A microplate assay to measure classical and alternative complement activity.

BACKGROUND: We developed and validated a kinetic microplate hemolytic assay (HA) to quantify classical and alternative complement activity in a single dilution of human plasma or serum. METHODS: The assay is based on monitoring hemolysis of sensitized sheep (or uncoated rabbit) red blood cells by means of a 96-well microplate reader. The activity of the calibrator was evaluated by reference to 200 healthy adults. The conversion of 50% hemolysis time into a percentage of activity was obtained using a calibration curve plotted daily. RESULTS: The linearity of the assay as well as interference (by hemolysis, bilrubinemia and lipemia) was assessed for classical pathway (CP). The within-day and the between-day precision was satisfactory regarding the performance of commercially available liposome immunoassay (LIA) and ELISA. Patients with hereditary or acquired complement deficiencies were detected (activity was measured <30%). We also provided a reference range obtained from 200 blood donors. The agreement of CP evaluated on samples from 48 patients was 94% with LIA and 87.5% with ELISA. The sensitivity of our assay was better than that of LIA, and the cost was lower than either LIA or ELISA. In addition, this assay was less time consuming than previously reported HAs. CONCLUSIONS: This assay allows the simultaneous measurement of 36 samples in duplicate per run of a 96-well plate. The use of a daily calibration curve allows standardization of the method and leads to good reproducibility. The same technique was also adapted for the quantification of alternative pathway (AP) activity.

Morphological and color differences between island and mainland populations in the Mexican red rump tarantula, Brachypelma vagans.

The introduction of species into new ecosystems, especially in small and isolated regions such as islands, offers an excellent opportunity to answer questions of the evolutionary processes occurring in natural conditions on a scale that could never be achieved in laboratory conditions. In this study, we examined the Mexican red rump tarantula Brachypelma vagans Ausserer (Mygalomorphae: Theraphosidae), a species that was introduced to Cozumel Island, Mexico, 40 years ago. This introduction provides an exceptional model to study effects such as morphological variation between island populations and those on the mainland in open habitats facing the island. Intraspecific variation related to the color polymorphism was compared. The aim of this study was to determine the phenotypic differences between continental populations of B. vagans and the introduced population on Cozumel Island. Phenotypic difference was evaluated using two approaches: 1) comparison of the morphometric measurements of adult and juvenile individuals at the local scale and between continental and island populations, and 2) comparison of individual color polymorphism between mainland and island populations. Two locations were sampled within the continental part of the Yucatan peninsula and two on the island of Cozumel. The number of samples analyzed at each site was 30 individuals. The morphometric results showed significant differences between continental and island populations, with bigger individuals on the island. In addition, three new variations of the typical color pattern of B. vagans recorded so far were observed. This study opens the door to further investigations to elucidate the origin of the phenotypic variation of the isolated individuals on Cozumel Island. Also, the widest range of color morphs found for a tarantula species is reported.

Biodegradation of organic compounds during co-composting of olive oil mill waste and municipal solid waste with added rock phosphate.

Liquid and solid olive oil mill waste was treated by com posting in a mixture with the organic part of municipal solid waste and rock phosphate. The transformations that occurred during the process were evaluated by physical, chemical and spectroscopic analyses. After five months of com posting, the final compost presented a C/N ratio under 20, an NH4+/NO3(-)] ratio under 1 and a pH around neutral. A high level of organic matter decomposition paralleled a notable abatement of phenols and lipids. The results show the effective dissolution of mineral elements during composting. This transformation was followed by Fourier transform infrared which showed a decrease in the absorption bands of aliphatic bonds (2925 and 2855 cm(-1)) and carbonyls of carboxylic origin (1740 cm (-1)). In addition to the increase in humic substances and the improvement of germination indices, the parameters studied confirm the stability and the maturity of the composts. The absence of phytotoxicity opens the way to agricultural spreading.

Influence of the MHC genotype on the progression of experimental SIV infection in the Mauritian cynomolgus macaque.

Experimental infection of Mauritian cynomolgus macaques by simian immunodeficiency virus is a representative model of HIV infection, currently in favour for evaluating the efficacy of new preventive or curative treatments. Extensive studies of major histocompatibility complex (MHC) polymorphism by microsatellites revealed seven haplotypes (H1-H7). We present statistical evidence of the influence of MHC polymorphism on the set-point plasma viral load (PVL). Our analysis was based on the study of 45 Mauritian cynomolgus macaques inoculated by intravenous or intrarectal injection of a 50 AID50 dose of the SIVmac251 virus. The animals received no treatment before or after the inoculation. MHC polymorphism was investigated by means of 20 microsatellites distributed across the MHC and by DRB genotyping using the DGGE sequencing method. Statistical analysis with UNPHASED: software revealed that two markers located in the class IB region significantly influenced the Log PVL and that three class IB haplotypes were significantly associated with lower (H2 or H6) or higher (H4) set-point Log PVL values. Although the impact of MHC on Log PVL was found to be low (around one Log10), it is important to dispose of animals paired for their MHC genotypes, each animal tested for a given treatment and its untreated control, to minimize the influence of the MHC and clearly reveal the effect of the treatment.

