Predicting Perfect Adaptation Motifs in Reaction Kinetic Networks

Adaptation and compensation mechanisms are important to keep organisms fit in a changing environment. “Perfect adaptation” describes an organism’s response to an external stepwise perturbation by resetting some of its variables precisely to their original preperturbation values. Examples of perfect adaptation are found in bacterial chemotaxis, photoreceptor responses, or MAP kinase activities. Two concepts have evolved for how perfect adaptation may be understood. In one approach, so-called “robust perfect adaptation”, the adaptation is a network property (due to integral feedback control), which is independent of rate constant values. In the other approach, which we have termed “nonrobust perfect adaptation”, a fine-tuning of rate constant values is needed to show perfect adaptation. Although integral feedback describes robust perfect adaptation in general terms, it does not directly show where in a network perfect adaptation may be observed. Using control theoretic methods, we are able to predict robust perfect adaptation sites within reaction kinetic networks and show that a prerequisite for robust perfect adaptation is that the network is open and irreversible. We applied the method on various reaction schemes and found that new (robust) perfect adaptation motifs emerge when considering suggested models of bacterial and eukaryotic chemotaxis

Litters of chimeric founder pups obtained from C57BL/6N-EGFP-mCherry ESCs by microaggregation methods.

Pups labeled ES60, ES90, ES150, and ES300 were derived from embryos aggregated in the microdevice with 60, 90, 150, and 300 ESCs, respectively. ESV60 pups were derived from embryos aggregated with 60 ESCs in the bottom of a V-bottomed 96-well culture plate. All pups obtained are pictured except for one ES150 pup that had died.</p

Generation of completely ESC-derived chimeric mice by the microdevice aggregation method.

Generation of completely ESC-derived chimeric mice by the microdevice aggregation method.</p

Embryo culture test in the microdevice.

Six pronuclear-stage embryos were cultured in the microdevice. After two days of culture, the embryos were alive and had developed normally. Embryos were observed from the top of the microdevice using a stereoscopic microscope.</p

Design parameters used to estimate the gravity and surface tension forces on the medium in the device.

Design parameters used to estimate the gravity and surface tension forces on the medium in the device.</p

Newly designed device.

(A) Cross-sectional schematic view of the cell-aggregation device and the procedure for recovering the ESC-fused embryo using the device. (B) Image of designed devices set on a polystyrene 96-well plate. (C) Metal mold for polystyrene casting. (D) Picture of a fabricated device. (E) A polystyrene 96-well plate equipped with ESC aggregation devices. (F) An enlarged side-view picture of polystyrene devices installed on the 96-well plate.</p

F1 pups obtained by mating a completely ESC-derived male (judged by coat color) with an ICR female (shown in pictures, with a white coat).

Left: F1 pups from ID ES90-#2. Right: F1 pups from ID ES300-#1.</p

Conventional 8-cell injection method, conventional aggregation method, and newly developed microaggregation method for obtaining 100% embryonic-stem-cell (ESC)-derived mice.

(A) Injection method: completely separated ESCs are microinjected into 8-cell-stage embryos. (B) Conventional aggregation method: a clump of ESCs subjected to partial trypsinization are co-cultured with 8-cell-stage embryos with the zona-pellucida removed, in a dent made in the bottom of a culture dish. (C) Microaggregation method: ESCs and 8-cell-stage embryos, both prepared as in B, are separately introduced from the top of the device into a micro-hanging drop and co-cultured. In all three methods, chimeric embryos are cultured until the blastocyst stage and then transplanted into the uterus of recipient mice.</p

Germline transmission results of F0 ESC-derived mice with a completely black coat.

Germline transmission results of F0 ESC-derived mice with a completely black coat.</p

Non-Enzymatic DNA Cleavage Reaction Induced by 5-Ethynyluracil in Methylamine Aqueous Solution and Application to DNA Concatenation

<div><p>DNA can be concatenated by hybridization of DNA fragments with protruding single-stranded termini. DNA cleavage occurring at a nucleotide containing a DNA base analogue is a useful method to obtain DNA with designed protruding termini. Here, we report a novel non-enzymatic DNA cleavage reaction for DNA concatenation. We found that DNA is cleaved at a nucleotide containing 5-ethynyluracil in a methylamine aqueous solution to generate 5′-phosphorylated DNA fragment as a cleavage product. We demonstrated that the reaction can be applied to DNA concatenation of PCR-amplified DNA fragments. This novel non-enzymatic DNA cleavage reaction is a simple practical approach for DNA concatenation.</p></div