Unravelling interspecies interactions across heterogeneities in complex biofilm communities

The importance of microbial biofilms has been well-recognized for several decades, and focus is now shifting towards investigating multispecies biofilm communities rather than mono- or dual-species biofilms. Therefore, the demand for techniques that provide a sufficient amount of information at adequate resolution is increasing. One major challenge for multispecies studies is that diversity and spatial organization often lead to a high degree of spatial and chemical heterogeneity. Many current approaches do not account for such heterogeneity and therefore only provide average information (−omics techniques in particular), which could obscure important information about the community. Here, we bring attention to the issues of heterogeneity when analysing synthetic multi-species biofilms, in vitro, and the importance of multi-scale approaches. We provide an overview of current and newer approaches that can be applied to biofilm communities, in order to elucidate interactions at the appropriate scale.     

Subtype-specific, bi-component inhibition of SK channels by low internal pH

The effects of low intracellular pH (pH(i) 6.4) on cloned small-conductance Ca2+-activated K+ channel currents of all three subtypes (SK1, SK2, and SK3) were investigated in HEK293 cells using the patch-clamp technique. In 400 nM internal Ca2+ [Ca2+]i, all subtypes were inhibited by pH(i) 6.4 in the order of sensitivity: SK1>SK3>SK2. The inhibition increased with the transmembrane voltage. In saturating internal Ca2+, the inhibition was abolished for SK1-3 channels at negative potentials, indicating a [Ca2+]i-dependent mode of inhibition. Application of 50 microM 1-ethyl-2-benzimidazolone was able to potentiate SK3 current to the same extent as at neutral pH(i). We conclude that SK1-3 all are inhibited by low pH(i). We suggest two components of inhibition: a [Ca2+]i-dependent component, likely involving the SK beta-subunits calmodulin, and a voltage-dependent component, consistent with a pore-blocking effect. This pH(i)-dependent inhibition can be reversed pharmacologically.     

Effects of human deafness gamma-actin mutations (DFNA20/26) on actin function

Six point mutations in non-muscle gamma-actin at the DFNA20/26 locus cause autosomal dominant nonsyndromic hearing loss. The molecular basis for the hearing loss is unknown. We have engineered each gamma-actin mutation into yeast actin to investigate the effects of these mutations on actin function in vivo and in vitro. Cells expressing each of the mutant actins as the sole actin in the cell were viable. Four of the six mutant strains exhibited significant growth deficiencies in complete medium and an inability to grow on glycerol as the sole carbon source, implying a mitochondrial defect(s). These four strains exhibited abnormal mitochondrial morphology, although the mtDNA was retained. All of the mutant cells exhibited an abnormally high percentage of fragmented/non-polarized actin cables or randomly distributed actin patches. Five of the six mutants displayed strain-specific vacuole morphological abnormalities. Two of the purified mutant actins exhibited decreased thermal stability and increased rates of nucleotide exchange, indicative of increased protein flexibility. V370A actin alone polymerized abnormally. It aggregated in low ionic strength buffer and polymerized faster than wild-type actin, probably in part because of enhanced nucleation. Mixtures of wild-type and V370A actins displayed kinetic properties in proportion to the mole fraction of each actin in the mixture. No dominant effect of the mutant actin was observed. Our results suggest that a major factor in the deafness caused by these mutations is an altered ability of the actin filaments to be properly regulated by actin-binding proteins rather than an inability to polymerize.     

Infection Paradox: High Abundance but Low Impact of Freshwater Benthic Viruses

The discovery of an abundant and diverse virus community in oceans and lakes has profoundly reshaped ideas about global carbon and nutrient fluxes, food web dynamics, and maintenance of microbial biodiversity. These roles are exerted through massive viral impact on the population dynamics of heterotrophic bacterioplankton and primary producers. We took advantage of a shallow wetland system with contrasting microhabitats in close proximity to demonstrate that in marked contrast to pelagic systems, viral infection, determined directly by transmission electron microscopy, and consequently mortality of prokaryotes were surprisingly low in benthic habitats in all seasons. This was true even though free viruses were abundant throughout the year and bacterial infection and mortality rates were high in surrounding water. The habitats in which we found this pattern include sediment, decomposing plant litter, and biofilms on aquatic vegetation. Overall, we detected viruses in only 4 of a total of ~15,000 bacterial cells inspected in these three habitats; for comparison, nearly 300 of ~5,000 cells suspended in the water column were infected. The strikingly low incidence of impact of phages in the benthos may have important implications, since a major portion of microbial biodiversity and global carbon and nutrient turnover are associated with surfaces. Therefore, if failure to infect benthic bacteria is a widespread phenomenon, then the global role of viruses in controlling microbial diversity, food web dynamics, and biogeochemical cycles would be greatly diminished compared to predictions based on data from planktonic environments.     

