Microbial Colonization of Beech and Spruce Litter—Influence of Decomposition Site and Plant Litter Species on the Diversity of Microbial Community

The present study was conducted to investigate the effect of decomposition site and plant litter species on the colonizing microbial communities. For this, litter bag technique using beech and spruce litter was combined with RNA-based fingerprinting and cloning. Litter bags were incubated for 2 and 8 weeks in the A_h horizon of beech and beech–spruce mixed forest sites. Although sugars and starch were rapidly lost, lignin content increased by more than 40% for beech and more than doubled for spruce litter at both soil sites at the end of the experiment. Denaturing gradient gel electrophoresis analysis of 16S and 18S rRNA RT–PCR products was used for screening of differences between bacterial and fungal communities colonizing the two litter types. Development of the microbial community over time was observed to be specific for each litter type and decomposition site. RT–PCR products from both litter types incubated in beech–spruce mixed forest site were also cloned to identify the bacterial and fungal colonizers. The 16S rRNA clone libraries of beech litter were dominated by γ-proteobacterial members, whereas spruce libraries were mainly composed of α-, β-, and γ-proteobacterial members. Ascomycota members dominated the 18S rRNA clone libraries. Clones similar to Zygomycota were absent from spruce, whereas those similar to Basidiomycota and Glomeromycota were absent from beech libraries. Selective effects of litter quality were observed after 8 weeks. The study provides an insight into the bacterial and fungal communities colonizing beech and spruce litter, and the importance of litter quality and decomposition site as key factors in their development and succession.     

Performance of Multiplex Cytokine Assays in Serum and Saliva among Community-Dwelling Postmenopausal Women

Multiplexing arrays increase the throughput and decrease sample requirements for studies employing multiple biomarkers. The goal of this project was to examine the performance of Multiplex arrays for measuring multiple protein biomarkers in saliva and serum. Specimens from the OsteoPerio ancillary study of the Women’s Health Initiative Observational Study were used. Participants required the presence of at least 6 teeth and were excluded based on active cancer and certain bone issues but were not selected on any specific condition. Quality control (QC) samples were created from pooled serum and saliva. Twenty protein markers were measured on five multiplexing array panels. Sample pretreatment conditions were optimized for each panel. Recovery, lower limit of quantification (LLOQ) and imprecision were determined for each analyte. Statistical adjustment at the plate level was used to reduce imprecision estimates and increase the number of usable observations. Sample pre-treatment improved recovery estimates for many analytes. The LLOQ for each analyte agreed with manufacturer specifications except for MMP-1 and MMP-2 which were significantly higher than reported. Following batch adjustment, 17 of 20 biomarkers in serum and 9 of 20 biomarkers in saliva demonstrated acceptable precision, defined as <20% coefficient of variation (<25% at LLOQ). The percentage of cohort samples having levels within the reportable range for each analyte varied from 10% to 100%. The ratio of levels in saliva to serum varied from 1∶100 to 28∶1. Correlations between saliva and serum were of moderate positive magnitude and significant for CRP, MMP-2, insulin, adiponectin, GM-CSF and IL-5. Multiplex arrays exhibit high levels of analytical imprecision, particularly at the batch level. Careful sample pre-treatment can enhance recovery and reduce imprecision. Following statistical adjustments to reduce batch effects, we identified biomarkers that are of acceptable quality in serum and to a lesser degree in saliva using Multiplex arrays.     

