Novel protein candidates for serodiagnosis of African animal trypanosomosis: Evaluation of the diagnostic potential of lysophospholipase and glycerol kinase from Trypanosoma brucei

African trypanosomosis, a parasitic disease caused by protozoan parasites transmitted by tsetse flies, affects both humans and animals in sub-Saharan Africa. While the human form (HAT) is now limited to foci, the animal form (AAT) is widespread and affects the majority of sub-Saharan African countries, and constitutes a real obstacle to the development of animal breeding. The control of AAT is hampered by a lack of standardized and easy-to used diagnosis tools. This study aimed to evaluate the diagnostic potential of TbLysoPLA and TbGK proteins from Trypanosoma brucei brucei for AAT serodiagnosis in indirect ELISA using experimental and field sera, individually, in combination, and associated with the BiP C-terminal domain (C25) from T. congolense. These novel proteins were characterized in silico, and their sequence analysis showed strong identities with their orthologs in other trypanosomes (more than 60% for TbLysoPLA and more than 82% for TbGK). TbLysoPLA displays a low homology with cattle (<35%) and Piroplasma (<15%). However, TbGK shares more than 58% with cattle and between 45–55% with Piroplasma. We could identify seven predicted epitopes on TbLysoPLA sequence and 14 potential epitopes on TbGK. Both proteins were recombinantly expressed in Escherichia coli. Their diagnostic potential was evaluated by ELISA with sera from cattle experimentally infected with T. congolense and with T.b. brucei, sera from cattle naturally infected with T. congolense, T. vivax and T.b. brucei. Both proteins used separately had poor diagnostic performance. However, used together with the BiP protein, they showed 60% of sensitivity and between 87–96% of specificity, comparable to reference ELISA tests. In conclusion, we showed that the performance of the protein combinations is much better than the proteins tested individually for the diagnosis of AAT.     

Mycobacterial manipulation of the host cell

Phagosome biogenesis, the process by which macrophages neutralize ingested pathogens and initiate antigen presentation, has entered the field of cellular mycobacteriology research largely owing to the discovery 30 years ago that phagosomes harboring mycobacteria are refractory to fusion with lysosomes. In the past decade, the use of molecular genetics and biology in different model systems to study phagosome biogenesis have made significant advances in understanding subtle mechanisms by which mycobacteria inhibit the maturation of its phagosome. Thus, we are beginning to appreciate the extent to which these pathogens are able to interfere with innate immune responses and manipulate defense mechanisms to enhance their survival within the human host cell. Here, we summarize current knowledge about phagosome maturation arrest in infected macrophages and the subsequent attenuation of the macrophage-initiated adaptive anti-mycobacterial immune defenses.     

Rapid assay for plasma chlorambucil and phenyl acetic mustard using reversed-phase liquid chromatography

A rapid assay for chlorambucil, a drug used for the treatment of chronic lymphocytic leukemia, and its major metabolite is described. Chromatographic behaviour of the two compounds on two different reversed-phase columns is discussed, as well as the kinetics of their hydrolysis in aqueous medium. The developed analysis can be applied to the determination of the plasma levels of the drug and its metabolite. No sample preparation is required and the spectrophotometric detection affords the sensitivity in the picomole range. Total analysis time is between 10 and 15 min.     

Phosphorylation of serine 323 of ASB2α is pivotal for the targeting of filamin A to degradation

ASB proteins are the specificity subunits of cullin5-RING E3 ubiquitin ligases (CRL5) that play roles in ubiquitin-mediated protein degradation. However, how their activity is regulated remains poorly understood. Here, we unravel a novel mechanism of regulation of a CRL5 through phosphorylation of its specificity subunit ASB2α. Indeed, using mass spectrometry, we showed for the first time that ASB2α is phosphorylated and that phosphorylation of serine-323 (Ser-323) of ASB2α is crucial for the targeting of the actin-binding protein filamin A (FLNa) to degradation. Mutation of ASB2α Ser-323 to Ala had no effect on intrinsic E3 ubiquitin ligase activity of ASB2α but abolished the ability of ASB2α to induce degradation of FLNa. In contrast, the ASB2α Ser-323 to Asp phosphomimetic mutant induced acute degradation of FLNa. Moreover, inhibition of the extracellular signal-regulated kinases 1 and 2 (Erk1/2) activity reduced ASB2α-mediated FLNa degradation. We further showed that the subcellular localization of ASB2α to actin-rich structures is dependent on ASB2α Ser-323 phosphorylation and propose that the interaction with FLNa depends on the electrostatic potential redistribution induced by the Ser-323 phosphate group. Taken together, these data unravel an important mechanism by which ASB2α-mediated FLNa degradation can be regulated.     

