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The modern nitrogen cycle consists of a web of microbially mediated redox transformations. Among the most crucial reactions in this cycle is the oxidation of ammonia to nitrite, an obligately aerobic process performed by a limited number of lineages of bacteria (AOB) and archaea (AOA). As this process has an absolute requirement for O 2 , the timing of its evolution-especially as it relates to the Great Oxygenation Event ~ 2.3 billion years ago-remains contested and is pivotal to our understanding of nutrient cycles. To estimate the antiquity of bacterial ammonia oxidation, we performed phylogenetic and molecular clock analyses of AOB. Surprisingly, bacterial ammonia oxidation appears quite young, with crown group clades having originated during Neoproterozoic time (or later) with major radiations occurring during Paleozoic time. These results place the evolution of AOB broadly coincident with the pervasive oxygenation of the deep ocean. The late evolution AOB challenges earlier interpretations of the ancient nitrogen isotope record, predicts a more substantial role for AOA during Precambrian time, and may have implications for understanding of the size and structure of the biogeochemical nitrogen cycle through geologic time.

Human life intimately depends on plants for food, biomaterials, health, energy, and a sustainable environment. Various plants have been genetically improved mostly through breeding, along with limited modification via genetic engineering, yet they are still not able to meet the ever-increasing needs, in terms of both quantity and quality, resulting from the rapid increase in world population and expected standards of living. A step change that may address these challenges would be to expand the potential of plants using biosystems design approaches. This represents a shift in plant science research from relatively simple trial-and-error approaches to innovative strategies based on predictive models of biological systems. Plant biosystems design seeks to accelerate plant genetic improvement using genome editing and genetic circuit engineering or create novel plant systems through de novo synthesis of plant genomes. From this perspective, we present a comprehensive roadmap of plant biosystems design covering theories, principles, and technical methods, along with potential applications in basic and applied plant biology research. We highlight current challenges, future opportunities, and research priorities, along with a framework for international collaboration, towards rapid advancement of this emerging interdisciplinary area of research. Finally, we discuss the importance of social responsibility in utilizing plant biosystems design and suggest strategies for improving public perception, trust, and acceptance., Competing Interests: The authors declare that they have no conflicts of interest regarding the publication of this article., (Copyright © 2020 Xiaohan Yang et al.)

With its ability to catabolize a wide variety of carbon sources and a growing engineering toolkit, Pseudomonas putida KT2440 is emerging as an important chassis organism for metabolic engineering. Despite advances in our understanding of the organism, many gaps remain in our knowledge of the genetic basis of its metabolic capabilities. The gaps are particularly noticeable in our understanding of both fatty acid and alcohol catabolism, where many paralogs putatively coding for similar enzymes coexist, making biochemical assignment via sequence homology difficult. To rapidly assign function to the enzymes responsible for these metabolisms, we leveraged random barcode transposon sequencing (RB-Tn-Seq). Global fitness analyses of transposon libraries grown on 13 fatty acids and 10 alcohols produced strong phenotypes for hundreds of genes. Fitness data from mutant pools grown on fatty acids of varying chain lengths indicated specific enzyme substrate preferences and enabled us to hypothesize that DUF1302/DUF1329 family proteins potentially function as esterases. From the data, we also postulate catabolic routes for the two biogasoline molecules isoprenol and isopentanol, which are catabolized via leucine metabolism after initial oxidation and activation with coenzyme A (CoA). Because fatty acids and alcohols may serve as both feedstocks and final products of metabolic-engineering efforts, the fitness data presented here will help guide future genomic modifications toward higher titers, rates, and yields. IMPORTANCE To engineer novel metabolic pathways into P. putida , a comprehensive understanding of the genetic basis of its versatile metabolism is essential. Here, we provide functional evidence for the putative roles of hundreds of genes involved in the fatty acid and alcohol metabolism of the bacterium. These data provide a framework facilitating precise genetic changes to prevent product degradation and to channel the flux of specific pathway intermediates as desired., (Copyright © 2020 American Society for Microbiology.)

Global demand for food and bioenergy production has increased rapidly, while the area of arable land has been declining for decades due to damage caused by erosion, pollution, sea level rise, urban development, soil salinization, and water scarcity driven by global climate change. In order to overcome this conflict, there is an urgent need to adapt conventional agriculture to water-limited and hotter conditions with plant crop systems that display higher water-use efficiency (WUE). Crassulacean acid metabolism (CAM) species have substantially higher WUE than species performing C 3 or C 4 photosynthesis. CAM plants are derived from C 3 photosynthesis ancestors. However, it is extremely unlikely that the C 3 or C 4 crop plants would evolve rapidly into CAM photosynthesis without human intervention. Currently, there is growing interest in improving WUE through transferring CAM into C 3 crops. However, engineering a major metabolic plant pathway, like CAM, is challenging and requires a comprehensive deep understanding of the enzymatic reactions and regulatory networks in both C 3 and CAM photosynthesis, as well as overcoming physiometabolic limitations such as diurnal stomatal regulation. Recent advances in CAM evolutionary genomics research, genome editing, and synthetic biology have increased the likelihood of successful acceleration of C 3 -to-CAM progression. Here, we first summarize the systems biology-level understanding of the molecular processes in the CAM pathway. Then, we review the principles of CAM engineering in an evolutionary context. Lastly, we discuss the technical approaches to accelerate the C 3 -to-CAM transition in plants using synthetic biology toolboxes., Competing Interests: The authors declare that there is no conflict of interest regarding the publication of this article., (Copyright © 2020 Guoliang Yuan et al.)

Rubisco sustains the biosphere through the fixation of CO 2 into biomass. In plants and cyanobacteria, form I Rubisco is structurally comprised of large and small subunits, whereas all other Rubisco forms lack small subunits. The rise of the form I complex through the innovation of small subunits represents a key, yet poorly understood, transition in Rubisco's evolution. Through metagenomic analyses, we discovered a previously uncharacterized clade sister to form I Rubisco that evolved without small subunits. This clade diverged before the evolution of cyanobacteria and the origin of the small subunit; thus, it provides a unique reference point to advance our understanding of form I Rubisco evolution. Structural and kinetic data presented here reveal how a proto-form I Rubisco assembled and functioned without the structural stability imparted from small subunits. Our findings provide insight into a key evolutionary transition of the most abundant enzyme on Earth and the predominant entry point for nearly all global organic carbon.

