A simple enzymatic assay for the quantification of C1-specific cellulose oxidation by lytic polysaccharide monooxygenases.

OBJECTIVE: The development of an enzymatic assay for the specific quantification of the C1-oxidation product, i.e. gluconic acid of cellulose active lytic polysaccharide monooxygenases (LPMOs). RESULTS: In combination with a ß-glucosidase, the spectrophotometrical assay can reliably quantify the specific C1- oxidation product of LPMOs acting on cellulose. It is applicable for a pure cellulose model substrate as well as lignocellulosic biomass. The enzymatic assay compares well with the quantification performed by HPAEC-PAD. In addition, we show that simple boiling is not sufficient to inactivate LPMOs and we suggest to apply a metal chelator in addition to boiling or to drastically increase pH for proper inactivation. CONCLUSIONS: We conclude that the versatility of this simple enzymatic assay makes it useful in a wide range of experiments in basic and applied LPMO research and without the need for expensive instrumentation, e.g. HPAEC-PAD.

Proteomic profile of Piper tuberculatum (Piperaceae) / Perfil proteômico e metabólico de Piper tuberculatum (Piperaceae)

Abstract Piper tuberculatum (Piperaceae) is a species that accumulates especially amides as secondary metabolites and several biological activities was previously reported. In this article, we report a proteomic study of P. tuberculatum. Bidimensional electrophoresis (2D SDS-PAGE) and mass spectrometry (ESI-Q-TOF) were used in this study. Over a hundred spots and various peptides were identified in this species and the putative functions of these peptides related to defense mechanism as biotic and abiotic stress were assigned. The information presented extend the range of molecular information of P. tuberculatum.

Resumo Piper tuberculatum (Piperaceae) é uma espécie que acumula especialmente amidas como metabólitos secundários e diversas atividades biológicas dessa espécie foram relatadas anteriormente. No presente artigo, relatamos um estudo proteômico dessa espécie. Eletroforese bidimensional (2D SDS-PAGE) e espectrometria de massas (ESI-Q-TOF) foram utilizadas nesse estudos. Mais de cem spots e vários peptídeos foram identificados nesta espécie e as funções putativas desses peptídeos relacionadas a mecanismo de defesa como estresse biótico e abiótico foram atribuídos. As informações apresentadas ampliam a gama de informações moleculares dessa espécie.

Proteomic profile of Piper tuberculatum (Piperaceae).

Piper tuberculatum (Piperaceae) is a species that accumulates especially amides as secondary metabolites and several biological activities was previously reported. In this article, we report a proteomic study of P. tuberculatum. Bidimensional electrophoresis (2D SDS-PAGE) and mass spectrometry (ESI-Q-TOF) were used in this study. Over a hundred spots and various peptides were identified in this species and the putative functions of these peptides related to defense mechanism as biotic and abiotic stress were assigned. The information presented extend the range of molecular information of P. tuberculatum.

Proteomic profile of Piper tuberculatum (Piperaceae)

Abstract Piper tuberculatum (Piperaceae) is a species that accumulates especially amides as secondary metabolites and several biological activities was previously reported. In this article, we report a proteomic study of P. tuberculatum. Bidimensional electrophoresis (2D SDS-PAGE) and mass spectrometry (ESI-Q-TOF) were used in this study. Over a hundred spots and various peptides were identified in this species and the putative functions of these peptides related to defense mechanism as biotic and abiotic stress were assigned. The information presented extend the range of molecular information of P. tuberculatum.

Resumo Piper tuberculatum (Piperaceae) é uma espécie que acumula especialmente amidas como metabólitos secundários e diversas atividades biológicas dessa espécie foram relatadas anteriormente. No presente artigo, relatamos um estudo proteômico dessa espécie. Eletroforese bidimensional (2D SDS-PAGE) e espectrometria de massas (ESI-Q-TOF) foram utilizadas nesse estudos. Mais de cem spots e vários peptídeos foram identificados nesta espécie e as funções putativas desses peptídeos relacionadas a mecanismo de defesa como estresse biótico e abiótico foram atribuídos. As informações apresentadas ampliam a gama de informações moleculares dessa espécie.

