A Rapid Ribosome Profiling Method Elucidates Chloroplast Ribosome Behavior in Vivo

The profiling of ribosome footprints by deep sequencing has revolutionized the analysis of translation by mapping ribosomes with high resolution on a genome-wide scale. We present a variation on this approach that offers a rapid and cost-effective alternative for the genome-wide profiling of chloroplast ribosomes. Ribosome footprints from leaf tissue are hybridized to oligonucleotide tiling microarrays of the plastid ORFeome and report the abundance and translational status of every chloroplast mRNA. Each assay replaces several time-consuming traditional methods while also providing information that was previously inaccessible. To illustrate the utility of the approach, we show that it detects known defects in chloroplast gene expression in several nuclear mutants of maize (Zea mays) and that it reveals previously unsuspected defects. Furthermore, it provided firm answers to several lingering questions in chloroplast gene expression: (1) the overlapping atpB/atpE open reading frames, whose translation had been proposed to be coupled, are translated independently in vivo; (2) splicing is not a prerequisite for translation initiation on an intron-containing chloroplast RNA; and (3) a feedback control mechanism that links the synthesis of ATP synthase subunits in Chlamydomonas reinhardtii does not exist in maize. An analogous approach is likely to be useful for studies of mitochondrial gene expression

Efficacy and safety of the anti-IL-12/23 p40 monoclonal antibody, ustekinumab, in patients with active psoriatic arthritis despite conventional non-biological and biological anti-tumour necrosis factor therapy: 6-month and 1-year results of the phase 3, multicentre, double-blind, placebo-controlled, randomised PSUMMIT 2 trial

Objective: Assess ustekinumab efficacy (week 24/week 52) and safety (week 16/week 24/week 60) in patients with active psoriatic arthritis (PsA) despite treatment with conventional and/or biological anti-tumour necrosis factor (TNF) agents. Methods: In this phase 3, multicentre, placebo-controlled trial, 312 adults with active PsA were randomised (stratified by site, weight (≤100 kg/>100 kg), methotrexate use) to ustekinumab 45 mg or 90 mg at week 0, week 4, q12 weeks or placebo at week 0, week 4, week 16 and crossover to ustekinumab 45 mg at week 24, week 28 and week 40. At week 16, patients with <5% improvement in tender/swollen joint counts entered blinded early escape (placebo→45 mg, 45 mg→90 mg, 90 mg→90 mg). The primary endpoint was ≥20% improvement in American College of Rheumatology (ACR20) criteria at week 24. Secondary endpoints included week 24 Health Assessment Questionnaire-Disability Index (HAQ-DI) improvement, ACR50, ACR70 and ≥75% improvement in Psoriasis Area and Severity Index (PASI75). Efficacy was assessed in all patients, anti-TNF-naïve (n=132) patients and anti-TNF-experienced (n=180) patients. Results: More ustekinumab-treated (43.8% combined) than placebo-treated (20.2%) patients achieved ACR20 at week 24 (p<0.001). Significant treatment differences were observed for week 24 HAQ-DI improvement (p<0.001), ACR50 (p≤0.05) and PASI75 (p<0.001); all benefits were sustained through week 52. Among patients previously treated with ≥1 TNF inhibitor, sustained ustekinumab efficacy was also observed (week 24 combined vs placebo: ACR20 35.6% vs 14.5%, PASI75 47.1% vs 2.0%, median HAQ-DI change −0.13 vs 0.0; week 52 ustekinumab-treated: ACR20 38.9%, PASI75 43.4%, median HAQ-DI change −0.13). No unexpected adverse events were observed through week 60. Conclusions: The interleukin-12/23 inhibitor ustekinumab (45/90 mg q12 weeks) yielded significant and sustained improvements in PsA signs/symptoms in a diverse population of patients with active PsA, including anti-TNF-experienced PsA patients

Complete chloroplast genome sequence of Holoparasite Cistanche Deserticola (Orobanchaceae) reveals gene loss and horizontal gene transfer from Its host Haloxylon Ammodendron (Chenopodiaceae)