Plants produce a wealth of biologically active compounds, many of which are used to defend themselves from various pests and pathogens. We explore the possibility of expanding upon the natural chemical diversity of plants and create molecules that have enhanced properties, by engineering metabolic pathways new to nature. We rationally broaden the set of primary metabolites that can be utilized by the core biosynthetic pathway of the natural biopesticide, brassinin, producing in planta a novel class of compounds that we call crucifalexins. Two of our new-to-nature crucifalexins are more potent antifungals than brassinin and, in some instances, comparable to commercially used fungicides. Our findings highlight the potential to push the boundaries of plant metabolism for the biosynthesis of new biopesticides., Competing Interests: The authors declare the following competing financial interest(s): J.D.K. has a financial interest in Amyris, Lygos, Demetrix, Constructive Biology, Maple Bio, and Napigen. The research described in this publication is not related to the work of these companies., (Copyright © 2020 American Chemical Society.)

Background: Despite the latest advancements in metabolic engineering for genome editing and characterization of host performance, the successful development of robust cell factories used for industrial bioprocesses and accurate prediction of the behavior of microbial systems, especially when shifting from laboratory-scale to industrial conditions, remains challenging. To increase the probability of success of a scale-up process, data obtained from thoroughly performed studies mirroring cellular responses to typical large-scale stimuli may be used to derive crucial information to better understand potential implications of large-scale cultivation on strain performance. This study assesses the feasibility to employ a barcoded yeast deletion library to assess genome-wide strain fitness across a simulated industrial fermentation regime and aims to understand the genetic basis of changes in strain physiology during industrial fermentation, and the corresponding roles these genes play in strain performance., Results: We find that mutant population diversity is maintained through multiple seed trains, enabling large scale fermentation selective pressures to act upon the community. We identify specific deletion mutants that were enriched in all processes tested in this study, independent of the cultivation conditions, which include MCK1, RIM11, MRK1, and YGK3 that all encode homologues of mammalian glycogen synthase kinase 3 (GSK-3). Ecological analysis of beta diversity between all samples revealed significant population divergence over time and showed feed specific consequences of population structure. Further, we show that significant changes in the population diversity during fed-batch cultivations reflect the presence of significant stresses. Our observations indicate that, for this yeast deletion collection, the selection of the feeding scheme which affects the accumulation of the fermentative by-product ethanol impacts the diversity of the mutant pool to a higher degree as compared to the pH of the culture broth. The mutants that were lost during the time of most extreme population selection suggest that specific biological processes may be required to cope with these specific stresses., Conclusions: Our results demonstrate the feasibility of Bar-seq to assess fermentation associated stresses in yeast populations under industrial conditions and to understand critical stages of a scale-up process where variability emerges, and selection pressure gets imposed. Overall our work highlights a promising avenue to identify genetic loci and biological stress responses required for fitness under industrial conditions.

Agricultural biotechnology strategies often require the precise regulation of multiple genes to effectively modify complex plant traits. However, most efforts are hindered by a lack of characterized tools that allow for reliable and targeted expression of transgenes. We have successfully engineered a library of synthetic transcriptional regulators that modulate expression strength in planta. By leveraging orthogonal regulatory systems from Saccharomyces spp., we have developed a strategy for the design of synthetic activators, synthetic repressors, and synthetic promoters and have validated their use in Nicotiana benthamiana and Arabidopsis thaliana. This characterization of contributing genetic elements that dictate gene expression represents a foundation for the rational design of refined synthetic regulators. Our findings demonstrate that these tools provide variation in transcriptional output while enabling the concerted expression of multiple genes in a tissue-specific and environmentally responsive manner, providing a basis for generating complex genetic circuits that process endogenous and environmental stimuli.

Agrobacterium fabrum ARqua1 is a hybrid of Agrobacterium fabrum C58C bearing the megaplasmid pRiA4b. ARqua1 is used by many plant researchers to generate transgenic roots. The draft genome of ARqua1 includes a 249,350-bp contig that likely covers all of pRiA4b, and it will be a valuable resource to plant biologists., (Copyright © 2020 Thompson et al.)

Despite intensive study, plant lysine catabolism beyond the 2-oxoadipate (2OA) intermediate remains unvalidated. Recently we described a missing step in the D-lysine catabolism of Pseudomonas putida in which 2OA is converted to D-2-hydroxyglutarate (2HG) via hydroxyglutarate synthase (HglS), a DUF1338 family protein. Here we solve the structure of HglS to 1.1 Å resolution in substrate-free form and in complex with 2OA. We propose a successive decarboxylation and intramolecular hydroxylation mechanism forming 2HG in a Fe(II)- and O 2 -dependent manner. Specificity is mediated by a single arginine, highly conserved across most DUF1338 proteins. An Arabidopsis thaliana HglS homolog coexpresses with known lysine catabolism enzymes, and mutants show phenotypes consistent with disrupted lysine catabolism. Structural and biochemical analysis of Oryza sativa homolog FLO7 reveals identical activity to HglS despite low sequence identity. Our results suggest DUF1338-containing enzymes catalyze the same biochemical reaction, exerting the same physiological function across bacteria and eukaryotes.

Agrobacterium tumefaciens is an important tool in plant biotechnology due to its natural ability to transfer DNA into the genomes of host plants. Genetic manipulations of A. tumefaciens have yielded considerable advances in increasing transformational efficiency in a number of plant species and cultivars. Moreover, there is overwhelming evidence that modulating the expression of various mediators of A. tumefaciens virulence can lead to more successful plant transformation; thus, the application of synthetic biology to enable targeted engineering of the bacterium may enable new opportunities for advancing plant biotechnology. In this review, we highlight engineering targets in both A. tumefaciens and plant hosts that could be exploited more effectively through precision genetic control to generate high-quality transformation events in a wider range of host plants. We then further discuss the current state of A. tumefaciens and plant engineering with regard to plant transformation and describe how future work may incorporate a rigorous synthetic biology approach to tailor strains of A. tumefaciens used in plant transformation., Competing Interests: The authors declare that there is no conflict of interest regarding the publication of this article., (Copyright © 2020 Mitchell G. Thompson et al.)

