Evaluation of antibacterial activity induced by Staphylococcus aureus and Ent A in the hemolymph of Spodoptera littoralis

The problem of antibiotic resistance considers one of the most dangerous challenges facing the medical field. So, it is necessary to find substitutions to conventional antibiotics. Antimicrobial peptides (AMPs) are a bio-functional derivative that have been observed as one of the important solutions to such upcoming crisis. Owing to their role as the first line of defense against bacteria, fungi, and viruses. This study was conducted to induce the immune response of Spodoptera littoralis larvae by inoculation of sub lethal doses of Staphylococcus aureus and its enterotoxin. Since Staphylococcal enterotoxin A (SEA) considers the major causative agents of Staphylococcal food poisoning, our study oriented to purify and characterize this toxin to provoke its role in yielding AMPs with broad spectrum antimicrobial activity. A great fluctuation was recorded in the biochemical properties of immunized hemolymph not only in the total protein content but also protein banding pattern. Protein bands of ∼22 kDa (attacin-like) and ∼15 kDa (lysozyme-like) were found to be common between the AMPs induced as a result of both treatments. While protein bands of molecular weight ∼70 kDa (phenoloxidase-like) and ∼14 kDa (gloverin-like) were found specific for SEA treatment. Chromatographic analysis using HPLC for the induced AMPs showed different types of amino acids appeared with differences in their quantities and velocities. These peptides exhibited noticeable antimicrobial activity against certain Gram-positive and Gram-negative bacteria. In conclusion, the antimicrobial potential of the antimicrobial peptides (AMP) induced in the larval hemolymph of S. littoralis will be a promising molecule for the development of new therapeutic alternatives.     

Electrospun bioactive mats enriched with Ca-polyphosphate/retinol nanospheres as potential wound dressing

BackgroundWhile electrospun materials have been frequently used in tissue engineering no wound dressings exist that significantly improved wound healing effectively.MethodsWe succeeded to fabricate three-dimensional (3D) electrospun poly(D,l-lactide) (PLA) fiber mats into which nanospheres, formed from amorphous calcium polyphosphate (polyP) nanoparticles (NP) and encapsulated retinol (“retinol/aCa-polyP-NS” nanospheres [NS]), had been incorporated.ResultsExperiments with MC3T3-E1 cells revealed that co-incubation of the cells with Ca-polyP together with retinol (or incubation with retinol/aCa-polyP-NS) resulted in a significant synergistic effect on cell growth compared with particle-free polyP complexed with Ca²⁺ or amorphous Ca-polyP NPs and retinol alone. Incubation of the cells in the presence of the retinol/aCa-polyP NSs also caused a significant increase of the expression levels of the genes encoding for the fatty acid binding protein 4 (FABP4), as well as of the genes encoding for leptin and the leptin receptor. In contrast, the single components, soluble Na-polyP, complexed to Ca²⁺, or retinol-free aCa-polyP NPs, and retinol, had no significant effect on the expression of these genes.ConclusionsThese results indicate that the PLA fibers, supplemented with aCa-polyP-NP or retinol/aCa-polyP-NS, elicit morphogenetic activity, suggesting that these fiber mats, along with the antibacterial effect of polyP, have a beneficial potential as wound dressings combining antimicrobial and regenerative (wound healing) properties.General significanceThe PLA-based fiber mats, containing retinol and polyP nanoparticles, provide promising bioactive meshes that are urgently needed as dressings for chronic wounds.     

Dynamics of K Ion Conduction through Kv1.2

The crystallographic structure of a potassium channel, Kv1.2, in an open state makes it feasible to simulate entire K⁺ ion permeation events driven by a voltage bias and, thereby, elucidate the mechanism underlying ion conduction and selectivity of this type of channel. This Letter demonstrates that molecular dynamics simulations can provide movies of the overall conduction of K⁺ ions through Kv1.2. As suggested earlier, the conduction is concerted in the selectivity filter, involving 2-3 ions residing mainly at sites identified previously by crystallography and modeling. The simulations reveal, however, the jumps of ions between these sites and identify the sequence of multi-ion configurations involved in permeation.     

Fast Food Intake Increases the Incidence of Metabolic Syndrome in Children and Adolescents: Tehran Lipid and Glucose Study

The aim of the study was to evaluate the association between fast food consumption and incidence of metabolic syndrome (MetS) and its components among children and adolescents over a 3.6 year follow-up. Dietary data of 424 healthy subjects, aged 6–18 years, was collected using a valid and reliable food frequency questionnaire. Metabolic syndrome was defined according to the Cook et al criteria. Consumption of fast foods including hamburgers, sausages, bologna (beef), and fried potatoes was calculated and further categorized to quartiles. Multiple logistic regression models were used to estimate the incidence of MetS and its components in each quartile of fast food intake. The incidence of MetS was 11.3% after a 3.6 year follow up. In the fully adjusted model, compared to the lowest quartile of fast food intake, individuals in the highest had odds ratios of 2.96 (95% CI: 1.02–8.63; P for trend<0.001), 2.82 (95% CI: 1.01–7.87; P for trend = 0.037), and 2.58 (95% CI: 1.01–6.61; P for trend = 0.009) for incidence of MetS, hypertriglyceridemia, and abdominal obesity, respectively. No significant association was found between fast food intakes and other components of MetS. Fast food consumption is associated with the incidence of MetS, abdominal obesity, and hypertriglyceridemia in Tehranian children and adolescents.     

