Leishmanicidal activity of amphotericin B encapsulated in PLGA–DMSA nanoparticles to treat cutaneous leishmaniasis in C57BL/6 mice

The major goal of this work was to design a new nanoparticle drug delivery system for desoxycholate amphotericin B (D-AMB), based on controlled particle size, looking for the most successful release of the active agents in order to achieve the best site-specific action of the drug at the therapeutically optimal rate and dose regimen. For this, AMB nanoencapsulated in poly(lactic-co-glycolic acid) (PLGA) and dimercaptosuccinic acid (DMSA) nanoparticles (Nano-D-AMB) has been developed, and its efficacy was evaluated in the treatment of experimental cutaneous leishmaniasis in C57BL/6 mice, to test if our nano-drug delivery system could favor the reduction of the dose frequency required to achieve the same therapeutic level of free D-AMB, and so, an extended dosing interval. Magnetic citrate-coated maghemite nanoparticles were added to this nanosystem (Nano-D-AMB-MG) aiming to increase controlled release of AMB by magnetohyperthermia. Female mice (N = 6/group) were infected intradermally in the right footpad with promastigotes of Leishmania amazonensis in the metacyclic phase, receiving the following intraperitoneal treatments: 1% PBS for 10 consecutive days; D-AMB at 2 mg/kg/day for 10 days (totalizing 20 mg/kg/animal); Nano-D-AMB and Nano-D-AMB-MG at 6 mg/kg on the 1st, 4th and 7th days and at 2 mg/kg on the 10th day, also totalizing 20 mg/kg/animal by treatment end. The Nano-D-AMB-MG group was submitted to an AC magnetic field, allowing the induction of magnetohyperthermia. The evaluations were through paw diameter measurements; parasite number and cell viability were investigated by limiting dilution assay. D-AMB-coated PLGA–DMSA nanoparticles showed the same efficacy as free D-AMB to reduce paw diameter; however, the Nano-D-AMB treatment also promoted a significantly greater reduction in parasite number and cell viability compared with free D-AMB. The nano-drug AMB delivery system appeared more effective than free D-AMB therapy to reduce the dose frequency required to achieve the same therapeutic level. It thus favors a longer interval between doses, as expected with development of a new nano drug delivery system, and may be useful in the treatment of many different pathologies, from cancer to neurodegenerative diseases.     

High levels of Cellular Prion Protein improve astrocyte development

Prion protein (PrP^C) has neuroprotective functions and herein we demonstrate that astrocytes from PrP^C-over-expressing mice are more resistant to induced cell death than wild-type astrocytes. The Stress-Inducible-Protein 1 (STI1), a PrP^C ligand, prevents cell death in both wild-type and PrP^C-over-expressing astrocytes through the activation of protein-kinase-A. Cultured embryonic astrocytes and brain extracts from PrP^C-over-expressing mice show higher glial fibrillary acidic protein expression and reduced vimentin and nestin levels when compared to wild-type astrocytes, suggesting faster astrocyte maturation in the former mice. Our data indicate that PrP^C levels modulate astrocyte development, and that PrP^C–STI1 interaction contributes to protect against astrocyte death.     

Osmotic Challenge Drives Rapid and Reversible Chromatin Condensation in Chondrocytes

Changes in extracellular osmolality have been shown to alter gene expression patterns and metabolic activity of various cell types, including chondrocytes. However, mechanisms by which physiological or pathological changes in osmolality impact chondrocyte function remain unclear. Here we use quantitative image analysis, electron microscopy, and a DNase I assay to show that hyperosmotic conditions (>400 mOsm/kg) induce chromatin condensation, while hypoosmotic conditions (100 mOsm/kg) cause decondensation. Large density changes (p < 0.001) occur over a very narrow range of physiological osmolalities, which suggests that chondrocytes likely experience chromatin condensation and decondensation during a daily loading cycle. The effect of changes in osmolality on nuclear morphology (p < 0.01) and chromatin condensation (p < 0.001) also differed between chondrocytes in monolayer culture and three-dimensional agarose, suggesting a role for cell adhesion. The relationship between condensation and osmolality was accurately modeled by a polymer gel model which, along with the rapid nature of the chromatin condensation (<20 s), reveals the basic physicochemical nature of the process. Alterations in chromatin structure are expected to influence gene expression and thereby regulate chondrocyte activity in response to osmotic changes.     

