A modular microfluidic bioreactor to investigate plant cell-cell interactions.

Plants produce a wide variety of secondary metabolites, which often are of interest to pharmaceutical and nutraceutical industry. Plant-cell cultures allow producing these metabolites in a standardised manner, independently from various biotic and abiotic factors difficult to control during conventional cultivation. However, plant-cell fermentation proves to be very difficult, since these chemically complex compounds often result from the interaction of different biosynthetic pathways operating in different cell types. To simulate such interactions in cultured cells is a challenge. Here, we present a microfluidic bioreactor for plant-cell cultivation to mimic the cell-cell interactions occurring in real plant tissues. In a modular set-up of several microfluidic bioreactors, different cell types can connect through a flow that transports signals or metabolites from module to module. The fabrication of the chip includes hot embossing of a polycarbonate housing and subsequent integration of a porous membrane and in-plane tube fittings in a two-step ultrasonic welding process. The resulting microfluidic chip is biocompatible and transparent. Simulation of mass transfer for the nutrient sucrose predicts a sufficient nutrient supply through the membrane. We demonstrate the potential of this chip for plant cell biology in three proof-of-concept applications. First, we use the chip to show that tobacco BY-2 cells in suspension divide depending on a "quorum-sensing factor" secreted by proliferating cells. Second, we show that a combination of two Catharanthus roseus cell strains with complementary metabolic potency allows obtaining vindoline, a precursor of the anti-tumour compound vincristine. Third, we extend the approach to operationalise secretion of phytotoxins by the fungus Neofusicoccum parvum as a step towards systems to screen for interorganismal chemical signalling.

Author Correction: Microbial spatial footprint as a driver of soil carbon stabilization.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Microbial spatial footprint as a driver of soil carbon stabilization.

Increasing the potential of soil to store carbon (C) is an acknowledged and emphasized strategy for capturing atmospheric CO2. Well-recognized approaches for soil C accretion include reducing soil disturbance, increasing plant biomass inputs, and enhancing plant diversity. Yet experimental evidence often fails to support anticipated C gains, suggesting that our integrated understanding of soil C accretion remains insufficient. Here we use a unique combination of X-ray micro-tomography and micro-scale enzyme mapping to demonstrate for the first time that plant-stimulated soil pore formation appears to be a major, hitherto unrecognized, determinant of whether new C inputs are stored or lost to the atmosphere. Unlike monocultures, diverse plant communities favor the development of 30-150 µm pores. Such pores are the micro-environments associated with higher enzyme activities, and greater abundance of such pores translates into a greater spatial footprint that microorganisms make on the soil and consequently soil C storage capacity.

Initial Bacterial Adhesion Properties of Anodically Oxidized Ti6Al4V.

This paper reports about the initial interaction of bacteria with anodically oxidized Ti6Al4V for the use as dental implant abutment surfaces. Ti6Al4V samples are anodically oxidized in hydrofluoric acid using different voltages. The resulting nanotopographies are characterized by atomic force microscopy, scanning electron microscopy and contact angle measurements. The topographies reach from micro-porous structures with small nanoporosities on top to fully hexagonally aligned nanotubes. For initial bacterial adhesion tests, Escherichia coli and Staphylococcus aureus are used. Samples are incubated for 2 h and afterwards non-adherent cells are washed off. The results of live/dead staining and cell counts are presented. Gram-negative and Gram-positive strains show different behavior in respect to total number of initially adherent cells on different micro/nanotopographies. The observed reduction of adhered microorganisms is mainly based on underlying microporous topographies.

Functional Evaluation of Three Manure-Borne Indicator Bacteria Release Models with Multiyear Field Experiment Data.

Modeling the fate and transport of Escherichia coli is of substantial interest because of how this organism serves as an indicator of fecal contamination in microbial water quality assessment. The efficacy of models used to assess the export of E. coli from agricultural fields is dependent, in part, on submodels they utilize to simulate E. coli release from land-applied manure and animal waste. Although several release submodels have been proposed, they have only been evaluated and compared with data from laboratory or small plot E. coli release experiments. Our objective was to evaluate and compare performances of three manure-borne bacteria release submodels at field-scale: exponential release (EM), two-parametric Bradford and Schijven (B-S), and two-parametric Vadas-Kleinman-Sharpley (VKS); each was independently incorporated and tested as a submodel within the export model KINEROS2/STWIR, using E. coli. Dairy manure was uniformly applied via surface broadcasting once a year for six consecutive years on a 0.28 ha experimental field site. Two irrigation events followed each application: the first immediately followed the initial application and the second occurred one week later. Manure and soil samples were collected before and after irrigation, respectively, and manure, soil, and edge-of-field runoff samples were analyzed for E. coli. Model performance was evaluated with the Akaike criterion, coefficients of determination (R2), and root mean squared errors (RMSE) values. The percentage of exported manure-borne E. coli varied from 0.1% to 10% in most cases, generally reflecting the lag time between initiation of irrigation and initiation ofedge-of-field runoff. The export model performed better when using the VKS submodel which was preferred in 55% of cases. The B-S and EM submodels were preferred in 27% and 18% of cases, respectively. Two-parametric submodels were ultimately preferred over the single parameter submodel.

