Microcarrier-based fluorescent yeast estrogen screen assay for fast determination of endocrine disrupting compounds.

The presence of endocrine-disrupting compounds (EDCs) in water poses a significant threat to human and animal health, as recognized by regulatory agencies throughout the world. The Yeast Estrogen Screen (YES) assay is an excellent method to evaluate the presence of these compounds in water due to its simplicity and capacity to assess the bioaccessible forms/fractions of these compounds. In the presence of a compound with estrogenic activity, Saccharomyces cerevisiae cells, containing a lacZ reporter gene encoding the enzyme ß-galactosidase, are induced, the enzyme is synthesised, and released to the extracellular medium. In this work, a YES-based approach encompassing the use of a lacZ reporter gene modified strain of S. cerevisiae, microcarriers as solid support, and a fluorescent substrate, fluorescein di-ß-d-galactopyranoside, is proposed, allowing for the assessment of EDCs' presence after only 2 h of incubation. The proposed method provided an EC50 of 0.17 ± 0.03 nM and an LLOQ of 0.03 nM, expressed as 17ß-estradiol. The assessment of different EDCs provided EC50 values between 0.16 and 1.2 × 103 nM. After application to wastewaters, similar results were obtained for EDCs screening, much faster, compared to the conventional 45 h spectrophotometric procedure using a commercial kit, showing potential for onsite high-throughput screening of environmental contamination.

Greenhouse Gas Emissions and Crossbred Cow Milk Production in a Silvopastoral System in Tropical Mexico.

In Mexico, pasture degradation is associated with extensive pastures; additionally, under these conditions, livestock activities contribute considerably to greenhouse gas (GHG) emissions. Among the options to improve grazing systems and reduce GHG emissions, silvopastoral systems (SPS) have been recommended. The objectives of this work were to quantify the N outflow in a soil-plant-animal interface, as well as the CH4 emissions and milk production in an SPS with woody legumes (Leucaena leucocephala) that is associated with stargrass (Cynodon nlemfuensis). This was then compared with stargrass in a monoculture system (MS) in the seasons (dry and rainy period) over a two-year period. Dung was collected from the animals of each of the grazing systems and applied fresh to the land plots. Fresh dung and urine were collected from the cows of each grazing system and were applied to the experimental plots. In addition, the soil CH4 and N2O contents were measured to quantify the emissions. Average milk yield by seasons was similar: MS (7.1 kg per animal unit (AU)/day-1) and SPS (6.31 kg per AU/day-1). Cows in the MS had a mean N intake of 171.9 g/UA day-1 without seasonal variation, while the SPS animals' mean N intake was 215.7 g/UA day-1 for both seasons. For the urine applied to soil, the N2O outflow was higher in the MS (peak value = 1623.9 µg N-N2O m-2 h-1). The peak value for the SPS was 755.9 µg of N-N2O m-2 h-1. The N2O emissions were higher in the rainy season (which promotes denitrification). The values for the feces treatment were 0.05% (MS) and 0.01% (SPS). The urine treatment values were 0.52% (MS) and 0.17% (SPS). The emissions of CH4 showed that the feces of the SPS systems resulted in a higher accumulation of gas in the rainy season (29.8 g C ha-1), followed by the feces of the MS system in the dry season (26.0 g C ha-1). Legumes in the SPS helped to maintain milk production, and the N2O emissions were lower than those produced by the MS (where the pastures were fertilized with N).

Exploiting Kinetic Features of ORAC Assay for Evaluation of Radical Scavenging Capacity.

