Molecular methods resolve the bacterial composition of natural marine biofilms on galvanically coupled stainless steel cathodes

Navy vessels consist of various metal alloys and biofilm accumulation at the metal surface is thought to play a role in influencing metal deterioration. To develop better strategies to monitor and control metallic biofilms, it is necessary to resolve the bacterial composition within the biofilm. This study aimed to determine if differences in electrochemical current could influence the composition of dominant bacteria in a metallic biofilm, and if so, determine the level of resolution using metagenomic amplicon sequencing. Current was generated by creating galvanic couples between cathodes made from stainless steel and anodes made from carbon steel, aluminum, or copper nickel and exposing them in the Delaware Bay. Stainless steel cathodes (SSCs) coupled to aluminum or carbon steel generated a higher mean current (0.39 mA) than that coupled to copper nickel (0.17 mA). Following 3 months of exposure, the bacterial composition of biofilms collected from the SSCs was determined and compared. Dominant bacterial taxa from the two higher current SSCs were different from that of the low-current SSC as determined by DGGE and verified by Illumina DNA-seq analysis. These results demonstrate that electrochemical current could influence the composition of dominant bacteria in metallic biofilms and that amplicon sequencing is sufficient to complement current methods used to study metallic biofilms in marine environments.     

Adhesion of Desulfovibrio desulfuricans and Pseudomonas fluorescens to mild steel surfaces

The adhesion of micro-organisms to metal surfaces has been shown to be important in the corrosion process, but the cell surface structures participating in this adhesion have not previously been identified. Evidence is presented that a bacterial substance taking part in the initial adhesion of Pseudomonas fluorescens and Desulfovibrio desulfuricans (New Jersey) to mild steel is polysaccharide in nature. It is likely that this is present in the outer membrane of the bacterial cells as lipopolysaccharide.     

Bioelectric potential gradients may initiate cell cycling: ELF and zeta potential gradients may mimic this effect

When a number of experimental studies in bioelectromagnetics were reviewed, those in which weak, exogenous extremely low frequency (ELF) fields were applied in fixed juxtaposition to their target tissues, were found to initiate mitogenesis or mitogenesis-related signals more successfully than when the target tissue moved freely during the irradiation. It is suggested that ELF fields in fixed juxtaposition to their target tissue and implanted foreign bodies or endogenous tissues with a significant zeta potential, mimic bioelectric fields generated at wounds. When the potential is high enough, they assist healing by moving cells into the wound and stimulating quiescent cells at the wound margin to cycle. Electrophoresis may help the initial migration of cells into the wound to form a clot, and migration of fibroblasts and epithelial cells from the wound margin. When exposed for a long time in a fixed juxtaposition to a potential gradient too weak to show in situ microelectrophoresis along the cell membrane surface, surface particles may coalesce to form microclusters, where like-charged surface particles are in close proximity, and growth factor receptor oligomerization and other cycle-initiating reactions are facilitated. Bioelectromagnetics 18:341–348, 1997. © 1997 Wiley-Liss, Inc.     

Anaesthetic impairment of immune function is mediated via GABA(A) receptors

Background

GABA_A receptors are members of the Cys-loop family of neurotransmitter receptors, proteins which are responsible for fast synaptic transmission, and are the site of action of wide range of drugs [1]. Recent work has shown that Cys-loop receptors are present on immune cells, but their physiological roles and the effects of drugs that modify their function in the innate immune system are currently unclear [2]. We are interested in how and why anaesthetics increase infections in intensive care patients; a serious problem as more than 50% of patients with severe sepsis will die [3]–[6]. As many anaesthetics act via GABA_A receptors [7], the aim of this study was to determine if these receptors are present on immune cells, and could play a role in immunocompromising patients.