Factors associated with effective reassessment of antibiotic therapy on day 3.

UNLABELLED: Reassessment of antibiotic therapy (RA) after 3 days is constitutive of French antibiotic stewardship. This delay is required because of the need for clinical reappraisal and for obtaining microbiological data. Our aim was to determine the factors associated with an effective RA. PATIENTS AND METHOD: A prospective study was made in a 350-bed general hospital in which all prescriptions are computerized and validated daily by prescribers. All curative antibiotic therapies were reassessed during 4 weeks. RA was defined as effective if the initial antibiotic treatment was modified. All clinical, biological, and radiological data having contributed to the initial prescription and to RA were recorded during bedside visit with the prescribers, two hospital physicians and one infectious diseases specialist. RESULTS: In one month, 148 antibiotic treatments were reassessed. Pulmonary, digestive, and urinary infections accounted for two thirds of the cases. An effective RA was recorded in 28 cases (19%) and associated with hospitalization in the ICU (P=0.001), imaging supporting the diagnosis (P=0.016), and persistence or aggravation of clinical signs (P=0.007). Microbiological findings were not contributive to an effective RA. CONCLUSION: RA was associated to hospitalization in the ICU, to an inflammatory syndrome, and to the clinical outcome after 3 days. These results should help to improve the implementation of infectious diseases advice.

Proteomic analysis of Porphyromonas gingivalis exposed to nicotine and cotinine.

BACKGROUND AND OBJECTIVE: Smokers are more predisposed than nonsmokers to infection with Porphyromonas gingivalis, one of the most important pathogens involved in the onset and development of periodontitis. It has also been observed that tobacco, and tobacco derivatives such as nicotine and cotinine, can induce modifications to P. gingivalis virulence. However, the effect of the major compounds derived from cigarettes on expression of protein by P. gingivalis is poorly understood. Therefore, this study aimed to evaluate and compare the effects of nicotine and cotinine on the P. gingivalis proteomic profile. MATERIAL AND METHODS: Total proteins of P. gingivalis exposed to nicotine and cotinine were extracted and separated by two-dimensional electrophoresis. Proteins differentially expressed were successfully identified through liquid chromatography-mass spectrometry and primary sequence databases using MASCOT search engine, and gene ontology was carried out using DAVID tools. RESULTS: Of the approximately 410 protein spots that were reproducibly detected on each gel, 23 were differentially expressed in at least one of the treatments. A particular increase was seen in proteins involved in metabolism, virulence and acquisition of peptides, protein synthesis and folding, transcription and oxidative stress. Few proteins showed significant decreases in expression; those that did are involved in cell envelope biosynthesis and proteolysis and also in metabolism. CONCLUSION: Our results characterized the changes in the proteome of P. gingivalis following exposure to nicotine and cotinine, suggesting that these substances may modulate, with minor changes, protein expression. The present study is, in part, a step toward understanding the potential smoke-pathogen interaction that may occur in smokers with periodontitis.

Antimicrobial stewardship program and quality of antibiotic prescriptions.

OBJECTIVE: National recommendations have been issued to define which optimal organization in hospitals could improve the quality of antibiotic prescription. Our aim was to check whether there was a link between applying these national recommendations and the quality of antibiotic prescriptions. PATIENTS AND METHODS: A prospective study was carried out in three French regional hospitals (A to C), to assess how recommendations were applied. Hospital organization was measured with the ICATB score (antimicrobial stewardship index) and the appropriateness of antibiotic prescription was assessed by an audit during 1 week by two investigators, who shadowed physicians during bedside visits, in various medical and surgical departments. RESULTS: There was a considerable difference in the organization of these three hospitals in terms of computerized prescriptions, formulary restriction, availability of recommendations, and antibiotic consumption defined as delivered daily-dose. Institution A had strictly followed recommendations for hospital organization, but these were less observed in institution B and C. The prevalence of antibiotic treatment was comparable in the three hospitals, and concerned over 25% of patients. In institution A, 60% of antibiotic prescriptions were inadequate, 23% were not appropriate and 17% were optimal. In institution B, these figures reached 36%, 34%, and 34%, while in institution C they reached 25%, 55%, and 20%, respectively. CONCLUSION: There is no clear link between applying national recommendations for antibiotic prescription and optimization of hospital organization and the quality of antibiotic prescriptions.