Matrix notation for efficient development of first-principles models within PAT applications: integrated modeling of antibiotic production with Streptomyces coelicolor

A matrix notation coupled to macroscopic principles is introduced as a means to develop first- principles models in an efficient and structured way within PAT applications. The notation was evaluated for developing an integrated biological, chemical (pH modeling) and physical (gas-liquid exchange) model for describing antibiotic production with Streptomyces coelicolor in batch fermentations. The model provided statistically adequate fits to all the monitored macroscopic biological, chemical and physical data of the process, except the phosphate uptake dynamics. This phosphate discrepancy is hypothesized to result from the internal storage of phosphate as polyphosphate prior to the exponential growth phase. The antibiotic production was associated with the stationary phase and its kinetics was adequately described using a modified Luedeking-Piret equation. Further, the maintenance was best described by employing a combination of Pirt and Herbert models, a result that was supported by a model-based hypothesis testing. Overall the process knowledge currently incorporated in the model is believed to be useful both for process optimization purposes and for further testing of hypotheses aiming at improving the mechanistic understanding of antibiotic production with S. coelicolor. Last but not least, the matrix notation is believed to be a promising supporting tool for efficient development and communication of complex dynamic models within a PAT framework.     

Optic nerve histopathology in a case of Wolfram Syndrome: A mitochondrial pattern of axonal loss

Mitochondrial dysfunction in Wolfram Syndrome (WS) is controversial and optic neuropathy, a cardinal clinical manifestation, is poorly characterized. We here describe the histopathological features in postmortem retinas and optic nerves (ONs) from one patient with WS, testing the hypothesis that mitochondrial dysfunction underlies the pathology. Eyes and retrobulbar ONs were obtained at autopsy from a WS patient, and compared with those of a Leber hereditary optic neuropathy (LHON) patient and one healthy control. Retinas were stained with hematoxylin & eosin for general morphology and ONs were immunostained for myelin basic protein (MBP). Immunostained ONs were examined in four “quadrants”: superior, inferior, nasal, and temporal. The WS retinas displayed a severe loss of retinal ganglion cells in the macular region similar to the LHON retina, but not in the control. The WS ONs, immunostained for MBP, revealed a zone of degeneration in the temporal and inferior quadrants. This pattern was similar to that seen in the LHON ONs but not in the control. Thus, the WS patient displayed a distinct pattern of optic atrophy observed bilaterally in the temporal and inferior quadrants of the ONs. This arrangement of axonal degeneration, involving primarily the papillomacular bundle, closely resembled LHON and other mitochondrial optic neuropathies, supporting that mitochondrial dysfunction underlies its pathogenesis.     

IL-17/Th17 Pathway Is Activated in Acne Lesions

The mechanisms of inflammation in acne are currently subject of intense investigation. This study focused on the activation of adaptive and innate immunity in clinically early visible inflamed acne lesions and was performed in two independent patient populations. Biopsies were collected from lesional and non-lesional skin of acne patients. Using Affymetrix Genechips, we observed significant elevation of the signature cytokines of the Th17 lineage in acne lesions compared to non-lesional skin. The increased expression of IL-17 was confirmed at the RNA and also protein level with real-time PCR (RT-PCR) and Luminex technology. Cytokines involved in Th17 lineage differentiation (IL-1β, IL-6, TGF-β, IL23p19) were remarkably induced at the RNA level. In addition, proinflammatory cytokines and chemokines (TNF-α, IL-8, CSF2 and CCL20), Th1 markers (IL12p40, CXCR3, T-bet, IFN-γ), T regulatory cell markers (Foxp3, IL-10, TGF-β) and IL-17 related antimicrobial peptides (S100A7, S100A9, lipocalin, hBD2, hBD3, hCAP18) were induced. Importantly, immunohistochemistry revealed significantly increased numbers of IL-17A positive T cells and CD83 dendritic cells in the acne lesions. In summary our results demonstrate the presence of IL-17A positive T cells and the activation of Th17-related cytokines in acne lesions, indicating that the Th17 pathway is activated and may play a pivotal role in the disease process, possibly offering new targets of therapy.     