Flamholz and Shih explain how photosynthetic organisms on earth have evolved carbon dioxide concentrating mechanisms to contend with an increased abundance of oxygen in the atmosphere. This critical innovation has occurred numerous times and comes in two basic flavors., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Coproduction of high-value bioproducts at biorefineries is a key factor in making biofuels more cost-competitive. One strategy for generating coproducts is to directly engineer bioenergy crops to accumulate bioproducts in planta that can be fractionated and recovered at biorefineries. Here, we develop quantitative insights into the relationship between bioproduct market value and target accumulation rates by investigating a set of industrially relevant compounds already extracted from plant sources with a wide range of market prices and applications, including <$10/kg (limonene, latex, and polyhydroxybutyrate [PHB]), $10 to $100/kg (cannabidiol), and >$100/kg (artemisinin). These compounds are used to identify a range of mass fraction thresholds required to achieve net economic benefits for biorefineries and the additional amounts needed to reach a target $2.50/gal biofuel selling price, using cellulosic ethanol production as a test case. Bioproduct market prices and recovery costs determine the accumulation threshold; we find that moderate- to high-value compounds (i.e., cannabidiol and artemisinin) offer net economic benefits at accumulation rates of just 0.01% dry weight (dwt) to 0.02 dwt%. Lower-value compounds, including limonene, latex, and PHB, require at least an order-of-magnitude greater accumulation to overcome additional extraction and recovery costs (0.3 to 1.2 dwt%). We also find that a diversified approach is critical. For example, global artemisinin demand could be met with fewer than 10 biorefineries, while global demand for latex is equivalent to nearly 180 facilities. Our results provide a roadmap for future plant metabolic engineering efforts aimed at increasing the value derived from bioenergy crops., Competing Interests: The authors declare no competing interest., (Copyright © 2020 the Author(s). Published by PNAS.)

Ideotypes are theoretical archetypes of crops which serve as a practical framework for plant breeders to critically evaluate what traits they should be targeting for specific applications. With advances in plant biotechnology and a growing urgency to adopt more sustainable practices across our economy, new uses for crops as bioenergy feedstocks may pivot our definition of an ideal crop that is engineered for biomass and bioenergy production, in contrast to food production. Although there is a plethora of specific applications to which plant engineering efforts can contribute, here we highlight recent advances in two broad areas of research: increasing available plant biomass and engineering production of higher value co-products., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

A significant bottleneck in synthetic biology involves screening large genetically encoded libraries for desirable phenotypes such as chemical production. However, transcription factor-based biosensors can be leveraged to screen thousands of genetic designs for optimal chemical production in engineered microbes. In this study we characterize two glutarate sensing transcription factors (CsiR and GcdR) from Pseudomonas putida . The genomic contexts of csiR homologues were analyzed, and their DNA binding sites were bioinformatically predicted. Both CsiR and GcdR were purified and shown to bind upstream of their coding sequencing in vitro . CsiR was shown to dissociate from DNA in vitro when exogenous glutarate was added, confirming that it acts as a genetic repressor. Both transcription factors and cognate promoters were then cloned into broad host range vectors to create two glutarate biosensors. Their respective sensing performance features were characterized, and more sensitive derivatives of the GcdR biosensor were created by manipulating the expression of the transcription factor. Sensor vectors were then reintroduced into P. putida and evaluated for their ability to respond to glutarate and various lysine metabolites. Additionally, we developed a novel mathematical approach to describe the usable range of detection for genetically encoded biosensors, which may be broadly useful in future efforts to better characterize biosensor performance.

The fixation of inorganic carbon species like CO 2 to more reduced organic forms is one of the most fundamental processes of life as we know it. Although several carbon fixation pathways are known to exist, on Earth today nearly all global carbon fixation is driven by the Calvin cycle in oxygenic photosynthetic plants, algae, and Cyanobacteria. At other times in Earth history, other organisms utilizing different carbon fixation pathways may have played relatively larger roles, with this balance shifting over geological time as the environmental context of life has changed and evolutionary innovations accumulated. Among the most dramatic changes that our planet and the biosphere have undergone are those surrounding the rise of O 2 in our atmosphere-first during the Great Oxygenation Event at ∼2.3 Ga, and perhaps again during Neoproterozoic or Paleozoic time. These oxygenation events likely represent major step changes in the tempo and mode of biological productivity as a result of the increased productivity of oxygenic photosynthesis and the introduction of O 2 into geochemical and biological systems, and likely involved shifts in the relative contribution of different carbon fixation pathways. Here, we review what is known from both the rock record and comparative biology about the evolution of carbon fixation pathways, their contributions to primary productivity through time, and their relationship to the evolving oxygenation state of the fluid Earth following the evolution and expansion of oxygenic photosynthesis., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Metabolism drives life; thus, understanding how and when various branches of metabolism evolved provides a critical piece to understanding how life has integrated itself into the geochemical cycles of our planet over billions of years. Although the most transformative metabolisms that have significantly altered the trajectory of Earth are inherently linked to primary metabolism, natural products that stem from specialized metabolic pathways are also key components to many auxiliary facets of life. Cyanobacteria are primarily known as the original inventors of oxygenic photosynthesis, using sunlight to split water to create our dioxygen-filled atmosphere; however, many of them also have evolved to produce small molecules that function as sunscreens to protect themselves from ultraviolet radiation. Determining when cyanobacteria first evolved the ability to biosynthesize such compounds is an important piece to understanding the rise of oxygen and the eventual success of the phylum., (Copyright © 2019 Shih.)

The original version of this Article contained errors in Fig. 4. In panel a, the labels 'F420-reducing NiFe hydrogenase (group 3a)' and 'Group 2 NiFe hydrogenase' were misplaced. These errors have been corrected in both the PDF and HTML versions of the Article.

The evolution of aerobic respiration was likely linked to the origins of oxygenic Cyanobacteria. Close phylogenetic neighbors to Cyanobacteria, such as Margulisbacteria (RBX-1 and ZB3), Saganbacteria (WOR-1), Melainabacteria and Sericytochromatia, may constrain the metabolic platform in which aerobic respiration arose. Here, we analyze genomic sequences and predict that sediment-associated Margulisbacteria have a fermentation-based metabolism featuring a variety of hydrogenases, a streamlined nitrogenase, and electron bifurcating complexes involved in cycling of reducing equivalents. The genomes of ocean-associated Margulisbacteria encode an electron transport chain that may support aerobic growth. Some Saganbacteria genomes encode various hydrogenases, and others may be able to use O 2 under certain conditions via a putative novel type of heme copper O 2 reductase. Similarly, Melainabacteria have diverse energy metabolisms and are capable of fermentation and aerobic or anaerobic respiration. The ancestor of all these groups may have been an anaerobe in which fermentation and H 2 metabolism were central metabolic features. The ability to use O 2 as a terminal electron acceptor must have been subsequently acquired by these lineages.

Summary: Genome-resolved metagenomics allows the construction of draft microbial genomes from short-read shotgun metagenomics (Metagenome-Assembled Genomes, or MAGs); however, even high-quality MAGs are typically somewhat incomplete and contain a small amount of contaminant sequence, making accurate prediction of metabolic potential challenging. Here, we describe MetaPOAP, an algorithm for probabalistic assessment of the statistical likelihoods for the presence or absence of metabolic pathways in MAGs., Availability and Implementation: MetaPOAP is available as Python scripts on GitHub or from the Fischer lab webpage, https://github.com/lmward/MetaPOAP., Supplementary Information: Supplementary data are available at Bioinformatics online.