The central function of chloroplasts is to carry out photosynthesis, and its gene content and structure are highly conserved across land plants. Parasitic plants, which have reduced photosynthetic ability, suffer gene losses from the chloroplast (cp) genome accompanied by the relaxation of selective constraints. Compared with the rapid rise in the number of cp genome sequences of photosynthetic organisms, there are limited data sets from parasitic plants. The authors report the complete sequence of the cp genome of Cistanche deserticola, a holoparasitic desert species belonging to the family Orobanchaceae

Lesson learned during the designing and construction phases of the ORC-plus thermal energy storage system of 20 MWH

A limitation to the expansion of medium sized Concentrating Solar Power (CSP) plants is represented by the lack of technical solutions of Thermal Energy Storage (TES) systems specialized for this size of CSP plants and validated in a relevant industrial environment. In order to make the medium sized CSP systems more competitive than PV systems, the TES system has to allow the operators of the solar power plant to adjust the electricity production for matching consumer demand, so enabling the sale of electricity during peak demand periods for boosting plant revenues. At the same time, there is the need of lowering the TES system weight in the capital costs of the overall system. The ORC-PLUS (Organic Rankine Cycle - Prototype Link to Storage Unit) Project aims to realize a demonstrator of a TES system optimized for a medium sized CSP plant coupled with an ORC (Organic Rankine Cycle) turbine of 1 MWe. In this paper, the difficulties encountered during the manufacturing and commissioning operations of the TES system and their potential impact on capital costs will be reported

Structure of the actively translating plant 80S ribosome at 2.2 Å resolution

In plant cells, translation occurs in three compartments: the cytosol, the plastids and the mitochondria. While the structures of the (prokaryotic-type) ribosomes in plastids and mitochondria are well characterized, high-resolution structures of the eukaryotic 80S ribosomes in the cytosol have been lacking. Here the structure of translating tobacco (Nicotiana tabacum) 80S ribosomes was solved by cryo-electron microscopy with a global resolution of 2.2 Å. The ribosome structure includes two tRNAs, decoded mRNA and the nascent peptide chain, thus providing insights into the molecular underpinnings of the cytosolic translation process in plants. The map displays conserved and plant-specific rRNA modifications and the positions of numerous ionic cofactors, and it uncovers the role of monovalent ions in the decoding centre. The model of the plant 80S ribosome enables broad phylogenetic comparisons that reveal commonalities and differences in the ribosomes of plants and those of other eukaryotes, thus putting our knowledge about eukaryotic translation on a firmer footing

Multiple Checkpoints for the Expression of the Chloroplast-Encoded Splicing Factor MatK

The chloroplast genome of land plants contains only a single gene for a splicing factor, Maturase K (MatK). To better understand the regulation of matK gene expression, we quantitatively investigated the expression of matK across tobacco (Nicotiana tabacum) development at the transcriptional, posttranscriptional, and protein levels. We observed striking discrepancies of MatK protein and matK messenger RNA levels in young tissue, suggestive of translational regulation or altered protein stability. We furthermore found increased matK messenger RNA stability in mature tissue, while other chloroplast RNAs tested showed little changes. Finally, we quantitatively measured MatK-intron interactions and found selective changes in the interaction of MatK with specific introns during plant development. This is evidence for a direct role of MatK in the regulation of chloroplast gene expression via splicing. We furthermore modeled a simplified matK gene expression network mathematically. The model reflects our experimental data and suggests future experimental perturbations to pinpoint regulatory checkpoints