Bioethanol production with carboxymethylcellulase of Pseudomonas poae using castor bean (Ricinus communis L.) cake

Increased consumption of fossil fuels is an emerging problem. Scientists look for the existence of other alternatives to fossil fuels, including so-called renewable energy. Accordingly, we report the production of bio-ethanol from the remnants of castor oil bean seed cake (CBC) by the carboxymethylcellulase enzyme (CMCase). A bacterial strain isolated from rice straw showing higher CMCase activity was identified. The 16S rRNA result showed a 93% homology with the 16SrRNA gene sequences of Pseudomonas poae RE11-1-14, the strain was identified as Pseudomonas poae AB3. In addition, our results showed that the highest enzyme activity was achieved after 48 h and inoculum size of 3.7 × 10⁵ CFU. The optimum temperature, pH and Carboxymethylcellulose (CMC) concentration for the highest enzyme activity was 25 °C, pH 7 and 10 g/l respectively. Furthermore, The CMCase was purified by ammonium sulphate at a concentration of 60%. The SDS-PAGE of the purified enzyme showed a molecular weight of 88 kDa. Additionally, the (CBC) was hydrolyzed by the purified CMCase at the enzyme optimum conditions. The results showed the liberation of 5.2 g/L of reducing sugar by using dinitrosalicylic acid (DNS) assay. Finally, the total sugar produces 35 g/L after 48 h when Saccharomyces cerevisiae was used as a fermentation agent. Hence for the first time, we have been successfully able to produce bioethanol from CBC with CMCase of Pseudomonas poae.     

Conformational heterogeneity of α-synuclein in membrane

α-Synuclein (αS) is a natively disordered protein in solution, thought to be involved in the fusion of neurotransmitter vesicles to cellular membranes during neurotransmission. Monomeric αS has been previously characterized in two distinct membrane-associated conformations: a broken-helix structure, and an extended helix. By employing atomistic molecular dynamics and a novel membrane representation with significantly enhanced lipid mobility (HMMM), we investigate the process of spontaneous membrane binding of αS and the conformational dynamics of monomeric αS in its membrane-bound form.     

Validation and application of a highly specific and sensitive ELISA for the estimation of cortisone in saliva, urine and in vitro cell-culture media by using a novel antibody

It is generally acknowledged that local tissue concentrations of cortisol and cortisone are modulated by site-specific actions of 11β-hydroxysteroid dehydrogenase (11β-HSD) isoenzymes 1 and 2. Cortisone, the inactive metabolite of cortisol is produced by 11βHSD type 2. To assess 11β-HSD types 1 and 2 activities, the cortisol/cortisone ratio has to be accurately determined. Immunoassays to measure cortisone levels are not widely available and tend to lack specificity. The aim of this project was to develop a highly specific and sensitive ELISA method for the estimation of free cortisone levels in urine, saliva and in vitro media samples without chromatographic separation. Antibodies against cortisone were raised in rabbits using cortisone-3-CMO-KLH as immunogen. HRP-goat anti-rabbit IgG conjugate was used as enzyme tracer. Cross-reactivities of the untreated cortisone antiserum with major interfering steroids were minimal except for cortisol (3.15%). However, following an immune-affinity purification of the antibodies using CNBr-activated sepharose-cortisol-3-CMO-BSA, cross-reactivity of the purified cortisone antibody with cortisol was reduced to 0.27%. The minimum detection limit of cortisone ELISA was 28 pg/mL (77.7 pM). The validity of the cortisone ELISA was confirmed by the excellent correlation obtained before and after an HPLC fractionation step (Y=1.09X-0.21, R2=0.98). Intra-assay and inter-assay imprecision were 5.5-11.7% and 8.7-12.8% CV, respectively. Using this assay, salivary cortisone levels showed a circadian rhythm in men and women (11.2±7.3 nM at 08.00 h and 5.1±3.6 nM at 18.00 h), and the levels were reduced following liquorice ingestion. In media of adrenocortical H295 cell line incubations, basal cortisone levels were 4.24±0.22 nM that increased to 8.6±1.2 nM post forskolin treatment. Urinary free cortisone excretion levels in healthy subjects were 56.66±36.9 nmol/day. In human volunteers following ingestion of green coffee bean extract for 2 weeks, urinary free cortisol excretion reduced significantly from 66.67±22.3 to 42.66±17.5 nmol/day (p=0.02) and the cortisol/cortisone ratio from 2.04±1.33 to 1.49±1.13, p=0.05. In conclusion, a simple and highly specific and sensitive ELISA has been developed and applied to estimate cortisone levels in biological fluids and culture media.     

Molecular basis for the activation of a catalytic asparagine residue in a self-cleaving bacterial autotransporter

Autotransporters are secreted proteins produced by pathogenic Gram-negative bacteria. They consist of a membrane-embedded β-domain and an extracellular passenger domain that is sometimes cleaved and released from the cell surface. We solved the structures of three noncleavable mutants of the autotransporter EspP to examine how it promotes asparagine cyclization to cleave its passenger. We found that cyclization is facilitated by multiple factors. The active-site asparagine is sterically constrained to conformations favorable for cyclization, while electrostatic interactions correctly orient the carboxamide group for nucleophilic attack. During molecular dynamics simulations, water molecules were observed to enter the active site and to form hydrogen bonds favorable for increasing the nucleophilicity of the active-site asparagine. When the activated asparagine attacks its main-chain carbonyl carbon, the resulting oxyanion is stabilized by a protonated glutamate. Upon cleavage, this proton could be transferred to the leaving amine group, helping overcome a significant energy barrier. Together, these findings provide insight into factors important for asparagine cyclization, a mechanism broadly used for protein cleavage.