Principal Findings

We demonstrate, using RT-PCR, that monocytes express GABA_A receptors constructed of α1, α4, β2, γ1 and/or δ subunits. Whole cell patch clamp electrophysiological studies show that GABA can activate these receptors, resulting in the opening of a chloride-selective channel; activation is inhibited by the GABA_A receptor antagonists bicuculline and picrotoxin, but not enhanced by the positive modulator diazepam. The anaesthetic drugs propofol and thiopental, which can act via GABA_A receptors, impaired monocyte function in classic immunological chemotaxis and phagocytosis assays, an effect reversed by bicuculline and picrotoxin.

Significance

Our results show that functional GABA_A receptors are present on monocytes with properties similar to CNS GABA_A receptors. The functional data provide a possible explanation as to why chronic propofol and thiopental administration can increase the risk of infection in critically ill patients: their action on GABA_A receptors inhibits normal monocyte behaviour. The data also suggest a potential solution: monocyte GABA_A receptors are insensitive to diazepam, thus the use of benzodiazepines as an alternative anesthetising agent may be advantageous where infection is a life threatening problem.     

Assessment of the Impact of Potential Tetracycline Exposure on the Phenotype of Aedes aegypti OX513A: Implications for Field Use

Background

Aedes aegypti is the primary vector of dengue fever, a viral disease which has an estimated incidence of 390 million infections annually. Conventional vector control methods have been unable to curb the transmission of the disease. We have previously reported a novel method of vector control using a tetracycline repressible self-limiting strain of Ae. aegypti OX513A which has achieved >90% suppression of wild populations.

Methodology/Principal Findings

We investigated the impact of tetracycline and its analogues on the phenotype of OX513A from the perspective of possible routes and levels of environmental exposure. We determined the minimum concentration of tetracycline and its analogues that will allow an increased survivorship and found these to be greater than the maximum concentration of tetracyclines found in known Ae. aegypti breeding sites and their surrounding areas. Furthermore, we determined that OX513A parents fed tetracycline are unable to pre-load their progeny with sufficient antidote to increase their survivorship. Finally, we studied the changes in concentration of tetracycline in the mass production rearing water of OX513A and the developing insect.

Conclusion/Significance

Together, these studies demonstrate that potential routes of exposure of OX513A individuals to tetracycline and its analogues in the environment are not expected to increase the survivorship of OX513A.     

Empirical and deterministic accuracies of across-population genomic prediction

Background

Differences in linkage disequilibrium and in allele substitution effects of QTL (quantitative trait loci) may hinder genomic prediction across populations. Our objective was to develop a deterministic formula to estimate the accuracy of across-population genomic prediction, for which reference individuals and selection candidates are from different populations, and to investigate the impact of differences in allele substitution effects across populations and of the number of QTL underlying a trait on the accuracy.

Methods

A deterministic formula to estimate the accuracy of across-population genomic prediction was derived based on selection index theory. Moreover, accuracies were deterministically predicted using a formula based on population parameters and empirically calculated using simulated phenotypes and a GBLUP (genomic best linear unbiased prediction) model. Phenotypes of 1033 Holstein-Friesian, 105 Groninger White Headed and 147 Meuse-Rhine-Yssel cows were simulated by sampling 3000, 300, 30 or 3 QTL from the available high-density SNP (single nucleotide polymorphism) information of three chromosomes, assuming a correlation of 1.0, 0.8, 0.6, 0.4, or 0.2 between allele substitution effects across breeds. The simulated heritability was set to 0.95 to resemble the heritability of deregressed proofs of bulls.

Results

Accuracies estimated with the deterministic formula based on selection index theory were similar to empirical accuracies for all scenarios, while accuracies predicted with the formula based on population parameters overestimated empirical accuracies by ~25 to 30%. When the between-breed genetic correlation differed from 1, i.e. allele substitution effects differed across breeds, empirical and deterministic accuracies decreased in proportion to the genetic correlation. Using a multi-trait model, it was possible to accurately estimate the genetic correlation between the breeds based on phenotypes and high-density genotypes. The number of QTL underlying the simulated trait did not affect the accuracy.

