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.     

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.     

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.     

AL Amyloid Imaging and Therapy with a Monoclonal Antibody to a Cryptic Epitope on Amyloid Fibrils

The monoclonal antibody 2A4 binds an epitope derived from a cleavage site of serum amyloid protein A (sAA) containing a -Glu-Asp- amino acid pairing. In addition to its reactivity with sAA amyloid deposits, the antibody was also found to bind amyloid fibrils composed of immunoglobulin light chains. The antibody binds to synthetic fibrils and human light chain (AL) amyloid extracts with high affinity even in the presence of soluble light chain proteins. Immunohistochemistry with biotinylated 2A4 demonstrated positive reaction with ALκ and ALλ human amyloid deposits in various organs. Surface plasmon resonance analyses using synthetic AL fibrils as a substrate revealed that 2A4 bound with a K_D of ∼10 nM. Binding was inhibited in the presence of the –Glu-Asp- containing immunogen peptide. Radiolabeled 2A4 specifically localized with human AL amyloid extracts implanted in mice (amyloidomas) as evidenced by single photon emission (SPECT) imaging. Furthermore, co-localization of the radiolabeled mAb with amyloid was shown in biodistribution and micro-autoradiography studies. Treatment with 2A4 expedited regression of ALκ amyloidomas in mice, likely mediated by the action of macrophages and neutrophils, relative to animals that received a control antibody. These data indicate that the 2A4 mAb might be of interest for potential imaging and immunotherapy in patients with AL amyloidosis.     

Candidatus Bartonella merieuxii,a Potential New Zoonotic Bartonella Species in Canids from Iraq

Bartonellae are emerging vector-borne pathogens infecting erythrocytes and endothelial cells of various domestic and wild mammals. Blood samples were collected from domestic and wild canids in Iraq under the United States Army zoonotic disease surveillance program. Serology was performed using an indirect immunofluorescent antibody test for B. henselae, B. clarridgeiae, B. vinsonii subsp. berkhoffii and B. bovis. Overall seroprevalence was 47.4% in dogs (n = 97), 40.4% in jackals (n = 57) and 12.8% in red foxes (n = 39). Bartonella species DNA was amplified from whole blood and representative strains were sequenced. DNA of a new Bartonella species similar to but distinct from B. bovis, was amplified from 37.1% of the dogs and 12.3% of the jackals. B. vinsonii subsp. berkhoffii was also amplified from one jackal and no Bartonella DNA was amplified from foxes. Adjusting for age, the odds of dogs being Bartonella PCR positive were 11.94 times higher than for wild canids (95% CI: 4.55–31.35), suggesting their role as reservoir for this new Bartonella species. This study reports on the prevalence of Bartonella species in domestic and wild canids of Iraq and provides the first detection of Bartonella in jackals. We propose Candidatus Bartonella merieuxii for this new Bartonella species. Most of the Bartonella species identified in sick dogs are also pathogenic for humans. Therefore, seroprevalence in Iraqi dog owners and bacteremia in Iraqi people with unexplained fever or culture negative endocarditis requires further investigation as well as in United States military personnel who were stationed in Iraq. Finally, it will also be essential to test any dog brought back from Iraq to the USA for presence of Bartonella bacteremia to prevent any accidental introduction of a new Bartonella species to the New World.