Lead uptake, toxicity, and detoxification in plants.

Lead has gained considerable attention as a persistent toxic pollutant of concern,partly because it has been prominent in the debate concerning the growing anthropogenic pressure on the environment. The purpose of this review is to describe how plants take lead up and to link such uptake to the ecotoxicity of lead in plants.Moreover, we address the mechanisms by which plants or plant systems detoxify lead.Lead has many interesting physico-chemical properties that make it a very useful heavy metal. Indeed, lead has been used by people since the dawn of civilization.Industrialization, urbanization, mining, and many other anthropogenic activities have resulted in the redistribution of lead from the earth's crust to the soil and to the environment.Lead forms various complexes with soil components, and only a small fraction of the lead present as these complexes in the soil solution are phyto available. Despite its lack of essential function in plants, lead is absorbed by them mainly through the roots from soil solution and thereby may enter the food chain. The absorption of lead by roots occurs via the apoplastic pathway or via Ca2+-permeable channels.The behavior of lead in soil, and uptake by plants, is controlled by its speciation and by the soil pH, soil particle size, cation-exchange capacity, root surface area,root exudation, and degree of mycorrhizal transpiration. After uptake, lead primarily accumulates in root cells, because of the blockage by Casparian strips within the endodermis. Lead is also trapped by the negative charges that exist on roots' cell walls.Excessive lead accumulation in plant tissue impairs various morphological, physiological, and biochemical functions in plants, either directly or indirectly, and induces a range of deleterious effects. It causes phytotoxicity by changing cell membrane permeability, by reacting with active groups of different enzymes involved in plant metabolism and by reacting with the phosphate groups of ADP or ATP,and by replacing essential ions. Lead toxicity causes inhibition of ATP production, lipid peroxidation, and DNA damage by over production of ROS. In addition, lead strongly inhibits seed germination, root elongation, seedling development, plant growth, transpiration, chlorophyll production, and water and protein content. The negative effects that lead has on plant vegetative growth mainly result from the following factors: distortion of chloroplast ultrastructure, obstructed electron transport,inhibition of Calvin cycle enzymes, impaired uptake of essential elements, such as Mg and Fe, and induced deficiency of CO2 resulting from stomatal closure.Under lead stress, plants possess several defense strategies to cope with lead toxicity. Such strategies include reduced uptake into the cell; sequestration of lead into vacuoles by the formation of complexes; binding of lead by phytochelatins,glutathione, and amino acids; and synthesis of osmolytes. In addition, activation of various antioxidants to combat increased production of lead-induced ROS constitutes a secondary defense system.

Human fibroblasts share immunosuppressive properties with bone marrow mesenchymal stem cells.

INTRODUCTION: Bone marrow mesenchymal stem cells (BM-MSCs) and adipose tissue-derived stem cells share immunosuppressive capacities, suggesting that the latter could be a general property of stromal cells. METHODS: To check this hypothesis, we compared human BM-MSC and fibroblasts for their in vitro multi-potentiality, expandability and their immunomodulatory properties under normalized optimized culture conditions. RESULTS: We report that, unlike BM-MSCs, fibroblasts cannot differentiate in vitro into adipocytes and osteoblasts and differ from BM-MSCs by the expression of membrane CD106, CD10 and CD26 and by the expression of collagen VII mRNA. Like BM-MSCs, fibroblasts are unable to provoke in vitro allogeneic reactions, but strongly suppress lymphocyte proliferation induced by allogeneic mixed lymphocyte culture (MLC) or mitogens. We show that fibroblasts' immunosuppressive capacity is independent from prostaglandin E2, IL-10 and the tryptophan catabolising enzyme indoleamine 2,3-dioxygenase and is not abrogated after the depletion of CD8+ T lymphocytes, NK cells and monocytes. CONCLUSION: Finally, fibroblasts and BM-MSCs act at an early stage through blockage of lymphocyte activation, as demonstrated by down-regulation of GZMB (granzyme B) and IL2RA (CD25) expression.

