Carbon-11 Reveals Opposing Roles of Auxin and Salicylic Acid in Regulating Leaf Physiology, Leaf Metabolism, and Resource Allocation Patterns that Impact Root Growth in Zea mays

Auxin (IAA) is an important regulator of plant development and root differentiation. Although recent studies indicate that salicylic acid (SA) may also be important in this context by interfering with IAA signaling, comparatively little is known about its impact on the plant’s physiology, metabolism, and growth characteristics. Using carbon-11, a short-lived radioisotope (t 1/2 = 20.4 min) administered as 11CO2 to maize plants (B73), we measured changes in these functions using SA and IAA treatments. IAA application decreased total root biomass, though it increased lateral root growth at the expense of primary root elongation. IAA-mediated inhibition of root growth was correlated with decreased 11CO2 fixation, photosystem II (PSII) efficiency, and total leaf carbon export of 11C-photoassimilates and their allocation belowground. Furthermore, IAA application increased leaf starch content. On the other hand, SA application increased total root biomass, 11CO2 fixation, PSII efficiency, and leaf carbon export of 11C-photoassimilates, but it decreased leaf starch content. IAA and SA induction patterns were also examined after root-herbivore attack by Diabrotica virgifera to place possible hormone crosstalk into a realistic environmental context. We found that 4 days after infestation, IAA was induced in the midzone and root tip, whereas SA was induced only in the upper proximal zone of damaged roots. We conclude that antagonistic crosstalk exists between IAA and SA which can affect the development of maize plants, particularly through alteration of the root system’s architecture, and we propose that the integration of both signals may shape the plant’s response to environmental stress.

Growth at moderately elevated temperature alters the physiological response of the photosynthetic apparatus to heat stress in pea (Pisum sativum L.) leaves

The impact of heat stress on the functioning of the photosynthetic apparatus was examined in pea (Pisum sativum L.) plants grown at control (25 °C; 25 °C-plants) or moderately elevated temperature (35 °C; 35 °C-plants). In both types of plants net photosynthesis (Pn) decreased with increasing leaf temperature (LT) and was more than 80% reduced at 45 °C as compared to 25 °C. In the 25 °C-plants, LTs higher than 40 °C could result in a complete suppression of Pn. Short-term acclimation to heat stress did not alter the temperature response of Pn. Chlorophyll a fluorescence measurements revealed that photosynthetic electron transport (PET) started to decrease when LT increased above 35 °C and that growth at 35 °C improved the thermal stability of the thylakoid membranes. In the 25 °C-plants, but not in the 35 °C-plants, the maximum quantum yield of the photosystem II primary photochemistry, as judged by measuring the Fv/Fm ratio, decreased significantly at LTs higher than 38 °C. A post-illumination heat-induced reduction of the plastoquinone pool was observed in the 25 °C-plants, but not in the 35 °C-plants. Inhibition of Pn by heat stress correlated with a reduction of the activation state of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco). Western-blot analysis of Rubisco activase showed that heat stress resulted in a redistribution of activase polypeptides from the soluble to the insoluble fraction of extracts. Heat-dependent inhibition of Pn and PET could be reduced by increasing the intercellular CO2 concentration, but much more effectively so in the 35 °C-plants than in the 25 °C-plants. The 35 °C-plants recovered more efficiently from heat-dependent inhibition of Pn than the 25 °C-plants. The results show that growth at moderately high temperature hardly diminished inhibition of Pn by heat stress that originated from a reversible heat-dependent reduction of the Rubisco activation state. However, by improving the thermal stability of the thylakoid membranes it allowed the photosynthetic apparatus to preserve its functional potential at high LTs, thus minimizing the after-effects of heat stress.

