Dansyl-triazole-based fluorescent macrocycle for selective detection of nitro-antibiotic drugs and protein interaction.

A novel dansyl-triazole-based fluorescent macrocycle with high Stokes shift and positive solvatochromism was developed. This is an excellent fluorescence sensor for selective detection of nitro-containing antibiotics and other nitro-heteroaromatics. Detection was possible in real samples/paper strips in submicromolar concentration. The interaction of the macrocycle with multiple proteins exhibited its bioactivity.

Postoperative troponin release is associated with major adverse cardiovascular events in the first year after noncardiac surgery.

INTRODUCTION: Troponin elevations after intermediate-to-high risk noncardiac surgery are common and can predict mortality. However, the prognostic value for early and late major adverse cardiovascular events (MACE) is less well investigated. The authors evaluated the relationship between postoperative troponin release and MACE in the first year after noncardiac surgery. METHODS: This observational cohort registry comprised data of patients aged ≥60 years undergoing intermediate-to-high risk noncardiac surgery between July 2012 and 2015, at the Erasmus University Medical Center, Rotterdam, the Netherlands. High-sensitivity troponin T was measured on day 1 to 3 after surgery. Peak troponin values were divided into four categories: <14 ng·L-1, 14-49 ng·L-1, 50-149 ng·L-1 and ≥150 ng·L-1. The primary endpoint MACE was defined as the occurrence of myocardial infarction, angina, revascularization therapy or cerebrovascular accident in the first year after surgery. The incidence of MACE and all-cause mortality was calculated using Kaplan-Meier estimates. Cox regression was used to estimate risks for both endpoints. RESULTS: In total, 3085 patients were included for analyses and peak troponin elevation above 14 ng·L-1 was present in 1678 (54.4%) patients. The overall incidence for one-year MACE was 5.8% (3.4%, 6.1%, 10.4% and 40.6% per increasing troponin category) with adjusted HR (95% CI) 1.32 (0.85-2.06), 2.53 (1.42-4.53) and 10.24 (5.91-17.75) for the consecutive increasing categories. One-year mortality occurred in 14.6% and showed a similar stepwise increase with adjusted HR (95% CI) 1.25 (0.98-1.60), 2.39 (1.72-3.32) and 3.79 (2.60-5.54). CONCLUSION: Our dataset demonstrates a graded relationship between postoperative troponin release and occurrence of MACE in the first year after intermediate-to-high risk noncardiac surgery.

Prognostic value of postoperative high-sensitivity troponin T in patients with different stages of kidney disease undergoing noncardiac surgery.

BACKGROUND: Emerging evidence suggests that postoperative troponin release is a strong and independent predictor of short-term mortality. However, evaluating elevated troponins in patients with chronic kidney disease (CKD) is still controversial and is often disregarded. This study examines morbidity along with short- and long-term mortality risk associated with elevated high-sensitivity troponin T (hsTnT) in patients with different stages CKD undergoing noncardiac surgery. METHODS: This observational cohort comprised 3262 patients aged ≥60 yr who underwent noncardiac surgery. Postoperative hsTnT concentrations were divided into normal [<14 ng l-1 (reference)], low (14-49 ng l-1), moderate (50-149 ng l-1), and high (≥150 ng l-1) groups. A threshold of 50 ng l-1 was used to dichotomize hsTnT. The study endpoints were 30-day and long-term all-cause mortality, and postoperative myocardial infarction. RESULTS: Postoperative hsTnT was associated with a stepwise increase in 30-day and long-term mortality risk: low hsTnT adjusted hazard ratio (HR) 1.4 [95% confidence interval (CI): 1.1-1.7], moderate hsTnT adjusted HR 3.1 (95% CI: 2.3-4.3), high hsTnT adjusted HR 5.5 (95% CI: 3.6-8.4). Postoperative hsTnT ≥50 ng l-1 was associated with 30-day and long-term mortality risk for each stage of CKD. Elevated troponin concentrations in severe CKD (estimated glomerular filtration rate <30 mL min-1 1.73 m-2), however, did not predict short-term death. CONCLUSIONS: Elevated postoperative hsTnT is associated with a dose-dependent increase in 30-day and long-term mortality risk in each stage of CKD with an estimated glomerular filtration rate ≥30 ml min-1 1.73 m-2.

