Initial characterization of an autoclaved Toxoplasma vaccine in mice

Toxoplasmosis is a zoonotic protozoal disease that has a major significance from the perspectives of public health and veterinary medicine. Therefore, an obvious long-term goal of many scientists would be the development of an effective vaccine. In this study, autoclaved vaccine was evaluated for its ability to protect mice against Toxoplasma gondii RH challenge as an acute infection model. Results showed that autoclaved Toxoplasma vaccine (ATV) when combined with BCG as an adjuvant was effective in triggering cell mediated immunity as shown by a significant increase in the percentage of splenic CD8+ T-lymphocytes. Following challenge, death of mice vaccinated with ATV was delayed for nine days. There was a significant decrease in parasite density in different organs, and a marked reduction of pathological changes in the liver suggesting that significant immune responses were mounted following vaccination. Future studies are warranted to test the vaccine against challenge with brain cysts as a chronic infection model and to evaluate it with other recent immunization strategies that can further enhance its immunogenicity.     

Distinct intracellular motifs of Delta mediate its ubiquitylation and activation by Mindbomb1 and Neuralized

DSL proteins are transmembrane ligands of the Notch receptor. They associate with a RING (really interesting new gene) family E3 ubiquitin ligase, either Neuralized (Neur) or Mindbomb 1 (Mib1), as a prerequisite to signaling. Although Neur and Mib1 stimulate internalization of DSL ligands, it is not known how ubiquitylation contributes to signaling. We present a molecular dissection of the intracellular domain (ICD) of Drosophila melanogaster Delta (Dl), a prototype DSL protein. Using a cell-based assay, we detected ubiquitylation of Dl by both Neur and Mib1. The two enzymes use distinct docking sites and displayed different acceptor lysine preferences on the Dl ICD. We generated Dl variants that selectively perturb its interactions with Neur or Mib1 and analyzed their signaling activity in two in vivo contexts. We found an excellent correlation between the ability to undergo ubiquitylation and signaling. Therefore, ubiquitylation of the DSL ICD seems to be a necessary step in the activation of Notch.     

Biophysical characterization of gold nanoparticles-loaded liposomes

Gold nanoparticles were prepared and loaded into the bilayer of dipalmitoylphosphatidylcholine (DPPC) liposomes, named as gold-loaded liposomes. Biophysical characterization of gold-loaded liposomes was studied by transmission electron microscopy (TEM) and Fourier transform infrared (FTIR) spectroscopy as well as turbidity and rheological measurements. FTIR measurements showed that gold nanoparticles made significant changes in the frequency of the CH₂ stretching bands, revealing that gold nanoparticles increased the number of gauche conformers and create a conformational change within the acyl chains of phospholipids. The transmission electron micrographs (TEM) revealed that gold nanoparticles were loaded in the liposomal bilayer. The zeta potential of DPPC liposomes had a more negative value after incorporating of Au NPs into liposomal membranes. Turbidity studies revealed that the loading of gold nanoparticles into DPPC liposomes results in shifting the temperature of the main phase transition to a lower value. The membrane fluidity of DPPC bilayer was increased by loading the gold nanoparticles as shown from rheological measurements. Knowledge gained in this study may open the door to pursuing liposomes as a viable strategy for Au NPs delivery in many diagnostic and therapeutic applications.     

Proteomic profiling of aging in the mouse heart: Altered expression of mitochondrial proteins

Using two-dimensional gel electrophoresis and liquid chromatography-tandem mass spectrometry, we have used a systems biology approach to study the molecular basis of aging of the mouse heart. We have identified 8 protein spots whose expression is up-regulated due to aging and 36 protein spots whose expression is down-regulated due to aging (p0.05 as judged by Wilcoxon Rank Sum test). Among the up-regulated proteins, we have characterized 5 protein spots and 2 of them, containing 3 different enzymes, are mitochondrial proteins. Among the down-regulated proteins, we have characterized 27 protein spots and 16 of them are mitochondrial proteins. Mitochondrial damage is believed to be a key factor in the aging process. Our current study provides molecular evidence at the level of the proteome for the alteration of structural and functional parameters of the mitochondria that contribute to impaired activity of the mouse heart due to aging.     

Interactions of Bovine Brain Tubulin with Pyridostigmine Bromide and N,N′-Diethyl-m-Toluamide

Pyridostigmine bromide (PB), an inhibitor of acetylcholinesterase, has been used as a prophylactic for nerve gas poisoning. N,N-diethyl-m-toluamide (DEET) is the active ingredient in most insect repellents and is thought to interact synergistically with PB. Since PB can inhibit the binding of organophosphates to tubulin and since organophosphates inhibit microtubule assembly, we decided to examine the effects of PB and DEET on microtubule assembly as well as their interactions with tubulin, the subunit protein of microtubules. We found that PB binds to tubulin with an apparent K _d of about 60 M. PB also inhibits microtubule assembly in vitro, although at higher concentrations PB induces formation of tubulin aggregates of high absorbance. Like PB, DEET is a weak inhibitor of microtubule assembly and also induces formation of tubulin aggregates. Many tubulin ligands stabilize the conformation of tubulin as measured by exposure of sulfhydryl groups and hydrophobic areas and stabilization of colchicine binding. PB appears to have very little effect on tubulin conformation, and DEET appears to have no effect. Neither compound interferes with colchicine binding to tubulin. Our results raise the possibility that PB and DEET may exert some of their effects in vivo by interfering with microtubule assembly or function, although high intracellular levels of these compounds would be required.     

