Sustained oscillations in extended genetic oscillatory systems

Various dynamic cellular behaviors have been successfully modeled in terms of elementary circuitries showing particular characteristics such as negative feedback loops for sustained oscillations. Given, however, the increasing evidences indicating that cellular components do not function in isolation but form a complex interwoven network, it is still unclear to what extent the conclusions drawn from the elementary circuit analogy hold for systems that are highly interacting with surrounding environments. In this article, we consider a specific example of genetic oscillator systems, the so-called repressilator, as a starting point toward a systematic investigation into the dynamic consequences of the extension through interlocking of elementary biological circuits. From in silico analyses with both continuous and Boolean dynamics approaches to the four-node extension of the repressilator, we found that 1), the capability of sustained oscillation depends on the topology of extended systems; and 2), the stability of oscillation under the extension also depends on the coupling topology. We then deduce two empirical rules favoring the sustained oscillations, termed the coherent coupling and the homogeneous regulation. These simple rules will help us prioritize candidate patterns of network wiring, guiding both the experimental investigations for further physiological verification and the synthetic designs for bioengineering.     

High-density transient gene expression in suspension-adapted 293 EBNA1 cells

Large-scale transient gene expression (TGE) in mammalian cells is an attractive method to rapidly produce recombinant proteins for pre-clinical studies, with some processes reported to reach 100 L. However, the yield remains low, hardly over 20 mg protein/L, mainly because the current TGEs have been performed at low cell density (approximately 5 x 10(5) cells/mL). In this study, the strategy to improve TGE focuses on facilitating transfection at high cell density. A high-density perfusion culture of 293 EBNA1 cells was established in 2-L bioreactor using Freestyle 293 expression medium (Invitrogen, Singapore) to grow the cells for transfection. Transfection was then carried out at 1 x 10(7) cells/mL using polyethylenimine (PEI) as DNA carrier, at the optimized conditions of 6 microg DNA/10(7) cells and 1:3 DNA to PEI mass ratio. During the post-transfection phase, 80.8 mg/L of the model protein, EPO was obtained at day 5.5 post-transfection (130 mg total EPO production) using a fed-batch culture mode. In comparison, perfusion cultures using an enriched SFM II medium resulted in a longer post-transfection production phase (8 days), and 227 mg of EPO was produced in 10.7 L medium, showing that high-density TGE enables the production of several hundreds of milligrams of protein in a 2 L bioreactor. In addition, a protocol for economical plasmid preparation based on anion exchange was also established to satisfy TGE's demand in terms of quality and quantity. To the best of our knowledge, this is the first report of transient transfections at a high cell density of up to 1 x 10(7) cells/mL.     

Growth-promoting effects of lactoferrin on L. acidophilus and Bifidobacterium spp.

We investigated the effects of lactoferrin on the growth of L. acidophilus CH-2, Bifidobacterium breve ATCC 15700, B. longum ATCC 15707, B. infantis ATCC 15697, and B. bifidum ATCC 15696. The growth of L. acidophilus was stimulated by bovine holo-lactoferrin but not by apo-lactoferrin. With bifidobacteria, bovine lactoferrin stimulated growth of three strains: B. breve, B. infantis and B. bifidum under certain conditions. Both apoprotein and holoprotein had similar effects. However, B. longum growth was not affected by lactoferrin. Thus, the mechanism of stimulating growth of bifidobacteria may be different from that of L. acidophilus. By far-western blotting using biotinylated lactoferrin and horseradish peroxidase-conjugated streptavidin, lactoferrin-binding proteins were detected in the membrane protein fraction of L. acidophilus, B. bifidum, B. infantis and B. breve. The molecular weights of lactoferrin-binding proteins of L. acidophilus were estimated from SDS-polyacrylamide gel electrophoresis to be 27, 41 and 67 kDa, and those of the three bifidobacterial strains were estimated to be 67-69 kDa. However, no such lactoferrin-binding components were detected in the membrane fraction of B. longum. It is interesting that the appearance of lactoferrin-binding proteins in the membrane fraction of these species corresponds to their growth stimulation by lactoferrin.     

Osmotic stress induces inactivation of photosynthesis in guard cell protoplasts of Vicia leaves.