Expression of Xylella fastidiosa fimbrial and afimbrial proteins during biofilm formation.

Complete sequencing of the Xylella fastidiosa genome revealed characteristics that have not been described previously for a phytopathogen. One characteristic of this genome was the abundance of genes encoding proteins with adhesion functions related to biofilm formation, an essential step for colonization of a plant host or an insect vector. We examined four of the proteins belonging to this class encoded by genes in the genome of X. fastidiosa: the PilA2 and PilC fimbrial proteins, which are components of the type IV pili, and XadA1 and XadA2, which are afimbrial adhesins. Polyclonal antibodies were raised against these four proteins, and their behavior during biofilm development was assessed by Western blotting and immunofluorescence assays. In addition, immunogold electron microscopy was used to detect these proteins in bacteria present in xylem vessels of three different hosts (citrus, periwinkle, and hibiscus). We verified that these proteins are present in X. fastidiosa biofilms but have differential regulation since the amounts varied temporally during biofilm formation, as well as spatially within the biofilms. The proteins were also detected in bacteria colonizing the xylem vessels of infected plants.

[Prospective evaluation of antibiotic combinations in a general hospital]. / Evaluation prospective des associations antibiotiques d'un centre hospitalier général.

INTRODUCTION: We previously reported a 30% rate of inadequate antibiotic therapy in a general hospital with optimal organization. This data led to implement a systematic weekly infectious diseases consultation. We report an evaluation of antibiotic combinations. PATIENTS AND METHODS: The infectious diseases consultation was scheduled half-a-day per week. Antibiotic combinations were collected by the pharmacist via computerized prescriptions. Discussion with the managing physician was systematic in order to evaluate the adequacy of the treatment both in terms of diagnosis and therapy. RESULTS: For 9 months, 381 patients were prescribed 486 antibiotic combinations, among which 116 were evaluated. The infectious diseases specialist suggested a similar diagnosis in 71 antibiotic treatments (61%), the diagnosis appeared doubtful in 36 cases (31%), and a true diagnostic discordance was noted in nine cases. The discussion between specialist and managing physician suggested that the antibiotic combination was justified in 35%, of limited usefulness in 22%, and inadequate in 43% of the cases. There was a significant correlation between the result of the discussion and the accuracy of the antibiotic combination (p<0.001). Respiratory infections were the main reason for inadequate or limited usefulness of antibiotic combinations (30/49, 61%). CONCLUSION: Computerized prescriptions allow the evaluation of antibiotic therapy even when the infectious diseases specialist intervention is short. The discussion with the managing physician on diagnosis and treatment appears to play a central role for a better use of antibiotherapy.

Left ventricular hypertrophy and nocturnal hypoxemia in hemodialysis patients.

OBJECTIVE: Nocturnal hypoxemia has recently been proposed as a cardiovascular risk factor in patients with chronic renal failure. In this study we have tested the hypothesis that this disturbance is associated with left ventricular hypertrophy (LVH) in dialysis patients. METHODS: During a mid-week non-dialysis day, 38 hemodialysis patients underwent continuous monitoring of arterial O2 saturation (SaO2) during night-time as well as 24 h ambulatory blood pressure monitoring and echocardiography. RESULTS: Eighteen patients had one or more episodes of O2 desaturation during night-time (average: 21 episodes; range 1 to 120) while the other 20 had no episode. Neither day-time arterial pressure nor heart rate were significantly associated with nocturnal hypoxemia. However there was a significant correlation between the night/day systolic ratio and the severity of hypoxemia during night-time (r = 0.36, P = 0.03). On multivariate analysis, nocturnal hypoxemia proved to be the stronger independent predictor of relative wall thickness, mean wall thickness and left ventricular mass index, suggesting that nocturnal O2 desaturation is linked to concentric hypertrophy and to concentric geometry of the left ventricle. Accordingly, the proportion of patients with such geometric alteration was higher (chi2 = 4.1, P = 0.04) in patients with a pulse oximetry severity score > 50th percentile [15 of 19 (79%)] than in those below this threshold [nine of 19 (47%)]. CONCLUSIONS: Nocturnal hypoxemia is an important correlate of LVH in hemodialysis patients. Such an association is largely independent of arterial pressure. These data further underscore the importance of disturbed respiratory control as a cardiovascular risk factor in dialysis patients.