Isolation, purification and characterization of a pH tolerant and temperature stable proteinaceous protease inhibitor from marine Pseudomonas mendocina.

OBJECTIVES: An extracellular protease inhibitor (BTPI-301) of trypsin was purified and characterized from an isolate of Pseudomonas mendocina. RESULTS: BTPI-301was purified to homogeneity by (NH4)2SO4, precipitation, DEAE Sepharose and CNBr-activated Sepharose chromatography. Homogeneity was proved by native PAGE and SDS-PAGE. The intact molecular mass was 11567 Da by MALDI-TOF analysis. BTPI-301was a competitive inhibitor with a Ki of 3.5 × 10-10 M. It was stable and active at pH 4-12 and also at 4-90 °C for 1 h. Peptide mass fingerprinting by MALDI revealed that the BTPI-301 is a new inhibitor not reported so far with protease inhibitory activity. The pI of the inhibitor was 3.8. The stoichiometry of trypsin-BTPI-301 interaction is 1:1. The inhibitor was specific towards trypsin. CONCLUSION: A pH tolerant and thermostable protease inhibitor BTPI-301 active against trypsin was purified and characterized from P. mendocina that could be developed and used as biopreservative as well as biocontrol agent.

Off-site primary percutaneous coronary intervention in a new centre is safe: comparing clinical outcomes with a hospital with surgical backup.

OBJECTIVES: To evaluate the procedural and clinical outcomes of a new primary percutaneous coronary intervention (PPCI) centre without surgical back-up (off-site PCI) and to investigate whether these results are comparable with a high volume on-site PCI centre in the Netherlands. BACKGROUND: Controversy remains about the safety and efficacy of PPCI in off-site PCI centres. METHODS: We retrospectively analysed clinical and procedural data as well as 6­month follow-up of 226 patients diagnosed with ST-elevated myocardial infarction (STEMI) who underwent PPCI at VieCuri Medical Centre Venlo and 115 STEMI patients who underwent PPCI at Catharina Hospital Eindhoven. RESULTS: PPCI patients in VieCuri Medical Centre had similar procedural and clinical outcomes to those in Catharina Hospital. Overall there were no significant differences. The occurrence of procedural complications was low in both groups (8.4 % VieCuri vs. 12.3 % Catharina Hospital). In the VieCuri group there was one procedural-related death. No patients in either group needed emergency surgery. At 30 days, 17 (7.9 %) patients in the VieCuri group and 9 (8.1 %) in the Catharina Hospital group had a major adverse cardiac event. CONCLUSION: Performing PPCI in an off-site PCI centre is safe and effective. The study results show that the procedural and clinical outcomes of an off-site PPCI centre are comparable with an on-site high-volume PPCI centre.

Delays in the treatment of patients with acute coronary syndrome: Focus on pre-hospital delays and non-ST-elevated myocardial infarction.

Delays in patients suspected of acute coronary syndrome (ACS) should be kept as short as possible to reduce complications and mortality. In this review we discuss the substantial pre-hospital delays of ST-elevated myocardial infarction (STEMI) patients as well as non-STEMI patients. The pre-hospital delays include patient, doctor and emergency medical transport (EMT) delay. Patient delay is among the longest in the pre-hospital chain of ACS patients. Interventions as mass media campaigns or individual education programs have not yet shown much improvement. Patients with chest pain most often contact the general practitioner (GP) instead of the recommended EMT, increasing delays as well. To decrease the delays by referring all patients promptly and without restriction to the emergency department (ED) is not feasible. Up to 80% of the patients with chest pain do not have a cardiac diagnosis and thus referral of all these patients would result in overcrowding of the ED. Triage is therefore crucial. Triage of patients with chest pain is therefore imperative and there is a great need of (validated) triage tools.

Trypsin inhibitor from edible mushroom Pleurotus floridanus active against proteases of microbial origin.

