The S100A8/A9 protein as a partner for the cytosolic factors of NADPH oxidase activation in neutrophils.

In a previous study, the S100A8/A9 protein, a Ca2+- and arachidonic acid-binding protein, abundant in neutrophil cytosol, was found to potentiate the activation of the redox component of the O2- generating oxidase in neutrophils, namely the membrane-bound flavocytochrome b, by the cytosolic phox proteins p67phox, p47phox and Rac (Doussière J., Bouzidi F. and Vignais P.V. (2001) Biochem. Biophys. Res. Commun.285, 1317-1320). This led us to check by immunoprecipitation and protein fractionation whether the cytosolic phox proteins could bind to S100A8/A9. Following incubation of a cytosolic extract from nonactivated bovine neutrophil with protein A-Sepharose bound to anti-p67phox antibodies, the recovered immunoprecipitate contained the S100 protein, p47phox and p67phox. Cytosolic protein fractionation comprised two successive chromatographic steps on hydroxyapatite and DEAE cellulose, followed by isoelectric focusing. The S100A8/A9 heterodimeric protein comigrated with the cytosolic phox proteins, and more particularly with p67phox and Rac2, whereas the isolated S100A8 protein displayed a tendancy to bind to p47phox. Using a semirecombinant cell-free system of oxidase activation consisting of recombinant p67phox, p47phox and Rac2, neutrophil membranes and arachidonic acid, we found that the S100A8/A9-dependent increase in the elicited oxidase activity corresponded to an increase in the turnover of the membrane-bound flavocytochrome b, but not to a change of affinity for NADPH or O2. In the absence of S100A8/A9, oxidase activation departed from michaelian kinetics above a critical threshold concentration of cytosolic phox proteins. Addition of S100A8/A9 to the cell-free system rendered the kinetics fully michaelian. The propensity of S100A8/A9 to bind the cytosolic phox proteins, and the effects of S100A8/A9 on the kinetics of oxidase activation, suggest that S100A8/A9 might be a scaffold protein for the cytosolic phox proteins or might help to deliver arachidonic acid to the oxidase, thus favoring the productive interaction of the cytosolic phox proteins with the membrane-bound flavocytochrome b.     

Different rates of ultraviolet-induced DNA damage in the epidermis and dermis of a platyfish model for carcinogenesis

The purpose of this study was to compare fluence-response relationships for the production of cyclobutane pyrimidine dimers in epidermal or dermal DNA of platyfishXiphophorus hybrids irradiated with UVB, and to determine photoreactivation from black light on dimers producedin situ. This was accomplished by quantitative gel electrophoresis of unlabeled DNA following extraction of the DNA and treatment with an enzyme specific for the detection of pyrimidine dimers. The dermis was the target tissue for UV-induced DNA damage inXiphophorus hybrid fish skin. Shapes of dimer-fluence response data following filtered sunlamp irradiation ( > 290 nm) or monochromatic wavelength 302 nm in the epidermis or dermis were different. In the epidermis there was an initial steep upward slope followed by a plateau, whereas in the dermis a linear relationship was observed. The final values of dimers at the high doses were, however, nearly equal in the epidermis and dermis exposed to either radiation. These differences in fluence-response relationships are probably attributable to the intertwining of the epidermis and to the shielding effect of the epidermal layer, with scales leading to a heterogenous population of cells which are exposed to different UV doses. Photoreversal of dimers was readily observed by black light irradiation in both epidermis and dermis irradiated with either > 290 nm or 302 nm.This research was supported by the Office of Health and Environmental Research of the U.S. Department of EnergyThe author is recipient of the National Academy of Sciences' Kobelt Fund Grant     

Intérêt de l’imagerie hybride TEMP-TDM dans l’exploration des infections

Single-Photon Emission Computed Tomography-Computerized Tomography (SPECT-CT) is a new hybrid technique which offers new diagnostical capabilities in daily nuclear medicine practice. This technique not only allows to acquire fusioned anatomic and functional images in the same time, but also, it increases sensitivity and accuracy of SPECT thanks to attenuation and scattering corrections got from transmission data. Until now, SPECT-CT data have been mainly obtained in oncology and cardiology, but now, many authors use it in many scan studies and particularly for infectious diseases. In inflammatory bowel diseases, SPECT-CT seems to increase diagnostical performances and to modify management of many patients. In suspected vascular sepsis, SPECT-CT could increase sensitivity of white blood cell scintigraphy but also its specificity thanks to spatial resolution of CT. In osteoarticular sepsis, SPECT-CT has the advantage to distinguish osteomyelitis from soft tissue infection and to guide biopsies. Nevertheless, in the light of PET-CT works, SPECT-CT development will probably modify nuclear medicine practice and many studies have to be conducted to highlight consensual procedure guidelines.     