Herbicide accumulation and evolution in reservoir sediments.

The aim of the present study was to understand the effect of reservoir configurations on sediment pesticide fate. Two dams were selected on the River Garonne, in southwest France: Carbonne and Golfech, both with reservoirs subject to accumulation of herbicide-contaminated sediment. They are situated upstream and downstream respectively of an agricultural and urban area: the Mid-Garonne. The results presented include pesticide concentrations and C/N ratios in the smaller sediment particles (<2 mm) and values of oxygenation and herbicide concentrations in the water. The dynamic behaviour of sediment in the reservoirs is discussed. The present study shows that the theoretical lifespan (weak remanence in vitro) and the results actually observed in the sediment are conflicting. Pesticide contamination in Carbonne indicates conservation, even accumulation, of herbicide molecules while in Golfech transformation processes clearly dominate. The hydromorphological position of Golfech reservoir, i.e. located at the junction of two rivers with contrasting hydrological regimes and very different oxygenation conditions, leads to accelerated pesticide desorption or degradation. Unfortunately, this configuration is rare.

Bioavailability of Cu and Zn in raw and anaerobically digested pig slurry.

The impact of anaerobic digestion on the bioavailability of copper and zinc from pig slurry was assessed. Both chemical and biological approaches were used independently on raw slurry (RS) and anaerobically digested pig slurry (DS). This work, using ultracentrifugation pellets from the same pig slurry before and after an anaerobic treatment, confirmed that Cu and Zn behave differently in terms of bioavailability, and contrasting results were obtained by chemical and biological assessments. A chemical approach combined a preliminary study of the pH effect on particulate/dissolved metal partitioning, sequential extraction, and biochemical fractionation. This approach tended to show a lower mobility of metals from digested slurry (DS). A biological approach was carried out with Zea mays and Vicia faba to study Cu and Zn uptake in soil amended with RS or DS. This assay could not differentiate the two slurries.

Thymic output and peripheral T lymphocyte subsets in relapsing--remitting multiple sclerosis patients treated or not by IFN-beta.

We explored the parameters of central and peripheral tolerance in patients with stable relapsing-remitting multiple sclerosis, treated or not with IFN-beta. TREC-positive T cells were lower in patients compared with controls, mainly in CD4+ subset, compatible with a thymus dysfunction or an expansion of peripheral lymphocytes. Compared to controls, the frequency of activated CD4+CD25+ T cells was higher in patients without modification of the CD4+CD25(high) T cell proportion. The IFN-beta-treatment did not modify the TREC-positive cell frequency nor the naive/memory T cell subset percentage but was associated with lower blood lymphocyte count and a lower frequency of CD4+CD45RC(high) subset.

Particle size and metal distributions in anaerobically digested pig slurry.

Particle size distribution and trace element patterns were studied in a full-scale anaerobic digestion plant treating pig slurry. Mass balance was established for major (N, P, K, Ca, Fe, Mg and S) and minor (Al, Cu, Mn and Zn) elements. Most of the elements were conserved through the process but part of the P, Ca, Mg and Mn was deposited as crystals lining the digester. In the dry matter of the slurry, Cu and Zn occurred at between 170 and 2600 mg kg(-1) due to pig diet supplements. Analyses of particle size distributions in raw and digested slurries showed a general shift in distribution towards larger sizes due to degradation of small and easily degradable particles as well as formation of large microbial filaments. Graded sieving of digested slurry showed metals to be mainly present on 3-25 microm particles. Less than 2% Cu and Zn was removed by passage through a 250 microm rotary screen.

Aerobic biodegradation of sludge from the effluent of a vegetable oil processing plant mixed with household waste: physical-chemical, microbiological, and spectroscopic analysis.

Sludge from a sewage treatment plant dealing with the effluent produced during the processing of crude vegetable oil (Lesieur-Cristal, Morocco) was composted in two mixtures (M1 and M2) with household waste obtained from landfill. The different physico-chemical characteristics of the final composts after 5 months of composting were, for M1 and M2, respectively: pH: 8.5 and 7.08; C/N: 10 and 16; proportion of decomposition: 78% and 55%, NH(4)(+)/NO(3)(-): 0.78 and 1.02. Monitoring the levels of lipid and total polyphenols showed a reduction of 81% and 72% for lipids and of 75% and 76% for polyphenols in M1 and M2, respectively. These reductions were paralleled by a rise in the humic acid content to reach 22 and 36mg/g, respectively. Overall, these results were confirmed by the FTIR spectroscopy study of the two mixtures. For M1, the FTIR spectra taken at different stages showed that during composting, biodegradation of the aliphatic compounds occurred as the proportion of aromatic structures increased. The transformations observed qualitatively were then confirmed quantitatively by the changes occurring in the various absorption ratios during composting. Mixture M2, however, presented strong absorbance of aliphatic compounds. These results were statistically confirmed by correlation tests and principal components analysis, which confirmed the maturity of the two composts, M1 having matured more than M2.

