Probing Arabidopsis Chloroplast Diacylglycerol Pools by Selectively Targeting Bacterial Diacylglycerol Kinase to Suborganellar Membranes

Diacylglycerol (DAG) is an intermediate in metabolism of both triacylglycerols and membrane lipids. Probing the steady-state pools of DAG and understanding how they contribute to the synthesis of different lipids is important when designing plants with altered lipid metabolism. However, traditional methods of assaying DAG pools are difficult, because its abundance is low and because fractionation of subcellular membranes affects DAG pools. To manipulate and probe DAG pools in an in vivo context, we generated multiple stable transgenic lines of Arabidopsis (Arabidopsis thaliana) that target an Escherichia coli DAGkinase (DAGK) to each leaflet of each chloroplast envelope membrane. E. coli DAGKis small, self inserts into membranes, and has catalytic activity on only one side of each membrane. By comparing whole-tissue lipid profiles between our lines, we show that each line has an individual pattern of DAG, phosphatidic acid, phosphatidylcholine, and triacylglycerol steady-state levels, which supports an individual function of DAG in each membrane leaflet. Furthermore, conversion of DAG in the leaflets facing the chloroplast intermembrane space by DAGK impairs plant growth. As a result of DAGK presence in the outer leaflet of the outer envelope membrane, phosphatidic acid accumulation is not observed, likely because it is either converted into other lipids or removed to othermembranes. Finally, we use the outer envelope-targeted DAGK line as a tool to probe the accessibility of DAG generated in response to osmotic stress

Route prediction from trip observations,”

ABSTRACT This paper develops and tests algorithms for predicting the end-to-end route of a vehicle based on GPS observations of the vehicle's past trips. We show that a large portion of a typical driver's trips are repeated. Our algorithms exploit this fact for prediction by matching the first part of a driver's current trip with one of the set of previously observed trips. Rather than predicting upcoming road segments, our focus is on making long term predictions of the route. We evaluate our algorithms using a large corpus of real world GPS driving data acquired from observing over 250 drivers for an average of 15.1 days per subject. Our results show how often and how accurately we can predict a driver's route as a function of the distance already driven

The effect of non-medical factors on variations in the performance of colonoscopy among different health care settings

Background: Previous studies in the literature have shown significant variations in colonoscopy performance, even when medical factors are taken into account. This study aimed to examine the role of non-medical factors (i.e. embodied in health care system design) as possible contributors to variations in colonoscopy performance. Methods: We used patient data from a multicenter observational study conducted between 2000 and 2002 in 21 centers across 11 western countries. Variability was captured through two performance outcomes (diagnostic yield and colonoscopy withdrawal time), jointly studied as dependent variables using a multilevel two-equation system. Results: Results showed that open-access systems and high-volume colonoscopy centers were independently associated with a higher likelihood of detecting significant lesions and higher withdrawal durations. Fee for service (FFS) payment was associated with shorter withdrawal durations, and had an indirect negative impact on the diagnostic yield. Teaching centers exhibited lower detection rates and higher withdrawal times. Conclusions: Our results suggest that gate-keeping colonoscopy is likely to miss patients with significant lesions and that developing specialized colonoscopy units is important to improve performance. Results also suggest that FFS may result in a lower quality of care in colonoscopy practice and highlight that longer withdrawal times do not necessarily mean higher quality in teaching-centers.Medical Practice Variation (MPV), performance, non-medical factors, panel two-equation linear-probit model, colonoscopy

Gastroenterologists overestimate the appropriateness of colonoscopies they perform: an international observational study

