Adaptation of anaerobic biomass to saline conditions: Role of compatible solutes and extracellular polysaccharides

This study investigated the role of compatible solutes, extracellular polysaccharides (EPS), and nutrients on anaerobic biomass when stressed with salinity. When 1 mM of osmoregulants glycine betaine, α-glutamate and β-glutamate were added separately to serum bottles containing biomass not adapted to sodium, and fed with glucose and 35 g NaCl/L, all the compatible solutes were found to alleviate sodium inhibition, although glycine betaine was found to be the most effective. The effect of glycine betaine on different anaerobic bacterial groups under salinity stress was monitored using VFAs, and showed that methanogens were more protected than propionate utilisers. Moreover, the addition of 1 mM of glycine betaine to anaerobic biomass not adapted to salinity resulted in significantly higher methane production rates compared with anaerobic biomass that was exposed for 4 weeks to 35 g NaCl/L. Interestingly, under saline batch conditions when the medium was replaced totally the culture produced less methane than when only new substrate was added due to compatible solutes cycling between the media and the cell. The elimination of macronutrients from the medium was found to have a more pronounced negative effect on biomass under saline compared with nonsaline conditions, and because of the synthesis of N-compatible solutes sufficient nutrients should always be present. On the other hand, the absence from the medium of micronutrients did not further reduce biomass activity under salinity. Finally, a higher production of EPS was obtained from biomass exposed to higher salt concentrations, and its composition was found to change under different saline conditions and time. As a result, biomass under saline conditions had a slightly higher mean flock size compared with the biomass that was not subjected to salt.     

Differentiation of adult-type Leydig cells occurs in gonadotrophin-deficient mice

During mammalian testis development distinct generations of fetal and adult Leydig cells arise. Luteinising hormone (LH) is required for normal adult Leydig cell function and for the establishment of normal adult Leydig cell number but its role in the process of adult Leydig cell differentiation has remained uncertain. In this study we have examined adult Leydig cell differentiation in gonadotrophin-releasing hormone (GnRH)-null mice which are deficient in circulating gonadotrophins. Adult Leydig cell differentiation was assessed by measuring expression of mRNA species encoding four specific markers of adult Leydig cell differentiation in the mouse. Each of these markers (3β-hydroxysteroid dehydrogenase type VI (3βHSD VI), 17β-hydroxysteroid dehydrogenase type III (17βHSD III), prostaglandin D (PGD)-synthetase and oestrogen sulphotransferase (EST)) is expressed only in the adult Leydig cell lineage in the normal adult animal. Real-time PCR studies showed that all four markers are expressed in adult GnRH-null mice. Localisation of 3βHSD VI and PGD-synthetase expression by in situ hybridisation confirmed that these genes are expressed in the interstitial tissue of the GnRH-null mouse. Treatment of animals with human chorionic gonadotrophin increased expression of 3βHSD VI and 17βHSD III within 12 hours further indicating that differentiated, but unstimulated cells already exist in the GnRH-null mouse. Thus, while previous studies have shown that LH is required for adult Leydig cell proliferation and activity, results from the present study show that adult Leydig cell differentiation will take place in animals deficient in LH.     

Coextraction during reactive extraction of phenylalanine using Aliquat 336: modeling extraction equilibrium

Reactive liquid-liquid extraction can be used to recover hydrophilic fermentation products that would not otherwise partition into nonpolar solvents through an ion-exchange reaction at the two-phase interface. However, the ion-exchange reagent may not be specific to the solute of interest and other compounds present may also be extracted. In this study, the effect on solute extraction of other compounds present in the extraction medium was investigated for phenylalanine extraction using Aliquat 336. The extent of extraction at equilibrium was modeled using the equilibrium constants for the reactions present in the process. The interaction of different species within a multicomponent medium was examined using the model and experimental results. It was found that the extent of extraction and coextraction is controlled by the thermodynamics of each extraction reaction and, due to the formation of a common product, the interaction between each of the reactions. The main competition to reactive extraction will come from hydrophobic anionic compounds that will be present in fermentation broth.     

N332-Directed Broadly Neutralizing Antibodies Use Diverse Modes of HIV-1 Recognition: Inferences from Heavy-Light Chain Complementation of Function

Dozens of broadly neutralizing HIV-1 antibodies have been isolated in the last few years from the sera of HIV-1-infected individuals. Only a limited number of regions on the HIV-1 spike, however, are recognized by these antibodies. One of these regions (N332) is characterized by an N-linked glycan at residue 332 on HIV-1 gp120 and is recognized by antibody 2G12 and by the recently reported antibodies PGT121-137, the latter isolated from three donors. To investigate the diversity in mode of antibody recognition at the N332 site, we used functional complementation between antibody heavy and light chains as a means of assessing similarity in mode of recognition. We examined a matrix of 12 PGT-heavy chains with each of 12 PGT-light chains. Expression in 96-well format for the 144 antibodies (132 chimeric and 12 wild-type) was generally consistent (58±10 µg/ml). In contrast, recognition of HIV-1 gp120 was bimodal: when the source of heavy and light chains was from the same donor, recognition was good; when sources of heavy and light chains were from different donors, recognition was poor. Moreover, neutralization of HIV-1 strains SF162.LS and TRO.11 generally followed patterns of gp120 recognition. These results are consistent with published sequence, mutational, and structural findings, all of which indicate that N332-directed neutralizing antibodies from different donors utilize different modes of recognition, and provide support for a correlation between functional complementation of antibody heavy and light chains and similarity in antibody mode of recognition. Overall, our results add to the growing body of evidence that the human immune system is capable of recognizing the N332-region of HIV-1 gp120 in diverse ways.     

