Combination of inflammation-related cytokines promotes long-term muscle stem cell expansion

Muscle stem cells (MuSCs, satellite cells) are the major contributor to muscle regeneration. Like most adult stem cells, long-term expansion of MuSCs in vitro is difficult. The in vivo muscle regeneration abilities of MuSCs are quickly lost after culturing in vitro, which prevents the potential applications of MuSCs in cell-based therapies. Here, we establish a system to serially expand MuSCs in vitro for over 20 passages by mimicking the endogenous microenvironment. We identified that the combination of four pro-inflammatory cytokines, IL-1α, IL-13, TNF-α, and IFN-γ, secreted by T cells was able to stimulate MuSC proliferation in vivo upon injury and promote serial expansion of MuSCs in vitro. The expanded MuSCs can replenish the endogenous stem cell pool and are capable of repairing multiple rounds of muscle injuries in vivo after a single transplantation. The establishment of the in vitro system provides us a powerful method to expand functional MuSCs to repair muscle injuries.     

Comparison of the effect of hormones on the hormone synthesis of Tetrahymena in medium or salt solution

Tetrahymena pyriformis was maintained in TYM (tryptone‐yeast medium) as well as in Losina salt solution. One hour treatment of 10^?15 M histamine, serotonin or insulin was given before the histamine, serotonin, triiodothyronine and adrenocorticotropin contents of the cells were measured by flow cytometry after immunocytochemical staining. Maintenance in salt solution increased the hormone level in the cells, and use of the treatment hormone treatments further increased the endogenous hormone content relative to that in medium. The cells in salt mimic better the natural conditions, which means that the effects of hormones under more natural conditions are expressed to a greater extent than the exogenously given hormones in TYM typically used under laboratory conditions. Intercellular hormonal communication between the cells of a Tetrahymena population might assist in the survival of the individual cells.     

A high-throughput media design approach for high performance mammalian fed-batch cultures

An innovative high-throughput medium development method based on media blending was successfully used to improve the performance of a Chinese hamster ovary fed-batch medium in shaking 96-deepwell plates. Starting from a proprietary chemically-defined medium, 16 formulations testing 43 of 47 components at 3 different levels were designed. Media blending was performed following a custom-made mixture design of experiments considering binary blends, resulting in 376 different blends that were tested during both cell expansion and fed-batch production phases in one single experiment. Three approaches were chosen to provide the best output of the large amount of data obtained. A simple ranking of conditions was first used as a quick approach to select new formulations with promising features. Then, prediction of the best mixes was done to maximize both growth and titer using the Design Expert software. Finally, a multivariate analysis enabled identification of individual potential critical components for further optimization. Applying this high-throughput method on a fed-batch, rather than on a simple batch, process opens new perspectives for medium and feed development that enables identification of an optimized process in a short time frame.     

Real time monitoring of biofilm development under flow conditions in porous media

Biofilm growth can impact the effectiveness of industrial processes that involve porous media. To better understand and characterize how biofilms develop and affect hydraulic properties in porous media, both spatial and temporal development of biofilms under flow conditions was investigated in a translucent porous medium by using Pseudomonas fluorescens HK44, a bacterial strain genetically engineered to luminesce in the presence of an induction agent. Real-time visualization of luminescent biofilm growth patterns under constant pressure conditions was captured using a CCD camera. Images obtained over 8 days revealed that variations in bioluminescence intensity could be correlated to biofilm cell density and hydraulic conductivity. These results were used to develop a real-time imaging method to study the dynamic behavior of biofilm evolution in a porous medium, thereby providing a new tool to investigate the impact of biological fouling in porous media under flow conditions.     

Principles and approach to developing mammalian cell culture media for high cell density perfusion process leveraging established fed‐batch media

Perfusion medium was successfully developed based on our fed‐batch platform basal and feed media. A systematic development approach was undertaken by first optimizing the ratios of fed‐batch basal and feed media followed by targeted removal of unnecessary and redundant components. With this reduction in components, the medium could then be further concentrated by 2× to increase medium depth. The medium osmolality was also optimized where we found ～360 mOsm/kg was desirable resulting in a residual culture osmolality of ～300 mOsm/kg for our cell lines. Further building on this, the amino acids Q, E, N, and D were rebalanced to reduce lactate and ammonium levels, and increase the cell‐specific productivity without compromising on cell viability while leaving viable cell density largely unaffected. Further modifications were also made by increasing certain important vitamin and lipid concentrations, while eliminating other unnecessary vitamins. Overall, an effective perfusion medium was developed with all components remaining in the formulation understood to be important and their concentrations increased to improve medium depth. The critical cell‐specific perfusion rate using this medium was then established for a cell line of interest to be 0.075 nL/cell‐day yielding 1.2 g/L‐day at steady state. This perfusion process was then successfully scaled up to a 100 L single‐use bioreactor with an ATF6 demonstrating similar performance as a 2 L bioreactor with an ATF2. Large volume handling challenges in our fed‐batch facility were overcome by developing a liquid medium version of the powder medium product contained in custom totes for plug‐and‐play use with the bioreactor. © 2017 American Institute of Chemical Engineers Biotechnol. Prog., 33:891–901, 2017     

Estimating the maintenance energy and biomass concentration of Saccharomyces cerevisiae by continuous calorimetry

Continuous calorimetry has been applied to monitoring the heat evolution of Saccharomyces cerevisiae grown on d-glucose. The heat evolution, together with the energy and carbon balances, was used to evaluate the energetic efficiency of biomass, by-product biosynthesis, fermentative heat evolution as well as the maintenance energy of S. cerevisiae in ‘aerobic fermentation’ and ‘aerobic respiration’. In aerobic fermentation, under catabolite repression, the fraction of substrate energy converted to heat evolution, maintenance requirement, and biomass decreased with the increase of d-glucose concentration. The fraction of substrate energy converted to ethanol is the highest value and it could contribute up to 70% of the total substrate energy. In aerobic respiration, 43% of the total substrate energy was evolved as heat. While 50% of the total substrate energy was converted into biomass, only 7% of the total substrate energy was used for maintenance functions. The maintenance energy coefficient of S. cerevisiae was determined to be 0.427 MJ kg^?1 cell h^?1 (0.102 kcal g^?1 cell h^?1). For the first time, heat evolution together with yield-maintenance energy was used to predict biomass concentration during the fed-batch cultivation of S. cerevisiae.     