Inhibition of photosynthesis by high temperature in oak (Quercus pubescens L.) leaves grown under natural conditions closely correlates with a reversible heat-dependent reduction of the activation state of ribulose-1,5-bisphosphate carboxylase/oxygenase

Inhibition of the net photosynthetic CO2 assimilation rate (Pn) by high temperature was examined in oak (Quercus pubescens L.) leaves grown under natural conditions. Combined measurements of gas exchange and chlorophyll (Chl) a fluorescence were employed to differentiate between inhibition originating from heat effects on components of the thylakoid membranes and that resulting from effects on photosynthetic carbon metabolism. Regardless of whether temperature was increased rapidly or gradually, Pn decreased with increasing leaf temperature and was more than 90% reduced at 45 °C as compared to 25 °C. Inhibition of Pn by heat stress did not result from reduced stomatal conductance (gs), as heat-induced reduction of gs was accompanied by an increase of the intercellular CO2 concentration (Ci). Chl a fluorescence measurements revealed that between 25 and 45 °C heat-dependent alterations of thylakoid-associated processes contributed only marginally, if at all, to the inhibition of Pn by heat stress, with photosystem II being remarkably well protected against thermal inactivation. The activation state of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) decreased from about 90% at 25 °C to less than 30% at 45 °C. Heat stress did not affect Rubisco per se, since full activity could be restored by incubation with CO2 and Mg2+. Western-blot analysis of leaf extracts disclosed the presence of two Rubisco activase polypeptides, but heat stress did not alter the profile of the activase bands. Inhibition of Pn at high leaf temperature could be markedly reduced by artificially increasing Ci. A high Ci also stimulated photosynthetic electron transport and resulted in reduced non-photochemical fluorescence quenching. Recovery experiments showed that heat-dependent inhibition of Pn was largely, if not fully, reversible. The present results demonstrate that in Q. pubescens leaves the thylakoid membranes in general and photosynthetic electron transport in particular were well protected against heat-induced perturbations and that inhibition of Pn by high temperature closely correlated with a reversible heat-dependent reduction of the Rubisco activation state.

Primer extension based quantitative polymerase chain reaction reveals consistent differences in the methylation status of the MGMT promoter in diffusely infiltrating gliomas (WHO grade II-IV) of adults

Diffusely infiltrating gliomas (WHO grade II-IV) are the most common primary brain tumours in adults. These tumours are not amenable to cure by surgery alone, so suitable biomarkers for adjuvant modalities are required to guide therapeutic decision-making. Epigenetic silencing of the O(6)-methylguanine-DNA methyltransferase (MGMT) gene by promoter methylation has been associated with longer survival of patients with high-grade gliomas who receive alkylating chemotherapy; and molecular testing for the methylation status of the MGMT promoter sequence is regarded as among the most relevant of such markers. We have developed a primer extension-based assay adapted to formalin-fixed paraffin-embedded tissues that enables quantitative assessment of the methylation status of the MGMT promoter. The assay is very sensitive, highly reproducible, and provides valid test results in nearly 100% of cases. Our results indicate that oligodendrogliomas, empirically known to have a relatively favourable prognosis, are also the most homogeneous entities in terms of MGMT promoter methylation. Conversely, astrocytomas, which are more prone to spontaneous progression to higher grade malignancy, are significantly more heterogeneous. In addition, we show that the degree of promoter methylation correlates with the prevalence of loss of heterozygosity on chromosome arm 1p in the oligodendroglioma group, but not the astrocytoma group. Our results may have potentially important implications for clinical molecular diagnosis.

Evaluation of the diagnostic value of serologic microagglutination testing and a polymerase chain reaction assay for diagnosis of acute leptospirosis in dogs in a referral center.

OBJECTIVE To determine the diagnostic value of a serologic microagglutination test (MAT) and a PCR assay on urine and blood for the diagnosis of leptospirosis in dogs with acute kidney injury (AKI). DESIGN Cross-sectional study. Animals-76 dogs with AKI in a referral hospital (2008 to 2009). PROCEDURES Dogs' leptospirosis status was defined with a paired serologic MAT against a panel of 11 Leptospira serovars as leptospirosis-associated (n = 30) or nonleptospirosis-associated AKI (12). In 34 dogs, convalescent serologic testing was not possible, and leptospirosis status was classified as undetermined. The diagnostic value of the MAT single acute or convalescent blood sample was determined in dogs in which leptospirosis status could be classified. The diagnostic value of a commercially available genus-specific PCR assay was evaluated by use of 36 blood samples and 20 urine samples. RESULTS Serologic acute testing of an acute blood sample had a specificity of 100% (95% CI, 76% to 100%), a sensitivity of 50% (33% to 67%), and an accuracy of 64% (49% to 77%). Serologic testing of a convalescent blood sample had a specificity of 92% (65% to 99%), a sensitivity of 100% (87% to 100%), and an accuracy of 98% (88% to 100%). Results of the Leptospira PCR assay were negative for all samples from dogs for which leptospirosis status could be classified. CONCLUSIONS AND CLINICAL RELEVANCE Serologic MAT results were highly accurate for diagnosis of leptospirosis in dogs, despite a low sensitivity for early diagnosis. In this referral setting of dogs pretreated with antimicrobials, testing of blood and urine samples with a commercially available genus-specific PCR assay did not improve early diagnosis.