Numerous studies have estimated plant and animal diversification dynamics; however, no comparable rigorous estimates exist for bacteria-the most ancient and widespread form of life on Earth. Here, we analyse phylogenies comprising up to 448,112 bacterial lineages to reconstruct global bacterial diversification dynamics. To handle such large phylogenies, we developed methods based on the statistical properties of infinitely large trees. We further analysed sequencing data from 60 environmental studies to determine the fraction of extant bacterial diversity missing from the phylogenies-a crucial parameter for estimating speciation and extinction rates. We estimate that there are about 1.4-1.9 million extant bacterial lineages when lineages are defined by 99% similarity in the 16S ribosomal RNA gene, and that bacterial diversity has been continuously increasing over the past 1 billion years (Gyr). Recent bacterial extinction rates are estimated at 0.03-0.05 per lineage per million years (lineage -1  Myr -1 ), and are only slightly below estimated recent bacterial speciation rates. Most bacterial lineages ever to have inhabited this planet are estimated to be extinct. Our findings disprove the notion that bacteria are unlikely to go extinct, and provide a valuable perspective on the evolutionary history of a domain of life with a sparse and cryptic fossil record.

Humans have domesticated many plant species as indispensable sources of food, materials, and medicines. The dawning era of synthetic biology represents a means to further refine, redesign, and engineer crops to meet various societal and industrial needs. Current and future endeavors will utilize plants as the foundation of a bio-based economy through the photosynthetic production of carbohydrate feedstocks for the microbial fermentation of biofuels and bioproducts, with the end goal of decreasing our dependence on petrochemicals. As our technological capabilities improve, metabolic engineering efforts may expand the utility of plants beyond sugar feedstocks through the direct production of target compounds, including pharmaceuticals, renewable fuels, and commodity chemicals. However, relatively little work has been done to fully realize the potential in redirecting central carbon metabolism in plants for the engineering of novel bioproducts. Although our ability to rationally engineer and manipulate plant metabolism is in its infancy, I highlight some of the opportunities and challenges in applying synthetic biology towards engineering plant primary metabolism., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Background: We previously developed several strategies to engineer plants to produce cost-efficient biofuels from plant biomass. Engineered Arabidopsis plants with low xylan and lignin content showed normal growth and improved saccharification efficiency under standard growth conditions. However, it remains to be determined whether these engineered plants perform well under drought stress, which is the primary source of abiotic stress in the field., Results: Upon exposing engineered Arabidopsis plants to severe drought, we observed better survival rates in those with a low degree of xylan acetylation, low lignin, and low xylan content compared to those in wild-type plants. Increased pectic galactan content had no effect on drought tolerance. The drought-tolerant plants exhibited low water loss from leaves, and drought-responsive genes ( RD29A , RD29B , DREB2A ) were generally up-regulated under drought stress, which did not occur in the well-watered state. When compared with the wild type, plants with low lignin due to expression of QsuB, a 3-dehydroshikimate dehydratase, showed a stronger response to abscisic acid (ABA) in assays for seed germination and stomatal closure. The low-lignin plants also accumulated more ABA in response to drought than the wild-type plants. On the contrary, the drought tolerance in the engineered plants with low xylan content and low xylan acetylation was not associated with differences in ABA content or response compared to the wild type. Surprisingly, we found a significant increase in galactose levels and sugar released from the low xylan-engineered plants under drought stress., Conclusions: This study shows that plants engineered to accumulate less lignin or xylan are more tolerant to drought and activate drought responses faster than control plants. This is an important finding because it demonstrates that modification of secondary cell walls does not necessarily render the plants less robust in the environment, and it shows that substantial changes in biomass composition can be achieved without compromising plant resilience.

Competing Interests: Competing interests: None declared.

The evolutionary mechanisms behind the extant distribution of photosynthesis is a point of substantial contention. Hypotheses range from the presence of phototrophy in the last universal common ancestor and massive gene loss in most lineages, to a later origin in Cyanobacteria followed by extensive horizontal gene transfer into the extant phototrophic clades, with intermediate scenarios that incorporate aspects of both end-members. Here, we report draft genomes of 11 Chloroflexi: the phototrophic Chloroflexia isolate Kouleothrix aurantiaca as well as 10 genome bins recovered from metagenomic sequencing of microbial mats found in Japanese hot springs. Two of these metagenome bins encode photrophic reaction centers and several of these bins form a metabolically diverse, monophyletic clade sister to the Anaerolineae class that we term Candidatus Thermofonsia. Comparisons of organismal (based on conserved ribosomal) and phototrophy (reaction center and bacteriochlorophyll synthesis) protein phylogenies throughout the Chloroflexi demonstrate that two new lineages acquired phototrophy independently via horizontal gene transfer (HGT) from different ancestral donors within the classically phototrophic Chloroflexia class. These results illustrate a complex history of phototrophy within this group, with metabolic innovation tied to HGT. These observations do not support simple hypotheses for the evolution of photosynthesis that require massive character loss from many clades; rather, HGT appears to be the defining mechanic for the distribution of phototrophy in many of the extant clades in which it appears.

Background: Second-generation biofuels produced from biomass can help to decrease dependency on fossil fuels, bringing about many economic and environmental benefits. To make biomass more suitable for biorefinery use, we need a better understanding of plant cell wall biosynthesis. Increasing the ratio of C6 to C5 sugars in the cell wall and decreasing the lignin content are two important targets in engineering of plants that are more suitable for downstream processing for second-generation biofuel production., Results: We have studied the basic mechanisms of cell wall biosynthesis and identified genes involved in biosynthesis of pectic galactan, including the GALS1 galactan synthase and the UDP-galactose/UDP-rhamnose transporter URGT1. We have engineered plants with a more suitable biomass composition by applying these findings, in conjunction with synthetic biology and gene stacking tools. Plants were engineered to have up to fourfold more pectic galactan in stems by overexpressing GALS1, URGT1, and UGE2, a UDP-glucose epimerase. Furthermore, the increased galactan trait was engineered into plants that were already engineered to have low xylan content by restricting xylan biosynthesis to vessels where this polysaccharide is essential. Finally, the high galactan and low xylan traits were stacked with the low lignin trait obtained by expressing the QsuB gene encoding dehydroshikimate dehydratase in lignifying cells., Conclusion: The results show that approaches to increasing C6 sugar content, decreasing xylan, and reducing lignin content can be combined in an additive manner. Thus, the engineered lines obtained by this trait-stacking approach have substantially improved properties from the perspective of biofuel production, and they do not show any obvious negative growth effects. The approach used in this study can be readily transferred to bioenergy crop plants.