Probabilistic disease classification of expression-dependent proteomic data from mass spectrometry of human serum.

We have developed an algorithm called Q5 for probabilistic classification of healthy versus disease whole serum samples using mass spectrometry. The algorithm employs principal components analysis (PCA) followed by linear discriminant analysis (LDA) on whole spectrum surface-enhanced laser desorption/ionization time of flight (SELDI-TOF) mass spectrometry (MS) data and is demonstrated on four real datasets from complete, complex SELDI spectra of human blood serum. Q5 is a closed-form, exact solution to the problem of classification of complete mass spectra of a complex protein mixture. Q5 employs a probabilistic classification algorithm built upon a dimension-reduced linear discriminant analysis. Our solution is computationally efficient; it is noniterative and computes the optimal linear discriminant using closed-form equations. The optimal discriminant is computed and verified for datasets of complete, complex SELDI spectra of human blood serum. Replicate experiments of different training/testing splits of each dataset are employed to verify robustness of the algorithm. The probabilistic classification method achieves excellent performance. We achieve sensitivity, specificity, and positive predictive values above 97% on three ovarian cancer datasets and one prostate cancer dataset. The Q5 method outperforms previous full-spectrum complex sample spectral classification techniques and can provide clues as to the molecular identities of differentially expressed proteins and peptides.     

Pseudomonas caspiana sp. nov., a citrus pathogen in the Pseudomonas syringae phylogenetic group

In a screening by multilocus sequence analysis of Pseudomonas strains isolated from diverse origins, 4 phylogenetically closely related strains (FBF58, FBF102^T, FBF103, and FBF122) formed a well-defined cluster in the Pseudomonas syringae phylogenetic group. The strains were isolated from citrus orchards in northern Iran with disease symptoms in the leaves and stems and its pathogenicity against citrus plants was demonstrated. The whole genome of the type strain of the proposed new species (FBF102^T = CECT 9164^T = CCUG 69273^T) was sequenced and characterized. Comparative genomics with the 14 known Pseudomonas species type strains of the P. syringae phylogenetic group demonstrated that this strain belonged to a new genomic species, different from the species described thus far. Genome analysis detected genes predicted to be involved in pathogenesis, such as an atypical type 3 secretion system and two type 6 secretion systems, together with effectors and virulence factors. A polyphasic taxonomic characterization demonstrated that the 4 plant pathogenic strains represented a new species, for which the name Pseudomonas caspiana sp. nov. is proposed.     

Aflatoxin B<Subscript>1</Subscript> levels in groundnut products from local markets in Zambia

In Zambia, groundnut products (milled groundnut powder, groundnut kernels) are mostly sold in under-regulated markets. Coupled with the lack of quality enforcement in such markets, consumers may be at risk to aflatoxin exposure. However, the level of aflatoxin contamination in these products is not known. Compared to groundnut kernels, milled groundnut powder obscures visual indicators of aflatoxin contamination in groundnuts such as moldiness, discoloration, insect damage or kernel damage. A survey was therefore conducted from 2012 to 2014, to estimate and compare aflatoxin levels in these products (n = 202), purchased from markets in important groundnut growing districts and in urban areas. Samples of whole groundnut kernels (n = 163) and milled groundnut powder (n = 39) were analysed for aflatoxin B₁ (AFB₁) by competitive enzyme-linked immunosorbent assay (cELISA). Results showed substantial AFB₁ contamination levels in both types of groundnut products with maximum AFB₁ levels of 11,100 μg/kg (groundnut kernels) and 3000 μg/kg (milled groundnut powder). However, paired t test analysis showed that AFB₁ contamination levels in milled groundnut powder were not always significantly higher (P > 0.05) than those in groundnut kernels. Even for products from the same vendor, AFB₁ levels were not consistently higher in milled groundnut powder than in whole groundnut kernels. This suggests that vendors do not systematically sort out whole groundnut kernels of visually poor quality for milling. However, the overall contamination levels of groundnut products with AFB₁ were found to be alarmingly high in all years and locations. Therefore, solutions are needed to reduce aflatoxin levels in such under-regulated markets.     

