Peroxidase from Astragalus maritimus: purification and properties

A peroxidase has been purified to homogeneity from Astragalus maritimus seeds using ammonium sulfate precipitation and chromatography on DEAE-cellulose and hydroxylapatite. The purification obtained was 255 fold. The enzyme preparations were homogenous by the criteria of SDS-PAGE and analytical gel electrofocusing. The protein contained 0.11% of iron that corresponds to a minimum molecular size of 50,700. Determinations of molecular size by SDS-PAGE gave values of 48,000 +/- 1,000 while the one obtained by Sephadex gel filtration was 49,000. The pH optimum of the enzyme was 6.0. The activation energy was estimated to be 6 Kcal/mol. The prosthetic group was shown to be ferriprotoporphyrin IX. The presence of 13% neutral sugars was found. The spectrophotometric analysis showed the presence, in the visible region, of absorption maxima at 403, 490 and 633 nm. The Rz value (A403/A275) was 2.7.     

Antiproliferative Activity and VEGF Expression Reduction in MCF7 and PC-3 Cancer Cells by Paclitaxel and Imatinib Co-encapsulation in Folate-Targeted Liposomes

Co-encapsulation of anticancer drugs paclitaxel and imatinib in nanocarriers is a promising strategy to optimize cancer treatment. Aiming to combine the cytotoxic and antiangiogenic properties of the drugs, a liposome formulation targeted to folate receptor co-encapsulating paclitaxel and imatinib was designed in this work. An efficient method was optimized for the synthesis of the lipid anchor DSPE-PEG(2000)-folic acid (FA). The structure of the obtained product was confirmed by RMN, FT-IR, and ESI-MS techniques. A new analytical method was developed and validated for simultaneous quantification of the drugs by liquid chromatography. Liposomes, composed of phosphatidylcholine, cholesterol, and DSPE-mPEG(2000), were prepared by extrusion. Their surface was modified by post-insertion of DSPE-PEG(2000)-FA. Reaction yield for DSPE-PEG(2000)-FA synthesis was 87%. Liposomes had a mean diameter of 122.85 ± 1.48 nm and polydispersity index of 0.19 ± 0.01. Lyophilized formulations remained stable for 60 days in terms of size and drug loading. FA-targeted liposomes had a higher effect on MCF7 cell viability reduction (p < 0.05) when compared with non-targeted liposomes and free paclitaxel. On PC-3 cells, viability reduction was greater (p < 0.01) when cells were exposed to targeted vesicles co-encapsulating both drugs, compared with the non-targeted formulation. VEGF gene expression was reduced in MCF7 and PC-3 cells (p < 0.0001), with targeted vesicles exhibiting better performance than non-targeted liposomes. Our results demonstrate that multifunctional liposomes associating molecular targeting and multidrug co-encapsulation are an interesting strategy to achieve enhanced internalization and accumulation of drugs in targeted cells, combining multiple antitumor strategies.     

In vitro antibiofilm and anti‐adhesion effects of magnesium oxide nanoparticles against antibiotic resistant bacteria

The aim of the current investigation was to determine the antibacterial and antibiofilm potential of MgO nanoparticles (NPs) against antibiotic‐resistant clinical strains of bacteria. MgO NPs were synthesized by a wet chemical method and further characterized by scanning electron microscopy and energy dispersive X‐ray. Antibacterial activity was determined by broth microdilution and agar diffusion methods. The Bradford method was used to assess cellular protein leakage as a result of loss of membrane integrity. Microtiter plate assay following crystal violet staining was employed to determine the effect of MgO NPs on biofilm formation and removal of established biofilms. MIC values ranged between 125 and 500 μg/mL. Moreover, treatment with MgO NPs accelerated rate of membrane disruption, measured as a function of leakage of cellular proteins. Leakage of cellular protein content was greater among gram‐negative bacteria. Cell adherence assay indicated 25.3–49.8% inhibition of bacterial attachment to plastic surfaces. According to a static biofilm method, MgO NPs reduced biofilm formation potential from 31% to 82.9% in a time‐dependent manner. Moreover, NPs also significantly reduced the biomass of 48, 72, 96 and 120 hr old biofilms (P < 0.05). Cytotoxicity experiments using a neutral red assay revealed that MgO NPs are non‐toxic to HeLa cells at concentrations of 15–120 μg/mL. These data provide in vitro scientific evidence that MgO NPs are effective and safe antibiofilm agents that inhibit adhesion, biofilm formation and removal of established biofilms of multidrug‐resistant bacteria.