BACKGROUND AND STUDY AIM: Little is known about how gastroenterologists perceive the appropriateness of colonoscopies they perform. The objective of this study was to compare the appropriateness and necessity of colonoscopies as assessed by an expert panel and by the gastroenterologists performing the colonoscopies. METHODS: This observational study included 21 centers in 11 countries. Patients referred for colonoscopy were consecutively included. Appropriateness and necessity of colonoscopies were independently rated on a 9-point scale by the gastroenterologists performing them and by an expert panel using a validated method (RAND). The differences between the ratings from the two groups were examined. Results: 6004 patients were included in the study. Comparisons of ratings were possible for 5381 (89.6 %) patients. The gastroenterologists' mean appropriateness rating was 7.2 +/- 1.7, and the panel's mean appropriateness rating was 5.4 +/- 2.3 ( P &lt; 0.001). The percentages of indications rated inappropriate, uncertain, appropriate, and necessary were 4.1 %, 23.8 %, 14.2 %, and 58.0 % for the gastroenterologists and 27.2 %, 26.7 %, 25.0 %, and 21.1 % for the panel, respectively. Agreement between the two groups' ratings was poor (28.8 %, kappa = 0.11). Differences between the two groups' ratings decreased with increasing patient age, decreasing health status, and decreasing expertise level of the referring physician. However, the gastroenterologists produced consistently higher ratings. CONCLUSIONS: Compared with an expert panel, gastroenterologists tend to overestimate the appropriateness of colonoscopies they perform. Except for well-delineated reasons, participating gastroenterologists weighed patient characteristics differently from the panel when judging appropriateness. Ways to increase the prospective use of appropriateness criteria in order to improve appropriateness and reduce overuse of colonoscopies should be examined further. [Authors]]]> Colonoscopy ; Gastroenterology oai:serval.unil.ch:BIB_6852 2022-05-07T01:19:36Z <oai_dc:dc xmlns:dc="http://purl.org/dc/elements/1.1/" xmlns:xs="http://www.w3.org/2001/XMLSchema" xmlns:xsi="http://www.w3.org/2001/XMLSchema-instance" xmlns:oai_dc="http://www.openarchives.org/OAI/2.0/oai_dc/" xsi:schemaLocation="http://www.openarchives.org/OAI/2.0/oai_dc/ http://www.openarchives.org/OAI/2.0/oai_dc.xsd"> https://serval.unil.ch/notice/serval:BIB_6852 Prospective randomized comparison of imipenem-cilastatin and piperacillin-tazobactam in nosocomial pneumonia or peritonitis. Jaccard, C Troillet, N Harbarth, S Zanetti, G Aymon, D Schneider, R Chioléro, R Ricou, B Romand, J Huber, O Ambrosetti, P Praz, G Lew, D Bille, J Glauser, MP Cometta, A info:eu-repo/semantics/article article 1998 Antimicrob Agents Chemother, vol. 42, pp. 2966-72 oai:serval.unil.ch:BIB_6852615BE8BB 2022-05-07T01:19:36Z <oai_dc:dc xmlns:dc="http://purl.org/dc/elements/1.1/" xmlns:xs="http://www.w3.org/2001/XMLSchema" xmlns:xsi="http://www.w3.org/2001/XMLSchema-instance" xmlns:oai_dc="http://www.openarchives.org/OAI/2.0/oai_dc/" xsi:schemaLocation="http://www.openarchives.org/OAI/2.0/oai_dc/ http://www.openarchives.org/OAI/2.0/oai_dc.xsd"> https://serval.unil.ch/notice/serval:BIB_6852615BE8BB Smoked cannabis and doping control: looking for the wrong target analyte? Brenneisen, R. Meyer, P. Chtioui, H. Saugy, M. Schweizer, C. Kamber, M. info:eu-repo/semantics/conferenceObject inproceedings 2009-08 Annales de toxicologie analytique, vol. 21, pp. S25 Goullé, Jean-Pierre (ed.) info:eu-repo/semantics/altIdentifier/isbn/0768-598X <![CDATA[Introduction: Since 2004, cannabis is prohibited by the World Anti-Doping Agency (WADA) for all sports in competition. In the years since then, about half of all positive doping cases in Switzerland have been related to cannabis consumption. In most cases, the athletes plausibly claim to have consumed cannabis several days or even weeks before competition and only for recreational purposes not related to competition. In doping analysis, the target analyte in urine samples is 11-nor-delta-9-tetrahydrocannabinol- 9-carboxylic acid (THC-COOH), the reporting threshold for laboratories is 15 ng/mL. However, the wide detection window of this long-term THC metabolite in urine does not allow a conclusion concerning the time of consumption or the impact on the physical performance. Aim: The purpose of the present pharmacokinetic study on volunteers was to evaluate target analytes with shorter urinary excretion time. Subsequently, urines from athletes tested positive for cannabis should be reanalyzed including these analytes. Methods: In an one-session clinical trial (approved by IRB, Swissmedic, and Federal Office of Public Health), 12 healthy, male volunteers (age 26 ± 3 yrs, BMI 24 ± 2 kg/m2) with cannabis experience (&gt; once/month) smoked a Cannabis cigarette standardized to 70 mg THC/cigarette (Bedrobinol® 7%, Dutch Office for Medicinal Cannabis) following a paced-puffing procedure. Plasma and urine was collected up to 8 h and 11 days, respectively. Total THC, 11-hydroxy-THC (THC-OH), and THC-COOH were determined after enzymatic hydrolyzation followed by SPE and GC/MS-SIM. The limit of quantitation (LOQ) for all analytes was 0.1 ng/mL. Visual analog scales (VAS) and vital functions were used for monitoring psychological and somatic side-effects at every timepoint of specimen collection (up to 480 min). Results: Eight puffs delivered a mean THC dose of 45 mg. Mean plasma levels of total THC, THC-OH and THC-COOH were measured in the range of 0.1-20.9, 0.1-1.8, and 1.8-7.5 ng/mL, respectively. Peak concentrations were observed at 5, 10, and 90 min. Mean urine levels were measured in the range of 0.1-0.7, 0.10-6.2, and 0.1-13.4 ng/mL, respectively. The detection windows were 2-8, 2-96, and 2-120 h. No or only mild effects were observed, such as dry mouth, sedation, and tachycardia. Besides high to very high THC-COOH levels (0-978 ng/mL), THC (0.1-24 ng/mL) and THC-OH (1-234 ng/mL) were found in 90 and 96% of the cannabis-positive urines from athletes. Conclusion: Instead of or in addition to THC-COOH, the pharmacologically active THC and THC-OH should be the target analytes for doping urine analysis. This would allow the estimation of more recent Cannabis consumption, probably influencing performance during competition. Keywords: cannabis, doping, clinical trial, plasma and urine levels, athlete's sample