Comprehensive In Vitro Toxicity Testing of a Panel of Representative Oxide Nanomaterials: First Steps towards an Intelligent Testing Strategy

Nanomaterials (NMs) display many unique and useful physico-chemical properties. However, reliable approaches are needed for risk assessment of NMs. The present study was performed in the FP7-MARINA project, with the objective to identify and evaluate in vitro test methods for toxicity assessment in order to facilitate the development of an intelligent testing strategy (ITS). Six representative oxide NMs provided by the EC-JRC Nanomaterials Repository were tested in nine laboratories. The in vitro toxicity of NMs was evaluated in 12 cellular models representing 6 different target organs/systems (immune system, respiratory system, gastrointestinal system, reproductive organs, kidney and embryonic tissues). The toxicity assessment was conducted using 10 different assays for cytotoxicity, embryotoxicity, epithelial integrity, cytokine secretion and oxidative stress. Thorough physico-chemical characterization was performed for all tested NMs. Commercially relevant NMs with different physico-chemical properties were selected: two TiO₂ NMs with different surface chemistry – hydrophilic (NM-103) and hydrophobic (NM-104), two forms of ZnO – uncoated (NM-110) and coated with triethoxycapryl silane (NM-111) and two SiO₂ NMs produced by two different manufacturing techniques – precipitated (NM-200) and pyrogenic (NM-203). Cell specific toxicity effects of all NMs were observed; macrophages were the most sensitive cell type after short-term exposures (24-72h) (ZnO>SiO₂>TiO₂). Longer term exposure (7 to 21 days) significantly affected the cell barrier integrity in the presence of ZnO, but not TiO₂ and SiO₂, while the embryonic stem cell test (EST) classified the TiO₂ NMs as potentially ‘weak-embryotoxic’ and ZnO and SiO₂ NMs as ‘non-embryotoxic’. A hazard ranking could be established for the representative NMs tested (ZnO NM-110 > ZnO NM-111 > SiO₂ NM-203 > SiO₂ NM-200 > TiO₂ NM-104 > TiO₂ NM-103). This ranking was different in the case of embryonic tissues, for which TiO₂ displayed higher toxicity compared with ZnO and SiO₂. Importantly, the in vitro methodology applied could identify cell- and NM-specific responses, with a low variability observed between different test assays. Overall, this testing approach, based on a battery of cellular systems and test assays, complemented by an exhaustive physico-chemical characterization of NMs, could be deployed for the development of an ITS suitable for risk assessment of NMs. This study also provides a rich source of data for modeling of NM effects.     

Fluctuations in glucose availability prevent global proteome changes and physiological transition during prolonged chemostat cultivations of Saccharomyces cerevisiae

Chemostat cultivation mode imposes selective pressure on the cells, which may result in slow adaptation in the physiological state over time. We applied a two-compartment scale-down chemostat system imposing feast–famine conditions to characterize the long-term (100 s of hours) response of Saccharomyces cerevisiae to fluctuating glucose availability. A wild-type strain and a recombinant strain, expressing an insulin precursor, were cultured in the scale-down system, and analyzed at the physiological and proteomic level. Phenotypes of both strains were compared with those observed in a well-mixed chemostat. Our results show that S. cerevisiae subjected to long-term chemostat conditions undergoes a global reproducible shift in its cellular state and that this transition occurs faster and is larger in magnitude for the recombinant strain including a significant decrease in the expression of the insulin product. We find that the transition can be completely avoided in the presence of fluctuations in glucose availability as the strains subjected to feast–famine conditions under otherwise constant culture conditions exhibited constant levels of the measured proteome for over 250 hr. We hypothesize possible mechanisms responsible for the observed phenotypes and suggest experiments that could be used to test these mechanisms.