Various lines of evidence from both comparative biology and the geologic record make it clear that the biochemical machinery for anoxygenic photosynthesis was present on early Earth and provided the evolutionary stock from which oxygenic photosynthesis evolved ca. 2.3 billion years ago. However, the taxonomic identity of these early anoxygenic phototrophs is uncertain, including whether or not they remain extant. Several phototrophic bacterial clades are thought to have evolved before oxygenic photosynthesis emerged, including the Chloroflexi, a phylum common across a wide range of modern environments. Although Chloroflexi have traditionally been thought to be an ancient phototrophic lineage, genomics has revealed a much greater metabolic diversity than previously appreciated. Here, using a combination of comparative genomics and molecular clock analyses, we show that phototrophic members of the Chloroflexi phylum are not particularly ancient, having evolved well after the rise of oxygen ( ca. 867 million years ago), and thus cannot be progenitors of oxygenic photosynthesis. Similarly, results show that the carbon fixation pathway that defines this clade-the 3-hydroxypropionate bicycle-evolved late in Earth history as a result of a series of horizontal gene transfer events, explaining the lack of geological evidence for this pathway based on the carbon isotope record. These results demonstrate the role of horizontal gene transfer in the recent metabolic innovations expressed within this phylum, including its importance in the development of a novel carbon fixation pathway., Competing Interests: The authors declare no conflict of interest.

The rise of oxygen ca. 2.3 billion years ago (Ga) is the most distinct environmental transition in Earth history. This event was enabled by the evolution of oxygenic photosynthesis in the ancestors of Cyanobacteria. However, long-standing questions concern the evolutionary timing of this metabolism, with conflicting answers spanning more than one billion years. Recently, knowledge of the Cyanobacteria phylum has expanded with the discovery of non-photosynthetic members, including a closely related sister group termed Melainabacteria, with the known oxygenic phototrophs restricted to a clade recently designated Oxyphotobacteria. By integrating genomic data from the Melainabacteria, cross-calibrated Bayesian relaxed molecular clock analyses show that crown group Oxyphotobacteria evolved ca. 2.0 billion years ago (Ga), well after the rise of atmospheric dioxygen. We further estimate the divergence between Oxyphotobacteria and Melainabacteria ca. 2.5-2.6 Ga, which-if oxygenic photosynthesis is an evolutionary synapomorphy of the Oxyphotobacteria-marks an upper limit for the origin of oxygenic photosynthesis. Together, these results are consistent with the hypothesis that oxygenic photosynthesis evolved relatively close in time to the rise of oxygen., (© 2016 John Wiley & Sons Ltd.)

The advent and growth of synthetic biology has demonstrated its potential as a promising avenue of research to address many societal needs. However, plant synthetic biology efforts have been hampered by a dearth of DNA part libraries, versatile transformation vectors and efficient assembly strategies. Here, we describe a versatile system (named jStack) utilizing yeast homologous recombination to efficiently assemble DNA into plant transformation vectors. We demonstrate how this method can facilitate pathway engineering of molecules of pharmaceutical interest, production of potential biofuels and shuffling of disease-resistance traits between crop species. Our approach provides a powerful alternative to conventional strategies for stacking genes and traits to address many impending environmental and agricultural challenges., Competing Interests: D.L. has financial interests in Afingen. The remaining authors declare no competing financial interests.

The Green Revolution has fuelled an exponential growth in human population since the mid-20th century. Due to population growth, food and energy demands will soon surpass supply capabilities. To overcome these impending problems, significant improvements in genetic engineering will be needed to complement breeding efforts in order to accelerate the improvement of agronomical traits. The new field of plant synthetic biology has emerged in recent years and is expected to support rapid, precise, and robust engineering of plants. In this review, we present recent advances made in the field of plant synthetic biology, specifically in genome editing, transgene expression regulation, and bioenergy crop engineering, with a focus on traits related to lignocellulose, oil, and soluble sugars. Ultimately, progress and innovation in these fields may facilitate the development of beneficial traits in crop plants to meet society's bioenergy needs., (© 2016 The Authors. The Plant Journal published by Society for Experimental Biology and John Wiley & Sons Ltd.)

The antiquity and global abundance of the enzyme, RuBisCO, attests to the crucial and longstanding role it has played in the biogeochemical cycles of Earth over billions of years. The counterproductive oxygenase activity of RuBisCO has persisted over billions of years of evolution, despite its competition with the carboxylase activity necessary for carbon fixation, yet hypotheses regarding the selective pressures governing RuBisCO evolution have been limited to speculation. Here we report the resurrection and biochemical characterization of ancestral RuBisCOs, dating back to over one billion years ago (Gyr ago). Our findings provide an ancient point of reference revealing divergent evolutionary paths taken by eukaryotic homologues towards improved specificity for CO2, versus the evolutionary emphasis on increased rates of carboxylation observed in bacterial homologues. Consistent with these distinctions, in vivo analysis reveals the propensity of ancestral RuBisCO to be encapsulated into modern-day carboxysomes, bacterial organelles central to the cyanobacterial CO2 concentrating mechanism.

Life has been built on the evolution and innovation of microbial metabolisms. Even with our scant understanding of the full diversity of microbial life, it is clear that microbes have become integral components of the biogeochemical cycles that drive our planet. The antiquity of life further suggests that various microbial metabolisms have been core and essential to global elemental cycling for a majority of Earth's history., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