Reaction of Copper(II) Chloroacetate with Pyrazole. Synthesis of a One-Dimensional Coordination Polymer and Unexpected Dehydrochlorination Reaction

The reaction of copper(II) chloroacetate (1d) with pyrazole (Hpz) mainly yielded the mononuclear compound [Cu(μ-ClCH2COO)2(Hpz)2] (2m), which self-assembled generating a one-dimensional coordination polymer. Moreover, the concomitant isolation of the tetranuclear [{Cu2(μ-pz)(μ-OCH2COO)(Hpz)(MeOH)}2(μ-ClCH2COO)2] (3t) and hexanuclear [{Cu3(μ3-OH)(μ-pz)3(Hpz)2}2(μ-ClCH2COO)2](Cl)2 (4h) species evidenced the occurrence of a peculiar, previously unreported, dehydrochlorination reaction and the formation of the trinuclear triangular moiety [Cu3(μ3-OH)(μ-pz)3]. Theoretical calculations based on density functional theory including solvation effects indicate a possible pathway for the formation of 3t. Interestingly, besides the energy minimum corresponding to 3t, a further relative energy minimum is found for a species which can be considered a possible reaction intermediate.

Local Heat Application for the Treatment of Buruli Ulcer: Results of a Phase II Open Label Single Center Non Comparative Clinical Trial.

BACKGROUND Buruli ulcer (BU) is a necrotizing skin disease most prevalent among West African children. The causative organism, Mycobacterium ulcerans, is sensitive to temperatures above 37°C. We investigated the safety and efficacy of a local heat application device based on phase change material. METHODS In a phase II open label single center noncomparative clinical trial (ISRCTN 72102977) under GCP standards in Cameroon, laboratory confirmed BU patients received up to 8 weeks of heat treatment. We assessed efficacy based on the endpoints 'absence of clinical BU specific features' or 'wound closure' within 6 months ("primary cure"), and 'absence of clinical recurrence within 24 month' ("definite cure"). RESULTS Of 53 patients 51 (96%) had ulcerative disease. 62% were classified as World Health Organization category II, 19% each as category I and III. The average lesion size was 45 cm(2). Within 6 months after completion of heat treatment 92.4% (49 of 53, 95% confidence interval [CI], 81.8% to 98.0%) achieved cure of their primary lesion. At 24 months follow-up 83.7% (41 of 49, 95% CI, 70.3% to 92.7%) of patients with primary cure remained free of recurrence. Heat treatment was well tolerated; adverse effects were occasional mild local skin reactions. CONCLUSIONS Local thermotherapy is a highly effective, simple, cheap and safe treatment for M. ulcerans disease. It has in particular potential as home-based remedy for BU suspicious lesions at community level where laboratory confirmation is not available. CLINICAL TRIALS REGISTRATION ISRCT 72102977.

Safety and Efficacy of Ranibizumab in Diabetic Macular Edema (RESOLVE Study): A 12-month, randomized, controlled, double-masked, multicenter phase II study

The expression of vascular endothelial growth factor (VEGF) is elevated in diabetic macular edema (DME). Ranibizumab binds to and inhibits multiple VEGF variants. We investigated the safety and efficacy of ranibizumab in DME involving the foveal center.

