Thermal radiation absorbed by dairy cows in pasture

The goal of the present paper was to assess a method for estimating the thermal radiation absorbed by dairy cows (0.875 Holstein–0.125 Guzerath) on pasture. A field test was conducted with 472 crossbred dairy cows in three locations of a tropical region. The following environmental data were collected: air temperature, partial vapour pressure, wind speed, black globe temperature, ground surface temperature and solar radiation. Average total radiation absorbed by animals was calculated as R_abs = 640.0 ±3.1 W.m ^{- 2} {R_{abs}} = 640.0 \pm 3.1\, W.{m^{ - 2}} . Absorbed short-wave radiation (solar direct, diffuse and reflected) averaged 297.9 ± 2.7 W m⁻²; long wave (from the sky and from terrestrial surfaces) averaged 342.1 ± 1.5 W m⁻². It was suggested that a new environmental measurement, the effective radiant heat load (ERHL), could be used to assess the effective mean radiant temperature ( T_\textmr^* ) \left( {T_{\text{mr}}^* } \right) . Average T_\textmr^* T_{\text{mr}}^* was 101.4 ± 1.2°C, in contrast to the usual mean radiant temperature, T_mr = 65.1 ±0.5^° C {T_{mr}} = 65.1 \pm 0.5^\circ C . Estimates of T_\textmr^* T_{\text{mr}}^* were considered as more reliable than those of T _mr in evaluating the thermal environment in the open field, because T _mr is almost totally associated only with long wave radiation.     

Phylogeographical, ecological and biological patterns shown by nuclear (ssrRNA and gGAPDH) and mitochondrial (Cyt b) genes of trypanosomes of the subgenus Schizotrypanum parasitic in Brazilian bats

The genetic diversity and phylogeographical patterns of Trypanosoma species that infect Brazilian bats were evaluated by examining 1043 bats from 63 species of seven families captured in Amazonia, the Pantanal, Cerrado and the Atlantic Forest biomes of Brazil. The prevalence of trypanosome-infected bats, as estimated by haemoculture, was 12.9%, resulting in 77 cultures of isolates, most morphologically identified as Trypanosoma cf. cruzi, classified by barcoding using partial sequences from ssrRNA gene into the subgenus Schizotrypanum and identified as T. cruzi (15), T. cruzi marinkellei (37) or T. cf. dionisii (25). Phylogenetic analyses using nuclear ssrRNA, glycosomal glyceraldehyde 3-phosphate dehydrogenase (gGAPDH) and mitochondrial cytochrome b (Cyt b) gene sequences generated three clades, which clustered together forming the subgenus Schizotrypanum. In addition to vector association, bat trypanosomes were related by the evolutionary history, ecology and phylogeography of the bats. Trypanosoma cf. dionisii trypanosomes (32.4%) infected 12 species from four bat families captured in all biomes, from North to South Brazil, and clustered with T. dionisii from Europe despite being separated by some genetic distance. Trypanosoma cruzi marinkellei (49.3%) was restricted to phyllostomid bats from Amazonia to the Pantanal (North to Central). Trypanosoma cruzi (18.2%) was found mainly in vespertilionid and phyllostomid bats from the Pantanal/Cerrado and the Atlantic Forest (Central to Southeast), with a few isolates from Amazonia.     

Binding of Azole Antibiotics to Staphylococcus aureus Flavohemoglobin Increases Intracellular Oxidative Stress