Protease inhibitors can be versatile tools mainly in the fields of medicine, agriculture and food preservative applications. Fungi have been recognized as sources of protease inhibitors, although there are only few such reports on mushrooms. This work reports the purification and characterization of a trypsin inhibitor from the fruiting body of edible mushroom Pleurotus floridanus (PfTI) and its effect on the activity of microbial proteases. The protease inhibitor was purified up to 35-fold by DEAE-Sepharose ion exchange column, trypsin-Sepharose column and Sephadex G100 column. The isoelectric point of the inhibitor was 4.4, and its molecular mass was calculated as 37 kDa by SDS-PAGE and 38.3 kDa by MALDI-TOF. Inhibitory activity confirmation was by dot-blot analysis and zymographic activity staining. The specificity of the inhibitor toward trypsin was with Ki of 1.043 × 10(-10) M. The inhibitor was thermostable up to 90 °C with maximal stability at 30 °C, active over a pH range of 4-10 against proteases from Aspergillus oryzae, Bacillus licheniformis, Bacillus sp. and Bacillus amyloliquefaciens. Results indicate the possibility of utilization of protease inhibitor from P. floridanus against serine proteases.

The first year of the Venlo percutaneous coronary intervention program: procedural and 6-month clinical outcomes.

OBJECTIVES: Analysis of the first results of off-site percutaneous coronary interventions (PCI) and fractional flow reserve (FFR) measurements at VieCuri Medical Centre for Northern Limburg in Venlo. BACKGROUND: Off-site PCI is accepted in the European and American Cardiac Guidelines as the need for PCI increases and it has been proven to be a safe treatment option for acute coronary syndrome. METHODS: Retrospective cohort study reporting characteristics, PCI and FFR specifications, complications and 6-month follow-up for all consecutive patients from the beginning of off-site PCI in Venlo until July 2012. If possible, the data were compared with those of Medical Centre Alkmaar, the first off-site PCI centre in the Netherlands. RESULTS: Of the 333 patients, 19 (5.7 %) had a procedural complication. At 6 months, a major adverse cardiovascular event (MACE) occurred in 43 (13.1 %) patients. There were no deaths or emergency surgery related to the PCI or FFR procedures. There was no significant difference in occurrence of a MACE or adverse cerebral event between the Alkmaar and Venlo population in the 30-day follow-up. CONCLUSION: This study demonstrates off-site PCI at VieCuri Venlo to have a high success rate. Furthermore, there was a low complication rate, low MACE and no procedure-related mortality.

Production optimization and properties of beta glucosidases from a marine fungus Aspergillus-SA 58.

Aspergillus strain SA 58, showing considerable beta glucosidase production was selected as the potential strain. The fungus showed enzyme production in both acidic and alkaline pH. A temperature of 35 degrees C was found to be optimum for enzyme production. Maximum enzyme production was seen when pectin was used as the carbon source (80 U/ml). In solid-state fermentation, an enzyme production of 6200 U/g Initial Dry Substrate was noted. The strain produced two extra cellular enzymes and two intra cellular enzymes. For both the extra cellular enzymes (BGL A and BGL B), 60 degrees C was found to be optimum temperature for activity. BGL A showed an optimum pH of 4.0 while BGL B showed an optimum pH of 3.0 for activity. Both the enzymes showed a second peak of activity at pH 9.0. Both BGL A and BGL B showed high thermal stabilities with residual activities of 86% and 85% even after 6h of incubation at 50 degrees C.

Design of a 5' exonuclease-based real-time PCR assay for simultaneous detection of Bacillus licheniformis, members of the 'B. cereus group' and B. fumarioli in gelatine.

AIMS: The design of a fast, sensitive and specific detection method for Bacillus licheniformis, members of the 'B. cereus group' and B. fumarioli in gelatine. METHODS AND RESULTS: Specific Taqman probes were designed and tested in a real-time PCR setting. A specific fluorescent signal could be obtained for all gelatine isolates attributed to these species in one single real-time PCR reaction. After sample preparation, a gelatine sample spiked with 1 CFU provided enough template DNA for a significant signal. CONCLUSION: The potential of a real-time PCR assay for simultaneous detection of B. licheniformis, members of the 'B. cereus group' and B. fumarioli in gelatine is demonstrated. SIGNIFICANCE AND IMPACT OF THE STUDY: Implementation of the assay in gelatine producing plants may shorten delivery terms and inform on hazards to public health and suitable remediation procedures.

