and the excretion of lactate but no increase in the production of hydrogen. In addition the production of 3-hydroxybutyrate was identified in knock-down line cultures during the transition between microaerobic and anoxic conditions. Overall our results indicate that microRNA knock-down is a useful tool to manipulate anaerobic metabolism in C. reinhardtii., (Copyright © 2012 Elsevier B.V. All rights reserved.)

Background: Investigations into 'inappropriate' use of emergency health services are limited by the lack of definition of what constitutes a health emergency. Position papers from Australian and international sources emphasise the patient's right to access emergency healthcare, and the responsibility of emergency health care workers to provide treatment to all patients. However, discordance between the two perspectives remain, with literature labelling patient use of emergency health services as 'inappropriate'., Objective: To define a 'health emergency' and compare patient and health professionals perspectives., Method: A sample of 600 emergency department (ED) patients were surveyed about a recent health experience and asked to rate their perceived urgency. This rating was compared to their triage score allocated at the hospital ED., Results: No significant relationship was found between the two ratings of urgency (P=0.51). CONCLUSIONS; Differing definitions of a 'health emergency' may explain patient help-seeking behaviour when accessing emergency health resources including hospital ED and ambulance services. A new definition of health emergency that encapsulates the health professional and patient perspectives is proposed. An agreed definition of when emergency health resources should be used has the potential to improve emergency health services demand and patient flow issues, and optimise emergency health resource allocation.

The twin problems of energy security and global warming make hydrogen an attractive alternative to traditional fossil fuels with its combustion resulting only in the release of water vapor. Biological hydrogen production represents a renewable source of the gas and can be performed by a diverse range of microorganisms from strict anaerobic bacteria to eukaryotic green algae. Compared to conventional methods for generating H(2), biological systems can operate at ambient temperatures and pressures without the need for rare metals and could potentially be coupled to a variety of biotechnological processes ranging from desalination and waste water treatment to pharmaceutical production. Photobiological hydrogen production by microalgae is particularly attractive as the main inputs for the process (water and solar energy) are plentiful. This chapter focuses on recent developments in solar-driven H(2) production in green algae with emphasis on the model organism Chlamydomonas reinhardtii. We review the current methods used to achieve sustained H(2) evolution and discuss possible approaches to improve H(2) yields, including the optimization of culturing conditions, reducing light-harvesting antennae and targeting auxiliary electron transport and fermentative pathways that compete with the hydrogenase for reductant. Finally, industrial scale-up is discussed in the context of photobioreactor design and the future prospects of the field are considered within the broader context of a biorefinery concept., (Copyright © 2011 Elsevier Inc. All rights reserved.)

We have previously shown that a "reversed chloroquine (RCQ)" molecule, composed of a chloroquine-like moiety and a resistance reversal-like moiety, can overcome chloroquine resistance in P. falciparum ( Burgess , S. J. ; Selzer , A. ; Kelly , J. X. ; Smilkstein , M. J. ; Riscoe , M. K. ; Peyton , D. H. J. Med. Chem. 2006 , 49 , 5623 . Andrews , S. ; Burgess , S. J. ; Skaalrud , D. ; Kelly , J. X. ; Peyton , D. H. J. Med. Chem. 2010 , 53 , 916 ). Here, we present an investigation into the structure-activity relationship of the RCQ structures, resulting in an orally active molecule with good in vitro and in vivo antimalarial activity. We also present evidence of the mode of action, indicating that the RCQ molecules inhibit hemozoin formation in the parasite's digestive vacuole in a manner similar to that of chloroquine.

We have shown that "reversed chloroquine molecules" constructed from chloroquine-like and resistance "reversal-agent"-like cores can be powerful drugs against malaria ( J. Med. Chem. 2006 , 49 , 5623 - 5625 ). Several reversed chloroquines are now presented that probe parameters governing the activities against chloroquine-resistant and chloroquine-sensitive malaria strains. The design is tolerant to linker and reversal agent changes, but a piperazinyl group adjacent to the quinoline, at least for the group of compounds studied here, may be detrimental.

Human NKG2D/DAP10 is an activation receptor expressed by NK and subsets of T cells, whose ligands include MHC class I chain-related (MIC) protein A and protein B and UL16-binding proteins that are often up-regulated by stress or pathological conditions. DAP10 is required for NKG2D/DAP10 cell surface expression and signaling capacity. Little is known about the mechanisms that regulate DAP10 gene expression. We describe the existence of multiple transcriptional start sites upstream of DAP10 exon 1 and identify the location of the basic promoter upstream of these starting sites. The promoter is active in NK and CD8+ T cells, but not in CD4+ T cells. We demonstrate TCR-mediated up-regulation of DAP10 transcription and found that a 40 bp region within the DAP10 promoter, containing an Ap-1 binding site, is largely responsible for this increased transcription. Using pull-down and chromatin immunoprecipitation assays, we show that the DAP10 promoter interacts with Ap-1 transcription factors in primary CD8+ T and NK cells in vitro and in vivo. Overexpression of c-Jun or c-Fos in NK and T cells led to enhanced DAP10 promoter activity and DAP10 protein expression. Taken together, our data indicate that Ap-1 is an important transcription factor for regulating DAP10 gene expression in human NK and T cells, and that Ap-1 plays a key role in the transactivation of DAP10 promoter following TCR stimulation.

NK cells are critical components of our immune system functioning, in part, to recognize and then eradicate virally infected or tumorigenic cells without previous sensitization. One of the best-characterized activating receptors expressed on NK cells is the NKG2D receptor, which is capable of transmitting co-stimulatory signals by subsets of T cells. Viruses and tumors have evolved strategies to evade NKG2D-mediated immune recognition thus highlighting the importance of this receptor in immunity. This review will focus on the structure of NKG2D and its interaction with its diverse array of ligands, as well as highlighting current knowledge regarding NKG2D signal transduction and biological mechanisms that govern its cell surface expression. The impact that NKG2D has in disease pathologies is also assessed.