The analysis and interpretation of data retrieved from Oxygen Radical Absorbance Capacity (ORAC) assays represent a challenging task. ORAC indexes originate from different mathematical approaches often lacking correct elucidation of kinetic features concerning radical scavenging reactions by antioxidant compounds. In this work, the expression of ORAC values as area under fluorescein (FL) decay curves (AUC) and lag time are critically compared. This multi-parametric analysis showed the extension of radical scavenging reactions beyond the lag time period for caffeic acid, gallic acid, reduced glutathione and quercetin, extending their antioxidant protection of FL. Ethanol delayed the reaction of both FL and antioxidant compounds with free radical species generated from 2,2'-azobis(2-amidinopropane) dihydrochloride thermolysis. Trolox equivalent values, commonly used to express ORAC values, were more affected by the differences in radical scavenging kinetics between the reference and the tested antioxidant compounds when calculated from AUC than from lag time. These findings stressed the importance of choosing calibrator compounds presenting ORAC kinetics similar to samples to prevent biased estimation of the antioxidant capacity. Additionally, the framework proposed here provides a sustainable analytical method for the evaluation of antioxidant capacity, with an AGREE score of 0.73.

Bradyrhizobium occurrence in nodules of perennial horsegram.

The introduction of a forage legume into a tropical pasture should decrease the need for N fertilizer, provided biological N2 fixation (BNF) contributes enough to compensate for exported N. Macrotyloma axillare (perennial horsegram) is a suitable legume for composing mixed pastures, and our hypothesis is that the isolation of indigenous rhizobia from roots and rhizosphere is the way of achieving an efficient inoculant to maximize BNF to the legume. Nodules and rhizosphere soil taken from M. axillare grown in a mixed pasture with palisade grass were sampled and used in a trap host assay using Leonard jars containing a mixture of vermiculite and sand. A total of ten bacteria were initially isolated using this technique. The isolates were then used in two experiments to evaluate the inoculation responses on the perennial horsegram in greenhouse conditions to which nodulation, plant growth, and shoot N accumulation were measured. Phylogenetic analyses based on 16S rRNA and recA placed all strains within genus Bradyrhizobium, some of them not previously described. The best strain provided more than 120 nodules and more than 65 mg of nodules per plant. Strain BR14182 was considered the most promising given the high dry matter and N accumulation in plant shoots. This study provides the first analysis of Bradyrhizobium diversity nodulating M. axillare in Brazil and provided evidence of the role of inoculation in incrementing the plant-rhizobium symbiosis in a forage legume.

Flow-Based Dynamic Approach to Assess Bioaccessible Zinc in Dry Dog Food Samples.

This work proposes a simple and easy-to-use flow-through system for the implementation of dynamic extractions, aiming at the evaluation of bioaccessible zinc and the characterization of leaching kinetics in dry dog food samples. The kinetic profile of Zn extraction was determined by flame atomic absorption spectroscopy and the results were fitted in an exponential function (R2 > 0.960) compatible with a two first-order reactions model. Values of fast leachable Zn ranged from 83 ± 1 to 313 ± 5 mg of Zn per kg of sample, with associated rate constants ranging from 0.162 ± 0.004 to 0.290 ± 0.014 min-1. Similar results were observed compared to the static batch extraction. The percentage of bioaccessible Zn ranged from 49.0 to 70.0%, with an average value of 58.2% in relation to total Zn content. Principal component analysis regarding the variables fast leachable Zn, associated rate constant, total Zn, and market segment, has shown that 84.6% of variance is explained by two components, where the second component (24.0%) presented loadings only for the fast leachable Zn and associated rate constant. The proposed method is suitable for the fast evaluation (<1 h) of leaching kinetics and bioaccessibility in dry dog food.

Wash-Free Multiplexed Mix-and-Read Suspension Array Fluorescence Immunoassay for Anthropogenic Markers in Wastewater.

