Combined approaches provide an anatomical and transcriptomic fingerprint of maize cell wall digestibility

Understanding cell wall biosynthesis and degradation in grasses has become a major aim in plant biology. Although independent previous reports have focused on specific features that dictate cell wall digestibility, cytological, biochemical, and gene regulation parameters have never been integrated within the same study. Herein, we applied a combination of state-of-the-art technologies and different scales of observation on two maize lines that are characterized by highly contrasted forage digestibility. Comparative image analysis of internode sections allow to get an anatomical fingerprint associated with high digestibility: a thin peripheral rind of lignified parenchyma, small numerous vascular bundles, and low proportion of PeriVascular Sclerenchyma (PVS). This cell type patterning led to enhanced digestibility when internode sections were treated with Celluclast, a commercially cell wall degrading enzyme. At a lower scale of observation, Laser Capture Microdissection (LCM) followed by thioacidolysis of PVS revealed a higher proportion of Syringyl (S) unit lignins in the low digestible line while the high digestible line was p-Hydroxyphenyl (H)-rich. Moreover, cytological observation of internodes of the two lines point out that this difference in composition is associated with a delayed lignification of PVS. At the same time, comparative transcriptomics on internodes indicated differential expression of several genes encoding enzymes along the phenylpropanoid pathway and known cell wall-associated Transcription Factors (TFs). Together, these results give an integrative view of different factors which could aim in designing a maize silage ideotype and provide a novel set of potential regulatory genes controlling lignification in maize

Variation in Structure and Process of Care in Traumatic Brain Injury: Provider Profiles of European Neurotrauma Centers Participating in the CENTER-TBI Study.

INTRODUCTION: The strength of evidence underpinning care and treatment recommendations in traumatic brain injury (TBI) is low. Comparative effectiveness research (CER) has been proposed as a framework to provide evidence for optimal care for TBI patients. The first step in CER is to map the existing variation. The aim of current study is to quantify variation in general structural and process characteristics among centers participating in the Collaborative European NeuroTrauma Effectiveness Research in Traumatic Brain Injury (CENTER-TBI) study. METHODS: We designed a set of 11 provider profiling questionnaires with 321 questions about various aspects of TBI care, chosen based on literature and expert opinion. After pilot testing, questionnaires were disseminated to 71 centers from 20 countries participating in the CENTER-TBI study. Reliability of questionnaires was estimated by calculating a concordance rate among 5% duplicate questions. RESULTS: All 71 centers completed the questionnaires. Median concordance rate among duplicate questions was 0.85. The majority of centers were academic hospitals (n = 65, 92%), designated as a level I trauma center (n = 48, 68%) and situated in an urban location (n = 70, 99%). The availability of facilities for neuro-trauma care varied across centers; e.g. 40 (57%) had a dedicated neuro-intensive care unit (ICU), 36 (51%) had an in-hospital rehabilitation unit and the organization of the ICU was closed in 64% (n = 45) of the centers. In addition, we found wide variation in processes of care, such as the ICU admission policy and intracranial pressure monitoring policy among centers. CONCLUSION: Even among high-volume, specialized neurotrauma centers there is substantial variation in structures and processes of TBI care. This variation provides an opportunity to study effectiveness of specific aspects of TBI care and to identify best practices with CER approaches

Variation in general supportive and preventive intensive care management of traumatic brain injury: a survey in 66 neurotrauma centers participating in the Collaborative European NeuroTrauma Effectiveness Research in Traumatic Brain Injury (CENTER-TBI) study

Abstract Background General supportive and preventive measures in the intensive care management of traumatic brain injury (TBI) aim to prevent or limit secondary brain injury and optimize recovery. The aim of this survey was to assess and quantify variation in perceptions on intensive care unit (ICU) management of patients with TBI in European neurotrauma centers. Methods We performed a survey as part of the Collaborative European NeuroTrauma Effectiveness Research in Traumatic Brain Injury (CENTER-TBI) study. We analyzed 23 questions focused on: 1) circulatory and respiratory management; 2) fever control; 3) use of corticosteroids; 4) nutrition and glucose management; and 5) seizure prophylaxis and treatment. Results The survey was completed predominantly by intensivists (n = 33, 50%) and neurosurgeons (n = 23, 35%) from 66 centers (97% response rate). The most common cerebral perfusion pressure (CPP) target was > 60 mmHg (n = 39, 60%) and/or an individualized target (n = 25, 38%). To support CPP, crystalloid fluid loading (n = 60, 91%) was generally preferred over albumin (n = 15, 23%), and vasopressors (n = 63, 96%) over inotropes (n = 29, 44%). The most commonly reported target of partial pressure of carbon dioxide in arterial blood (PaCO2) was 36–40 mmHg (4.8–5.3 kPa) in case of controlled intracranial pressure (ICP) < 20 mmHg (n = 45, 69%) and PaCO2 target of 30–35 mmHg (4–4.7 kPa) in case of raised ICP (n = 40, 62%). Almost all respondents indicated to generally treat fever (n = 65, 98%) with paracetamol (n = 61, 92%) and/or external cooling (n = 49, 74%). Conventional glucose management (n = 43, 66%) was preferred over tight glycemic control (n = 18, 28%). More than half of the respondents indicated to aim for full caloric replacement within 7 days (n = 43, 66%) using enteral nutrition (n = 60, 92%). Indications for and duration of seizure prophylaxis varied, and levetiracetam was mostly reported as the agent of choice for both seizure prophylaxis (n = 32, 49%) and treatment (n = 40, 61%). Conclusions Practice preferences vary substantially regarding general supportive and preventive measures in TBI patients at ICUs of European neurotrauma centers. These results provide an opportunity for future comparative effectiveness research, since a more evidence-based uniformity in good practices in general ICU management could have a major impact on TBI outcome

