Metabolic acclimation to photosynthesis-associated stresses was examined in the thermophilic cyanobacterium Thermosynechococcus elongatus BP-1 using integrated computational and photobioreactor analyses. A genome-enabled metabolic model, complete with measured biomass composition, was analyzed using ecological resource allocation theory to predict and interpret metabolic acclimation to irradiance, O₂, and nutrient stresses. Reduced growth efficiency, shifts in photosystem utilization, changes in photorespiration strategies, and differing byproduct secretion patterns were predicted to occur along culturing stress gradients. These predictions were compared with photobioreactor physiological data and previously published transcriptomic data and found to be highly consistent with observations, providing a systems-based rationale for the culture phenotypes. The analysis also indicated that cyanobacterial stress acclimation strategies created niches for heterotrophic organisms and that heter... [more]

We have developed a closed-loop, high-throughput system that applies electrical stimulation and optical recording to facilitate the rapid characterization of extracellular, stimulus-evoked neuronal activity. In our system, a microelectrode array delivers current pulses to a dissociated neuronal culture treated with a calcium-sensitive fluorescent dye; automated real-time image processing of high-speed digital video identifies the neuronal response; and an optimized search routine alters the applied stimulus to achieve a targeted response. Action potentials are detected by measuring the post-stimulus, calcium-sensitive fluorescence at the neuronal somata. The system controller performs directed searches within the strength⁻duration (SD) stimulus-parameter space to build probabilistic neuronal activation curves. This closed-loop system reduces the number of stimuli needed to estimate the activation curves when compared to the more commonly used open-loop approach. This reduction allows t... [more]

Cross-linked starch nanocapsules (NCs) were synthesized by interfacial polymerization carried out using the inverse mini-emulsion technique. 2,4-toluene diisocyanate (TDI) was used as the cross-linker. The influence of TDI concentrations on the polymeric shell, particle size, and encapsulation efficiency of a hydrophilic dye, sulforhodamine 101 (SR 101), was investigated by Fourier transform infrared (FT-IR) spectroscopy, dynamic light scattering (DLS), and fluorescence measurements, respectively. The final NC morphology was confirmed by scanning electron microscopy. The leakage of SR 101 through the shell of NCs was monitored at 37 °C for seven days, and afterwards the NCs were redispersed in water. Depending on cross-linker content, permeable and impermeable NCs shell could be designed. Enzyme-triggered release of SR 101 through impermeable NC shells was investigated using UV spectroscopy with different α-amylase concentrations. Impermeable NCs shell were able to release their cargo... [more]

While many perceive mammalian cell culture-based manufacturing for biopharmaceuticals an established technology, numerous open questions remain to be solved. Genetic diversity and mutation rates in CHO cells have been underestimated since progeny of a clonal CHO cell become genetically diverse with each cell division. This is an important issue since products are made in bioreactors containing up to 1014 cells that have divided for weeks. Regulatory interest in “Proof of Clonality” is a misguided and misunderstood concern in this context. We revisit decades of research on scope and rate of genetic changes in CHO cells and suggest approaches to minimize trends for genomic instability when establishing reliable manufacturing processes. A concept is proposed for transfection-derived cell populations containing relatively stable (The term “stable” will always be used in a context of relative stability—considering time frames of weeks to months at best.) “CHO master sequence” genomes (conta... [more]

The realization of physiologically-relevant advanced in vitro models is not just related to the reproduction of a three-dimensional multicellular architecture, but also to the maintenance of a cell culture environment in which parameters, such as temperature, pH, and hydrostatic pressure are finely controlled. Tunable and reproducible culture conditions are crucial for the study of environment-sensitive cells, and can also be used for mimicking pathophysiological conditions related with alterations of temperature, pressure and pH. Here, we present the SUITE (Supervising Unit for In Vitro Testing) system, a platform able to monitor and adjust local environmental variables in dynamic cell culture experiments. The physical core of the control system is a mixing chamber, which can be connected to different bioreactors and acts as a media reservoir equipped with a pH meter and pressure sensors. The chamber is heated by external resistive elements and the temperature is controlled using a th... [more]