Smoking, blood pressure and serum albumin are major determinants of carotid atherosclerosis in dialysis patients. CREED Investigators. Cardiovascular Risk Extended Evaluation in Dialysis patients.

AIM: To investigate the relationship between carotid atherosclerosis and some major cardiovascular risk factors in uremic patients on chronic dialysis. METHODS: A cross-sectional study was carried out in 119 unselected dialysis patients (89 on hemodialysis and 30 on chronic ambulatory peritoneal dialysis, CAPD). Fasting blood sampling for serum lipids, albumin, hemoglobin, and echo-colour-Doppler evaluation of common carotid arteries were performed in all patients (during the non-dialysis day in hemodialysis patients). In hemodialysis patients BP was measured before and after dialysis; in CAPD patients home BP values were recorded during the month before the study day. RESULTS: Ninety-five patients had at least one plaque and 57 had at least four plaques. Thirty-eight had mild and eleven severe carotid stenosis. In multiple regression models, the mean internal diameter of carotid arteries was explained (R=0.52, P=0.0001) by systolic pressure (r=0.39), serum cholesterol (r=-0.28), age (r=0.27) and smoking (r=0.24) while the degree of carotid stenosis was predicted (R=0.39, P=0.0001) by age (r=0.36) and smoking (r=0.25). The number of atherosclerotic plaques was explained (R=0.51, P=0.0001) by age (r=0.36), smoking (r=0.25) and pulse pressure (r=0.20), serum albumin just failing to reach statistical significance (P = 0.06). However, serum albumin was a significant and independent predictor of the number of atherosclerotic plaques (r=-0.26) in hemodialysis patients (n=89). Sex, diabetes, Kt/V, duration of dialysis treatment, hemoglobin, serum calcium and phosphate did not add any predictive power to the models. CONCLUSIONS: In dialysis patients arterial pressure and smoking are associated with carotid atherosclerosis. Serum albumin appears to serve as an independent predictor of carotid atherosclerosis.

Nocturnal hypoxemia, night-day arterial pressure changes and left ventricular geometry in dialysis patients.

It is well established that nocturnal hypoxemia in sleep apnea causes an inversion of the circadian arterial pressure rhythm and triggers nocturnal hypertension. Since sleep apnea is very frequent in dialysis patients, we hypothesized that nocturnal hypoxemia may be a factor that contributes to alter the 24-hour arterial pressure profile in these patients. To test the hypothesis 32 dialysis patients underwent 24-hour blood pressure (BP) monitoring and continuous monitoring of arterial O2 saturation during the night-time. Hemodialysis patients were studied during the non-dialysis day. All patients underwent an echocardiographic study. Thirteen patients had no episode of nocturnal hypoxemia (group I), 7 had at least one episode overnight but less than 2 episodes/hr (group II) and 12 had > or = 2 episodes/hr (group III). The average daytime systolic pressure was similar in the three groups. However, the average nocturnal systolic pressure fell in the first group (-2.5 +/- 4.2%) and rose in the second (+2.0 +/- 3.6%) and in the third (+3.9 +/- 2.2%) group (one way ANOVA, P < 0.005). The relative wall thickness of the left ventricle (RWT) was significantly (P < 0.05) higher in group III than in group I, and in the aggregate (N = 32) there was an inverse relationship between average nocturnal SaO2 and RWT (r = -0.43, P = 0.015). The proportion of patients with concentric remodeling or concentric hypertrophy was higher (P = 0.05) in the group with a more severe degree of nocturnal hypoxemia (group III, 8 of 12) than in the other two groups (group I, 3 of 13; group II, 2 of 7). Nocturnal hypoxemia is associated with the "non-dipping" arterial pressure profile in dialysis patients. Disturbed respiratory control during the night may represent an important cardiovascular risk factor in dialysis patients.