Guard cell protoplasts isolated from Vicia leaves showed a strong suppression of the photosynthesis under hypotonic conditions, as reflected by changes in the chlorophyll fluorescence characteristics. The response was reversible as well. Mesophyll cell protoplasts did not show any lowering of photosynthetic activity under hypo-osmotic conditions. This result indicates that the response was guard cell specific.     

Production and purification of recombinant Blomia tropicalis group 5 allergen from Pichia pastoris culture

The gene of Blomia tropicalis group 5 allergen (Blo t 5) was cloned and expressed in Pichia pastoris KM71. Selected KM71 clones were cultivated in a fed-batch bioreactor feeding first glycerol then followed by methanol. Recombinant Blo t 5 constituted about 30% of the total broth protein after 60 h cultivation. The harvested broth was purified to >95% purity by a two-step anion exchange chromatography. The overall yield was 37 mg Blo t 5 per litre broth.     

A functional glycogen biosynthesis pathway in Lactobacillus acidophilus: expression and analysis of the glg operon

Glycogen metabolism contributes to energy storage and various physiological functions in some prokaryotes, including colonization persistence. A role for glycogen metabolism is proposed on the survival and fitness of Lactobacillus acidophilus, a probiotic microbe, in the human gastrointestinal environment. L. acidophilus NCFM possesses a glycogen metabolism (glg) operon consisting of glgBCDAP‐amy‐pgm genes. Expression of the glg operon and glycogen accumulation were carbon source‐ and growth phase‐dependent, and were repressed by glucose. The highest intracellular glycogen content was observed in early log‐phase cells grown on trehalose, which was followed by a drastic decrease of glycogen content prior to entering stationary phase. In raffinose‐grown cells, however, glycogen accumulation gradually declined following early log phase and was maintained at stable levels throughout stationary phase. Raffinose also induced an overall higher temporal glg expression throughout growth compared with trehalose. Isogenic ΔglgA (glycogen synthase) and ΔglgB (glycogen‐branching enzyme) mutants are glycogen‐deficient and exhibited growth defects on raffinose. The latter observation suggests a reciprocal relationship between glycogen synthesis and raffinose metabolism. Deletion of glgB or glgP (glycogen phosphorylase) resulted in defective growth and increased bile sensitivity. The data indicate that glycogen metabolism is involved in growth maintenance, bile tolerance and complex carbohydrate utilization in L. acidophilus.     

Verification of a virtual fields method to extract the mechanical properties of human optic nerve head tissues in vivo

We aimed to verify a custom virtual fields method (VFM) to estimate the patient-specific biomechanical properties of human optic nerve head (ONH) tissues, given their full-field deformations induced by intraocular pressure (IOP). To verify the accuracy of VFM, we first generated ‘artificial’ ONH displacements from predetermined (known) ONH tissue biomechanical properties using finite element analysis. Using such deformations, if we are able to match back the known biomechanical properties, it would indicate that our VFM technique is accurate. The peripapillary sclera was assumed anisotropic hyperelastic, while all other ONH tissues were considered isotropic. The simulated ONH displacements were fed into the VFM algorithm to extract back the biomechanical properties. The robustness of VFM was also tested against rigid body motions and noise added to the simulated displacements. Then, the computational speed of VFM was compared to that of a gold-standard stiffness measurement method (inverse finite element method or IFEM). Finally, as proof of principle, VFM was applied to IOP-induced ONH deformation data (obtained from one subject’s eye imaged with OCT), and the biomechanical properties of the prelamina and lamina cribrosa (LC) were extracted. From given ONH displacements, VFM successfully matched back the biomechanical properties of ONH tissues with high accuracy and efficiency. For all parameters, the percentage errors were less than 0.05%. Our method was insensitive to rigid body motions and was also able to recover the material parameters in the presence of noise. VFM was also found 125 times faster than the gold-standard IFEM. Finally, the estimated shear modulus for the prelamina and the LC of the studied subject’s eye were 33.7 and 63.5 kPa, respectively. VFM may be capable of measuring the biomechanical properties of ONH tissues with high speed and accuracy. It has potential in identifying patient-specific ONH biomechanical properties in the clinic if combined with optical coherence tomography.