Burkholderia sp. SCMS54 reduces cadmium toxicity and promotes growth in tomato

Cadmium (Cd) can enter soil through the use of fertilisers, calcareous, pesticides and industrial and/or domestic effluents. Cd can leach into groundwater and be taken up by plants, potentially leading to reductions in plant growth and yield. In soil, plant roots interact with heavy metal (HM)‐tolerant microorganisms that may promote plant growth. Soil microorganisms may also be able to solubilise or mobilise soil metals, thereby acting as bioremediators. A better understanding of the interaction among plants, metals, microorganisms and soil will lead to improved plant tolerance. Two multi‐tolerant bacteria from the Burkholderia genus were isolated from Cd‐contaminated and Cd‐uncontaminated soil of a coffee plantation. In addition to its high tolerance to Cd, the strain SCMS54 produces indole‐acetic acid (IAA), solubilises inorganic phosphate and produces siderophores, demonstrating its potential to contribute to beneficial plant–microorganism interactions. When interacting with tomato plants exposed to Cd, the bacterium led to decreases in plant peroxide and chlorosis levels, promoted relative plant growth and decreased the root absorption of Cd, resulting in increased plant tolerance to this highly toxic HM. The results indicated that the inoculation of tomato plants with Burkholderia sp. SCMS54 promotes better growth in plants cultivated in the presence of Cd. This phenomenon appears to be attributed to a mechanism that decreases Cd concentrations in the roots via a beneficial interaction between the bacteria and the plant roots.     

Effects of an evaporative cooling system on plasma cortisol, IGF-I, and milk production in dairy cows in a tropical environment

Access to an evaporative cooling system can increase production in dairy cows because of improved thermal comfort. This study aimed to evaluate the impact of ambient temperature on thermoregulation, plasma cortisol, insulin-like growth factor 1 (IGF-I), and productive status, and to determine the efficiency of an evaporative cooling system on physiological responses under different weather patterns. A total of 28 Holstein cows were divided into two groups, one with and the other without access to a cooling system with fans and mist in the free stall. The parameters were analyzed during morning (0700 hours) and afternoon milking (1430 hours) under five different weather patterns throughout the year (fall, winter, spring, dry summer, and rainy summer). Rectal temperature (RT), body surface temperature (BS), base of tail temperature (TT), and respiratory frequency (RF) were lower in the morning (P?<?0.01). The cooling system did not affect RT, and both the groups had values below 38.56 over the year (P?=?0.11). Cortisol and IGF-I may have been influenced by the seasons, in opposite ways. Cortisol concentrations were higher in winter (P?<?0.05) and IGF-I was higher during spring-summer (P?<?0.05). The air temperature and the temperature humidity index showed positive moderate correlations to RT, BS, TT, and RF (P?<?0.001). The ambient temperature was found to have a positive correlation with the physiological variables, independent of the cooling system, but cooled animals exhibited higher milk production during spring and summer (P?<?0.01).     

Growth hormone transgenesis affects osmoregulation and energy metabolism in zebrafish (Danio rerio)

Growth hormone (GH) transgenic fish are at a critical step for possible approval for commercialization. Since this hormone is related to salinity tolerance in fish, our main goal was to verify whether the osmoregulatory capacity of the stenohaline zebrafish (Danio rerio) would be modified by GH-transgenesis. For this, we transferred GH-transgenic zebrafish (T) from freshwater to 11 ppt salinity and analyzed survival as well as relative changes in gene expression. Results show an increased mortality in T versus non-transgenic (NT) fish, suggesting an impaired mechanism of osmotic acclimation in T. The salinity effect on expression of genes related to osmoregulation, the somatotropic axis and energy metabolism was evaluated in gills and liver of T and NT. Genes coding for Na⁺, K⁺-ATPase, H⁺-ATPase, plasma carbonic anhydrase and cytosolic carbonic anhydrase were up-regulated in gills of transgenics in freshwater. The growth hormone receptor gene was down-regulated in gills and liver of both NT and T exposed to 11 ppt salinity, while insulin-like growth factor-1 was down-regulated in liver of NT and in gills of T exposed to 11 ppt salinity. In transgenics, all osmoregulation-related genes and the citrate synthase gene were down-regulated in gills of fish exposed to 11 ppt salinity, while lactate dehydrogenase expression was up-regulated in liver. Na⁺, K⁺-ATPase activity was higher in gills of T exposed to 11 ppt salinity as well as the whole body content of Na⁺. Increased ATP content was observed in gills of both NT and T exposed to 11 ppt salinity, being statistically higher in T than NT. Taking altogether, these findings support the hypothesis that GH-transgenesis increases Na⁺ import capacity and energetic demand, promoting an unfavorable osmotic and energetic physiological status and making this transgenic fish intolerant of hyperosmotic environments.     