Early non-invasive cardiac output monitoring in hemodynamically unstable intensive care patients: A multi-center randomized controlled trial

Introduction Acute hemodynamic instability increases morbidity and mortality. We investigated whether early non-invasive cardiac output monitoring enhances hemodynamic stabilization and improves outcome. Methods A multicenter, randomized controlled trial was conducted in three European university hospital intensive care units in 2006 and 2007. A total of 388 hemodynamically unstable patients identified during their first six hours in the intensive care unit (ICU) were randomized to receive either non-invasive cardiac output monitoring for 24 hrs (minimally invasive cardiac output/MICO group; n = 201) or usual care (control group; n = 187). The main outcome measure was the proportion of patients achieving hemodynamic stability within six hours of starting the study. Results The number of hemodynamic instability criteria at baseline (MICO group mean 2.0 (SD 1.0), control group 1.8 (1.0); P = .06) and severity of illness (SAPS II score; MICO group 48 (18), control group 48 (15); P = .86)) were similar. At 6 hrs, 45 patients (22%) in the MICO group and 52 patients (28%) in the control group were hemodynamically stable (mean difference 5%; 95% confidence interval of the difference -3 to 14%; P = .24). Hemodynamic support with fluids and vasoactive drugs, and pulmonary artery catheter use (MICO group: 19%, control group: 26%; P = .11) were similar in the two groups. The median length of ICU stay was 2.0 (interquartile range 1.2 to 4.6) days in the MICO group and 2.5 (1.1 to 5.0) days in the control group (P = .38). The hospital mortality was 26% in the MICO group and 21% in the control group (P = .34). Conclusions Minimally-invasive cardiac output monitoring added to usual care does not facilitate early hemodynamic stabilization in the ICU, nor does it alter the hemodynamic support or outcome. Our results emphasize the need to evaluate technologies used to measure stroke volume and cardiac output--especially their impact on the process of care--before any large-scale outcome studies are attempted.

Analysis of lorazepam and its 30-glucuronide in human urine by capillary electrophoresis: evidence for the formation of two distinct diastereoisomeric glucuronides

Lorazepam (LOR) is a 3-hydroxy-1,4-benzodiazepine that is chiral and undergoes enantiomerization at room temperature. In humans, about 75% of the administered dose of LOR is excreted in the urine as its 30-glucuronide. CE-MS with negative ESI was used to confirm the presence of LOR-30-glucuronide in urines that stemmed from a healthy individual who ingested 1 or 2 mg LOR, whereas free LOR could be detected in extracts prepared from enzymatically hydrolyzed urines. As the 30-glucuronidation reaction occurs at the chiral center of the molecule, two diastereoisomers can theoretically be formed, molecules that can no longer interconvert. The stereoselective formation of LOR glucuronides in humans and in vitro was investigated. MEKC analysis of extracts of the nonhydrolyzed urines suggested the presence of the two different LOR glucuronides in the urine. The formation of the same two diastereoisomers was also observed in vitro employing incubations of LOR with human liver microsomes in the presence of uridine 5'-diphospho-glucuronic acid as coenzyme. The absence of other coenzymes excluded the formation of phase I or other phase II metabolites of LOR. Both results revealed a stereoselectivity, one diastereoisomer being formed in a higher amount than the other. After enzymatic hydrolysis using beta-glucuronidase, these peaks could not be detected any more. Instead, LOR was monitored. Analysis of the extracts prepared from enzymatically hydrolyzed urines by MEKC in the presence of 2-hydroxypropyl-beta-CD revealed the enantiomerization process of LOR (observation of two peaks of equal magnitude connected with a plateau zone). The data presented provide for the first time the evidence of the stereoselectivity of the LOR glucuronidation in humans.