Oncostatin M in bronchoalveolar lavage correlates with the severity of sarcoidosis.

BACKGROUND: Recent studies have shown that oncostatin M (OSM) might have a role in T cell-mediated inflammatory processes in which mast cells are also involved. Patients with severe sarcoidosis might develop fibrotic changes in the lung. We assessed whether there was a correlation between mast cells expressing OSM in bronchoalveolar lavage (BAL) and the severity of sarcoidosis. PATIENTS AND METHODS: Twelve consecutive patients with new diagnosis of sarcoidosis were eligible for the study. All underwent complete lung function tests, angiotensin converting enzyme (ACE), and bronchoscopy that included BAL and biopsies. Cytospins of BAL were prepared. All samples were incubated with the primary antibody rabbit anti-human c-kit, CD117 and stained for total mast cell count. The mouse anti-human Oncostatin M was applied and activated mast cells were counted. Clinical sarcoidosis parameters including ACE and lung functions were correlated with mast cells in BAL, as well as with OSM positive mast cells. RESULTS: FEV1 % was correlated with the percentage of activated mast cells, as well as with the percentage of OSM positive mast cells (r=0.61, p=0.033, 95% CI: 0.06-0.87; r=0.58, p=0.04, 95% CI: 0.015-0.86, respectively). FVC and FEV1/FVC correlated with activated mast cells (r=0.58, p=0.05; r=0.63, p=0.028, respectively). CONCLUSIONS: Direct correlation was found between clinical parameters including lung function tests (FEV1 and FVC) and OSM secretion from mast cells in patients with sarcoidosis. These findings suggest that mast cells and OSM have a role in sarcoidosis. Further studies to confirm these preliminary results are suggested.

Is routine bronchoscopic culture indicated in areas with low tuberculosis prevalence?

SETTING: Tertiary care university-affiliated medical centre. OBJECTIVE: To determine the value of routine culture of bronchoscopy samples for mycobacteria even when tuberculosis (TB) is not strongly suspected in low TB prevalence areas. DESIGN: A prospective study of 362 consecutive patients who underwent a bronchoscopy procedure. All demographic, clinical and computed tomography findings, and bacterial and mycobacterial culture results were collected. RESULTS: A total of 217 men and 145 women, with a mean age of 63 ± 15 years, were included in the study. All underwent bronchoscopy with routine culture for TB. Ten cultures (2.8%) grew mycobacteria: 2 (0.55%) Mycobacterium tuberculosis and 8 (2.2%) non-tuberculous mycobacteria (NTM). The NTM included M. avium complex (MAC) in six patients and M. simiae in two patients. Two patients had two different mycobacteria species: 1 patient with M. simiae and MAC and the other with TB and MAC. All eight patients were negative for microscopy. CONCLUSION: Based on our results, we suggest that routine culture of bronchial specimens for TB is not indicated in patients with a low clinical suspicion of active TB in countries with a low TB burden.

Modeling transport of Escherichia coli in a creek during and after artificial high-flow events: three-year study and analysis.

Escherichia coli is the leading indicator of microbial contamination of natural waters, and so its in-stream fate and transport needs to be understood to eventually minimize surface water contamination by microorganisms. To better understand mechanisms of E. coli release and transport from soil sediment in a creek the artificial high-water flow events were created by releasing 60-80 m(3) of city water on a tarp-covered stream bank in four equal allotments in July 2008, 2009 and 2010. A conservative tracer difluorobenzoic acid (DFBA) was added to the released water in 2009 and 2010. Water flow rate, E. coli and DFBA concentrations as well as water turbidity were monitored with automated samplers at three in-stream weirs. A one-dimensional model was applied to simulate water flow, and E. coli and DFBA transport during these experiments. The Saint-Venant equations were used to calculate water depth and discharge while a stream solute transport model accounted for release of bacteria by shear stress from bottom sediments, advection-dispersion, and exchange with transient storage (TS). Reach-specific model parameters were estimated by evaluating observed time series of flow rates and concentrations of DFBA and E. coli at all three weir stations. Observed DFBA and E. coli breakthrough curves (BTC) exhibited long tails after the water pulse and tracer peaks had passed indicating that transient storage (TS) might be an important element of the in-stream transport process. Comparison of simulated and measured E. coli concentrations indicated that significant release of E. coli continued when water flow returned to the base level after the water pulse passed and bottom shear stress was small. The mechanism of bacteria continuing release from sediment could be the erosive boundary layer exchange enhanced by changes in biofilm properties by erosion and sloughing detachment.