Objectives: Extracorporeal shock wave lithotripsy (ESWL) is a popular treatment for nephrolithiasis. We took advantage of noncontrast abdominal computed tomography (NCCT) to search the possible prognostic factors including abdominal fat distribution influencing stone-free rate., Methods: From August 2008 to August 2010, 145 patients who had renal calculus and had undergone ESWL were retrospectively reviewed. All of them received NCCT assessment before ESWL and were followed up after 1 month for stone clearance. These patients were divided into two groups: one was the stone-free group and the other was the residual-stone group. Affecting parameters included stone size, location, stone surface area, Hounsfield unit density (HU density), skin-to-stone distance (SSD), and abdominal fat area as analyzed between these two groups., Results: Of 145 patients, 70 were stone-free and 75 had residual stone after ESWL treatment and 1-month follow-up. From univariate analysis, stone size, HU density, SSD, and stone surface area were significant predicting factors for ESWL success. On multivariate analysis, the important factors influencing ESWL outcomes were HU density and stone surface area (odds ratio 1.002 vs. 77.18, respectively; P<.05). Abdominal fat accumulation and distribution had no significant difference between these two groups., Conclusion: This study revealed that stone size, HU density, SSD, and stone surface area were associated with stone-free rate after ESWL treatment. Therefore, these factors could be used to assess the feasibility of ESWL before deciding the treatment strategy. Abdominal fat distribution had no significant impact on ESWL outcome for renal stones., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Recent studies on the mechanisms by which topologically knotted proteins attain their natively knotted structures have intrigued theoretical and experimental biophysicists. Of particular interest is the finding that YibK and YbeA, two small trefoil knotted proteins, remain topologically knotted in their chemically denatured states. Using small-angle X-ray scattering (SAXS), we examine whether these chemically denatured knotted proteins are different from typical random coils. By revisiting the scaling law of radius of gyration (Rg) as a function of polypeptide chain length for chemically denatured proteins and natively folded proteins, we find that the chemically denatured knotted proteins in fact follow the same random coil-like behavior, suggesting that the formation of topological protein knots do not necessarily require global compaction while the loosely knotted polypeptide chains are capable of maintaining the correct chirality without defined secondary or tertiary structures.

The invention of oxygenic photosynthesis by cyanobacteria 2.4 billion years ago forever transformed Earth. This biogeochemical shift set into motion the evolution of subsequent microbial metabolisms and lifestyles. A new study provides a novel approach in piecing together evidence for how this evolutionary transition may have occurred., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

Urolithiasis is a common disease of the urinary system. Extracorporeal shockwave lithotripsy (SWL) has become one of the standard treatments for renal and ureteral stones; however, the success rates range widely and failure of stone disintegration may cause additional outlay, alternative procedures, and even complications. We used the data available from noncontrast abdominal computed tomography (NCCT) to evaluate the impact of stone parameters and abdominal fat distribution on calculus-free rates following SWL. We retrospectively reviewed 328 patients who had urinary stones and had undergone SWL from August 2012 to August 2013. All of them received pre-SWL NCCT; 1 month after SWL, radiography was arranged to evaluate the condition of the fragments. These patients were classified into stone-free group and residual stone group. Unenhanced computed tomography variables, including stone attenuation, abdominal fat area, and skin-to-stone distance (SSD) were analyzed. In all, 197 (60%) were classified as stone-free and 132 (40%) as having residual stone. The mean ages were 49.35 ± 13.22 years and 55.32 ± 13.52 years, respectively. On univariate analysis, age, stone size, stone surface area, stone attenuation, SSD, total fat area (TFA), abdominal circumference, serum creatinine, and the severity of hydronephrosis revealed statistical significance between these two groups. From multivariate logistic regression analysis, the independent parameters impacting SWL outcomes were stone size, stone attenuation, TFA, and serum creatinine. [Adjusted odds ratios and (95% confidence intervals): 9.49 (3.72-24.20), 2.25 (1.22-4.14), 2.20 (1.10-4.40), and 2.89 (1.35-6.21) respectively, all p < 0.05]. In the present study, stone size, stone attenuation, TFA and serum creatinine were four independent predictors for stone-free rates after SWL. These findings suggest that pretreatment NCCT may predict the outcomes after SWL. Consequently, we can use these predictors for selecting the optimal treatment for patients with urinary stones., (Copyright © 2014. Published by Elsevier Taiwan.)

Global photosynthetic productivity is limited by the enzymatic assimilation of CO2 into organic carbon compounds. Ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO), the carboxylating enzyme of the Calvin-Benson cycle, poorly discriminates between CO2 and O2, leading to photorespiration and the loss of fixed carbon and nitrogen. With the advent of synthetic biology, it is now feasible to design, synthesize, and introduce biochemical pathways in vivo. We engineered a synthetic photorespiratory bypass based on the 3-hydroxypropionate bi-cycle into the model cyanobacterium, Synechococcus elongatus sp. PCC 7942. The heterologously expressed cycle is designed to function as both a photorespiratory bypass and an additional CO2-fixing pathway, supplementing the Calvin-Benson cycle. We demonstrate the function of all six introduced enzymes and identify bottlenecks to be targeted in subsequent bioengineering. These results have implications for efforts to improve photosynthesis and for the "green" production of high value products of biotechnological interest.

It is often suggested that horizontal gene transfer is so ubiquitous in microbes that the concept of a phylogenetic tree representing the pattern of vertical inheritance is oversimplified or even positively misleading. "Universal proteins" have been used to infer the organismal phylogeny, but have been criticized as being only the "tree of one percent." Currently, few options exist for those wishing to rigorously assess how well a universal protein phylogeny, based on a relative handful of well-conserved genes, represents the phylogenetic histories of hundreds of genes. Here, we address this problem by proposing a visualization method and a statistical test within a Bayesian framework. We use the genomes of marine cyanobacteria, a group thought to exhibit substantial amounts of HGT, as a test case. We take 379 orthologous gene families from 28 cyanobacteria genomes and estimate the Bayesian posterior distributions of trees - a "treecloud" - for each, as well as for a concatenated dataset based on putative "universal proteins." We then calculate the average distance between trees within and between all treeclouds on various metrics and visualize this high-dimensional space with non-metric multidimensional scaling (NMMDS). We show that the tree space is strongly clustered and that the universal protein treecloud is statistically significantly closer to the center of this tree space than any individual gene treecloud. We apply several commonly-used tests for incongruence/HGT and show that they agree HGT is rare in this dataset, but make different choices about which genes were subject to HGT. Our results show that the question of the representativeness of the "tree of one percent" is a quantitative empirical question, and that the phylogenetic central tendency is a meaningful observation even if many individual genes disagree due to the various sources of incongruence.

Biological soil crusts (BSCs) cover extensive portions of the earth's deserts. In order to survive desiccation cycles and utilize short periods of activity during infrequent precipitation, crust microorganisms must rely on the unique capabilities of vegetative cells to enter a dormant state and be poised for rapid resuscitation upon wetting. To elucidate the key events involved in the exit from dormancy, we performed a wetting experiment of a BSC and followed the response of the dominant cyanobacterium, Microcoleus vaginatus, in situ using a whole-genome transcriptional time course that included two diel cycles. Immediate, but transient, induction of DNA repair and regulatory genes signaled the hydration event. Recovery of photosynthesis occurred within 1 h, accompanied by upregulation of anabolic pathways. Onset of desiccation was characterized by the induction of genes for oxidative and photo-oxidative stress responses, osmotic stress response and the synthesis of C and N storage polymers. Early expression of genes for the production of exopolysaccharides, additional storage molecules and genes for membrane unsaturation occurred before drying and hints at preparedness for desiccation. We also observed signatures of preparation for future precipitation, notably the expression of genes for anaplerotic reactions in drying crusts, and the stable maintenance of mRNA through dormancy. These data shed light on possible synchronization between this cyanobacterium and its environment, and provides key mechanistic insights into its metabolism in situ that may be used to predict its response to climate, and or, land-use driven perturbations.