Effects of rapeseed meal-glucosinolates on thyroid metabolism and feed utilization in rainbow trout.

Two rapeseed meals (RM1 and RM2), containing glucosinolates at a concentration of 26 and 40 micromol/g, respectively, were incorporated at increasing levels (10, 20, and 30% for RM1 and 30 and 50% for RM2) in diets of juvenile rainbow trout. Disturbances in the thyroid axis appeared after 14 days of feeding (with a dietary incorporation level of 10%). The dietary supplementation with T(3) or iodine induced an increase in plasma T(3) levels, compared to that in fish fed the RM diets, and reduced the deleterious effect of RM on growth. When trout were reared in seawater, there was also a slight increase in thyroid hormone levels. TSH treatment had no effect on the thyroid hormone plasma levels. The incorporation of 30% of RM1, which induced a lower dietary content of toxic compounds than RM2, led to a rapid decrease of plasma T(4) and T(3) levels, but growth was affected only after 6 months of feeding. During these studies, the deiodinase activities responded in a complex manner to restore plasma and tissue levels of T(3).

Dietary low-glucosinolate rapeseed meal affects thyroid status and nutrient utilization in rainbow trout (Oncorhynchus mykiss).

Two rapeseed (Brassica napus) meals, RM1 and RM2, with two levels of glucosinolates (GLS; 5 and 41 mumol/g DM respectively) were incorporated at the levels of 300 and 500 g/kg of the diets of juvenile rainbow trout (Oncorhynchus mykiss) in replacement of fish meal, and compared with a fish-meal-based diet. A decrease in the digestibility of the DM, protein, gross energy and P was observed with high-rapeseed meal (RM) incorporation. In trout fed on RM-based diets, growth performance was reduced even after only 3 weeks of feeding. Feed efficiency was adversely affected by RM and GLS intake. Protein and energy retention coefficients were significantly lower in fish fed on the diet containing the higher level of GLS. P retention was significantly lower with all the RM-based diets than with the fish-meal diet. Irrespective of the degree of growth inhibition, fish fed on RM-based diets exhibited similar typical features of hypothyroid condition due to GLS intake, expressed by lower plasma levels of triiodothyronine and especially thyroxine and a hyperactivity of the thyroid follicles. This hypothyroidal condition led to a strong adjustment of the deiodinase activities in the liver, the kidney and the brain. A significant increase of the outer ring deiodinase activities (deiodinases type I and II respectively) and a decrease of the inner ring deiodinase activity (deiodinase type III) were observed. It is concluded that the observed growth depression could be attributed to the concomitant presence of GLS, depressing the thyroid function, and of other antinutritional factors affecting digestibility and the metabolic utilization of dietary nutrients and energy.

Inhibition and activation of the thyroidal axis by the adrenal axis in vertebrates.

Hormones of the adrenal or interrenal axis and stress situations which induce elevated glucocorticoid plasma levels (e.g. handling and starvation), inhibit thyroid function in growing and adult vertebrates. However, data indicate that during foetal and embryonic development (mammals and birds) or during larval growth and metamorphosis (fish and amphibians), the adrenal axis may stimulate thyroid function. Recent findings have provided some information concerning this stimulatory interference of the adrenal axis. In amphibians corticotropin releasing hormone and not thyrotropin releasing hormone is thyrotropic during metamorphosis, thus providing the substrate T4 necessary for T3 production. Other data indicate that the increase in plasma T3 at metamorphic climax may be the result of an inhibition of the T3 degrading activity, rather than stimulation of the T4 into T3 converting activity, and that glucocorticoids may be responsible for this. Also, in the chick embryo glucocorticoids effectively increase plasma T3 concentration by reducing the hepatic T3 degrading activity, whereas corticotropin releasing hormone also induces an elevation in the thyrotropin plasma levels and hence raises T4 concentrations which may function as a substrate for T3 production.

Feeding behaviour and food utilisation in tilapia, Oreochromis niloticus: effect of sex ratio and relationship with the endocrine status.