Optimized protocol for the pilot-scale preparation of fungal cellulase

Summary A 25-l scale protocol is devised for the optimal secretion and recovery of fungal cellulase. Using a selected higher yieldingTrichoderma viride SMC strain, a protocol consisted of: a) an optimized production medium rich in microcrystalline cellulose (MCC), fortified with 1% (w/v) ammonium sulphate, 0.5% (w/v) soybean flour, 0.1% (v/v) Tween-80 and other trace nutrients; b) optimized physical parameters of production, such as an inoculum containing a homogeneous suspension of 6×10⁷ conidia per 1,28±1°C, pH 4.0±0.5, 300±20 rpm, 11000±1000 l/h aeration, and 170–220 h duration; c) optimal recovery through a filter press (450 l/h rate of filtration) followed by precipitation with 2.5–3.0 volumes of acetone (15°C and basket centrifugation (27°C, 1700 rpm)); and d) vacuum drying (35°C, 4–6 h). This afforded 70% recovery of cellulase in the form of white fluffy powder containing 20000±2000 carboxy methyl cellulase and 1000±50 units filter paperase per g activities, with raw material cost of US$ 8–10 per million carboxy methyl cellulase units. During storage for 18 months at 4°C, ambient temperature and 37°C, the cellulase preparation was found to retain 100, 75 and 60% of its initial activity, respectively.     

Modeling the temperature response of nitrification

To model nitrification rates in soils, it is necessary to have equations that accurately describe the effect of environmental variables on nitrification rates. A variety of equations have been used previously to describe the effect of temperature on rates of microbial processes. It is not clear which of these best describes the influence of temperature on nitrification rates in soil. I compared five equations for describing the effects of temperature on nitrification in two soils with very different temperature optima from a California oak woodland-annual grassland. The most appropriate equation depended on the range of temperatures being evaluated. A generalized Poisson density function best described temperature effects on nitrification rates in both soils over the range of 5 to 50 °C; however, the Arrhenius equation best described temperature effects over the narrower range of soil temperatures that normally occurs in the ecosystem (5 to 28 °C). Temperature optima for nitrification in most of the soils were greater than even the highest soil temperatures recorded at the sites. A model accounting for increased maintenance energy requirements at higher temperatures demonstrates how net energy production, rather than the gross energy production from nitrification, is maximized during adaptation by nitrifier populations to soil temperatures.     

An in vitro urinary tract catheter system to investigate biofilm development in catheter-associated urinary tract infections

Biofilm development in urinary tract catheters is an often underestimated problem. However, this form of infection leads to high mortality rates and causes significant costs in health care. Therefore, it is important to analyze these biofilms and establish avoiding strategies. In this study a continuous flow-through system for the cultivation of biofilms under catheter-associated urinary tract infection conditions was established and validated. The in vitro urinary tract catheter system implies the composition of urine (artificial urine medium), the mean volume of urine of adults (1 mL min^-1), the frequently used silicone catheter (foley silicon catheter) as well as the infection with uropathogenic microorganisms like Pseudomonas aeruginosa. Three clinical isolates from urine of catheterized patients were chosen due to their ability to form biofilms, their mobility and their cell surface hydrophobicity. As reference strain P. aeruginosa PA14 has been used. Characteristic parameters as biofilm thickness, specific biofilm growth rate and substrate consumption were observed. Biofilm thicknesses varied from 105 ± 16 μm up to 246 ± 67 μm for the different isolates. The specific biofilm growth rate could be determined with a non invasive optical biomass sensor. This sensor allows online monitoring of the biofilm growth in the progress of the cultivation.     

Phospholipase D1 downregulation by α-synuclein: Implications for neurodegeneration in Parkinson's disease

We have previously shown that phospholipase D (PLD) pathways have a role in neuronal degeneration; in particular, we found that PLD activation is associated with synaptic injury induced by oxidative stress. In the present study, we investigated the effect of α-synuclein (α-syn) overexpression on PLD signaling. Wild Type (WT) α-syn was found to trigger the inhibition of PLD1 expression as well as a decrease in ERK1/2 phosphorylation and expression levels. Moreover, ERK1/2 subcellular localization was shown to be modulated by WT α-syn in a PLD1-dependent manner. Indeed, PLD1 inhibition was found to alter the neurofilament network and F-actin distribution regardless of the presence of WT α-syn. In line with this, neuroblastoma cells expressing WT α-syn exhibited a degenerative-like phenotype characterized by a marked reduction in neurofilament light subunit (NFL) expression and the rearrangement of the F-actin organization, compared with either the untransfected or the empty vector-transfected cells. The gain of function of PLD1 through the overexpression of its active form had the effect of restoring NFL expression in WT α-syn neurons. Taken together, our findings reveal an unforeseen role for α-syn in PLD regulation: PLD1 downregulation may constitute an early mechanism in the initial stages of WT α-syn-triggered neurodegeneration.