Degradation of polyesters by a novel marine Nocardiopsis aegyptia sp. nov.: application of Plackett-Burman experimental design for the improvement of PHB depolymerase activity

This is the first report on the degradation of poly(3-hydroxybutyrate) (PHB), and its copolymers poly(3-hydroxyvalerate) P(3HB-co-10-20% HV) by Nocardiopsis aegyptia, a new species isolated from marine seashore sediments. The strain excreted an extracellular PHB depolymerase and grew efficiently on PHB or its copolymers as the sole carbon sources. The degradation activity was detectable by the formation of a transparent clearing zone around the colony on an agar Petri plate after 25 days, or a clearing depth under the colony in test tubes within 3 weeks. The previous techniques proved that the bacterium was able to assimilate the monomeric components of the shorter alkyl groups of the polymers. Nocardiopsis aegyptia hydrolyzed copolymers 10-20% PHBV more rapidly than the homopolymer PHB. The bacterial degradation of the naturally occurring sheets of poly(3-hydroxybutyrate), and its copolymer poly(3-hydroxybutyrate-co-3-hydroxyvalerate) was observed by scanning electron microscopy (SEM). The samples were degraded at the surface and proceeded to the inner part of the materials. Clear morphological alterations of the polymers were noticed, indicating the degradative capability of the bacterium. Plackett-Burman statistical experimental design has been employed to optimize culture conditions for maximal enzyme activity. The main factors that had significant positive effects on PHB depolymerase activity of Nocardiopsis aegyptia were sodium gluconate, volume of medium/flask and age of inoculum. On the other hand, MgSO4.7H2O, KH2PO4, K2HPO4 and NH4NO3 exhibited negative effects. Under optimized culture conditions, the highest activity (0.664 U/mg protein) was achieved in a medium predicted to be near optimum containing (in g/L): PHB, 0.5; C6H11O7Na, 7.5; MgSO4.7H2O, 0.35; K2HPO4, 0.35; NH4NO3, 0.5; KH2PO4, 0.35; malt extract, 0.5 and prepared with 50% seawater. The medium was inoculated with 1% (v/v) spore suspension of 7 days old culture. Complete clarity of the medium was achieved after 3 days at 30 degrees C.     

Enhancing the Therapeutic Efficacy of Tamoxifen Citrate Loaded Span-Based Nano-Vesicles on Human Breast Adenocarcinoma Cells

Serious adverse effects and low selectivity to cancer cells are the main obstacles of long term therapy with Tamoxifen (Tmx). This study aimed to develop Tmx-loaded span-based nano-vesicles for delivery to malignant tissues with maximum efficacy. The effect of three variables on vesicle size (Y₁), zeta potential (Y₂), entrapment efficiency (Y₃) and the cumulative percent release after 24 h (Y₄) were optimized using Box-Behnken design. The optimized formula was prepared and tested for its stability in different storage conditions. The observed values for the optimized formula were 310.2 nm, ??42.09 mV, 75.45 and 71.70% for Y₁, Y₂, Y₃, and Y₄, respectively. The examination using electron microscopy confirmed the formation of rounded vesicles with distinctive bilayer structure. Moreover, the cytotoxic activity of the optimized formula on both breast cancer cells (MCF-7) and normal cells (BHK) showed enhanced selectivity (9.4 folds) on cancerous cells with IC₅₀ values 4.7?±?1.5 and 44.3?±?1.3 μg/ml on cancer and normal cells, respectively. While, free Tmx exhibited lower selectivity (2.5 folds) than optimized nano-vesicles on cancer cells with IC₅₀ values of 9.0?±?1.1 μg/ml and 22.5?±?5.3 μg/ml on MCF-7 and BHK cells, respectively. The promising prepared vesicular system, with greater efficacy and selectivity, provides a marvelous tool to overcome breast cancer treatment challenges.     

Impact of Apo E gene polymorphism on HCV therapy related outcome in a cohort of HCV Egyptian patients

The functional apolipoprotein E (Apo E) gene polymorphism could be used as a determinant of outcome of HCV infection. This study aimed to demonstrate the impact of Apo E genotype on the response to HCV combined therapy. Material and methods: The study has been implemented on 125 individuals with persistent HCV infection and 120 cases with sustained virologic response (SVR). All participants were genotyped for ApoE gene polymorphism by a real-time quantitative PCR (qPCR). Results: Statistically significant differences were demonstrated regarding the Apo E genotypes between the two groups (P-value?<?.001) where the frequency of E3E3 was significantly higher among the chronic HCV-patients while E3E4 and E4E4 genotypes frequencies were higher among the SVR-subjects group and E3E3 genotype was associated with increased risk of chronicity (OR 4.7; 95% CI 1.9–12.1, P-value?<?.001). Moreover, There were statically significant differences regarding E3 and E4 alleles frequencies, where E3 allele display a higher frequency among the chronic HCV-patient group while the SVR-subjects group showed higher frequency of E4 allele and the carriers of E3 allele have 1.4 times more risk to develop chronicity than those with E4 allele (OR 1.4; 95% CI 1.0–2.0, P-value?<?.05). Meanwhile the protective E2 allele was absent in all infected participants. Conclusion: This study supports the hypothesis of the protective impact of Apo E4 allele that favors viral clearance of HCV infection and its recovery after combined therapy, while the Apo E3 allele is considered as a particular risk factor for the chronicity in HCV patients and resistance to therapy. Whereas the Apo E2 allele confers a resistance to HCV infection at a time of exposure.