Sarcoidosis-associated pulmonary hypertension in patients with near-normal lung function.

SETTING: Tertiary care medical centre in Israel. BACKGROUND: Pulmonary hypertension (PH) is a predictor of poor outcome in patients with sarcoidosis. Early diagnosis may improve outcome. OBJECTIVE: To determine factors that might contribute to the early diagnosis of PH in sarcoidosis patients with near normal lung function tests. DESIGN: Retrospective patient review. METHODS: Data from 127 patients with sarcoidosis and near normal lung function tests (forced vital capacity > 70%, forced expiratory volume in 1 second > 70% and diffusion capacity of carbon monoxide [D(LCO)] > 60%), who underwent high resolution computed tomography (HRCT) scan, the 6-minute walk distance (6MWD) test and echocardiogram were analysed. Demographic, clinical and HRCT findings were compared between patients with and those without PH. RESULTS: Thirty-six patients (28.3%) had PH. Patients with PH tended to have lower D(LCO) (68% ± 8 vs. 75% ± 17, P = 0.038), 6MWD (308 m ± 106 vs. 486 m ± 99, P = 0.009) and exercise saturation (91 ± 4 vs. 95 ± 3, P = 0.0001) compared to those without PH. HRCT patterns in PH showed higher frequencies of interstitial thickening (P = 0.004), ground glass appearance (P = 0.01) and fibrosis (P = 0.032). In logistic regression, only 6MWD was predictive of PH (P = 0.005, 95%CI 0.970-0.995). CONCLUSION: Physiological and radiographic characteristics appeared to differentiate patients with PH from those without. Physicians should be aware of PH in patients with sarcoidosis, even in those with near normal lung function.

Effect of biofilm in irrigation pipes on microbial quality of irrigation water.

AIMS: The focus of this work was to investigate the contribution of native Escherichia coli to the microbial quality of irrigation water and to determine the potential for contamination by E. coli associated with heterotrophic biofilms in pipe-based irrigation water delivery systems. METHODS AND RESULTS: The aluminium pipes in the sprinkler irrigation system were outfitted with coupons that were extracted before each of the 2-h long irrigations carried out with weekly intervals. Water from the creek water and sprinklers, residual water from the previous irrigation and biofilms on the coupons were analysed for E. coli. High E. coli concentrations in water remaining in irrigation pipes between irrigation events were indicative of E. coli growth. In two of the four irrigations, the probability of the sample source, (creek vs sprinkler), being a noninfluential factor, was only 0.14, that is, source was an important factor. The population of bacteria associated with the biofilm on pipe walls was estimated to be larger than that in water in pipes in the first three irrigation events and comparable to one in the fourth event. CONCLUSION: Biofilm-associated E. coli can affect microbial quality of irrigation water and, therefore, should not be neglected when estimating bacterial mass balances for irrigation systems. SIGNIFICANCE AND IMPACT OF THE STUDY: This work is the first peer-reviewed report on the impact of biofilms on microbial quality of irrigation waters. Flushing of the irrigation system may be a useful management practice to decrease the risk of microbial contamination of produce. Because microbial water quality can be substantially modified while water is transported in an irrigation system, it becomes imperative to monitor water quality at fields, rather than just at the intake.

Survival of manure-borne E. coli in streambed sediment: effects of temperature and sediment properties.