The feeding behaviour of male monosex, female monosex, and mixed groups of Oreochromis niloticus was studied under conditions of self-feeding. Feeding activity was observed almost exclusively during the light period. The food intake pattern was similar whatever the sex ratio, and voluntary food intake (VFI) appeared lower in the male monosex groups than in the others. Male monosex groups displayed higher specific growth rates (SGR) and a lower food conversion ratio than female monosex and mixed groups. The SGR of males was higher in the monosex than in the mixed groups, whereas females of mixed and monosex groups displayed no significant difference in SGR. The efficiency of food utilisation was also analysed: nutrient retention ratios were higher in male monosex than in female monosex and mixed groups. Males displayed a distinctly higher metabolic capacity. Differences in sex-related hormones (11 ketotestosterone = 11-KT, 17beta-Oestradiol = 17beta-E2) and a metabolic hormone (triiodothyronine = T3) were observed between males and females. The hypothesis of an involvement of these hormones in the higher metabolic capacity of males is discussed. The observed differences in feeding behaviour between the different groups also suggest an effect of social interactions on the efficiency of food conversion and thus on the differential growth of males and females.

Characterization of iodothyronine outer ring and inner ring deiodinase activities in the blue tilapia, Oreochromis aureus.

The presence of iodothyronine deiodinases was investigated in the different tissues of blue tilapia (Oreochromis aureus), and their biochemical properties were compared with those of mammalian deiodinases. High-Km rT3 outer ring deiodination (ORD) was observed in tilapia kidney, low-Km T4 ORD in liver, and low-Km T3 inner ring deiodination (IRD) in brain and gill. The rT3 ORD activity in tilapia kidney has a very similar substrate specificity as rat liver type I iodothyronine deiodinase but is much less sensitive to inhibition by propylthiouracil, iodoacetic acid, and aurothioglucose. Tilapia liver T4 ORD activity and tilapia brain and gill T3 IRD activities show very similar substrate specificities as well as similar inhibitor sensitivities as rat type II and type III iodothyronine deiodinase, respectively. The optimal pH of the tilapian enzymes is 6-7, and the optimal incubation temperature is approximately 37 C. All tilapia deiodinases are stimulated by dithiothreitol, but the optimal DTT concentrations are generally lower than those required by the corresponding rat enzymes. The apparent Km values of the various tilapia deiodinases for their preferred substrate are in the same range as for the corresponding rat enzymes. Based on these findings, we conclude that fish deiodinases are more similar to mammalian deiodinases than generally accepted.

Consequences of food restriction on short-term growth variation and on plasma circulating hormones in Oreochromis niloticus in relation to sex.

In tilapia, there is a sex-related growth difference between males and females. This study tried to detect any correlation between the somatic growth and the plasma endocrine status. For this, individually marked (Floytags) male and female tilapia (BW 82 +/- 10 g) were either starved or fed on different daily food rations (1, 2, or 3% of the biomass) during 15 days. We have found that specific growth rates (SGR) were positively and significantly related to feeding levels. Growth hormone (GH) plasma levels tended to increase with the decrease in food levels, and thus with the decrease in growth rate. No significant correlation was found between GH levels and SGR. Triiodothyronine (T3) levels in well-fed fish were higher than those in restricted fish (0 and 1%), but no differences in thyroxine (T4) levels were observed. No significant relationship was found between plasma levels of steroid hormones and feeding ration, even though 11-ketotestosterone (11-KT) levels tended to increase with the ration in fed males. SGR were not significantly different between males and females at the same feeding level, but taken as a whole, they were significantly different in favor of males (P < 0.05). There was no important difference in GH levels between the two sexes. Steroid hormones were, in general, higher in males for 11-KT and in females for 17 beta-estradiol (17 beta-E2). Males and females exhibited significant differences in T3 levels (respectively 4.25 +/- 0.18 and 2.71 +/- 0.09 pmol/ml), whatever the food ration, but no significant differences in T4 levels were observed except in the high-ration group. The correlation between T3 levels and SGR was low but stronger in males (r2 = 0.21; n = 90) than in females (r2 = 0.10; n = 105). The slope of the log-log regression of T3 levels with body weight was much lower in females (b = 0.87) than in males (b = 1.31). This relationship suggests the involvement of T3 in tilapia growth and probably in the differential growth between males and females. In both males and females, a significant but low correlation was observed between T3 and 11-KT levels (respectively r2 = 0.12; n = 82 and r2 = 0.08; n = 89), while no correlation was found between the levels of T3 and 17 beta-E2. T3 plasma levels were found to be the most different parameter between males and females. This hormone seemed to be involved in the control of somatic growth, and could explain the differential growth rate between males and females.