Current sedation and monitoring practice for colonoscopy: an international observational study (EPAGE)

BACKGROUND AND STUDY AIMS: Sedation and monitoring practice during colonoscopy varies between centers and over time. Knowledge of current practice is needed to ensure quality of care and help focus future research. The objective of this study was to examine sedation and monitoring practice in endoscopy centers internationally. PATIENTS AND METHODS: This observational study included consecutive patients referred for colonoscopy at 21 centers in 11 countries. Endoscopists reported sedation and monitoring practice, using a standard questionnaire for each patient. RESULTS: 6004 patients were included in this study, of whom 53 % received conscious/moderate sedation during colonoscopy, 30 % received deep sedation, and 17 % received no sedation. Sedation agents most commonly used were midazolam (47 %) and opioids (33 %). Pulse oximetry was done during colonoscopy in 77 % of patients, blood pressure monitoring in 34 %, and electrocardiography in 24 %. Pulse oximetry was most commonly used for moderately sedated patients, while blood pressure monitoring and electrocardiography were used predominantly for deeply sedated patients. Sedation and monitoring use ranged from 0 % to 100 % between centers. Oxygen desaturation (&lt;/= 85 %) occurred in 5 % of patients, of whom 80 % were moderately sedated. On average, three staff members were involved in procedures. An anesthesiologist was present during 27 % of colonoscopies, and during 85 % of colonoscopies using deep sedation. CONCLUSIONS: Internationally, sedation and monitoring practice during colonoscopy varied widely. Moderate sedation was the most common sedation method used and electronic monitoring was used in three-quarters of patients. Deep sedation tended to be more resource-intensive, implying a greater use of staff and monitoring. [Authors]]]> Colonoscopy ; Conscious Sedation ; Hypnotics and Sedatives ; Monitoring, Physiologic ; Physician's Practice Patterns oai:serval.unil.ch:BIB_254F2604090A 2022-05-07T01:13:03Z <oai_dc:dc xmlns:dc="http://purl.org/dc/elements/1.1/" xmlns:xs="http://www.w3.org/2001/XMLSchema" xmlns:xsi="http://www.w3.org/2001/XMLSchema-instance" xmlns:oai_dc="http://www.openarchives.org/OAI/2.0/oai_dc/" xsi:schemaLocation="http://www.openarchives.org/OAI/2.0/oai_dc/ http://www.openarchives.org/OAI/2.0/oai_dc.xsd"> https://serval.unil.ch/notice/serval:BIB_254F2604090A Simulating fully-integrated hydrological dynamics in complex Alpine headwaters info:doi:10.31223/X5RG7Q info:eu-repo/semantics/altIdentifier/doi/10.31223/X5RG7Q https://doi.org/10.31223/X5RG7Q James, Thornton René, Therrien Grégoire, Mariéthoz Niklas, Linde Philip, Brunner info:eu-repo/semantics/other misc 2021-02-26 eng oai:serval.unil.ch:BIB_255 2022-05-07T01:13:03Z <oai_dc:dc xmlns:dc="http://purl.org/dc/elements/1.1/" xmlns:xs="http://www.w3.org/2001/XMLSchema" xmlns:xsi="http://www.w3.org/2001/XMLSchema-instance" xmlns:oai_dc="http://www.openarchives.org/OAI/2.0/oai_dc/" xsi:schemaLocation="http://www.openarchives.org/OAI/2.0/oai_dc/ http://www.openarchives.org/OAI/2.0/oai_dc.xsd"> https://serval.unil.ch/notice/serval:BIB_255 High Pressure Techniques in Chemistry and Physics: Spectroscopic Measurements: NMR. Helm, L Powell, D.H. Merbach, A.E. info:eu-repo/semantics/bookPart incollection 1997 Practical Approach Series, pp. 187-216 oai:serval.unil.ch:BIB_2549 2022-05-07T01:13:03Z <oai_dc:dc xmlns:dc="http://purl.org/dc/elements/1.1/" xmlns:xs="http://www.w3.org/2001/XMLSchema" xmlns:xsi="http://www.w3.org/2001/XMLSchema-instance" xmlns:oai_dc="http://www.openarchives.org/OAI/2.0/oai_dc/" xsi:schemaLocation="http://www.openarchives.org/OAI/2.0/oai_dc/ http://www.openarchives.org/OAI/2.0/oai_dc.xsd"> https://serval.unil.ch/notice/serval:BIB_2549 Paul Tillich et l'histoire de l'art. Reymond, B info:eu-repo/semantics/article article 1997 RThPh, pp. 67-74 oai:serval.unil.ch:BIB_25490 2022-05-07T01:13:03Z <oai_dc:dc xmlns:dc="http://purl.org/dc/elements/1.1/" xmlns:xs="http://www.w3.org/2001/XMLSchema" xmlns:xsi="http://www.w3.org/2001/XMLSchema-instance" xmlns:oai_dc="http://www.openarchives.org/OAI/2.0/oai_dc/" xsi:schemaLocation="http://www.openarchives.org/OAI/2.0/oai_dc/ http://www.openarchives.org/OAI/2.0/oai_dc.xsd"> https://serval.unil.ch/notice/serval:BIB_25490 Actualité de la Convention européenne des droits de l'homme, mai 2002 - octobre 2002. Flauss, JF info:eu-repo/semantics/article article 2002 Actualité juridique de droit administratif, vol. 20, pp. 1277-1286 oai:serval.unil.ch:BIB_25491 2022-05-07T01:13:03Z <oai_dc:dc xmlns:dc="http://purl.org/dc/elements/1.1/" xmlns:xs="http://www.w3.org/2001/XMLSchema" xmlns:xsi="http://www.w3.org/2001/XMLSchema-instance" xmlns:oai_dc="http://www.openarchives.org/OAI/2.0/oai_dc/" xsi:schemaLocation="http://www.openarchives.org/OAI/2.0/oai_dc/ http://www.openarchives.org/OAI/2.0/oai_dc.xsd"> https://serval.unil.ch/notice/serval:BIB_25491 Le statut pénal du Président de la République et la Convention européenne des droits de l'homme. Flauss, JF info:eu-repo/semantics/article article 2002 Les Petites Affiches, vol. 261, pp. 4-10 oai:serval.unil.ch:BIB_25492 2022-05-07T01:13:03Z <oai_dc:dc xmlns:dc="http://purl.org/dc/elements/1.1/" xmlns:xs="http://www.w3.org/2001/XMLSchema" xmlns:xsi="http://www.w3.org/2001/XMLSchema-instance" xmlns:oai_dc="http://www.openarchives.org/OAI/2.0/oai_dc/" xsi:schemaLocation="http://www.openarchives.org/OAI/2.0/oai_dc/ http://www.openarchives.org/OAI/2.0/oai_dc.xsd"> https://serval.unil.ch/notice/serval:BIB_25492 Du droit international comparé de droits de l'homme dans la jurisprudence de la Cour européenne des droits de l'homme. Flauss, JF info:eu-repo/semantics/bookPart incollection 2002 Le rôle du droit comparé dans l'avènement du droit européen, vol. 43, pp. 159-182 Widmer, P. (ed.) oai:serval.unil.ch:BIB_25493 2022-05-07T01:13:03Z <oai_dc:dc xmlns:dc="http://purl.org/dc/elements/1.1/" xmlns:xs="http://www.w3.org/2001/XMLSchema" xmlns:xsi="http://www.w3.org/2001/XMLSchema-instance" xmlns:oai_dc="http://www.openarchives.org/OAI/2.0/oai_dc/" xsi:schemaLocation="http://www.openarchives.org/OAI/2.0/oai_dc/ http://www.openarchives.org/OAI/2.0/oai_dc.xsd"> https://serval.unil.ch/notice/serval:BIB_25493 Dualisme juridictionnel et Convention européenne des droits de l'homme. Flauss, JF info:eu-repo/semantics/bookPart incollection 2002 Liber amicorum J. Waline, Gouverner, administrer, juger, pp. 523-546 oai:serval.unil.ch:BIB_25494 2022-05-07T01:13:03Z <oai_dc:dc xmlns:dc="http://purl.org/dc/elements/1.1/" xmlns:xs="http://www.w3.org/2001/XMLSchema" xmlns:xsi="http://www.w3.org/2001/XMLSchema-instance" xmlns:oai_dc="http://www.openarchives.org/OAI/2.0/oai_dc/" xsi:schemaLocation="http://www.openarchives.org/OAI/2.0/oai_dc/ http://www.openarchives.org/OAI/2.0/oai_dc.xsd"> https://serval.unil.ch/notice/serval:BIB_25494 Pathologies infectieuses en réanimation Charbonneau, P Praz, G Glauser, M.P info:eu-repo/semantics/book book 2002 oai:serval.unil.ch:BIB_25495 2022-05-07T01:13:03Z <oai_dc:dc xmlns:dc="http://purl.org/dc/elements/1.1/" xmlns:xs="http://www.w3.org/2001/XMLSchema" xmlns:xsi="http://www.w3.org/2001/XMLSchema-instance" xmlns:oai_dc="http://www.openarchives.org/OAI/2.0/oai_dc/" xsi:schemaLocation="http://www.openarchives.org/OAI/2.0/oai_dc/ http://www.openarchives.org/OAI/2.0/oai_dc.xsd"> https://serval.unil.ch/notice/serval:BIB_25495 La primauté des droits de l'homme : licéité ou illicéité de l'intervention humanitaire. Flauss, JF info:eu-repo/semantics/bookPart incollection 2002 Le Kosovo et la Communauté internationale - L'histoire, l'actualité et l'avenir. Kosovo and the International Community, pp. 87-102 Tomuschat, C (ed.) oai:serval.unil.ch:BIB_254951546C6B 2022-05-07T01:13:03Z <oai_dc:dc xmlns:dc="http://purl.org/dc/elements/1.1/" xmlns:xs="http://www.w3.org/2001/XMLSchema" xmlns:xsi="http://www.w3.org/2001/XMLSchema-instance" xmlns:oai_dc="http://www.openarchives.org/OAI/2.0/oai_dc/" xsi:schemaLocation="http://www.openarchives.org/OAI/2.0/oai_dc/ http://www.openarchives.org/OAI/2.0/oai_dc.xsd"> https://serval.unil.ch/notice/serval:BIB_254951546C6B MR gated subtraction angiography: evaluation of lower extremities info:eu-repo/semantics/altIdentifier/pmid/3961174 Meuli, R. A. Wedeen, V. J. Geller, S. C. Edelman, R. R. Frank, L. R. Brady, T. J. Rosen, B. R. info:eu-repo/semantics/article article 1986-05 Radiology, vol. 159, no. 2, pp. 411-8 info:eu-repo/semantics/altIdentifier/pissn/0033-8419 <![CDATA[We report the first clinical experience with a new method for projective imaging of blood vessels (angiography) using magnetic resonance. Vascular contrast is produced noninvasively by the phase response of moving protons. Diastolic and systolic gated images produce, respectively, flow signal and flow void; the difference image is a map of the pulsatile flow: an arteriogram. Preliminary studies are presented of the lower extremities of one healthy volunteer and four patients (one each with occlusive disease, soft-tissue tumor, arteriovenous malformation, and venous femoral-popliteal graft). Patient data are compared with accompanying conventional arteriograms, and the new method is discussed