Escherichia coli bacteria are commonly used as indicator organisms to designate of impaired surface waters and to guide the design of management practices to prevent fecal contamination of water. Stream sediments are known to serve as a reservoir and potential source of fecal bacteria (E. coli) for stream water. In agricultural watersheds, substantial numbers of E. coli may reach surface waters, and subsequently be deposited into sediments, along with fecal material in runoff from land-applied manures, grazing lands, or wildlife excreta. The objectives of this work were (a) to test the hypothesis that E. coli survival in streambed sediment in the presence of manure material will be affected by sediment texture and organic carbon content and (b) to evaluate applicability of the exponential die-off equation to the E. coli survival data in the presence of manure material. Experiments were conducted at three temperatures (4 degrees C, 14 degrees C, and 24 degrees C) in flow-through chambers using sediment from three locations at the Beaverdam Creek Tributary in Beltsville, Maryland mixed with dairy manure slurry in the proportion of 1000:1. Indigenous E. coli populations in sediments ranged from ca. 10(1) to 10(3)MPNg(-1) while approx 10(3) manure-borne E. coli MPNg(-1) were added. E. coli survived in sediments much longer than in the overlaying water. The exponential inactivation model gave an excellent approximation of data after 6-16 days from the beginning of the experiment. Slower inactivation was observed with the increase in organic carbon content in sediments with identical granulometric composition. The increase in the content of fine particles and organic carbon in sediments led not only to the slower inactivation but also to lower sensitivity of the inactivation to temperature. Streambed sediment properties have to be documented to better evaluate the role of sediments as reservoirs of E. coli that can affect microbiological stream water quality during high flow events.

Uncertainty evaluation of coliform bacteria removal from vegetated filter strip under overland flow condition.

Vegetated filter strips (VFS) have become an important component of water quality improvement by reducing sediment and nutrients transport to surface water. This management practice is also beneficial for controlling manure-borne pathogen transport to surface water. The objective of this work was to assess the VFS efficiency and evaluate the uncertainty in predicting the microbial pollutant removal from overland flow in VFS. We used the kinematic wave overland flow model as implemented in KINEROS2 coupled with the convective-dispersive overland transport model which accounts for the reversible attachment-detachment and surface straining of infiltrating bacteria. The model was successfully calibrated with experimental data obtained from a series of simulated rainfall experiments at vegetated and bare sandy loam and clay loam plots, where fecal coliforms were released from manure slurry applied on the top of the plots. The calibrated model was then used to assess the sensitivity of the VFS efficiency to the model parameters, rainfall duration, and intensity for a case study with a 6-m VFS placed at the edge of 200-m long field. The Monte Carlo simulations were also performed to evaluate the uncertainty associated with the VFS efficiency given the uncertainty in the model parameters and key inputs. The VFS efficiency was found to be <95% in 25%, <75% in 23%, and <25% in 20% of cases. Relatively long high-intensity rainfalls, low hydraulic conductivities, low net capillary drives of soil, and high soil moisture contents before rainfalls caused the partial failure of VFS to retain coliforms from the infiltration excess runoff.

Comparison of release and transport of manure-borne Escherichia coli and enterococci under grass buffer conditions.

AIM: To test the hypothesis that Escherichia coli and enterococci bacteria have similar release rates and transport characteristics after being released from land-applied manure. METHODS AND RESULTS: Turfgrass soil sod was placed into 200 cm long boxes that had the top two 25 cm sections separated to monitor the release and infiltration of bacteria, which affected bacteria transport in the rest of the box. Dairy manure with added KBr was broadcast on the top two sections. Boxes with either live or dead grass stand were placed under a rainfall simulator for 90 min. Runoff and infiltration samples were collected and analysed for Br, E. coli, enterococci and turbidity. Significant differences in release kinetics of E. coli and enterococci were found. A change from first-order release kinetics to zero-order kinetics after 1 h of rainfall simulation was observed. CONCLUSION: Differences in release rates but not in the subsequent transport were observed for E. coli and enterococci. SIGNIFICANCE AND IMPACT OF THE STUDY: Because both E. coli and enterococci are currently used as indicator organisms for manure-borne pathogens, the differences in their release rates may affect the efficiency of using these organisms as indicators.

Rainfall-induced release of fecal coliforms and other manure constituents: comparison and modeling.

Modeling release of fecal coliforms is an important component of fate and transport simulations related to environmental water quality. Manure constituents other than fecal coliforms may serve as natural tracers of fecal contamination provided that their release from manure to runoff is similar to the fecal coliform release. The objectives of this work were to compare release of fecal coliforms (FC), chloride (Cl-), organic carbon (OC), and water-soluble phosphorus (P) from dissolving manure and to assess the performance of three models in describing the observed release. Bovine manure was applied on 0.5- by 0.3-m bare and vegetated subplots with 20% slope on sandy loam and clay loam soils. Concentrations of Cl-, FC, OC, and P were measured in runoff collected from troughs at the edges of the subplots at 5-min intervals during 1-h rainfall simulations. The one-parametric exponential model and two-parametric Vadas-Kleinman-Sharpley model and Bradford-Schijven model were fitted to the data. The Bradford-Schijven model had uncorrelated parameters, one of which was linearly related to the irrigation rate, and another parameter reflected the presence or the absence of vegetation. Kinetics of the FC release from manure was similar to the release kinetics of P and OC. The Bradford-Schijven model is recommended to simulate the release of manure constituents.