Epilepsy associated with hypocalcemia: description of a family.

The unusual occurrence of epilepsy and hypocalcemia in the same family is described. Epilepsy was present in three siblings (convulsive generalized in two females and partial in one male). All six family members had calcium ion, plasmatic phosphorus, plasmatic and erythrocytic potassium, urine calcium and phosphate concentration below the normal levels. Altered values of calcium-induced potassium membrane permeability were found in three subjects (two of them also affected by epilepsy). The assumption that alterations of membrane structure or function play an essential role in determining familial epilepsy and that the normal "set point" for calcium is genetically determined in familial hypocalcemia could explain the concomitance of both pathologies to some extent.

Complex partial status epilepticus simulating an ischemic insult: case report.

A patient presenting complex partial status epilepticus (CPSE) had a clinical history and neurological picture on admission that mimicked a cerebrovascular insult. On admission she was confused and totally unresponsive to verbal stimuli. EEG showed high voltage paroxysmal activity on the left hemisphere, prominent on the temporoccipital leads and tending to spread to the opposite regions. Intravenous diazepam led to resolution of the clinical and EEG picture within 24 hours, only a medium voltage slow wave focus being present on the left temporal regions. CPSE should be considered whenever a prolonged impairment of consciousness is present. Only accurate EEG recordings permit correct diagnosis in doubtful cases.

Synergistic anticonvulsant effect of valproic acid and ethosuximide on pentylenetetrazole-induced epileptic phenomena in rats.

The possible synergistic effect of valproic acid and ethosuximide in combination on pentylenetetrazole-induced epilepsy was investigated in rats. Valproic acid and ethosuximide administered intraperitoneally both showed dose-dependent anti-epileptic activity towards pentylenetetrazole-induced myoclonias and tonic-clonic seizures. The valproic acid-ethosuximide combination had a synergistic pharmacological effect. Against myoclonias combined valproic acid-ethosuximide produced a non-significant decrease in the effective dose of both drugs compared with treatment with either drug alone. In the case of tonic-clonic seizures the protective effect against the seizures was significantly increased by combined treatment compared with treatment with either drug alone. Neither plasma concentrations nor any other pharmacokinetic parameters were significantly changed when the same doses of valproic acid and ethosuximide were given, singly or in combination.

Effects of temperature on the regulation of photosynthetic carbon assimilation in leaves of maize and barley.

The aim of this work was to examine the effect of temperature in the range 5 to 30 ° C upon the regulation of photosynthetic carbon assimilation in leaves of the C4 plant maize (Zea mays L.) and the C3 plant barley (Hordeum vulgare L.). Measurements of the CO2-assimilation rate in relation to the temperature were made at high (735 µbar) and low (143 µbar) intercellular CO2 pressure in barley and in air in maize. The results show that, as the temperature was decreased, (i) in barley, pools of phosphorylated metabolites, particularly hexose-phosphate, ribulose 1,5-bisphosphate and fructose 1,6-bisphosphate, increased in high and low CO2; (ii) in maize, pools of glycerate 3-phosphate, triose-phosphate, pyruvate and phosphoenolpyruvate decreased, reflecting their role in, and dependence on, intercellular transport processes, while pools of hexose-phosphate, ribulose 1,5-bis phosphate and fructose 1,6-bisphosphate remained approximately constant; (iii) the redox state of the primary electron acceptor of photosystem II (QA) increased slightly in barley, but rose abruptly below 12° C in maize. Non-photochemical quenching of chlorophyll fluorescence increased slightly in barley and increased to high values below 20 ° C in maize. The data from barley are consistent with the development of a limitation by phosphate status at low temperatures in high CO2, and indicate an increasing regulatory importance for regeneration of ribulose 1,5-bisphosphate within the Calvin cycle at low temperatures in low CO2. The data from maize do not show that any steps of the C4 cycle are particularly cold-sensitive, but do indicate that a restriction in electron transport occurs at low temperature. In both plants the data indicate that regulation of product synthesis results in the maintenance of pools of Calvin-cycle intermediates at low temperatures.

Factors influencing the capacity for photosynthetic carbon assimilation in barley leaves at low temperatures.