Pharmaceuticals, certain food ingredients, and mammalian endogenous metabolic products in wastewater are mostly of human origin. They are anthropogenic markers. Proper knowledge of their levels in wastewater helps to track sources of pollutants in natural waters and allows for calculation of removal efficiencies in wastewater treatment plants. Here, we describe the development and application of an indirect competitive, multiplexing suspension array fluorescence immunoassay (SAFIA) for the detection of carbamazepine (CBZ), diclofenac (DCF), caffeine (CAF), and isolithocholic acid (ILA) in wastewater, covering those classes of anthropogenic markers. The assay consists of haptens covalently conjugated to fluorescence-encoded polystyrene core/silica shell microparticles to create a site for competitive binding of the antibodies (Abs). Bound Abs are then stained with fluorophore-labeled Abs. Encoding and signaling fluorescence of the particles are determined by an automated flow cytometer. For compatibility of the immunoassay with the 96-well microtiter plate format, a stop reagent, containing formaldehyde, is used. This enables a wash-free procedure while decreasing time-to-result. Detection limits of 140 ± 40 ng/L for CBZ, 180 ± 110 ng/L for CAF, 4 ± 3 ng/L for DCF, and 310 ± 70 ng/L for ILA are achieved, which meet the sensitivity criteria of wastewater analysis. We demonstrate the applicability of SAFIA to real wastewater samples from three different wastewater treatment plants, finding the results in good agreement with LC-MS/MS. Moreover, the accuracy in general exceeded that from classical ELISAs. We therefore propose SAFIA as a quick and reliable approach for wastewater analysis meeting the requirements for process analytical technology.

Fluxes of CO2, CH4, and N2O in tundra-covered and Nothofagus forest soils in the Argentinian Patagonia.

While most soils in periglacial environments present high fluxes of CO2 (FCO2), CH4 (FCH4), and N2O (FN2O), few of them have a tendency to drain greenhouse gases from the atmosphere. This study aimed to assess greenhouse gas fluxes at different sub-Antarctic sites and time periods (at the beginning of thaw and height of summer). To investigate the time of year effect on greenhouse gas emissions, FCO2, FCH4, and FN2O were measured at two sites tundra-covered (Ti and Th) and Nothofagus forest soil (Nf) on Monte Martial, at the southernmost tip of South America, Tierra del Fuego, Argentina. FCO2 ranged from 96.33 to 225.72 µg CO2 m-2 s-1 across all sites and periods, showing a positive correlation with soil temperature (Ts) (4.1 and 8.2 °C, respectively) (r2 > 0.7; p < 0.05). The highest values of FCO2 were found at Ti and Th (728.2 and 662.64 µg CO2 m-2 s-1, respectively), which were related to higher temperatures (8.2 and 8.6 °C, respectively) when compared to those of Nf. For FCH4, the capture (drain) occurred during both periods at Nf (-26 and -79 µg C-CH4 m-2 h-1) as well as Ti and Th (-21 and 12 µg C-CH4 m-2 h-1, respectively). FN2O also presented low values during both periods and showed a tendency to drain N2O from the atmosphere, especially at Nf (-2 µg N-N2O m-2 h-1). In addition, FN2O was slightly positive for Ti and Th (0.3 and 0.55 µg N-N2O m-2 h-1, respectively). Soil moisture did not show a correlation (p > 0.05) with the measured greenhouse gas fluxes. A scenario of increased temperatures might result in changes in the balance between the emissions and drains of these gases from soils, leading to higher emission values of CH4 and N2O, especially for tundra covered soils (Ti and Th), where the highest average fluxes and thermohydric variations were observed over the year.

Estimating 20-year land-use change and derived CO2 emissions associated with crops, pasture and forestry in Brazil and each of its 27 states.

Land-use change (LUC) in Brazil has important implications on global climate change, ecosystem services and biodiversity, and agricultural expansion plays a critical role in this process. Concerns over these issues have led to the need for estimating the magnitude and impacts associated with that, which are increasingly reported in the environmental assessment of products. Currently, there is an extensive debate on which methods are more appropriate for estimating LUC and related emissions and regionalized estimates are lacking for Brazil, which is a world leader in agricultural production (e.g. food, fibres and bioenergy). We developed a method for estimating scenarios of past 20-year LUC and derived CO2 emission rates associated with 64 crops, pasture and forestry in Brazil as whole and in each of its 27 states, based on time-series statistics and in accordance with most used carbon-footprinting standards. The scenarios adopted provide a range between minimum and maximum rates of CO2 emissions from LUC according to different possibilities of land-use transitions, which can have large impacts in the results. Specificities of Brazil, like multiple cropping and highly heterogeneous carbon stocks, are also addressed. The highest CO2 emission rates are observed in the Amazon biome states and crops with the highest rates are those that have undergone expansion in this region. Some states and crops showing large agricultural areas have low emissions associated, especially in southern and eastern Brazil. Native carbon stocks and time of agricultural expansion are the most decisive factors to the patterns of emissions. Some implications on LUC estimation methods and standards and on agri-environmental policies are discussed.