Appropriateness of colorectal cancer screening: appraisal of evidence by experts

Objectives. To evaluate how the level of evidence perceived by an international panel of experts was concordant with the level of evidence found in the literature, to compare experts perceived level of evidence to their appropriateness scores, and to compare appropriateness criteria for colonoscopy between experts and an evidence-based approach. Design. Comparison of expert panel opinions and systematic literature review regarding the level of evidence and appropriateness of colonoscopy indications. Participants. European Panel on the Appropriateness of Gastrointestinal Endoscopy multidisciplinary experts from 14 European countries. Main outcome measures. Concordance and weighted kappa coefficient between level of evidence as perceived by the experts' and that found in the literature, and between panel- and literature-based appropriateness categories. Results. Experts overestimated the level of published evidence of 57 indications. Concordance between the level of evidence perceived by the experts and the actual level of evidence found in the literature was 36% (weighted kappa 0.18). Indications for colonoscopy were reported to be appropriate, uncertain, and inappropriate by the experts in 54, 19, and 27% of the cases, and by the literature in 37, 46, and 17% of the cases. A 46% agreement (weighted kappa 0.29) was found between literature-based and experts' appropriateness criteria. Conclusions. Experts often overestimated the level of evidence on which they based their decisions. However, rarely did the experts' judgement completely disagree with the literature, although concordance between panel- and literature-based appropriateness was only fair. A more explicit discussion of existing evidence should be undertaken with the experts before they evaluate appropriateness criteri

A J-Like Protein Influences Fatty Acid Composition of Chloroplast Lipids in Arabidopsis