The fulvic acid fraction as it changes in the mature phase of vegetable oil-mill sludge and domestic waste composting.

Sludge resulting from the treatment of effluent from a vegetable oil mill, was composted mixed with domestic waste in a pile for five months. Different proportions of sludge and dry waste were mixed: M1 (1v/2v) and M2 (1v/1v). Monitoring different physical-chemical parameters showed the effect of the substrate on the microbiological activity and on the formation of fulvic acids, affecting the maturity of the final compost. Elemental analysis revealed that the fulvic acids of mixes M1 and M2 presented very low concentrations of carbon, hydrogen, and oxygen and a high level of nitrogen. The FTIR spectroscopy results showed a decrease during composting of the intensity of absorbance of the easily assimilable compounds that are predominant in the initial mixtures i.e. the carbohydrates (1170-1080 cm(-1)) in M1 and long aliphatic chains (2920 cm(-1)) in M2. For mix M1 there was enrichment in compounds bearing oxygen-containing moieties. In M2 it was the nitrogen-containing compounds (in the form of stable amides) which predominated at the end of composting. The first component of PCA analysis, PC1, accounted for 83% of the difference between two distinct groups of parameters governing degradation and restructuration of the fulvic acids during composting. PC2 (17%) explained the variance due to the level of free or less polycondensed compounds in the two mixtures. Oxidised polyphenolic and polysaccharide structures were the least free, or most polycondensed, in the fulvic structures of M1. In M2 fulvic acids however, it was the polyphenols and peptide structures that were involved in the bonding, most likely of the polyphenol-peptide type.

Physico-chemical analysis of tannery solid waste and structural characterization of its isolated humic acids after composting.

In Marrakech, solid by-products from tanneries are highly polluting, generating large amounts of nitrogenous and organic matter. In the present study composting is tested as a cost-effective method for waste management to overcome many of the environmental hazards and produce a stable, rich material for soil fertilization. Two composting trials were conducted after neutralization by ammonia or lime. The aim of the neutralization was to avoid the antimicrobial effects of the acidity in the tannery waste, thus ensuring correct composting. Different techniques such as elemental analysis and 13C NMR spectroscopy were applied to analyse humic acids isolated from raw and composted materials, and to monitor the process of tannery waste composting, and the stability and maturity of the final product according to the means of neutralization. Comparison of data showed similar behaviour in both trials, but the composting process appeared to be more complete following neutralization with lime. The C, H and N content decreased, while the O increased. The FTIR and 13C NMR spectra show the decrease of aliphatic compounds demonstrated by the reduction of absorbance around 2922cm(-1) and of the resonance in the C-alkyl area around 0-55ppm. The humic acids newly formed during composting were richer in the O-N alkyl and oxidized aromatic structures that increased almost twofold on composting after neutralization with lime. The first principal component axis PC1 (54%) separated C-aliphatic, C-carboxylic and other less stable and less polycondensed compounds such as polyphenols from the more polycondensed O-N alkyl and oxidized C-aromatic compounds.

Ship breaking or scuttling? A review of environmental, economic and forensic issues for decision support.

In a globalized world, the world trade fleet plays a pivotal role in limiting transport costs. But, the management of obsolete ships is an acute problem, with most Ship Recycling Facilities (SRF) situated in developing countries. They are renowned for their controversial work and safety conditions and their environmental impact. Paradoxically, dismantlement is paid for by the shipowners in accordance with international conventions therefore it is more profitable for them to sell off ships destined for scrapping. Scuttling, the alternative to scrapping, is assessed in the present review to compare the cost/benefit ratios of the two approaches. Although scrapping provides employment and raw materials - but with environmental, health and safety costs - scuttling provides fisheries and diving tourism opportunities but needs appropriate management to avoid organic and metal pollution, introduction of invasive species and exacerbation of coastal erosion. It is also limited by appropriate bottom depth, ship type and number. The present review inventories the environmental, health, safety, economic, and forensic aspects of each alternative.

Using metabarcoding to reveal and quantify plant-pollinator interactions.