Autophagy in osteoporosis: Relation to oxidative stress

Impaired autophagy and oxidative stress are implicated in the development of many diseases. This study aimed to investigate the involvement of autophagy represented by autophagy-related gene 7 (Atg7) and oxidative stress represented by superoxide dismutase 2 (SOD2) gene expression and enzyme activity in the pathogenesis of osteoporosis. Atg7 and SOD2 gene relative expression were evaluated by SYBR green quantitative real-time-polymerase chain reaction in the osteoporotic group (n = 26) versus the osteoporosis free group (n = 14). SOD2 enzyme activity was evaluated by colorimetric method in both study groups. Both Atg7 and SOD2 relative expression showed highly significant decrease (P < 0.01) between both groups. However, SOD2 enzyme activity showed no significant difference between the two groups. There was a significant direct correlation between Atg7 and SOD2 gene expression in both study groups. Atg7 relative expression showed significant ( P < 0.01) direct correlation with vitamin D serum levels and body mass index in osteoporotic group. In conclusion, both genes are involved in the pathogenesis of osteoporosis and this could be amenable to future therapeutic intervention.     

Dielectrical Properties of CeO2 Nanoparticles at Different Temperatures

A template-free precipitation method was used as a simple and low cost method for preparation of CeO₂ nanoparticles. The structure and morphology of the prepared nanoparticle samples were studied in detail using X-ray diffraction, Raman spectroscopy and Scanning Electron Microscopy (SEM) measurements. The whole powder pattern modelling (WPPM) method was applied on XRD data to accurately measure the crystalline domain size and their size distribution. The average crystalline domain diameter was found to be 5.2 nm, with a very narrow size distribution. UV-visible absorbance spectrum was used to calculate the optical energy band gap of the prepared CeO₂ nanoparticles. The FT-IR spectrum of prepared CeO₂ nanoparticles showed absorption bands at 400 cm^-1 to 450 cm^-1 regime, which correspond to CeO₂ stretching vibration. The dielectric constant (ε_r) and dielectric loss (tan δ) values of sintered CeO₂ compact consolidated from prepared nanoparticles were measured at different temperatures in the range from 298 K (room temperature) to 623 K, and at different frequencies from 1 kHz to 1 MHz.     

Occurrence and distribution of tomato seed-borne mycoflora in Saudi Arabia and its correlation with the climatic variables

One hundred samples of tomato seeds were collected in 2011 and 2012 from tomato-cultivated fields in Saudi Arabia and screened for their seed-borne mycoflora. A total of 30 genera and 57 species of fungi were recovered from the collected seed samples using agar plate and deep-freezing blotter methods. The two methods differed as regards the frequency of recovered seed-borne fungi. Seven fungi among those recovered from tomato seeds, which are known as plant pathogens, were tested for their pathogenicity and transmission on tomato seedlings. The recovery rate of these pathogens gradually decreased from root up to the upper stem, and did not reach to the stem apex. The distribution of tomato seed-borne fungi was also investigated throughout Saudi Arabia. In this concern, Al-Madena governorate recorded the highest incidence of fungal flora associated with tomato seeds. The impact of meteorological variables on the distribution of tomato seed-borne mycoflora was explored using the ordination technique (canonical correspondence analysis). Among all climatic factors, relative humidity was the most influential variable in this regard. Our findings may provide a valuable contribution to our understanding of future global disease change and may be used also to predict disease occurrence and fungal transfer to new uninfected areas.     

Adaptive Neuro-Fuzzy Appraisal of Plasmonic Studies on Morphology of Deposited Silver Thin Films Having Different Thicknesses

This work presents an experimental analysis on the tunable localized surface plasmon resonance (LSPR), obtained from deposited silver (Ag) thin films of various thicknesses. Silver thin films are prepared using electron beam deposition and undergo an annealing process at different temperatures to produce distinctive sizes of Ag metal nanoparticles (MNPs). The variability of structure sizes and shapes provides an effective means of tuning the position of the LSPR within a wide wavelength range. This paper provides an estimation of LSPR over a broad wavelength range by a process in which the resonance spectra of silver nanoparticles differing in thickness are simulated using an adaptive neuro-fuzzy inference system (ANFIS) method. The ANFIS methodology allows for estimation of sizes of granular structures formed on top of a wafer at certain temperatures, whereupon these intelligent estimators are implemented using MATLAB and their subsequent performances are investigated. The results presented in this paper show the effectiveness of the method of simulation.