Effect of manure on Escherichia coli attachment to soil.

Attachment of bacteria to soil is an important component of bacterial fate and transport. Escherichia coli are commonly used as indicators of fecal contamination in the environment. Despite the fact that E. coli are derived exclusively from feces or manure, effect of the presence of manure colloids on bacteria attachment to agricultural soils was never directly studied. The objective of this work was to evaluate the magnitude of the effect of manure on E. coli attachment to soil. Escherichia coli attachment to soil was studied in batch experiments with samples of loam and sandy clay loam topsoil that were taken in Pennsylvania and Maryland. Escherichia coli cells were added to the water-manure suspensions containing 0, 20, and 40 g L(-1) of filtered liquid bovine manure, which subsequently were equilibrated with air-dry sieved soil in different soil to suspension ratios. The Langmuir isotherm equation was fitted to data. Manure dramatically affected E. coli attachment to soil. Attachment isotherms were closer to linear without manure and were strongly nonlinear in the presence of manure. The maximum E. coli attachment occurred in the absence of manure. Increasing manure content generally resulted in decreased attachment.

Microthermoforming as a novel technique for manufacturing scaffolds in tissue engineering (CellChips).

The CellChip is a microstructured polymer scaffold, which favours a three-dimensional cultivation of cells within an array of cubic microcontainers. The manufacturing process used so far is microinjection moulding combined with laser-based perforation. In a first attempt to simplify the process, costly perforation was avoided by using commercially available, inexpensive microfiltration membranes for the bottom of the microcavities. Microthermoforming is a promising novel technique which allows the CellChip to be produced from thin film. Working pressures of approximately 4000 kPa were required for the adequate moulding of 50 microm thick films from three different polymers (polystyrene, polycarbonate, cyclo-olefin polymer). Integrating drafts and chamfers in micromoulds is not going to eliminate an uneven thickness profile, but reduces demoulding forces. Microthermoformed CellChips of polycarbonate were perforated by an ion track technique to guarantee a sufficient supply of medium and gases to the cells. The prestructured CellChips were irradiated with 1460 MeV xenon ions at a fluence of a few 10(6) ions/cm2. The tracks were etched in an aqueous solution of 5 N NaOH at 30 degrees C, which resulted in cylindrical pores approximately 2 microm in diameter. Microinjection-moulded, membrane-bonded and thermoformed CellChips were subjected to comparative examination for viability in a cell culture experiment with parenchymal liver cells (HepG2). The cells stayed viable over a period of more than 20 days. No significant differences in viability between injection-moulded, membrane-bonded, and thermoformed CellChips were observed.

[Lab-On-A- Chip--systems for biomedical research and diagnosis]. / Lab-On-A-Chip--Systeme für Die Biomedizinische Forschung und Diagnostik.

In today's biomedical research and diagnosis, a number of substances and agents have to be checked. Frequently, plastic micro titer plates are used for this purpose as large-area test platforms. For the first time, plastic micro titer plates with 96 identical microfluidic labon-a-chip structures for simultaneous capillary electrophoresis (CE) have now been produced using microtechnical fabrication methods. Such structures are suited for e.g. the separation of biomolecules. In completely sealed microfluidic channel systems, smallest sample volumes can be processed, separated, mixed with other substances, or detected. Due to the small channel dimensions, these microfluidic systems are characterized by very small sample volumes needed.

Miniaturized instrument systems for minimally invasive diagnosis and therapy.

Microsystems technologies allow to considerably improve the functionality of existing medical instruments and produce novel devices. Using extremely miniaturized operation systems based on micro-technically processed nickel-titanium alloys, minimally invasive therapeutic interventions can be accomplished in the most sensitive parts of the human body. This has not been possible so far. Fields of use presently comprise among others minimally invasive surgery, endoscopic neurosurgery, interventional cardiology, gynaecology, urology, and ophthalmology.

[Plastic micro-tips for drug delivery]. / Mikrospitzen aus Kunststoff für die Medikamentenapplikation.

Removal or exact transfer of minimum substance volumes from reservoirs or microfluidic systems may be accomplished by means of miniaturized tips with integrated through-going capillaries. Applications in biomedical engineering, e.g. for the application of drugs, or in life sciences, e.g. equipping of microarrays, require the use of disposable plastic products for hygienic reasons and reasons of costs. For this purpose, a method to fabricate microtips out of plastic by doublesided molding has been developed at the Forschungszentrum Karlsruhe.