Given the ongoing decline of both pollinators and plants, it is crucial to implement effective methods to describe complex pollination networks across time and space in a comprehensive and high-throughput way. Here we tested if metabarcoding may circumvent the limits of conventional methodologies in detecting and quantifying plant-pollinator interactions. Metabarcoding experiments on pollen DNA mixtures described a positive relationship between the amounts of DNA from focal species and the number of trnL and ITS1 sequences yielded. The study of pollen loads of insects captured in plant communities revealed that as compared to the observation of visits, metabarcoding revealed 2.5 times more plant species involved in plant-pollinator interactions. We further observed a tight positive relationship between the pollen-carrying capacities of insect taxa and the number of trnL and ITS1 sequences. The number of visits received per plant species also positively correlated to the number of their ITS1 and trnL sequences in insect pollen loads. By revealing interactions hard to observe otherwise, metabarcoding significantly enlarges the spatiotemporal observation window of pollination interactions. By providing new qualitative and quantitative information, metabarcoding holds great promise for investigating diverse facets of interactions and will provide a new perception of pollination networks as a whole.

Effect of Different Amendments on Growing of Canna indica L. Inoculated with AMF on Mining Substrate.

Canna indica L. (CiL) was used here in phytoremediation of mining soils. Our work evaluated the effect of AMF (i) on the growth and (ii) on the uptake of heavy metals (HM). The tests were conducted in the greenhouse on mining substrates collected from the Kettara mine (Morocco). The mine soil was amended by different proportions of agricultural soil and compost and then inoculated with two isolates of AMF (IN1) and (IN2) of different origins. After six months of culture, the results show that on mining soils (100%) only AMF (IN2) was able to colonize the roots of CiL with a frequency of 40±7% and an intensity of 6.5±1.5%. Also, the lowest values of shoot and root dry biomass are obtained on these mining soils with respectively 0.30 g and 0.27 g. In contrast, the accumulation of HM was higher and reached more than 50% of that contained in the mining soils, the highest values with 138 mg kg(-1) Cu2+, Zn2+ 270 mg kg(-1) and 1.38 mg kg(-1) Cd was recorded. These results indicate that the colonization of CiL roots by AMF (IN2) could significantly improve its potential to be used in phytoremediation of polluted soil.

Modulation of gene expression in CD4+ T lymphocytes following in vitro HIV infection: a comparison between human and chimpanzee.

Chimpanzees are susceptible to experimental infection by human deficiency virus (HIV)-1, but unlike humans, they exceptionally develop an immunodeficiency syndrome after HIV-1 inoculation. To explore the difference between human and chimpanzee, we analyzed the expression of 1547 genes of various functions in human or chimpanzee CD4+ lymphoblasts inoculated in vitro with HIV-1. We observed that, 1 day after HIV inoculation, fifty-eight genes were up-regulated in lymphoblasts of the three humans while their expression remained unchanged in lymphoblasts of the three chimpanzees. One gene is involved in adhesion of HIV (catenin-alpha), three in the immune response (semaphorin 4D, placental growth factor, IL-6), three in apoptosis (deleted in colorectal carcinoma, caspase 9 and FOXO1A). No difference between species was revealed for the expression of 373 genes related to glycosylation pathways. The in vitro human/chimpanzee comparison reveals new candidate genes up-regulated after inoculation with HIV-1 only in human lymphoblasts and which could be related to the higher sensitivity of human to HIV-induced AIDS.

Characterization of Freshwater Diatom Communities: Comparing Taxonomic and Genetic-Fingerprinting Approaches.

Benthic diatom assemblages from five sampling sites located on two rivers were characterized simultaneously by means of traditional microscopic observations and PCR-DGGE fingerprinting with primers specifically designed for Bacillariophyceae. Community structure, richness, and diversity assessed by both methods were compared. Diatom lists obtained from morphological identification were separated into subsets, depending on (i) the taxonomic level considered (genus, species, variety) and, for each of them, (ii) the relative abundance (RA) of each component (the whole data set, RA > 1%, RA > 2%). These data were then compared to genetic fingerprinting data. Clusters based on taxonomic composition and DGGE banding patterns were very similar, showing good correspondence of community structure between the two methods. Data were compared by linear regressions between indices (richness, diversity) and by Mantel tests on dissimilarity matrices generated for each community composition data set. Statistical analysis indicated that the most reliable correlations with fingerprinting were obtained for genera representing more than 1% RA or species representing more than 2% RA. The results reveal that the PCR-DGGE protocol described here offers a satisfactory alternative for performing preliminary screening of coarse differences in diatom global community structure between samples. It can be regarded as a good complement to taxonomic analyses, which still remain necessary to detect precise changes in richness and diversity, especially when considering species with low abundance in natural assemblages.