Programmable flow system for automation of oxygen radical absorbance capacity assay using pyrogallol red for estimation of antioxidant reactivity.

An automated oxygen radical absorbance capacity (ORAC) method based on programmable flow injection analysis was developed for the assessment of antioxidant reactivity. The method relies on real time spectrophotometric monitoring (540 nm) of pyrogallol red (PGR) bleaching mediated by peroxyl radicals in the presence of antioxidant compounds within the first minute of reaction, providing information about their initial reactivity against this type of radicals. The ORAC-PGR assay under programmable flow format affords a strict control of reaction conditions namely reagent mixing, temperature and reaction timing, which are critical parameters for in situ generation of peroxyl radical from 2,2'-azobis(2-amidinopropane) dihydrochloride (AAPH). The influence of reagent concentrations and programmable flow conditions on reaction development was studied, with application of 37.5 µM of PGR and 125 mM of AAPH in the flow cell, guaranteeing first order kinetics towards peroxyl radicals and pseudo-zero order towards PGR. Peroxyl-scavenging reactivity of antioxidants, bioactive compounds and phenolic-rich beverages was estimated employing the proposed methodology. Recovery assays using synthetic saliva provided values of 90 ± 5% for reduced glutathione. Detection limit calculated using the standard antioxidant compound Trolox was 8 µM. RSD values were <3.4 and <4.9%, for intra and inter-assay precision, respectively. Compared to previous batch automated ORAC assays, the developed system also accounted for high sampling frequency (29 h(-1)), low operating costs and low generation of waste.

Nitrous Oxide and Methane Fluxes Following Ammonium Sulfate and Vinasse Application on Sugar Cane Soil.

This study aimed to quantify nitrous oxide (N2O) and methane (CH4) emission/sink response from sugar cane soil treated with fertilizer nitrogen (N) and vinasse applied separately or in sequence, the latter being investigated with regard to the time interval between applications for a possible effect on emissions. The study was carried out in a traditional area of unburned sugar cane in São Paulo state, Brazil. Two levels of N fertilization (0 and 100 kg N ha(-1)) with no added vinasse and combined with vinasse additions at different times (100 m(-3) ha(-1) at 3 and 15 days after N fertilization) were evaluated. Methane and N2O fluxes were monitored for 211 days. On average, the soil was a sink for CH4, which was not affected by the treatments. Emissions of N2O were induced by N fertilizer and vinasse applications. For ammonium sulfate, 0.6% of the added N was emitted as N2O, while for vinasse, this ranged from 1.0 to 2.2%. Changes in N2O fluxes were detected the day after application of vinasse on the N fertilized areas, but although the emission factor (EF) was 34% greater, the EF was not significantly different from fertilizer N alone. Nevertheless, we recommend to not apply vinasse after N fertilization to avoid boosting N2O emissions.

A critical review of the function of neuromelanin and an attempt to provide a unified theory.