Chloroplasts and mitochondria descended from bacterial ancestors, but the dating of these primary endosymbiosis events remains very uncertain, despite their importance for our understanding of the evolution of both bacteria and eukaryotes. All phylogenetic dating in the Proterozoic and before is difficult: Significant debates surround potential fossil calibration points based on the interpretation of the Precambrian microbial fossil record, and strict molecular clock methods cannot be expected to yield accurate dates over such vast timescales because of strong heterogeneity in rates. Even with more sophisticated relaxed-clock analyses, nodes that are distant from fossil calibrations will have a very high uncertainty in dating. However, endosymbiosis events and gene duplications provide some additional information that has never been exploited in dating; namely, that certain nodes on a gene tree must represent the same events, and thus must have the same or very similar dates, even if the exact date is uncertain. We devised techniques to exploit this information: cross-calibration, in which node date calibrations are reused across a phylogeny, and cross-bracing, in which node date calibrations are formally linked in a hierarchical Bayesian model. We apply these methods to proteins with ancient duplications that have remained associated and originated from plastid and mitochondrial endosymbionts: the α and β subunits of ATP synthase and its relatives, and the elongation factor thermo unstable. The methods yield reductions in dating uncertainty of 14-26% while only using date calibrations derived from phylogenetically unambiguous Phanerozoic fossils of multicellular plants and animals. Our results suggest that primary plastid endosymbiosis occurred ∼900 Mya and mitochondrial endosymbiosis occurred ∼1,200 Mya.

A general approach to mimic the sensing scheme of allosteric enzymes is developed. Through the covalent labeling of a sulfonamide inhibitor to the enzyme HCAII via SNAP-tag protein, the enzyme is rendered inactive. Catalytic activation is triggered only when a protease is present to cleave the recognized peptide sequence.

Background and Purpose: Shockwave lithotripsy (SWL) is a widely used treatment for patients with renal and ureteral stones because of its noninvasive approach. Although minor complications occur in most patients, a relative severe complication, perirenal or subcapsular hematoma, may also occur. We evaluate the possible risk factors for perirenal hematoma after SWL., Patients and Methods: Between 2001 and 2011, a total of 10,887 SWL treatments were performed for urolithiasis. All SWL procedures were performed using a Siemens Lithostar multiline lithotripter at a frequency of 2/sec under intermittent fluoroscopic guidance. All these patients underwent outpatient treatment without general anesthesia, but pethidine was administered for pain control. Treatment episodes were retrospectively reviewed for medical history, patient age, sex, body mass index (BMI), mean arterial pressure at induction, location of stone, total number of shockwaves, and peak shockwave intensity. We also compared the hematoma group with the control group (no hematoma formation after SWL with matched age and sex) for various factors., Results: After 10,887 treatment episodes on a total of 6177 patients during this period, subcapsular or perirenal hematoma developed in 20 patients for a total incident rate of 0.32%. Eighteen patients had the symptom of flank pain, and 2 patients received a diagnosis accidentally without symptoms. Four patients received a blood transfusion because of low hemoglobulin concentration. All of them received conservative and supportive treatment without surgical exploration. Ten (50%) patients had a history of hypertension. Renal hematoma developed in 11 patients at the second or third SWL treatment. Hypertension, higher BMI, and larger stone size are predisposing risk factors (P=0.022, 0.026 and 0.026, respectively) for renal hematoma., Conclusions: Renal hematoma is a rare (incidence rate, 0.32%) but possibly lethal complication. The most common symptoms of renal hematoma are severe flank pain and hematuria. A history of hypertension and higher BMI are important predisposing factors to perirenal hematoma.

The cyanobacterial phylum encompasses oxygenic photosynthetic prokaryotes of a great breadth of morphologies and ecologies; they play key roles in global carbon and nitrogen cycles. The chloroplasts of all photosynthetic eukaryotes can trace their ancestry to cyanobacteria. Cyanobacteria also attract considerable interest as platforms for "green" biotechnology and biofuels. To explore the molecular basis of their different phenotypes and biochemical capabilities, we sequenced the genomes of 54 phylogenetically and phenotypically diverse cyanobacterial strains. Comparison of cyanobacterial genomes reveals the molecular basis for many aspects of cyanobacterial ecophysiological diversity, as well as the convergence of complex morphologies without the acquisition of novel proteins. This phylum-wide study highlights the benefits of diversity-driven genome sequencing, identifying more than 21,000 cyanobacterial proteins with no detectable similarity to known proteins, and foregrounds the diversity of light-harvesting proteins and gene clusters for secondary metabolite biosynthesis. Additionally, our results provide insight into the distribution of genes of cyanobacterial origin in eukaryotic nuclear genomes. Moreover, this study doubles both the amount and the phylogenetic diversity of cyanobacterial genome sequence data. Given the exponentially growing number of sequenced genomes, this diversity-driven study demonstrates the perspective gained by comparing disparate yet related genomes in a phylum-wide context and the insights that are gained from it.

Objectives: To assess the effects of abdominal fat on shock wave lithotripsy (SWL). We used pre-SWL unenhanced computed tomography (CT) to evaluate the impact of abdominal fat distribution and calculus characteristics on the outcome of SWL., Methods: One hundred and eighty-five patients with a solitary ureteric calculus treated with SWL were retrospectively reviewed. Each patient underwent unenhanced CT within 1 month before SWL treatment. Treatment outcomes were evaluated 1 month later. Unenhanced CT parameters, including calculus surface area, Hounsfield unit (HU) density, abdominal fat area and skin to calculus distance (SSD) were analysed., Results: One hundred and twenty-eight of the 185 patients were found to be calculus-free following treatment. HU density, total fat area, visceral fat area and SSD were identified as significant variables on multivariate logistic regression analysis. The receiver-operating characteristic analyses showed that total fat area, para/perirenal fat area and visceral fat area were sensitive predictors of SWL outcomes., Conclusion: This study revealed that higher quantities of abdominal fat, especially visceral fat, are associated with a lower calculus-free rate following SWL treatment. Unenhanced CT is a convenient technique for diagnosing the presence of a calculus, assessing the intra-abdominal fat distribution and thereby helping to predict the outcome of SWL., Key Points: • Unenhanced CT is now widely used to assess ureteric calculi. • The same CT protocol can provide measurements of abdominal fat distribution. • Ureteric calculi are usually treated by shock wave lithotripsy (SWL). • Greater intra-abdominal fat stores are generally associated with poorer SWL results.

