Redox-stratification controlled biofilm (ReSCoBi) for completely autotrophic nitrogen removal: the effect of co- versus counter-diffusion on reactor performance

A multi-population biofilm model for completely autotrophic nitrogen removal was developed and implemented in the simulation program AQUASIM to corroborate the concept of a redox-stratification controlled biofilm (ReSCoBi). The model considers both counter- and co-diffusion biofilm geometries. In the counter-diffusion biofilm, oxygen is supplied through a gas-permeable membrane that supports the biofilm while ammonia (NH(4)(+)) is supplied from the bulk liquid. On the contrary, in the co-diffusion biofilm, both oxygen and NH(4)(+) are supplied from the bulk liquid. Results of the model revealed a clear stratification of microbial activities in both of the biofilms, the resulting chemical profiles, and the obvious effect of the relative surface loadings of oxygen and NH(4)(+) (J(O(2))/J(NH(4)(+))) on the reactor performances. Steady-state biofilm thickness had a significant but different effect on T-N removal for co- and counter-diffusion biofilms: the removal efficiency in the counter-diffusion biofilm geometry was superior to that in the co-diffusion counterpart, within the range of 450-1,400 microm; however, the efficiency deteriorated with a further increase in biofilm thickness, probably because of diffusion limitation of NH(4)(+). Under conditions of oxygen excess (J(O(2))/J(NH(4)(+)) > 3.98), almost all NH(4)(+) was consumed by aerobic ammonia oxidation in the co-diffusion biofilm, leading to poor performance, while in the counter-diffusion biofilm, T-N removal efficiency was maintained because of the physical location of anaerobic ammonium oxidizers near the bulk liquid. These results clearly reveal that counter-diffusion biofilms have a wider application range for autotrophic T-N removal than co-diffusion biofilms.     

Genetic structure in a dynamic baboon hybrid zone corroborates behavioural observations in a hybrid population

Behaviour and genetic structure are intimately related: mating patterns and patterns of movement between groups or populations influence the movement of genetic variation across the landscape and from one generation to the next. In hybrid zones, the behaviour of the hybridizing taxa can also impact the incidence and outcome of hybridization events. Hybridization between yellow baboons and anubis baboons has been well documented in the Amboseli basin of Kenya, where more anubis-like individuals tend to experience maturational and reproductive advantages. However, it is unknown whether these advantages are reflected in the genetic structure of populations surrounding this area. Here, we used microsatellite genotype data to evaluate the structure and composition of baboon populations in southern Kenya. Our results indicate that, unlike for mitochondrial DNA, microsatellite-based measures of genetic structure concord with phenotypically based taxonomic distinctions and that the currently active hybrid zone is relatively narrow. Isolation with migration analysis revealed asymmetric gene flow in this region from anubis populations into yellow populations, in support of the anubis-biased phenotypic advantages observed in Amboseli. Populations that are primarily yellow but that receive anubis gene flow exhibit higher levels of genetic diversity than yellow populations far from the introgression front. Our results support previous work that indicates a long history of hybridization and introgression among East African baboons. Specifically, it suggests that anubis baboons are in the process of gradual range expansion into the range of yellow baboons, a pattern potentially explained by behavioural and life history advantages that correlate with anubis ancestry.     

In vivo imaging of immunotoxin treatment using Katushka-transfected A-431 cells in a murine xenograft tumour model

Purpose

Preclinical in vivo analyses of treatment responses are an important prerequisite to evaluate new therapeutics. Molecular in vivo imaging in the far red (FR)/near infra red (NIR) is a promising method, as it enables measurements at different time points in individual animals, thereby reducing the number of animals required, while increasing statistical significance. Here, we show the establishment of a method to monitor response to treatment using fluorescent cells, expressing the epidermal growth factor receptor (EGFR), a target already used in therapy.

Methods

We transfected A-431 tumour cells with the far red–emitting protein Katushka (Kat2), resulting in strong fluorescence allowing for the monitoring of tumour growth when implanted in BALB/c nu/nu mice with a CRi Maestro in vivo imager. We targeted A-431 cells with a previously reported immunotoxin (IT), consisting of the anti-EGFR antibody single-chain variable fragment (scFv) 425, fused to Pseudomonas aeruginosa Exotoxin A’ (ETA’). In addition, EGFR expression was verified using the 425(scFv) conjugated to a NIR dye BG-747 through a SNAP-tag linker.

Results

The results show the feasibility to evaluate response to treatment in vivo by FR imaging, while at the same location detecting EGFR expression. Treatment with 425(scFv)-ETA’ resulted in decelerated tumour growth, while not affecting the overall health of the animals. This is in contrast to treatment with Doxorubicin, which, although decreasing the tumour size, resulted in poor health.