Historical trends in frog populations in New Zealand based on public perceptions

Surveys were distributed to New Zealand land users in 1998 and 2008 to acquire information about New Zealand frogs with the aim of compiling and mapping their distribution and inferred population trends without costly and time-consuming field surveys. The overall frog population trend was reported as declining, with possible causes reported as an increase in agriculture, an increase in the distribution of predatory fish and disease. The resultant maps could be used for four main purposes: 1) to identify regions where Litoria populations are known to occur, which can be eliminated when considering suitable regions for translocation of Leiopelma; 2) to identify growing or stable populations of Litoria species, which may assist future disease surveys, population monitoring and to identify sources of genetic material that may serve as an Ark for declining Australian populations; 3) to highlight populations that are in decline to enable effective targeting of detailed disease studies; and 4) to approximate the stability of amphibian populations in the absence of more accurate, but costly, scientific monitoring.     

RNA-driven genetic changes in bacteria and in human cells

As recently demonstrated in the yeast Saccharomyces cerevisiae model organism using synthetic RNA-containing oligonucleotides (oligos), RNA can serve as a template for DNA synthesis at the chromosomal level during the process of double-strand break (DSB) repair. Herein we show that the phenomenon of RNA-mediated DNA modification and repair is not limited to yeast cells. A tract of six ribonucleotides embedded in single-strand DNA oligos corresponding to either lagging or leading strand sequences could serve as a template to correct a defective lacZ marker gene in the chromosome of the bacterium Escherichia coli. In order to test the capacity of RNA to modify DNA in mammalian cells, we utilized DNA oligos containing an embedded tract of six ribonucleotides, as well as oligos mostly made of RNA. These oligos were designed to repair a chromosomal break generated within a copy of the green fluorescent protein (GFP) gene randomly integrated into the genome of human HEK-293 cells. We show that these RNA-containing oligos can serve as templates to repair a DSB in human cells and can introduce base changes into genomic or plasmid DNA. In both E. coli and human cells, the strand bias of chromosomal gene correction by the single-strand RNA-containing oligos was the same as that obtained for the corresponding DNA molecules. Therefore, the RNA-containing oligos are not converted into a cDNA before annealing with complementary DNA. Overall, we demonstrate that in both bacterial and human cells, as in yeast, RNA sequences can have a direct role in DNA genetic modification and remodeling.     

The European Renal Genome Project: An Integrated Approach Towards Understanding the Genetics of Kidney Development and Disease

Rapid progress in genome research creates a wealth of information on the functional annotation of mammalian genome sequences. However, as we accumulate large amounts of scientific information we are facing problems of how to integrate and relate the data produced by various genomic approaches. Here, we propose the novel concept of an organ atlas where diverse data from expression maps to histological findings to mutant phenotypes can be queried, compared and visualized in the context of a three-dimensional reconstruction of the organ. We will seek proof of concept for the organ atlas by elucidating genetic pathways involved in development and pathophysiology of the kidney. Such a kidney atlas may provide a paradigm for a new systems-biology approach in functional genome research aimed at understanding the genetic bases of organ development, physiology and disease.Key Words: EuReGene, kidney, genome, development, pathophysiology, genetics     

Fluorescence Lifetime Readouts of Troponin-C-Based Calcium FRET Sensors: A Quantitative Comparison of CFP and mTFP1 as Donor Fluorophores

We have compared the performance of two Troponin-C-based calcium FRET sensors using fluorescence lifetime read-outs. The first sensor, TN-L15, consists of a Troponin-C fragment inserted between CFP and Citrine while the second sensor, called mTFP-TnC-Cit, was realized by replacing CFP in TN-L15 with monomeric Teal Fluorescent Protein (mTFP1). Using cytosol preparations of transiently transfected mammalian cells, we have measured the fluorescence decay profiles of these sensors at controlled concentrations of calcium using time-correlated single photon counting. These data were fitted to discrete exponential decay models using global analysis to determine the FRET efficiency, fraction of donor molecules undergoing FRET and calcium affinity of these sensors. We have also studied the decay profiles of the donor fluorescent proteins alone and determined the sensitivity of the donor lifetime to temperature and emission wavelength. Live-cell fluorescence lifetime imaging (FLIM) of HEK293T cells expressing each of these sensors was also undertaken. We confirmed that donor fluorescence of mTFP-TnC-Cit fits well to a two-component decay model, while the TN-L15 lifetime data was best fitted to a constrained four-component model, which was supported by phasor analysis of the measured lifetime data. If the constrained global fitting is employed, the TN-L15 sensor can provide a larger dynamic range of lifetime readout than the mTFP-TnC-Cit sensor but the CFP donor is significantly more sensitive to changes in temperature and emission wavelength compared to mTFP and, while the mTFP-TnC-Cit solution phase data broadly agreed with measurements in live cells, this was not the case for the TN-L15 sensor. Our titration experiment also indicates that a similar precision in determination of calcium concentration can be achieved with both FRET biosensors when fitting a single exponential donor fluorescence decay model to the fluorescence decay profiles. We therefore suggest that mTFP-based probes are more suitable for FLIM experiments than CFP-based probes.