A class of hybrid molecules which we term 'reversed chloroquines' (RCQs) was designed, and a prototype molecule, N'-(7-chloroquinolin-4-yl)-N-[3-(10,11-dihydrodibenzo[b,f]azepin-5-yl)propyl]-N-methylpropane-1,3-diamine (1), was synthesized and tested against both chloroquine-sensitive and chloroquine-resistant strains of Plasmodium falciparum. An in vitro assay against the two strains indicated that 1 was effective at low-nM concentrations against both strains. A preliminary study in mice demonstrated oral efficacy against P. chabaudi and the absence of obvious toxicity. The RCQ approach therefore appears to be feasible.

IL-21 is a recently described cytokine, produced by activated Th cells, that shares significant homology with members of the IL-2 family of cytokines. IL-21 mediates its biological effects via the IL-21R in conjunction with the common receptor gamma-chain that is also shared by members of the IL-2 family. We report that culture of human primary NK and CD8+ T cells with IL-21 in combination with IL-2 results in significant reduction of the cell surface expression of NKG2D, compared with that in cells treated with IL-2 alone. The reduced expression of NKG2D after IL-21 culture had functional consequences for NK cell function, as assessed by NKG2D-mediated redirected lysis assays and degranulation assays, compared with NK cells treated with IL-2 alone. IL-21-mediated NKG2D down-regulation in human NK cells correlated with a marked reduction of DNAX-activating protein of 10 kDa (DAP10) transcription in cells treated with IL-2 in combination with IL-21 compared with cells stimulated with only IL-2. This was attributed to a dramatic reduction in DAP10 promoter activity, as assessed by a DAP10 luciferase reporter construct. In contrast to NKG2D expression, IL-21 was able to induce the expression of the NK activation receptors NKp30 and 2B4 as well as the costimulatory receptor CD28 on CD8+ T cells. These data indicate that IL-21 is able to channel NK and CD8+ T cell function by altering the expression pattern of activation/costimulatory receptors.

The leukemic T cell line Jurkat is deficient in protein expression of the lipid phosphatases Src homology 2 domain containing inositol polyphosphate phosphatase (SHIP) and phosphatase and tensin homolog deleted on chromosome ten (PTEN). We examined whether the lack of expression of SHIP-1 and PTEN is shared by other leukemic T cell lines and PBLs. Analysis of a range of cell lines and PBLs revealed that unlike Jurkat cells, two other well-characterized T cell lines, namely CEM and MOLT-4 cells, expressed the 5'-phosphatase SHIP at the protein level. However, the 3-phosphatase PTEN was not expressed by CEM or MOLT-4 cells or Jurkat cells. The HUT78 cell line and PBLs expressed both SHIP and PTEN. Jurkat cells exhibited high basal levels of phosphatidylinositol 3,4,5-trisphosphate (PI(3,4,5)P(3); the lipid substrate for both SHIP and PTEN) as well as saturated protein kinase B (PKB) phosphorylation. Lower levels of PI(3,4,5)P(3) and higher levels of phosphatidylinositol 3,4-bisphosphate (PI(3,4)P(2)) as well as unsaturated constitutive phosphorylation of PKB were observed in CEM and MOLT-4 cells compared with Jurkat cells. In PBLs and HUT78 cells which express both PTEN and SHIP-1, there was no constitutive PI(3,4,5)P(3) or PKB phosphorylation, and receptor stimuli were able to elicit robust phosphorylation of PKB. Expression of a constitutively active SHIP-1 protein in Jurkat cells was sufficient to reduce both constitutive PKB membrane localization and PKB phosphorylation. Together, these data indicate important differences between T leukemic cells as well as PBLs, regarding expression of key lipid phosphatases. This study provides the first evidence that SHIP-1 can influence the constitutive levels of PI(3,4,5)P(3) and the activity of downstream phosphoinositide 3-kinase effectors in T lymphocytes.

The D-3 phosphoinositide lipids phosphatidylinositol 3,4, 5-trisphophate [PtdIns(3,4,5)P(3)] and phosphatidylinositol 3, 4-bisphosphate [PtdIns(3,4)P(2)] represent upstream components of a major signaling pathway that is strongly activated by the T cell costimulatory molecule CD28. A major route for degradation of PtdIns(3,4,5)P(3) (and hence, regulation of PtdIns(3,4,5)P(3)-driven effector pathways), involves its conversion to PtdIns(3,4)P(2) by the 145-kDa SH2-containing inositol (poly)phosphate 5-phosphatase (SHIP). In this study, we demonstrate using the murine T cell hybridoma DC27.1, that SHIP is strongly tyrosine phosphorylated after ligation of CD28 by either mAb or the natural ligand B7.1. Ligation of CD3 also stimulates SHIP tyrosine phosphorylation and an additive effect on tyrosine phosphorylation of SHIP is observed when both CD3 and CD28 are ligated. The tyrosine phosphorylation of SHIP in response to CD28 ligation correlates with a marked redistribution of SHIP from the cytosol to the plasma membrane, as well as an increase in the in vitro 5-phosphatase activity associated with SHIP immunoprecipitates derived from CD28-stimulated cells. However, we have been unable to detect a direct association between CD28 and SHIP, so the mechanisms by which CD28 exerts the observed effects on SHIP remain unclear. This is the first demonstration that SHIP is a biochemical target for CD28 and suggests that SHIP may be involved in the regulation of T cell activation.