14-3-3- and II/3-10-gene expression as molecular markers to address viability and growth activity of Echinococcus multilocularis metacestodes

The present study aimed to search for and characterize parasite molecules, whose expression levels correlate with the viability and growth activity of Echinococcus multilocularis metacestodes. We focused on the expression profiles of 2 parasite-derived genes, 14-3-3 and II/3-10, as putative molecular markers for viability and growth activity of the larval parasite. In experiments in vivo, gene expression levels of 14-3-3 and II/3-10 were relatively quantified by real-time reverse transcription-PCR using a housekeeping gene, beta-actin, as a reference reaction. All three reactions were compared with growth activity of the parasite developing in permissive nu/nu and in non-permissive wild type BALB/c mice. At 2 months p.i., the transcription level of 14-3-3 was significantly higher in parasites actively proliferating in nu/nu mice compared to parasites moderately growing in wild type mice. Immunoblotting experiments confirmed at the protein level that 14-3-3 was over-expressed in parasites derived from nu/nu mice at 2 months p.i. In vitro treatment of E. multilocularis with an anti-echinococcal drug nitazoxanide resulted in a significant decrease of both 14-3-3 and II/3-10 transcription levels found after 8 days of treatment, which correlated with the kinetics of a housekeeping gene, beta-actin. The conclusion is that 14-3-3, combined with II/3-10, exhibits good potential as a molecular marker to assess viability and growth activity of the parasite.

Supported Cu(II) polymer catalysts for aqueous phenol oxidation

Supported Cu(II) polymer catalysts were used for the catalytic oxidation of phenol at 30 degrees C and atmospheric pressure using air and H(2)O(2) as oxidants. Heterogenisation of homogeneous Cu(II) catalysts was achieved by adsorption of Cu(II) salts onto polymeric matrices (poly(4-vinylpyridine), Chitosan). The catalytic active sites were represented by Cu(II) ions and showed to conserve their oxidative activity in heterogeneous catalysis as well as in homogeneous systems. The catalytic deactivation was evaluated by quantifying released Cu(II) ions in solution during oxidation, from where Cu-PVP(25) showed the best leaching levels no more than 5 mg L(-1). Results also indicated that Cu-PVP(25) had a catalytic activity (56% of phenol conversion when initial Cu(II) catalytic content was 200 mg L(Reaction)(-1)) comparable to that of commercial catalysts (59% of phenol conversion). Finally, the balance between activity and copper leaching was better represented by Cu-PVP(25) due to the heterogeneous catalytic activity had 86% performance in the heterogeneous phase, and the rest on the homogeneous phase, while Cu-PVP(2) had 59% and CuO/gamma-Al(2)O(3) 68%.

Individual versus standard quality of life assessment in a phase II clinical trial in mesothelioma patients: feasibility and responsiveness to clinical changes

In patients with malignant pleural mesothelioma undergoing a multimodality therapy, treatment toxicity may outweigh the benefit of progression-free survival. The subjective experience across different treatment phases is an important clinical outcome. This study compares a standard with an individual quality of life (QoL) measure used in a multi-center phase II trial.

A multi-center retrospective analysis of treatment effects and quality of life in adult patients with cranial ependymomas

Long term quality of life data of adult patients harboring intracranial ependymomas have not been reported. The role of adjuvant radiation therapy in Grade II ependymomas is unclear and differs from study to study. We therefore sought to retrospectively analyze outcome and quality of life of adult patients that were operated on intracranial ependymomas at four different surgical centers in two countries. All patients were attempted to be contacted via telephone to assess quality of life (QoL) at the time of the telephone interview. The standard EORTC QoL Questionnaire C30 (EORTC QLQ-C30) and the EORTC QLQ-Brain Cancer Module (QLQ-BN20) were used. 64 adult patients with intracranial ependymomas were included in the study. The only factor that was associated with increased survival was age <55 years (p < 0.001). Supratentorial location was correlated with shorter progression free survival than infratentorial location (PFS; p = 0.048). In WHO Grade II tumors local irradiation did not lead to increased PFS (p = 0.888) or overall survival (p = 0.801). Even for incompletely resected Grade II tumors local irradiation did not lead to a benefit in PFS (p = 0.911). In a multivariate analysis of QoL, irradiated patients had significantly worse scores in the item "fatigue" (p = 0.037) than non-irradiated patients. Here we present QoL data of adult patients with intracranial ependymomas. Our data show that local radiation therapy may have long-term effects on patients' QoL. Since in the incompletely resected Grade II tumors local irradiation did not lead to a benefit in PFS in this retrospective study, prospective randomized studies are necessary. In addition to age, supratentorial tumor location is associated with a worse prognosis in adult ependymoma patients.