A comprehensive understanding of the lipid and fatty acid metabolic machinery is needed for optimizing production of oils and fatty acids for fuel, industrial feedstocks and nutritional improvement in plants. T-DNA mutants in the poorly annotated Arabidopsis thaliana gene At1g08640 were identified as containing moderately high levels (50–100%) of 16∶1Δ7 and 18∶1Δ9 leaf fatty acids and subtle decreases (5–30%) of 16∶3 and 18∶3 (http://www.plastid.msu.edu/). TLC separation of fatty acids in the leaf polar lipids revealed that the chloroplastic galactolipids monogalactosyldiacylglycerol (MGDG) and digalactosyldiacylglycerol (DGDG) were the main lipid types affected by this mutation. Analysis of the inferred amino acid sequence of At1g08640 predicted the presence of a transit peptide, three transmembrane domains and an N-terminal J-like domain, and the gene was named CJD1 for Chloroplast J-like Domain 1. GFP reporter experiments and in vitro chloroplast import assays demonstrated CJD1 is a chloroplast membrane protein. Screening of an Arabidopsis cDNA library by yeast-2-hybrid (Y2H) using the J-like domain of CJD1 as bait identified a plastidial inner envelope protein (Accumulation and Replication of Chloroplasts 6, ARC6) as the primary interacting partner in the Y2H assay. ARC6 plays a central role in chloroplast division and binds CJD1 via its own J-like domain along with an adjacent conserved region whose function is not fully known. These results provide a starting point for future investigations of how mutations in CJD1 affect lipid composition

Probing Arabidopsis Chloroplast Diacylglycerol Pools by Selectively Targeting Bacterial Diacylglycerol Kinase to Suborganellar Membranes

Diacylglycerol (DAG) is an intermediate in metabolism of both triacylglycerols and membrane lipids. Probing the steady-state pools of DAG and understanding how they contribute to the synthesis of different lipids is important when designing plants with altered lipid metabolism. However, traditional methods of assaying DAG pools are difficult, because its abundance is low and because fractionation of subcellular membranes affects DAG pools. To manipulate and probe DAG pools in an in vivo context, we generated multiple stable transgenic lines of Arabidopsis (Arabidopsis thaliana) that target an Escherichia coli DAGkinase (DAGK) to each leaflet of each chloroplast envelope membrane. E. coli DAGKis small, self inserts into membranes, and has catalytic activity on only one side of each membrane. By comparing whole-tissue lipid profiles between our lines, we show that each line has an individual pattern of DAG, phosphatidic acid, phosphatidylcholine, and triacylglycerol steady-state levels, which supports an individual function of DAG in each membrane leaflet. Furthermore, conversion of DAG in the leaflets facing the chloroplast intermembrane space by DAGK impairs plant growth. As a result of DAGK presence in the outer leaflet of the outer envelope membrane, phosphatidic acid accumulation is not observed, likely because it is either converted into other lipids or removed to othermembranes. Finally, we use the outer envelope-targeted DAGK line as a tool to probe the accessibility of DAG generated in response to osmotic stress

Low-coverage massively parallel pyrosequencing of cDNAs enables proteomics in non-model species: Comparison of a species-specific database generated by pyrosequencing with databases from related species for proteome analysis of pea chloroplast envelopes

Bräutigam A, Shrestha RP, Whitten D, et al. Low-coverage massively parallel pyrosequencing of cDNAs enables proteomics in non-model species: Comparison of a species-specific database generated by pyrosequencing with databases from related species for proteome analysis of pea chloroplast envelopes. Journal of Biotechnology. 2008;136(1-2):44-53.Proteomics is a valuable tool for establishing and comparing the protein content of defined tissues, cell types, or subcellular structures. Its use in non-model species is currently limited because the identification of peptides Critically depends on sequence databases. In this study, we explored the potential of a preliminary cDNA database for the non-model species Pisum sativum created by a small number of massively parallel pyrosequencing (MPSS) runs for its use in proteomics and compared it to comprehensive cDNA databases from Medicago truncatula and Arabidopsis thaliana created by Sanger sequencing. Each database was used to identify Proteins from a pea leaf chloroplast envelope preparation. It is shown that the pea database identified more proteins with higher accuracy, although the sequence quality was low and the sequence contigs were short compared to databases from model species. Although the number of identified proteins in non-species-specific databases could potentially be increased by lowering the threshold for Successful protein identifications, this strategy markedly increases the number of wrongly identified proteins. The identification rate with non-species-specific databases correlated with spectral abundance but not with the predicted membrane helix content, and Strong conservation is necessary but not sufficient for protein identification with a non-species-specific database. It is concluded that massively Parallel sequencing of cDNAs substantially increases the power Of proteomics in non-model species. (C) 2008 Elsevier B.V. All rights reserved