The neuroprotective effect of mesenchymal stem cells on an experimentally induced model for multiple sclerosis in mice

Multiple sclerosis (MS) is a chronic autoimmune demyelinating neurodegenerative central nervous system disorder. The aim of the present study was to investigate the prophylactic effect exerted by the one‐time intraperitoneal injection of mesenchymal stem cells (MSCs) 1 × 10⁶ and 14‐day intraperitoneal injection of methylprednisolone (MP) 40 mg/kg in an experimental autoimmune encephalomyelitis (EAE). EAE was induced by intradermal injection of rat spinal cord homogenate with complete Freund's adjuvant in Swiss mice. Results of MSCs and MP‐treated mice showed a significantly milder disease and fewer clinical scores compared to control mice. They suppressed tumor necrosis factor‐alpha and myeloperoxidase and increased interleukin 10, whereas thiobarbituric acid reactive substances and nitric oxide brain contents were reduced to comparable levels between treatment groups. Brain content of GSH was significantly higher in MSCs‐treated mice than control mice. It is evident that MSCs have relevant prophylactic effect in an animal model of MS and might represent a valuable tool for stem cell based therapy in MS.     

Preparation,Characterization, and Optimization of Folic Acid-Chitosan-Methotrexate Core-Shell Nanoparticles by Box-Behnken Design for Tumor-Targeted Drug Delivery

The objective of this study was to investigate the combined influence of independent variables in the preparation of folic acid-chitosan-methotrexate nanoparticles (FA-Chi-MTX NPs). These NPs were designed and prepared for targeted drug delivery in tumor. The NPs of each batch were prepared by coaxial electrospray atomization method and evaluated for particle size (PS) and particle size distribution (PSD). The independent variables were selected to be concentration of FA-chitosan, ratio of shell solution flow rate to core solution flow rate, and applied voltage. The process design of experiments (DOE) was obtained with three factors in three levels by Design expert software. Box-Behnken design was used to select 15 batches of experiments randomly. The chemical structure of FA-chitosan was examined by FTIR. The NPs of each batch were collected separately, and morphologies of NPs were investigated by field emission scanning electron microscope (FE-SEM). The captured pictures of all batches were analyzed by ImageJ software. Mean PS and PSD were calculated for each batch. Polynomial equation was produced for each response. The FE-SEM results showed the mean diameter of the core-shell NPs was around 304 nm, and nearly 30% of the produced NPs are in the desirable range. Optimum formulations were selected. The validation of DOE optimization results showed errors around 2.5 and 2.3% for PS and PSD, respectively. Moreover, the feasibility of using prepared NPs to target tumor extracellular pH was shown, as drug release was greater in the pH of endosome (acidic medium). Finally, our results proved that FA-Chi-MTX NPs were active against the human epithelial cervical cancer (HeLa) cells.     

Properties of bacteriophage T4 proteins deficient in replication repair

An epistasis group of mutations engendering increased sensitivity to diverse DNA-damaging agents was described previously in bacteriophage T4. These mutations are alleles of genes 32 and 41, which, respectively, encode a single-stranded DNA-binding protein (gp32) and the replicative DNA helicase (gp41). The mechanism by which the lethality of DNA damage is mitigated is unknown but seems not to involve the direct reversal of damage, excision repair, conventional recombination repair, or translesion synthesis. Here we explore the hypothesis that the mechanism involves a switch in DNA primer extension from the cognate template to an alternative template, the just-synthesized daughter strand of the other parental strand. The activities of the mutant proteins are reduced about 2-fold (for gp32) or 4-fold (for gp41) in replication complexes catalyzing coordinated synthesis of leading and lagging strands, in binding single-stranded DNA, promoting DNA annealing, and promoting branch migration. In striking contrast, the mutant proteins are strongly impaired in promoting template switching, thus supporting the hypothesis of survival by template switching.