This paper provides a critical review of the numerous and various biological functions so far attributed to neuromelanin and an attempt to provide a unified theory based on the peculiar physical and chemical properties of the black particle (the neuromelanin cage). It is stressed that neuromelanin is not homogeneous, as is commonly accepted, but is made up of different substrate specific black pigments formed by the oxidation of o.diphenols or other oxygenated precursors (substantia nigra melanin, locus coeruleus melanin, retinal pigmented epithelium or ocular melanin, inner-ear melanin, and so on). Ocular melanin is believed to protect the eye by trapping metals and free radicals. The paper shows that this unconfirmed mechanism is a rather fortuitous irreversible molecular accident, which at times may prove itself deleterious. Albinism often leads to deafness in animals, indicating a genetic correlation. These two conditions appear to be correlated at a molecular level to eye/ear pigmentation and suggest verifying this hypothesis in normal and albino human individuals. Skin and ocular melanin are chemically different. However, they are both involved in light absorption/dissipation. The black particle structure (melanin cage) is believed to be fundamental to this process because there is a common bioelectric mechanism. The latter is worth of further investigation. It is also proposed checking how ocular melanin dissipates the excessive absorbed light (as heat or as current?). It has been claimed that inner-ear melanin mutes acoustic waves. This paper suggests investigating the underlying mechanism and also studying whether this pigment is bio-electrically involved in audiology. According to numerous authors, substantia nigra melanin is only biological garbage. This view is rejected, and it is stressed that intracellular melanogenesis is a fundamental and genetically controlled physiological process. It has been repeatedly claimed that the binding of iron, heavy metals, free radicals and harmful chemicals by substantia nigra melanin is fundamental to body detoxification/protection. Presumably, such irreversible and generic binding mechanisms have no physiological foundation; it is suggested the alternative that, substantia nigra melanin acts as semiconductor, transmitting and modulating nervous impulses, in a reversible way. In fact, substantia nigra melanin is absent or significantly scarce in two conditions of life in which the coordination of movement is either inefficient (newborn babies) or strongly compromised (Parkinson). To check this assumption, further investigation of nucleus caudatus, putamen, globus pallidus, substantia nigra pars compacta and reticulata, nucleus hypothalamicus is recommended.

Tongue piercing. The new "rusty nail"?

BACKGROUND: Cephalic tetanus is a rare form of the tetanus caused primarily by wounds or other infectious processes involving the head and neck. This condition frequently progresses to the generalized form of tetanus with the attendant risks and complications. METHODS: A case report of a young female who developed an unusual form of tetanus after a tongue piercing is presented here. We discuss this disorder as it applies to the contemporary caregiver with a focus on its presentation and recognition. RESULTS: A delay in diagnosis of 13 days from presentation occurred. The patient had a slow, uneventful but incomplete recovery course. She never developed significant airway compromise, nor did she demonstrate any evidence of hemodynamic instability but continued to have right facial weakness up to 6 months after discharge. CONCLUSIONS: A few factors were identified that contributed to the significant delay in diagnosis. The unusual nature of the disease and a lowered index of suspicion on the part of the initial caregivers were probably the major causes. Fortunately, no major adverse sequelae resulted from the delay. However, if this case heralds the onset of a rise in the incidence of tetanus, early recognition and diagnosis would seem essential to avoid much of the morbidity and mortality associated with the disease.

Treatment of traumatic mandibular nonunion.

BACKGROUND AND OBJECTIVES: Nonunion is a complication in mandibular fractures. The causative factors include delay in treatment, infection, inadequate immobilization, and improper internal fixation; concomitant infection may be present. Pain, mobility of the fracture segments, and radiographic evidence of radiolucency did in diagnosis. METHODS AND MATERIALS: Three clinical cases are used to present the methods of treatment to manage nonunion following a gunshot wound, assault with a blunt object, and a fall. Treatment included antimicrobial therapy, fracture site debridement, segment immobilization, and bone grafting. Maxillomandibular fixation, debridement, and placement of a reconstruction plate were used in the first case; reconstruction plate, autogenous bone graft in a polyglactin resorbable mesh, and screw buttons in the second; and custom reconstruction plate and iliac crest bone graft in the third. RESULTS AND/OR CONCLUSIONS: All cases healed uneventfully. Due to rapid revascularization, use of autogenous cancellous bone grafts is preferred to cortical bone. Custom-molded polyglactin mesh provides control of the loose cancellous bone graft.