The aim of this work was to examine the effect upon photosynthetic capacity of short-term exposure (up to 10 h) to low temperatures (5° C) of darkened leaves of barley (Hordeum vulgare L.) plants. The carbohydrate content, metabolite status and the photosynthetic rate of leaves were measured at low temperature, high light and higher than ambient CO2. Under these conditions we could detect whether previous exposure of leaves to low temperature overcame the limitation by phosphate which occurs in leaves of plants not previously exposed to low temperatures. The rates of CO2 assimilation measured at 8° C differed by as much as twofold, depending upon the pretreatment. (i) Leaves from plants which had previously been darkened for 24 h had a low content of carbohydrate, had the lowest CO2-assimilation rates at low temperature, and photosynthesis was limited by carbohydrate, as shown by a large stimulation of photosynthesis by feeding glucose, (ii) Leaves from plants which had previously been illuminated for 24 h and which contained large carbohydrate reserves showed an accumulation of phosphorylated intermediates and higher CO2-assimilation rates at low temperature, but nevertheless remained limited by phosphate, (iii) Maximum rates of CO2 assimilation at low temperature were observed in leaves which had intermediate reserves of carbohydrate or in leaves which were rich in carbohydrate and which were also fed phosphate. It is suggested that carbohydrate reserves potentiate the system for the achievement of high rates of photosynthesis at low temperatures by accumulation of photosynthetic intermediates such as hexose phosphates, but that this potential cannot be realised if, at the same time, carbohydrate accumulation is itself leading to feedback inhibition of photosynthesis.

Influence of low temperature on respiration and contents of phosphorylated intermediates in darkened barley leaves.

The aim of this work was to examine the effect of exposure of leaves to low temperatures (5 degrees C) upon the contents of phosphorylated intermediates and respiration in darkened barley (Hordeum vulgare L.) plants which differed in their carbohydrate status. In leaves that had previously been illuminated for 24 hours, there was a large increase in amounts of phosphorylated metabolites at 5 degrees C during the first 3 hours of darkness, compared with control plants kept at 30 degrees C. Hexose phosphates accounted for about two-thirds of this increase, which reached a peak after about 3 hours. At higher temperatures, there was a peak in the amount of fructose 2,6-bisphosphate and the rate of respiration which accompanied the transient increase in phosphorylated intermediates. At 5 degrees C the increase in phosphorylated intermediates was not accompanied by appreciable changes in fructose 2,6-bisphosphate, and there was a rapid decline in the rate of respiration. Leaves that had previously been darkened for 24 hours and that were low in carbohydrate failed to accumulate phosphorylated intermediates when exposed to low temperatures. The results are discussed with respect to the acclimation of carbohydrate metabolism to low temperatures. The results suggest that respiratory carbohydrate metabolism is strictly controlled even when the carbohydrate supply and glycolytic intermediates are abundant. The possibility that accumulation of hexose phosphates may be involved in acclimation of metabolism to low temperature is discussed.

Phosphate sequestration by glycerol and its effects on photosynthetic carbon assimilation by leaves.

Glycerol induced a limitation on photosynthetic carbon assimilation by phosphate when supplied to leaves of barley (Hordeum vulgare L.) and spinach (Spinacia oleracea L.). This limitation by phosphate was evidenced by (i) reversibility of the inhibition of photosynthesis by glycerol by feeding orthophosphate (ii) a decrease in light-saturated rates of photosynthesis and saturation at a lower irradiance, (iii) the promotion of oscillations in photosynthetic CO2 assimilation and in chlorophyll fluorescence, (iv) decreases in the pools of hexose monophosphates and triose phosphates and increases in the ratio of glycerate-3-phosphate to triose phosphate, (v) decreased photochemical quenching of chlorophyll fluorescence, and increased non-photochemical quenching, specifically of the component which relaxed rapidly, indicating that thylakoid energisation had increased. In barley there was a massive accumulation of glycerol-3-phosphate and an increase in the period of the oscillations, but in spinach the accumulation of glycerol-3-phosphate was comparatively slight. The mechanism(s) by which glycerol feeding affects photosynthetic carbon assimilation are discussed in the light of these results.