Acclimation in plants – the Green Hub consortium

Acclimation is the capacity to adapt to environmental changes within the lifetime of an individual. This ability allows plants to cope with the continuous variation in ambient conditions to which they are exposed as sessile organisms. Because environmental changes and extremes are becoming even more pronounced due to the current period of climate change, enhancing the efficacy of plant acclimation is a promising strategy for mitigating the consequences of global warming on crop yields. At the cellular level, the chloroplast plays a central role in many acclimation responses, acting both as a sensor of environmental change and as a target of cellular acclimation responses. In this Perspective article, we outline the activities of the Green Hub consortium funded by the German Science Foundation. The main aim of this research collaboration is to understand and strategically modify the cellular networks that mediate plant acclimation to adverse environments, employing Arabidopsis, tobacco (Nicotiana tabacum) and Chlamydomonas as model organisms. These efforts will contribute to ‘smart breeding’ methods designed to create crop plants with improved acclimation properties. To this end, the model oilseed crop Camelina sativa is being used to test modulators of acclimation for their potential to enhance crop yield under adverse environmental conditions. Here we highlight the current state of research on the role of gene expression, metabolism and signalling in acclimation, with a focus on chloroplast‐related processes. In addition, further approaches to uncovering acclimation mechanisms derived from systems and computational biology, as well as adaptive laboratory evolution with photosynthetic microbes, are highlighted.Deutsche Forschungsgemeinschaft http://dx.doi.org/10.13039/501100001659Peer Reviewe

Acclimation in plants - the Green Hub consortium

Acclimation is the capacity to adapt to environmental changes within the lifetime of an individual. This ability allows plants to cope with the continuous variation in ambient conditions to which they are exposed as sessile organisms. Because environmental changes and extremes are becoming even more pronounced due to the current period of climate change, enhancing the efficacy of plant acclimation is a promising strategy for mitigating the consequences of global warming on crop yields. At the cellular level, the chloroplast plays a central role in many acclimation responses, acting both as a sensor of environmental change and as a target of cellular acclimation responses. In this Perspective article, we outline the activities of the Green Hub consortium funded by the German Science Foundation. The main aim of this research collaboration is to understand and strategically modify the cellular networks that mediate plant acclimation to adverse environments, employing Arabidopsis, tobacco (Nicotiana tabacum) and Chlamydomonas as model organisms. These efforts will contribute to 'smart breeding' methods designed to create crop plants with improved acclimation properties. To this end, the model oilseed crop Camelina sativa is being used to test modulators of acclimation for their potential to enhance crop yield under adverse environmental conditions. Here we highlight the current state of research on the role of gene expression, metabolism and signalling in acclimation, with a focus on chloroplast-related processes. In addition, further approaches to uncovering acclimation mechanisms derived from systems and computational biology, as well as adaptive laboratory evolution with photosynthetic microbes, are highlighted

Isolation and analysis of high quality nuclear DNA with reduced organellar DNA for plant genome sequencing and resequencing

<p>Abstract</p> <p>Background</p> <p>High throughput sequencing (HTS) technologies have revolutionized the field of genomics by drastically reducing the cost of sequencing, making it feasible for individual labs to sequence or resequence plant genomes. Obtaining high quality, high molecular weight DNA from plants poses significant challenges due to the high copy number of chloroplast and mitochondrial DNA, as well as high levels of phenolic compounds and polysaccharides. Multiple methods have been used to isolate DNA from plants; the CTAB method is commonly used to isolate total cellular DNA from plants that contain nuclear DNA, as well as chloroplast and mitochondrial DNA. Alternatively, DNA can be isolated from nuclei to minimize chloroplast and mitochondrial DNA contamination.</p> <p>Results</p> <p>We describe optimized protocols for isolation of nuclear DNA from eight different plant species encompassing both monocot and eudicot species. These protocols use nuclei isolation to minimize chloroplast and mitochondrial DNA contamination. We also developed a protocol to determine the number of chloroplast and mitochondrial DNA copies relative to the nuclear DNA using quantitative real time PCR (qPCR). We compared DNA isolated from nuclei to total cellular DNA isolated with the CTAB method. As expected, DNA isolated from nuclei consistently yielded nuclear DNA with fewer chloroplast and mitochondrial DNA copies, as compared to the total cellular DNA prepared with the CTAB method. This protocol will allow for analysis of the quality and quantity of nuclear DNA before starting a plant whole genome sequencing or resequencing experiment.</p> <p>Conclusions</p> <p>Extracting high quality, high molecular weight nuclear DNA in plants has the potential to be a bottleneck in the era of whole genome sequencing and resequencing. The methods that are described here provide a framework for researchers to extract and quantify nuclear DNA in multiple types of plants.</p