Preparation of antibodies and development of an enzyme-linked immunosorbent assay for determination of dealkylated hydroxytriazines

9 pages, 4 figures, 6 tables.-- PMID: 12502401 [PubMed].-- Available online Nov 22, 2002.The development of an indirect competitive enzyme-linked immunosorbent assay (ELISA) for dealkylated hydroxytriazines is reported here for the first time. The assay uses polyclonal antibodies raised against N-(4-amine-6-hydroxy-[1,3,5]triazin-2-yl)-4-aminobutanoic acid (hapten 2g) conjugated to keyhole limpet hemocyanin by the active ester method. The immunizing hapten was synthesized by first introducing the amino group to the triazine ring in a protected form in order to increase its solubility in organic media. Subsequent steps consisted of reacting this compound with an appropriate spacer arm, followed by removal of the protecting group in acidic media. The resulting assay uses a homologous competitor hapten coupled to conalbumin by the mixed anhydride method. Coating antigens prepared using a homologous covalent coupling procedure failed to produce competitive immunoassays. The assay tolerates media with high ionic strength (up to 70 mS cm-1) and basic pH values (7.5−9.5 units). Under the optimized conditions, this ELISA is specific for dealkylated hydroxytriazines, reaching suitable limits of detection.This work has been supported by the EC Program (Contract QLRT-2000-01670) and by CICYT (BIO2000-0351-P4-05 and AGL2001-5005-E).Peer reviewe

An attempt to characterize the human Chorionic Gonadotropin protein by reversed phase liquid chromatography coupled with high-resolution mass spectrometry at the intact level

For the first time, the human Chorionic Gonadotropin (hCG) hormone at the intact level was character-ized by reversed phase liquid chromatography (RPLC) coupled with high resolution mass spectrometry(HRMS). This heterodimeric protein is specific to human pregnancy, consists in an and a subunit, so-called hCG and hCG, respectively, and has 8 glycosylation sites leading to a high number of isoforms.First, the LC method was optimized to separate the largest number of isoforms and also to facilitate theMS ionization process and data treatment. The initial mobile phase composition, slope of the gradient,and column temperature were appropriately selected to maximize the number of separated isoforms.Moreover, the MS detection parameters were adjusted to i) promote the efficient transfer of the heaviestions, ii) avoid or limit the fragmentation of the ions and iii) improve the sensitivity. The repeatability ofthe final method in terms of retention times and peak areas was assessed. The method was next used tocharacterize two hCG-based drugs: Ovitrelle®(a recombinant hCG, r-hCG) and Pregnyl®(hCG isolatedfrom urine of pregnant women, u-hCG). After the deconvolution step, the analytical method did not allowto observe the isoforms of the hCG. This may be due to its dramatic higher heterogeneity induced byits 6 glycosylation sites and a lack of ionization in the MS source. Nevertheless, the results revealed thepresence of more than 30 hCG isoforms, which differ by their number and their nature in the two drugs.Then, the molecular weights of the N-glycans already described in the literature for hCG were compiledin a database to identify the hCG glycoforms by mass matching. This strategy was successfully appliedfor the identification of five glycoforms for both r-hCG and u-hCG. This work demonstrates for the firsttime the potential of RPLC-HRMS for the identification of the intact hCG glycoforms

An attempt to characterize the human Chorionic Gonadotropin protein by reversed phase liquid chromatography coupled with high-resolution mass spectrometry at the intact level

For the first time, the human Chorionic Gonadotropin (hCG) hormone at the intact level was character-ized by reversed phase liquid chromatography (RPLC) coupled with high resolution mass spectrometry(HRMS). This heterodimeric protein is specific to human pregnancy, consists in an and a subunit, so-called hCG and hCG, respectively, and has 8 glycosylation sites leading to a high number of isoforms.First, the LC method was optimized to separate the largest number of isoforms and also to facilitate theMS ionization process and data treatment. The initial mobile phase composition, slope of the gradient,and column temperature were appropriately selected to maximize the number of separated isoforms.Moreover, the MS detection parameters were adjusted to i) promote the efficient transfer of the heaviestions, ii) avoid or limit the fragmentation of the ions and iii) improve the sensitivity. The repeatability ofthe final method in terms of retention times and peak areas was assessed. The method was next used tocharacterize two hCG-based drugs: Ovitrelle®(a recombinant hCG, r-hCG) and Pregnyl®(hCG isolatedfrom urine of pregnant women, u-hCG). After the deconvolution step, the analytical method did not allowto observe the isoforms of the hCG. This may be due to its dramatic higher heterogeneity induced byits 6 glycosylation sites and a lack of ionization in the MS source. Nevertheless, the results revealed thepresence of more than 30 hCG isoforms, which differ by their number and their nature in the two drugs.Then, the molecular weights of the N-glycans already described in the literature for hCG were compiledin a database to identify the hCG glycoforms by mass matching. This strategy was successfully appliedfor the identification of five glycoforms for both r-hCG and u-hCG. This work demonstrates for the firsttime the potential of RPLC-HRMS for the identification of the intact hCG glycoforms