     

CoQ10 supplementation rescues nephrotic syndrome through normalization of H2S oxidation pathway

Nephrotic syndrome (NS), a frequent chronic kidney disease in children and young adults, is the most common phenotype associated with primary coenzyme Q₁₀ (CoQ₁₀) deficiency and is very responsive to CoQ₁₀ supplementation, although the pathomechanism is not clear. Here, using a mouse model of CoQ deficiency-associated NS, we show that long-term oral CoQ₁₀ supplementation prevents kidney failure by rescuing defects of sulfides oxidation and ameliorating oxidative stress, despite only incomplete normalization of kidney CoQ levels and lack of rescue of CoQ-dependent respiratory enzymes activities. Liver and kidney lipidomics, and urine metabolomics analyses, did not show CoQ metabolites. To further demonstrate that sulfides metabolism defects cause oxidative stress in CoQ deficiency, we show that silencing of sulfide quinone oxido-reductase (SQOR) in wild-type HeLa cells leads to similar increases of reactive oxygen species (ROS) observed in HeLa cells depleted of the CoQ biosynthesis regulatory protein COQ8A. While CoQ₁₀ supplementation of COQ8A depleted cells decreases ROS and increases SQOR protein levels, knock-down of SQOR prevents CoQ₁₀ antioxidant effects. We conclude that kidney failure in CoQ deficiency-associated NS is caused by oxidative stress mediated by impaired sulfides oxidation and propose that CoQ supplementation does not significantly increase the kidney pool of CoQ bound to the respiratory supercomplexes, but rather enhances the free pool of CoQ, which stabilizes SQOR protein levels rescuing oxidative stress.     

Efficiency of spinosad, <Emphasis Type="Italic">Bacillus thuringiensis</Emphasis> and <Emphasis Type="Italic">Trichogramma brassicae</Emphasis> against the tomato leafminer in greenhouse

The objective of this study was to evaluate the efficacy of three eco-friendly control agents, either singly or in a pairwise combination, for the control of the tomato leafminer, Tuta absoluta (Meyrick) (Lep: Gelechiidae). They include the naturally derived pesticide spinosad, a commercially available formulation of Bacillus thuringiensis var. Kurstaki (Bt), and a native population of Trichogramma brassicae Bezdenko (Hym: Trichogrammatidae). Tomato plants containing the T. absoluta were treated with one of the seven following treatments in a greenhouse: (1) a single release of T. brassicae against the eggs; (2) two applications of Bt (2 kg ha^?1); (3) and (4) one application of spinosad at two rates (60 and 120 g a.i. ha^?1); (5) T. brassicae release?+?Bt spray; (6) T. brassicae release?+?spinosad spray; and (7) spinosad spray?+?Bt spray. The highest mortality rate was recorded for the spinosad?+?Bt (88.33?±?1.43%) and T. brassicae?+?spinosad (78.33?±?3.74%) combinations, respectively; while the lowest mortality rate was obtained through the single application of T. brassicae (31.67?±?4.84%). Based on our results, the Bt and spinosad seem to be suitable candidates for combination with other biological and cultural techniques towards an integrated management of the tomato leafminer.     

Isolation and characterization of the mating-type locus of the barley pathogen Pyrenophora teres and frequencies of mating-type idiomorphs within and among fungal populations collected from barley landraces.

Pyrenophora teres f. sp. teres mating-type genes (MAT-1: 1190 bp; MAT-2: 1055 bp) have been identified. Their predicted proteins, measuring 379 and 333 amino acids, respectively, are similar to those of other Pleosporales, such as Pleospora sp., Cochliobolus sp., Alternaria alternata, Leptosphaeria maculans, and Phaeosphaeria nodorum. The structure of the MAT locus is discussed in comparison with those of other fungi. A mating-type PCR assay has also been developed; with this assay we have analyzed 150 isolates that were collected from 6 Sardinian barley landrace populations. Of these, 68 were P. teres f. sp. teres (net form; NF) and 82 were P. teres f. sp. maculata (spot form; SF). Within each mating type, the NF and SF amplification products were of the same length and were highly similar in sequence. The 2 mating types were present in both the NF and the SF populations at the field level, indicating that they have all maintained the potential for sexual reproduction. Despite the 2 forms being sympatric in 5 fields, no intermediate isolates were detected with amplified fragment length polymorphism (AFLP) analysis. These results suggest that the 2 forms are genetically isolated under the field conditions. In all of the samples of P. teres, the ratio of the 2 mating types was consistently in accord with the 1:1 null hypothesis. This ratio is expected when segregation distortion and clonal selection among mating types are absent or asexual reproduction is rare. Overall, sexual reproduction appears to be the major process that equalizes the frequencies of the 2 mating types within populations.     