Conclusions

We developed a novel method to non-invasively determine treatment responses by in vivo imaging of multiple parameters which showed the efficacy of 425(scFv)-ETA’.     

Intrinsic and extrinsic variables controlling the productivity of asexual populations of Nais spp, (Naididae,Oligochaeta)

The productivity of Nais spp. from periphyton of fishponds of the Dombes area (Ain) was studied in semi-natural conditions by cultivation of zooids in experimental glass enclosures immersed in situ and filled with pond water receiving injections of fertilizers (P₂O₅) and natural filtered periphyton extracts (particles < 70 µm). The growth rate of the experimental populations was not significantly affected by the concentration of fertilizers added to culture media. On the contrary, the water management of the culture media (as renewal or non-renewal of the water in experimental enclosures), the closing procedure of the enclosures and the load and composition of the nutritive substrate controlled the produced biomass. Temperature and food supply were the principal extrinsic variables controlling the asexual growth rate of the Nais species. The stolonization rate was analyzed as a biological parameter implicated in the instantaneous birth rate of zooids and the growth of naidid populations.     

Localized vs. systemic inflammation in guinea pigs: a role for prostaglandins at distinct points of the fever induction pathways?

In guinea pigs, dose-dependent febrile responses were induced by injection of a high (100 microg/kg) or a low (10 microg/kg) dose of bacterial lipopolysaccharide (LPS) into artificial subcutaneously implanted Teflon chambers. Both LPS doses further induced a pronounced formation of prostaglandin E(2) (PGE(2)) at the site of localized subcutaneous inflammation. Administration of diclofenac, a nonselective cyclooxygenase (COX) inhibitor, at different doses (5, 50, 500, or 5,000 microg/kg) attenuated or abrogated LPS-induced fever and inhibited LPS-induced local PGE(2) formation (5 or 500 microg/kg diclofenac). Even the lowest dose of diclofenac (5 microg/kg) attenuated fever in response to 10 microg/kg LPS, but only when administered directly into the subcutaneous chamber, and not into the site contralateral to the chamber. This observation indicated that a localized formation of PGE(2) at the site of inflammation mediated a portion of the febrile response, which was induced by injection of 10 microg/kg LPS into the subcutaneous chamber. Further support for this hypothesis derived from the observation that we failed to detect elevated amounts of COX-2 mRNA in the brain of guinea pigs injected subcutaneously with 10 microg/kg LPS, whereas subcutaneous injections of 100 microg/kg LPS, as well as systemic injections of LPS (intra-arterial or intraperitoneal routes), readily caused expression of the COX-2 gene in the guinea pig brain, as demonstrated by in situ hybridization. Therefore, fever in response to subcutaneous injection of 10 microg/kg LPS may, in part, have been evoked by a neural, rather than a humoral, pathway from the local site of inflammation to the brain.     

Arabidopsis myrosinases TGG1 and TGG2 have redundant function in glucosinolate breakdown and insect defense

In Arabidopsis and other Brassicaceae, the enzyme myrosinase (beta-thioglucoside glucohydrolase, TGG) degrades glucosinolates to produce toxins that deter herbivory. A broadly applicable selection for meiotic recombination between tightly linked T-DNA insertions was developed to generate Arabidopsis tgg1tgg2 double mutants and study myrosinase function. Glucosinolate breakdown in crushed leaves of tgg1 or tgg2 single mutants was comparable to that of wild-type, indicating redundant enzyme function. In contrast, leaf extracts of tgg1tgg2 double mutants had undetectable myrosinase activity in vitro, and damage-induced breakdown of endogenous glucosinolates was apparently absent for aliphatic and greatly slowed for indole glucosinolates. Maturing leaves of myrosinase mutants had significantly increased glucosinolate levels. However, developmental decreases in glucosinolate content during senescence and germination were unaffected, showing that these processes occur independently of TGG1 and TGG2. Insect herbivores with different host plant preferences and feeding styles varied in their responses to myrosinase mutations. Weight gain of two Lepidoptera, the generalist Trichoplusia ni and the facultative Solanaceae-specialist Manduca sexta, was significantly increased on tgg1tgg2 double mutants. Two crucifer-specialist Lepidoptera had differing responses. Whereas Plutella xylostella was unaffected by myrosinase mutations, Pieris rapae performed better on wild-type, perhaps due to reduced feeding stimulants in tgg1tgg2 mutants. Reproduction of two Homoptera, Myzus persicae and Brevicoryne brassicae, was unaffected by myrosinase mutations.     