Conclusions

The deterministic formula based on selection index theory estimated the accuracy of across-population genomic predictions well. The deterministic formula using population parameters overestimated the across-population genomic accuracy, but may still be useful because of its simplicity. Both formulas could accommodate for genetic correlations between populations lower than 1. The number of QTL underlying a trait did not affect the accuracy of across-population genomic prediction using a GBLUP method.     

Field performance of engineered male mosquitoes

Dengue is the most medically important arthropod-borne viral disease, with 50-100 million cases reported annually worldwide. As no licensed vaccine or dedicated therapy exists for dengue, the most promising strategies to control the disease involve targeting the predominant mosquito vector, Aedes aegypti. However, the current methods to do this are inadequate. Various approaches involving genetically engineered mosquitoes have been proposed, including the release of transgenic sterile males. However, the ability of laboratory-reared, engineered male mosquitoes to effectively compete with wild males in terms of finding and mating with wild females, which is critical to the success of these strategies, has remained untested. We report data from the first open-field trial involving a strain of engineered mosquito. We demonstrated that genetically modified male mosquitoes, released across 10 hectares for a 4-week period, mated successfully with wild females and fertilized their eggs. These findings suggest the feasibility of this technology to control dengue by suppressing field populations of A. aegypti.     

Open Field Release of Genetically Engineered Sterile Male Aedes aegypti in Malaysia

Background

Dengue is the most important mosquito-borne viral disease. In the absence of specific drugs or vaccines, control focuses on suppressing the principal mosquito vector, Aedes aegypti, yet current methods have not proven adequate to control the disease. New methods are therefore urgently needed, for example genetics-based sterile-male-release methods. However, this requires that lab-reared, modified mosquitoes be able to survive and disperse adequately in the field.

Methodology/Principal Findings

Adult male mosquitoes were released into an uninhabited forested area of Pahang, Malaysia. Their survival and dispersal was assessed by use of a network of traps. Two strains were used, an engineered ‘genetically sterile’ (OX513A) and a wild-type laboratory strain, to give both absolute and relative data about the performance of the modified mosquitoes. The two strains had similar maximum dispersal distances (220 m), but mean distance travelled of the OX513A strain was lower (52 vs. 100 m). Life expectancy was similar (2.0 vs. 2.2 days). Recapture rates were high for both strains, possibly because of the uninhabited nature of the site.

Conclusions/Significance

After extensive contained studies and regulatory scrutiny, a field release of engineered mosquitoes was safely and successfully conducted in Malaysia. The engineered strain showed similar field longevity to an unmodified counterpart, though in this setting dispersal was reduced relative to the unmodified strain. These data are encouraging for the future testing and implementation of genetic control strategies and will help guide future field use of this and other engineered strains.     

State-dependent Inhibition of TRPM2 Channel by Acidic pH

Transient receptor potential melastatin 2 (TRPM2) channel fulfills an important role in oxidative stress signaling in immune and other cells, to which local extracellular acidosis is known to occur under physiological or pathological conditions and impose significant effects on their functions. Here, we investigated whether the ADP-ribose-activated TRPM2 channel is a target for modulation by extracellular acidic pH by patch clamp recording of HEK293 cells expressing hTRPM2 channel. Induced whole cell or single channel currents were rapidly inhibited upon subsequent exposure to acidic pH. The inhibition in the steady state was complete, voltage-independent, and pH-independent in the range of pH 4.0–6.0. The inhibition was irreversible upon returning to pH 7.3, suggesting channel inactivation. In contrast, exposure of closed channels to acidic pH reduced the subsequent channel activation in a pH-dependent manner with an IC₅₀ for H⁺ of 20 μm (pH 4.7) and rendered subsequent current inhibition largely reversible, indicating differential or state-dependent inhibition and inactivation. Alanine substitution of residues in the outer vestibule of the pore including Lys⁹⁵² and Asp¹⁰⁰² significantly slowed down or reduced acidic pH-induced inhibition and prevented inactivation. The results suggest that acidic pH acts as a negative feedback mechanism where protons bind to the outer vestibule of the TRPM2 channel pore and inhibit the TRPM2 channels in a state-dependent manner.