In this work, we report that flavohemoglobin contributes to the azole susceptibility of Staphylococcus aureus. We first observed that deletion of the flavohemoglobin gene leads to an increase in the viability of imidazole-treated S. aureus cells and that reversion to the wild-type phenotype occurs upon expression of flavohemoglobin from a multicopy plasmid. Further spectroscopic analyses showed that miconazole, the most efficient azole antibiotic against S. aureus, ligates to heme of both oxidized and reduced flavohemoglobin. The binding of miconazole to oxidized flavohemoglobin, with an association constant of 1.7 × 10⁶ M⁻¹, typical of a tight, specific binding equilibrium, results in augmentation of the superoxide production by the enzyme. These results are corroborated by in vivo studies showing that imidazole-treated S. aureus cells expressing flavohemoglobin contain a larger amount of reactive oxygen species. Moreover, it was observed that the survival of miconazole-treated S. aureus internalized by murine macrophages is higher for cells lacking flavohemoglobin. Altogether, the present data revealed that in S. aureus, flavohemoglobin enhances the antimicrobial activity of imidazoles via an increase of intracellular oxidative stress.Staphylococcus aureus is an opportunistic pathogen responsible for a large number of human infections that cause systemic diseases from a mild to life-threatening character. The increasing incidence of methicillin-resistant S. aureus (MRSA) strains observed in the past few years makes S. aureus infections a leading threat to public health, causing more deaths in the United States and Europe than human immunodeficiency virus (AIDS) (11). Like other Gram-positive bacteria, staphylococci are sensitive to imidazoles (27). Imidazoles (such as clotrimazole, miconazole, ketoconazole, and sulconazole) (Fig. ​(Fig.1)1) represent one of the major classes of azole antifungal that are useful in the treatment of infections, including cutaneous and vaginal candidiasis (8). The activity of these antifungal drugs derives primarily from inhibition of the biosynthesis of ergosterol, an essential component of the fungal plasma membrane, at the level of lanosterol 14-alpha demethylase. Furthermore, in fungi and yeast, azole treatment leads to an increase in the endogenous production of reactive oxygen species (ROS) (12, 25). For example, in Candida albicans and Saccharomyces cerevisiae, the miconazole inhibition of cytochrome c oxidase, peroxidase, and catalase has been reported to be responsible for a high level of ROS production (3, 4). It has also been reported that clotrimazole inhibition of Plasmodium falciparum hemoperoxidase leads to ROS accumulation in this protozoan pathogen (26). For S. cerevisiae, C. albicans, and Escherichia coli, the action of imidazoles was also correlated with the inhibition of the nitric oxide (NO) scavenger activity of flavohemoglobin (7).Open in a separate windowFIG. 1.Structures of the azole (imidazole; 1,2,4-triazole) antibiotics investigated.Flavohemoglobins (Hmp) are widespread among bacteria and yeast and contain three domains: C-terminal NAD- and flavin adenine dinucleotide (FAD)-binding domains, which together constitute a ferredoxin-NADP⁺ oxidoreductase-like domain, and an N-terminal globin domain, which harbors a single B-type heme. The high-spin heme contains one axial histidine and binds small molecules like NO, carbon monoxide (CO), and dioxygen (O₂). The heme can also bind bulky aromatic bases, since it is inserted in a large hydrophobic pocket (7). We observed that the binding of imidazoles to S. aureus flavohemoglobin results in an increase in the amount of deleterious reactive oxygen species produced by flavohemoglobin that contributes to the bactericidal effect of azole antibiotics toward S. aureus.     

In vivo uptake of a haem analogue Zn protoporphyrin IX by the human malaria parasite P. falciparum‐infected red blood cells

The cellular traffic of haem during the development of the human malaria parasite Plasmodium falciparum, through the stages R (ring), T (trophozoite) and S (schizonts), was investigated within RBC (red blood cells). When Plasmodium cultures were incubated with a fluorescent haem analogue, ZnPPIX (Zn protoporphyrin IX) the probe was seen at the cytoplasm (R stage), and the vesicle‐like structure distribution pattern was more evident at T and S stages. The temporal sequence of ZnPPIX uptake byP. falciparum‐infected erythrocytes shows that at R and S stages, a time‐increase acquisition of the porphyrin reaches the maximum fluorescence distribution after 60 min; in contrast, at the T stage, the maximum occurs after 120 min of ZnPPIX uptake. The difference in time‐increase acquisition of the porphyrin is in agreement with a maximum activity of haem uptake at the T stage. To gain insights into haem metabolism, recombinant PfHO (P. falciparum haem oxygenase) was expressed, and the conversion of haem into BV (biliverdin) was detected. These findings point out that, in addition to haemozoin formation, the malaria parasite P. falciparum has evolved two distinct mechanisms for dealing with haem toxicity, namely, the uptake of haem into a cellular compartment where haemozoin is formed and HO activity. However, the low Plasmodium HO activity detected reveals that the enzyme appears to be a very inefficient way to scavenge the haem compared with the Plasmodium ability to uptake the haem analogue ZnPPIX and delivering it to the food vacuole.     