Norfloxacin Zn(II)-based complexes: acid base ionization constant determination, DNA and albumin binding properties and the biological effect against Trypanosoma cruzi

Zn(II) complexes with norfloxacin (NOR) in the absence or in the presence of 1,10-phenanthroline (phen) were obtained and characterized. In both complexes, the ligand NOR was coordinated through a keto and a carboxyl oxygen. Tetrahedral and octahedral geometries were proposed for [ZnCl₂(NOR)]·H₂O (1) and [ZnCl₂(NOR)(phen)]·2H₂O (2), respectively. Since the biological activity of the chemicals depends on the pH value, pH titrations of the Zn(II) complexes were performed. UV spectroscopic studies of the interaction of the complexes with calf-thymus DNA (CT DNA) have suggested that they can bind to CT DNA with moderate affinity in an intercalative mode. The interactions between the Zn(II) complexes and bovine serum albumin (BSA) were investigated by steady-state and time-resolved fluorescence spectroscopy at pH 7.4. The experimental data showed static quenching of BSA fluorescence, indicating that both complexes bind to BSA. A modified Stern–Volmer plot for the quenching by complex 2 demonstrated preferential binding near one of the two tryptophan residues of BSA. The binding constants obtained (K _b ) showed that BSA had a two orders of magnitude higher affinity for complex 2 than for 1. The results also showed that the affinity of both complexes for BSA was much higher than for DNA. This preferential interaction with protein sites could be important to their biological mechanisms of action. The analysis in vitro of the Zn(II) complexes and corresponding ligand were assayed against Trypanosoma cruzi, the causative agent of Chagas disease and the data showed that complex 2 was the most active against bloodstream trypomastigotes.     

Differences in Al tolerance between Plantago algarbiensis and P. almogravensis reflect their ability to respond to oxidative stress

We evaluated the impact of low pH and aluminum (Al) on the leaves and roots of Plantago almogravensis Franco and Plantago algarbiensis Samp., focusing on energy partitioning in photosystem II, H₂O₂ levels, lipid peroxidation, electrolyte leakage (EL), protein oxidation, total soluble protein content and antioxidant enzyme activities. In both species, Al triggered more changes in oxidative metabolism than low pH alone, particularly in the roots. We found that Al increased the levels of H₂O₂ in P. algarbiensis roots, but reduced the levels of H₂O₂ in P. almogravensis leaves and roots. Neither low pH nor Al affected the spatial heterogeneity of chlorophyll fluorescence, the maximum photochemical efficiency of PSII (F_v/F_m), the actual quantum efficiency of PSII (?_PSII) or the quantum yields of regulated (?_NPQ) and nonregulated (?_NO) energy dissipation, and there was no significant change in total soluble protein content and EL. In P. algarbiensis, Al increased the carbonyl content and the activities of superoxide dismutase (SOD) and catalase (CAT) in the roots, and also CAT, ascorbate peroxidase and guaiacol peroxidase activities in the leaves. In P. almogravensis, Al reduced the level of malondialdehyde in the roots as well as SOD activity in the leaves and roots. We found that P. almogravensis plantlets could manage the oxidative stress caused by low pH and Al, whereas the P. algarbiensis antioxidant system was unable to suppress Al toxicity completely, leading to the accumulation of H₂O₂ and consequential protein oxidation in the roots.     

A combinatorial approach to the structure elucidation of a pyoverdine siderophore produced by a Pseudomonas putida isolate and the use of pyoverdine as a taxonomic marker for typing P. putida subspecies

The structure of a pyoverdine produced by Pseudomonas putida, W15Oct28, was elucidated by combining mass spectrometric methods and bioinformatics by the analysis of non-ribosomal peptide synthetase genes present in the newly sequenced genome. The only form of pyoverdine produced by P. putida W15Oct28 is characterized to contain α-ketoglutaric acid as acyl side chain, a dihydropyoverdine chromophore, and a 12 amino acid peptide chain. The peptide chain is unique among all pyoverdines produced by Pseudomonas subspecies strains. It was characterized as –l-Asp-l-Ala-d-AOHOrn-l-Thr-Gly-c[l-Thr(O-)-l-Hse-d-Hya-l-Ser-l-Orn-l-Hse-l-Ser-O-]. The chemical formula and the detected and calculated molecular weight of this pyoverdine are: C₆₅H₉₃N₁₇O₃₂, detected mass 1624.6404 Da, calculated mass 1624.6245. Additionally, pyoverdine structures from both literature reports and bioinformatics prediction of the genome sequenced P. putida strains are summarized allowing us to propose a scheme based on pyoverdines structures as tool for the phylogeny of P. putida. This study shows the strength of the combination of in silico analysis together with analytical data and literature mining in determining the structure of secondary metabolites such as peptidic siderophores.     

Identification of Penicillin-binding proteins employing support vector machines and random forest

Penicillin-Binding Proteins are peptidases that play an important role in cell-wall biogenesis in bacteria and thus maintaining bacterial infections. A wide class of β-lactam drugs are known to act on these proteins and inhibit bacterial infections by disrupting the cell-wall biogenesis pathway. Penicillin-Binding proteins have recently gained importance with the increase in the number of multi-drug resistant bacteria. In this work, we have collected a dataset of over 700 Penicillin-Binding and non-Penicillin Binding Proteins and extracted various sequence-related features. We then created models to classify the proteins into Penicillin-Binding and non-binding using supervised machine learning algorithms such as Support Vector Machines and Random Forest. We obtain a good classification performance for both the models using both the methods.