Efficient Recombinant Parvovirus Production with the Help of Adenovirus-derived Systems

Rodent parvoviruses (PV) such as rat H-1PV and MVM, are small icosahedral, single stranded, DNA viruses. Their genome includes two promoters P4 and P38 which regulate the expression of non-structural (NS1 and NS2) and capsid proteins (VP1 and VP2) respectively¹. They attract high interest as anticancer agents for their oncolytic and oncosuppressive abilities while being non-pathogenic for humans². NS1 is the major effector of viral cytotoxicity³. In order to further enhance their natural antineoplastic activities, derivatives from these vectors have been generated by replacing the gene encoding for the capsid proteins with a therapeutic transgene (e.g. a cytotoxic polypeptide, cytokine, chemokine, tumour suppressor gene etc.)⁴. The recombinant parvoviruses (recPVs) vector retains the NS1/2 coding sequences and the PV genome telomeres which are necessary for viral DNA amplification and packaging. Production of recPVs occurs only in the producer cells (generally HEK293T), by co-transfecting the cells with a second vector (pCMV-VP) expressing the gene encoding for the VP proteins (Fig. 1)⁴. The recPV vectors generated in this way are replication defective. Although recPVs proved to possess enhanced oncotoxic activities with respect to the parental viruses from which they have been generated, their production remains a major challenge and strongly hampers the use of these agents in anti-cancer clinical applications.We found that introduction of an Ad-5 derived vector containing the E2a, E4(orf6) and the VA RNA genes (e.g. pXX6 plasmid) into HEK293T improved the production of recPVs by more than 10 fold in comparison to other protocols in use. Based on this finding, we have constructed a novel Ad-VP-helper that contains the genomic adenoviral elements necessary to enhance recPVs production as well as the parvovirus VP gene unit⁵. The use of Ad-VP-helper, allows production of rec-PVs using a protocol that relies entirely on viral infection steps (as opposed to plasmid transfection), making possible the use of cell lines that are difficult to transfect (e.g. NB324K) (Fig. 2). We present a method that greatly improves the amount of recombinant virus produced, reducing both the production time and costs, without affecting the quality of the final product⁵. In addition, large scale production of recPV (in suspension cells and bioreactors) is now conceivable.     

The Effects of Periphyton, Fish and Fertilizer Dose on Biological Processes Affecting Water Quality in Earthen Fish Ponds

The potential of periphyton-based aquaculture in South Asia is under investigation in an extensive research program. This paper is a further analysis of data from four experiments carried out in that framework, to explore periphyton, fish and fertilizer dose effects on water quality. Factor analysis and ANOVA models applied to a data matrix of water quality parameters in ponds with and without artificial substrates (bamboo poles and kanchi sticks), with and without fish (filter feeders catla and rohu, with and without bottom feeder kalbaush), and with a standard or 50% increased fertilizer dose, allowed us to identify the underlying ecological processes governing this novel periphyton-based pond system, and construct conceptual graphic models of the periphyton–environment relationships observed. We clearly established that the phosphorus flow is mainly linked to phytoplankton activity in the water column and decomposition on the pond bottom, while nitrogen flow is mainly linked to autotrophic (photosynthesis) and heterotrophic (decomposition and nitrification) processes that take place in the periphyton in addition to the water column and pond bottom. Consequently, disruption of the pond bottom by bottom feeding fish primarily promoted phosphate cycling and phytoplankton, while periphyton development on the supplied substrates and fertilization mainly improved oxygen balance and nitrogen related processes developing in the water column. The use of bamboo poles led to better results than kanchi sticks, related to the greater autotrophic periphyton development on bamboo and to the larger surface of bamboo poles that facilitate fish grazing and periphyton dislodgment that in turn have a renewal effect on periphyton. Stocking bottom feeding fish produces a fertilizing effect through the food web that benefits the filter-feeding fish and that makes it unnecessary to increase the dose of inorganic and organic fertilizers applied to the ponds. Thus, the output of this analysis will help the fish farmers in resource constrained countries to improve their production in periphyton-based ponds just by choosing bamboo substrates, stocking a bottom feeder fish together with the filter feeders, and saving money on fertilizers.     