We have investigated the effect of pressure on imidazolium C-H---O interactions in 1-ethyl-3-methylimidazolium bis(trifluoromethylsulfonyl)amide (EMI(+)TFSA(-))/L64 and EMI(+)TFSA(-)/1,4-dioxane mixtures. The addition of Pluronic L64 to EMI(+)TFSA(-) leads to appreciable changes in band frequencies and shapes of the imidazolium C-H stretching bands. A possible explanation is the formation of C-H---O interactions between imidazolium C-H groups and oxygen atoms of polyethylene oxides (PEOs). In other words, L64 can be added to change the relative contribution of the isolated and associated components of EMI(+)TFSA(-). In contrast to L64, the oxygen atoms of 1,4-dioxane cannot perturb the local structures of imidazolium C-H groups of EMI(+)TFSA(-) and the association configuration is still favored in the presence of 1,4-dioxane. As the pressure is elevated, 1,4-dioxane molecules tend to associate with themselves and TFSA(-) interacts with EMI(+) to form associated configurations. Our results suggest the formation of association between EMI(+) cation and L64 and the complexes are stable up to the pressure of 2.5 GPa.

Using high-pressure infrared methods, we have investigated close interactions of charge-enhanced C-H-O type in ionic liquid∕dimethyl sulfoxide (DMSO) mixtures. The solvation and association of the 1-butyl-3-methylimidazolium tetrafluoroborate (BMI(+)BF(4)(-)) and 1-butyl-2,3-dimethylimidazolium tetrafluoroborate (BMM(+)BF(4)(-)) in DMSO-d(6) were examined by analysis of C-H spectral features. Based on our concentration-dependent results, the imidazolium C-H groups are more sensitive sites for C-H-O than the alkyl C-H groups and the dominant imidazolium C-H species in dilute ionic liquid∕DMSO-d(6) should be assigned to the isolated (or dissociated) structures. As the dilute mixtures were compressed by high pressures, the loss in intensity of the bands attributed to the isolated structures was observed. In other words, high pressure can be used to perturb the association-dissociation equilibrium in the polar region. This result is remarkably different from what is revealed for the imidazolium C-H in the BMM(+)BF(4)(-)∕D(2)O mixtures. DFT-calculations are in agreement with our experimental results indicating that C(4)-H-O and C(5)-H-O interactions seem to play non-negligible roles for BMM(+)BF(4)(-)∕DMSO mixtures.

We have studied spectroscopic properties of the 16kDa red carotenoid protein (RCP), which is closely related to the orange carotenoid protein (OCP) from cyanobacteria. Both proteins bind the same chromophore, the carotenoid 3'-hydroxyechinenone (hECN), and the major difference between the two proteins is lack of the C-terminal domain in the RCP; this results in exposure of part of the carotenoid. The excited-state lifetime of hECN in the RCP is 5.5ps, which is markedly longer than in OCP (3.3ps) but close to 6ps obtained for hECN in organic solvent. This confirms that the binding of hECN to the C-terminal domain in the OCP changes conformation of hECN, thereby altering its excited-state properties. Hydrogen bonds between the C-terminal domain and the carotenoid are also absent in the RCP. This allows the conformation of hECN in the RCP to be similar to that in solution, which results in comparable excited-state properties of hECN in solution. The red-shift of the RCP absorption spectrum is most likely due to aggregation of RCP induced by hydrophobic nature of hECN that, when exposed to buffer, stimulates formation of assemblies minimizing contact of hECN with water. We suggest that the loss of the C-terminal domain renders the protein amphipathic, containing both hydrophobic (the exposed part of hECN) and hydrophilic (N-terminal domain) regions, and may help the RCP to interact with lipid membranes; exposed hECN can penetrate into the hydrophobic environment of the lipid membrane, possibly to provide additional photoprotection., (Copyright © 2010 Elsevier B.V. All rights reserved.)

In this report, we describe a 31-year-old man in whom acute pancreatitis was the initial feature of a subsequently diagnosed pancreatic adenocarcinoma with multiple metastases. He initially presented at our hospital with acute pancreatitis. Abdominal ultrasonography revealed a mildly dilated pancreatic duct and an enlarged pancreatic head. Although a follow-up abdominal ultrasonography revealed a progressively dilated pancreatic duct and a progressively enlarged pancreatic head, he refused further investigation and was lost to follow-up. Four months later, he returned to our hospital with relapsed acute pancreatitis. Obstructive jaundice was noted and drainage was performed. Because choledochoplasty with multiple balloon catheters was not fully effective, biliary tract bypass surgery was carried out. Intraoperative biopsy confirmed pancreatic adenocarcinoma with multiple metastases. The patient died of massive gastrointestinal bleeding a few weeks later. To our knowledge, this is the youngest case of pancreatic cancer with the uncommon initial presentation of acute pancreatitis reported in the literature. For a patient with acute pancreatitis, particularly recurrent episodes, but with no known risk factors for pancreatitis, a pancreatic neoplasm should be considered as a potential underlying cause, even in a young man., (Copyright 2010 Elsevier. Published by Elsevier B.V. All rights reserved.)

A significant fraction of a plant's nuclear genome encodes chloroplast-targeted proteins, many of which are devoted to the assembly and function of the photosynthetic apparatus. Using digital video imaging of chlorophyll fluorescence, we isolated proton gradient regulation 7 (pgr7) as an Arabidopsis thaliana mutant with low nonphotochemical quenching of chlorophyll fluorescence (NPQ). In pgr7, the xanthophyll cycle and the PSBS gene product, previously identified NPQ factors, were still functional, but the efficiency of photosynthetic electron transport was lower than in the wild type. The pgr7 mutant was also smaller in size and had lower chlorophyll content than the wild type in optimal growth conditions. Positional cloning located the pgr7 mutation in the At3g21200 (PGR7) gene, which was predicted to encode a chloroplast protein of unknown function. Chloroplast targeting of PGR7 was confirmed by transient expression of a GFP fusion protein and by stable expression and subcellular localization of an epitope-tagged version of PGR7. Bioinformatic analyses revealed that the PGR7 protein has two domains that are conserved in plants, algae, and bacteria, and the N-terminal domain is predicted to bind a cofactor such as FMN. Thus, we identified PGR7 as a novel, conserved nuclear gene that is necessary for efficient photosynthetic electron transport in chloroplasts of Arabidopsis.