Photophysical properties of hydroxyphenyl benzazoles and their applications as fluorescent probes to study local environment in DNA,protein and lipid

Fluorescence techniques have drawn increasing attention because they provide crucial information about molecular interactions in protein–ligand systems beyond that obtained by other methods. The advantage of fluorescence spectroscopy stems from the fact that the majority of molecules in biological systems do not exhibit fluorescence, making fluorescent probes useful with high sensitivity. Also, the fluorescence emission is highly sensitive to the local environment, providing a valuable tool to investigate the nature of binding sites in macromolecules. In this review, we discuss some of the important applications of a class of molecules that have been used as fluorescent probes in a variety of studies. Hydroxyphenyl benzazoles (HBXs) show distinct spectroscopic features that make them suitable probes for the study of certain biological mechanisms in DNA, protein and lipid. In particular, the complex photophysics of 2‐(2′‐hydroxyphenyl)benzoxazole (HBO) and the distinguished fluorescence signatures of its different tautomeric forms make this molecule a useful probe in several applications. Among these are probing the DNA local environment, study of the flexibility and specificity of protein‐binding sites, and detecting the heterogeneity and ionization ability of the head groups of different lipidic phases. The spectroscopy of HBX molecules and some of their chemically modified structures is also reviewed. Copyright © 2016 John Wiley & Sons, Ltd.     

A Randomized Controlled Double Blind Trial of Ciclosporin versus Prednisolone in the Management of Leprosy Patients with New Type 1 Reaction,in Ethiopia

Background

Leprosy Type 1 (T1R) reactions are immune-mediated events leading to nerve damage and preventable disability affecting hands, feet and eyes. Type 1 Reactions are treated with oral corticosteroids. There is little evidence on alternative treatments for patients who do not respond to steroids or experience steroid adverse effects. We report the results of a randomized controlled trial testing the efficacy and adverse effect profile of ciclosporin and prednisolone (CnP) in comparison to prednisolone only (P) in patients with new T1R in Ethiopia. Ciclosporin is a potent immunosuppressant. Outcomes were measured using a clinical severity score, recurrence rate, adverse events and quality of life.

Results

Seventy three patients with new T1R were randomized to receive CnP or P for 20 weeks. Recovery rates in skin signs was similar in both groups (91% vs 88%). Improvements in nerve function both, new and old, sensory (66% vs 49%) and motor (75% vs 74%) loss were higher (but not significantly so) in the patients on CnP. Recurrences rates of T1R (85%) were high in both groups, and recurrences occurred significantly earlier (8 weeks) in patients CnP, who needed 10% more additional prednisolone. Serious major and minor adverse events rates were similar in patients in the two treatment arms of the study. Both groups had a significant improvement in their quality of life after the study, measured by the SF-36.

Conclusions

This is the first double-blind RCT assessing ciclosporin, in the management of T1R in Africa. Ciclosporin could be a safe alternative second-line drug for patients with T1R who are not improving with prednisolone or are experiencing adverse events related to prednisolone. This study illustrates the difficulty in switching off leprosy inflammation. Better treatment agents for leprosy patients with reactions and nerve damage are needed.     

Comparison of Efficacy and Safety of Ciclosporin to Prednisolone in the Treatment of Erythema Nodosum Leprosum: Two Randomised,Double Blind,Controlled Pilot Studies in Ethiopia

Background

Erythema Nodosum Leprosum (ENL) is a serious complication of leprosy. It is normally treated with high dose steroids, but its recurrent nature leads to prolonged steroid usage and associated side effects. There is little evidence on the efficacy of alternative treatments for ENL, especially for patients who have become steroid resistant or have steroid side effects. These two pilot studies compare the efficacy and side effect profile of ciclosporin plus prednisolone against prednisolone alone in the treatment of patients with either new ENL or chronic and recurrent ENL.

Methods and Results

Thirteen patients with new ENL and twenty patients with chronic ENL were recruited into two double-blinded randomised controlled trials. Patients were randomised to receive ciclosporin and prednisolone or prednisolone treatment only. Patients with acute ENL had a delay of 16 weeks in the occurrence of ENL flare-up episode, with less severe flare-ups and decreased requirements for additional prednisolone. Patients with chronic ENL on ciclosporin had the first episode of ENL flare-up 4 weeks earlier than those on prednisolone, as well as more severe ENL flare-ups requiring 2.5 times more additional prednisolone. Adverse events attributable to prednisolone were more common that those attributable to ciclosporin.

Conclusions

This is the first clinical trial on ENL management set in the African context, and also the first trial in leprosy to use patients’ assessment of outcomes. Patients on ciclosporin showed promising results in the management of acute ENL in this small pilot study. But ciclosporin, did not appear to have a significant steroid–sparing effects in patients with chronic ENL, which may have been due to the prolonged use of steroids in these patients in combination with a too rapid decrease of steroids in patients given ciclosporin. Further research is needed to determine whether the promising results of ciclosporin in acute ENL can be reproduced on a larger scale.