Electrical stimulation is ubiquitous as a method for activating neuronal tissue, but there is still significant room for advancement in the ability of these electrical devices to implement smart stimulus waveform design to more selectively target populations of neurons. The capability of a device to encode more complicated and precise messages to a neuronal network greatly increases if the stimulus input space is broadened to include variable shaped waveforms and multiple stimulating electrodes. The relationship between a stimulating electrode and the activated population is unknown; a priori. For that reason, the population of excitable neurons must be characterized in real-time and for every combination of stimulating electrodes and neuronal populations. Our automated experimental system allows investigation into the stimulus-evoked neuronal response to a current pulse using dissociated neuronal cultures grown atop microelectrode arrays (MEAs). The studies presented here demonstrate... [more]

The gut microbiome is a highly complex microbial community that strongly impacts human health and disease. The two dominant phyla in healthy humans are Bacteroidetes and Firmicutes, with minor phyla such as Proteobacteria having elevated abundances in various disease states. While the gut microbiome has been widely studied, relatively little is known about the role of interspecies interactions in promoting microbiome stability and function. We developed a biofilm metabolic model of a very simple gut microbiome community consisting of a representative bacteroidete (Bacteroides thetaiotaomicron), firmicute (Faecalibacterium prausnitzii) and proteobacterium (Escherichia coli) to investigate the putative role of metabolic byproduct cross feeding between species on community stability, robustness and flexibility. The model predicted coexistence of the three species only if four essential cross-feeding relationships were present. We found that cross feeding allowed coexistence to be robustly... [more]

The process of oxygenic photosynthesis is robust and ubiquitous, relying centrally on input of light, carbon dioxide, and water, which in many environments are all abundantly available, and from which are produced, principally, oxygen and reduced organic carbon. However, photosynthetic machinery can be conflicted by the simultaneous presence of carbon dioxide and oxygen through a process sometimes called photorespiration. We present here a model of phototrophy, including competition for RuBisCO binding sites between oxygen and carbon dioxide, in a chemostat-based microbial population. The model connects to the idea of metabolic pathways to track carbon and degree of reduction through the system. We find decomposition of kinetics into elementary flux modes a mathematically natural way to study synchronization of mismatched rates of photon input and chemostat turnover. In the single species case, though total biomass is reduced by photorespiration, protection from excess light exposures... [more]

In this study, the effect of ultra-high-pressure homogenization (UHPH: 100 or 200 MPa at 25 °C), in comparison to colloid mill (CM: 5000 rpm at 20 °C) and conventional homogenization (CH: 15 MPa at 60 °C), on the stability of oil-in-water emulsions with different oil concentrations (10, 30 or 50 g/100 g) emulsified by whey protein isolate (4 g/100 g) was investigated. Emulsions were characterized for their microstructure, rheological properties, surface protein concentration (SPC), stability to creaming and oxidative stability under light (2000 lux/m²). UHPH produced emulsions containing lipid droplets in the sub-micron range (100⁻200 nm) and with low protein concentrations on droplet surfaces. Droplet size (d3.2, µm) was increased in CH and UHPH emulsions by increasing the oil concentration. CM emulsions exhibited Newtonian flow behaviour at all oil concentrations studied; however, the rheological behaviour of CH and UHPH emulsions varied from Newtonian flow (n ≈ 1) to shear-thinning... [more]

There is currently much interest in pomegranate juice because of the high content of phenolic compounds. Moreover, the interest in the separation of bioactive compounds from natural sources has remarkably grown. In this work, for the first time, the Punica granatum L. (pomegranate) juice—clarified by using polyvinylidene fluoride (PVDF) and polysulfone (PSU) hollow fiber (HF) membranes prepared in the laboratory—was screened for its antioxidant properties by using different in vitro assays, namely 2,2-diphenyl-1-picrylhydrazyl (DPPH), 2,2'-azino-bis-(3-ethylbenzothiazoline-6-sulphonic acid) (ABTS), Ferric Reducing Antioxidant Power (FRAP), and β-carotene bleaching tests, and for its potential inhibitory activity of the carbohydrate-hydrolysing enzymes, α-amylase and α-glucosidase. The effects of clarification on quality characteristics of the juice were also investigated in terms of total phenols, flavonoids, anthocyanins, and ascorbic acid. Experimental results indicated that PVDF mem... [more]