Quantification of five putative periodontal pathogens in female patients with and without chronic periodontitis by real-time polymerase chain reaction

Chronic periodontitis is a highly prevalent endogenous polymicrobial disease. To better understand the etiology of the disease a quantitative approach is mandatory and real-time PCR is the molecular technique currently preferred to achieve this purpose. Taking into account that such a kind of study is still scarce, we aimed to evaluate the association between periodontal microbiota and chronic periodontitis. A total of 60 low-income age-matched female adults, 30 with chronic periodontitis and 30 without periodontal disease, were enrolled. DNA obtained from subgingival specimens was used for quantification of Aggregatibacter actinomycetemcomitans, Eikenella corrodens, Fusobacterium nucleatum, Porphyromonas gingivalis, and Prevotella intermedia by real-time PCR. A. actinomycetemcomitans, E. corrodens, and F. nucleatum were detected in all subjects, P. gingivalis was observed in 70.0% and 46.6% and P. intermedia in 90.0% and 80.0% of chronic periodontitis patients and periodontally healthy subjects, respectively. P. gingivalis mean count was significantly higher in patients with chronic periodontitis than in periodontally healthy individuals. Accurate detection and quantification of five putative periodontal pathogens was feasible using a simple and fast real-time PCR protocol. Although P. gingivalis and P. intermedia have been found more commonly in chronic periodontitis patients, no statistical difference was observed between periodontally diseased and healthy groups. Quantitative data indicated association between P. gingivalis and chronic periodontitis. However, because of its uneven distribution, it should not be solely taken as a marker of periodontal status.     

Antiprotozoal,Schistosomicidal, and Antimicrobial Activities of the Essential Oil from the Leaves of Baccharis dracunculifolia

Baccharis dracunculifolia DC. (Asteraceae), popularly known as ‘alecrim do campo’, is a native plant from Brazil used in folk medicine as febrifuge, anti‐inflammatory, antiseptic, and to treat skin sores. Also, B. dracunculifolia is the most important plant source of the Brazilian green propolis, which is recognized for its antiseptic and antiprotozoal activities. This study aimed at investigating the in vitro antiprotozoal, schistosomicidal, and antimicrobial activities of the essential oil from the leaves of B. dracunculifolia. The essential oil was obtained by hydrodistillation and analyzed by GC and GC/MS, which allowed the identification of 14 compounds, mainly oxygenated sesquiterpenes, such as (E)‐nerolidol (33.51%) and spathulenol (16.24%). The essential oil showed activity against promastigote forms of Leishmania donovani, with IC₅₀ values of 42 μg/ml. The essential oil displayed high activity in the schistosomicidal assay, since all pairs of Schistosoma mansoni adult worms were dead after incubation with the essential oil (10, 50, and 100 μg/ml). B. dracunculifolia essential oil was neither cytotoxic against Vero cells, nor active in the antimicrobial and antiplasmodial assays.     

Analysis of protein glycation using phenylboronate acrylamide gel electrophoresis

The incorporation of the specialized carbohydrate affinity ligand methacrylamido phenylboronic acid in polyacrylamide gels for SDS‐PAGE analysis has been successful for the separation of carbohydrates and has here been adapted for the analysis of post‐translationally modified proteins. While conventional SDS‐PAGE analysis cannot distinguish between glycated and unglycated proteins, methacrylamido phenylboronate acrylamide gel electrophoresis (mP‐AGE) in low loading shows dramatic retention of δ‐gluconolactone modified proteins, while the mobility of the unmodified proteins remains unchanged. With gels containing 1% methacrylamido phenylboronate, mP‐AGE analysis of gluconoylated recombinant protein Sbi results in the retention of the modified protein at a position expected for a protein that has quadrupled its expected molecular size. Subsequently, mP‐AGE was tested on HSA, a protein that is known to undergo glycation under physiological conditions. mP‐AGE could distinguish between various carbohydrate‐protein adducts, using in vitro glycated HSA, and discriminate early from late glycation states of the protein. Enzymatically glycosylated proteins show no altered retention in the phenylboronate‐incorporated gels, rendering this method highly selective for glycated proteins. We reveal that a trident interaction between phenylboronate and the Amadori cis 1,2 diol and amine group provides the molecular basis of this specificity. These results epitomize mP‐AGE as an important new proteomics tool for the detection, separation, visualization and identification of protein glycation. This method will aid the design of inhibitors of unwanted carbohydrate modifications in recombinant protein production, ageing, diabetes, cardiovascular diseases and Alzheimer's disease.