Maternal hypertension induces tissue-specific modulations of the apelinergic system in the fetoplacental unit in rat

Apelin and its receptor APJ are expressed in fetal tissues but their function and regulation remain largely unknown. In rat, maternal treatment with a nitric oxide synthase inhibitor inducing hypertension was used to investigate apelin plasma levels in mother/fetus pairs and on the gene expression level of the apelin/APJ system in fetal tissues and placenta. At term, plasma levels of apelin were not modulated but APJ expression was increased in placenta and lung but reduced in heart. Apelin expression was increased only in the heart. We postulate that the apelinergic system may control fetal growth and cardiovascular functions in utero.     

Human RhAG ammonia channel is impaired by the Phe65Ser mutation in overhydrated stomatocytic red cells

In red cells, Rh-associated glycoprotein (RhAG) acts as an ammonia channel, as demonstrated by stopped-flow analysis of ghost intracellular pH (pH(i)) changes. Recently, overhydrated hereditary stomatocytosis (OHSt), a rare dominantly inherited hemolytic anemia, was found to be associated with a mutation (Phe65Ser or Ile61Arg) in RHAG. Ghosts from the erythrocytes of four of the OHSt patients with a Phe65Ser mutation were resealed with a pH-sensitive probe and submitted to ammonium gradients. Alkalinization rate constants, reflecting NH(3) transport through the channel and NH(3) diffusion unmediated by RhAG, were deduced from time courses of fluorescence changes. After subtraction of the constant value found for Rh(null) lacking RhAG, we observed that alkalinization rate constant values decreased ～50% in OHSt compared with those of controls. Similar RhAG expression levels were found in control and OHSt. Since half of the expressed RhAG in OHSt most probably corresponds to the mutated form of RhAG, as expected from the OHSt heterozygous status, this dramatic decrease can be therefore related to the loss of function of the Phe65Ser-mutated RhAG monomer.     

Genetic Analysis of Hispanic Individuals with Cystic Fibrosis

We have performed molecular genetic analyses of Hispanic individuals with cystic fibrosis (CF) in the southwestern United States. Of 129 CF chromosomes analyzed, only 46% (59/129) carry ΔF508. The G542X mutation was found on 5% (7/129) of CF chromosomes. The 3849+10kbC→T mutation, detected primarily in Ashkenazi Jews, was present on 2% (3/129). R1162X and R334W, mutations identified in Spain and Italy, each occurred on 1.6% (2/129) of CF chromosomes. W1282X and R553X were each detected once. G551D and N1303K were not found. Overall, screening for 22 or more mutations resulted in detection of only 58% of CF transmembrane conductance regulator gene mutations among Hispanic individuals. Analysis of KM19/XV2c haplotypes revealed an unusual distribution. Although the majority of ΔF508 mutations are on chromosomes of B haplotypes, the other CF mutations are on A and C haplotypes at higher-than-expected frequencies. These genetic analyses demonstrate significant differences between Hispanic individuals with CF and those of the general North American population. Assessment of carrier/affected risk in Hispanic CF individuals cannot, therefore, be based on the mutation frequencies found through studies of the general population but must be adjusted to better reflect the genetic makeup of this ethnic group. Further studies are necessary to identify the causative mutation(s) in this population and to better delineate genotype/phenotype correlations. These will enable counselors to provide more accurate genetic counseling.     

Synthesis and antileishmanial activities of 4,5-di-substituted acridines as compared to their 4-mono-substituted homologues

Newly synthesized 4,5-di-substituted acridines were assessed for in vitro antileishmanial activities as compared to those of their 4-mono-substituted homologues. Mono-substituted acridines exhibited a weak specificity for Leishmania parasites. Di-substituted acridines, on the contrary, displayed interesting amastigote-specific activities through a mechanism of action that might not involve intercalation to DNA. This antileishmanial property, associated with a low antiproliferative activity towards human cells, led to the identification of a new class of promising acridine derivatives such as 4,5-bis(hydroxymethyl)acridine with a nonclassical mechanism of action based on the inhibition of Leishmania internalization within macrophages. In the meantime, the effects of experimental lighting on the biological properties of acridines were assessed: experimental lighting did not significantly improve the antileishmanial activity of the compounds since it produced a greater toxicity against human cells.