Transmission of MRSA between companion animals and infected human patients presenting to outpatient medical care facilities

Methicillin-resistant Staphylococcus aureus (MRSA) is a significant pathogen in both human and veterinary medicine. The importance of companion animals as reservoirs of human infections is currently unknown. The companion animals of 49 MRSA-infected outpatients (cases) were screened for MRSA carriage, and their bacterial isolates were compared with those of the infected patients using Pulsed-Field Gel Electrophoresis (PFGE). Rates of MRSA among the companion animals of MRSA-infected patients were compared to rates of MRSA among companion animals of pet guardians attending a “veterinary wellness clinic” (controls). MRSA was isolated from at least one companion animal in 4/49 (8.2%) households of MRSA-infected outpatients vs. none of the pets of the 50 uninfected human controls. Using PFGE, patient-pets MRSA isolates were identical for three pairs and discordant for one pair (suggested MRSA inter-specie transmission p-value = 0.1175). These results suggest that companion animals of MRSA-infected patients can be culture-positive for MRSA, representing a potential source of infection or re-infection for humans. Further studies are required to better understand the epidemiology of MRSA human-animal inter-specie transmission.     

Monolysocardiolipins accumulate in Barth syndrome but do not lead to enhanced apoptosis

Barth syndrome (BTHS) is an X-linked recessive disorder that is biochemically characterized by low cellular levels of the mitochondrial phospholipid cardiolipin (CL). Previously, we discovered that the yeast disruptant of the TAZ ortholog in Saccharomyces cerevisiae not only displays CL deficiency but also accumulates monolysocardiolipins (MLCLs), which are intermediates in CL remodeling. Therefore, we set out to investigate whether MLCL accumulation also occurs in BTHS. Indeed, we observed MLCL accumulation in heart, muscle, lymphocytes, and cultured lymphoblasts of BTHS patients; however, only very low levels of these lysophospholipids were found in platelets and fibroblasts of these patients. Although the fatty acid composition of the MLCLs was different depending on the tissue source, it did parallel the fatty acid composition of the (remaining) CLs. The possible implications of these findings for the two reported CL remodeling mechanisms, transacylation and deacylation/reacylation, are discussed. Because MLCLs have been proposed to be involved in the initiation of apoptosome-mediated cell death by the sequestration of the proapoptotic protein (t)BH3-interacting domain death agonist (Bid) to the mitochondrial membrane, we used control and BTHS lymphoblasts to investigate whether the accumulation of MLCLs results in higher levels of apoptosis. We found no differences in susceptibility to death receptor-mediated apoptosis or in cellular distribution of Bid, cytochrome c, and other parameters, implying that MLCL accumulation does not lead to enhanced apoptosis in cultured BTHS lymphoblasts.     

Characterization of an Autotrophic Nitrogen-Removing Biofilm from a Highly Loaded Lab-Scale Rotating Biological Contactor

In this study, a lab-scale rotating biological contactor (RBC) treating a synthetic NH₄⁺ wastewater devoid of organic carbon and showing high N losses was examined for several important physiological and microbial characteristics. The RBC biofilm removed 89% ± 5% of the influent N at the highest surface load of approximately 8.3 g of N m⁻² day⁻¹, with N₂ as the main end product. In batch tests, the RBC biomass showed good aerobic and anoxic ammonium oxidation (147.8 ± 7.6 and 76.5 ± 6.4 mg of NH₄⁺-N g of volatile suspended solids [VSS]⁻¹ day⁻¹, respectively) and almost no nitrite oxidation (< 1 mg of N g of VSS⁻¹ day⁻¹). The diversity of aerobic ammonia-oxidizing bacteria (AAOB) and planctomycetes in the biofilm was characterized by cloning and sequencing of PCR-amplified partial 16S rRNA genes. Phylogenetic analysis of the clones revealed that the AAOB community was fairly homogeneous and was dominated by Nitrosomonas-like species. Close relatives of the known anaerobic ammonia-oxidizing bacterium (AnAOB) Kuenenia stuttgartiensis dominated the planctomycete community and were most probably responsible for anoxic ammonium oxidation in the RBC. Use of a less specific planctomycete primer set, not amplifying the AnAOB, showed a high diversity among other planctomycetes, with representatives of all known groups present in the biofilm. The spatial organization of the biofilm was characterized using fluorescence in situ hybridization (FISH) with confocal scanning laser microscopy (CSLM). The latter showed that AAOB occurred side by side with putative AnAOB (cells hybridizing with probe PLA46 and AMX820/KST1275) throughout the biofilm, while other planctomycetes hybridizing with probe PLA886 (not detecting the known AnAOB) were present as very conspicuous spherical structures. This study reveals that long-term operation of a lab-scale RBC on a synthetic NH₄⁺ wastewater devoid of organic carbon yields a stable biofilm in which two bacterial groups, thought to be jointly responsible for the high autotrophic N removal, occur side by side throughout the biofilm.