Hormonal profile of growing male and female diploids and triploids of the blue tilapia,Oreochromis aureus, reared in intensive culture.

Triploidy as a result of thermal shock exposure of fertilized eggs decreases the growth rate ofOreochromis aureus as compared to their diploid controls, but this is due to the higher female ratio present in triploids (86%) and the lower growth rate of females. When females and males are considered separately, the growth rate is not significantly different in diploids and triploids. Since triploidy results in a malfunctioning steroidogenesis in females (mainly testosterone (T) and 17ß-estradiol (E2)), but does not affect the growth rate, it is concluded that female gonadal steroids do not influence growth unless in pharmacological concentrations. These low levels of gonadal steroids are generally accompanied by higher levels of gonadotropin (GtH), but the difference is not always significant.Despite their lower growth rate diploid females have higher plasma concentrations of growth hormone (GH) during several months compared to the triploid females and diploid males. 3,5,3'-triiodo-L-thyronine (T3) levels, however, are comparable between diploid and triploid females (except for 1 month), but higher in diploid males in 4 of the 5 months studied. 11-ketotestosterone (11kT) is always higher in males. These results indicate that the higher growth rate of males may be related to the high circulating levels of T3 and 11kT.

Different thyroid hormone-deiodinating enzymes in tilapia (Oreochromis niloticus) liver and kidney.

Enzymes catalyzing the outer ring deiodination (ORD) of iodothyronines are important for the regulation of thyroid hormone bioactivity. We have studied ORD of thyroxine (T4) and 3,3',5'-triiodothyronine (rT3) in liver and kidney microsomes of fish, i.e. tilapia (Oreochromis niloticus). Tilapia kidney contains an enzyme which resembles the mammalian selenoenzyme type I iodothyronine deiodinase (ID-I) with respect to substrate preference (rT3 > T4) and high (approximately microM) Km values, but is much less sensitive to selenocysteine (Sec)-targeted inhibitors, including 6-propyl-2-thiouracil (PTU). In contrast, tilapia liver contains an enzyme very similar to mammalian type II deiodinase (ID-II) with respect to substrate preference (T4 > rT3), low (approximately nM) Km values, and lack of sensitivity to Sec inhibitors.

Evidence for the kidney as an important source of 5'-monodeiodination activity and stimulation by somatostatin in Oreochromis niloticus L.

Radioimmunoassay of 3,5,3'-triiodothyronine (T3) and thyroxine (T4) in the thyroidal region of mature male Oreochromis niloticus revealed stores of T4 but negligible levels of T3, yielding a very low T3/T4 ratio (0.3%). 5'-Deiodination (5-D) of T4 into T3 was examined in liver and kidney homogenates in vitro by radioimmunoassay of T3 with T4 as substrate. In both organs, the 5'-D activity was temperature dependent: at 4 degrees C, T3 production was below the level of detection and maximal in both tissues at 37 degrees C; and at 45 degrees C, the enzymatic activity was reduced. T3 production seemed to reach a plateau after 60 min of incubation. The reaction required exogenous thiol cofactor (dithiothreitol) and was inhibited partially or completely by propylthiouracil depending on the concentrations used. Hepatic and renal 5'-D activities were stimulated by somatostatin (SRIF) within 4 hr, but a subsequent increase in plasma T3 was observed only when SRIF was injected together with T4, while the magnitude of rT3 production decreased. It is concluded that almost all the circulating T3 is provided by peripheral T4 to T3 conversion since T3 RIA in thyroidal follicles demonstrated insignificant T3 production. The kidney may contain the large part of the functional deiodinase which converts T4 into T3. As in mammals and unlike in other fishes, there is not only 5'-D activity, but also 5-D activity, and both may be influenced by SRIF.