YoeB–YefM, the widespread type II toxin–antitoxin (TA) module, binds to its own promoter to autoregulate its transcription: repress or induce transcription under normal or stress conditions, respectively. It remains unclear how YoeB–YefM regulates its transcription depending on the YoeB to YefM TA ratio. We find that YoeB–YefM complex from S.aureus exists as two distinct oligomeric assemblies: heterotetramer (YoeB–YefM2–YoeB) and heterohexamer (YoeB–YefM2–YefM2–YoeB) with low and high DNA-binding affinities, respectively. Structures of the heterotetramer alone and heterohexamer bound to promoter DNA reveals that YefM C-terminal domain undergoes disorder to order transition upon YoeB binding, which allosterically affects the conformation of N-terminal DNA-binding domain. At TA ratio of 1:2, unsaturated binding of YoeB to the C-terminal regions of YefM dimer forms an optimal heterohexamer for DNA binding, and two YefM dimers with N-terminal domains dock into the adjacent major grooves of DNA to specifically recognize the 5′-TTGTACAN6AGTACAA-3′ palindromic sequence, resulting in transcriptional repression. In contrast, at TA ratio of 1:1, binding of two additional YoeB molecules onto the heterohexamer induces the completely ordered conformation of YefM and disassembles the heterohexamer into two heterotetramers, which are unable to bind the promoter DNA optimally due to steric clashes, hence derepresses TA operon transcription.

Mesenchymal stem cells (MSCs) are adult stem cells with a self-renewal and multipotent capability and express extensively in multitudinous tissues. We found that water channel aquaporin-5 (AQP5) is expressed in bone marrow-derived MSCs (BMMSCs) in the plasma membrane pattern. BMMSCs from AQP5 -/ -mice showed significantly lower plasma membrane water permeability than those from AQP5 + / + mice. In characterizing the cultured BMMSCs from AQP5-/ -and AQP5 + / + mice, we found no obvious differences in morphology and proliferation between the 2 genotypes. However, the multiple differentiation capacity was significantly higher in AQP5 -/ -than AQP5 + / + BMMSCs as revealed by representative staining by Oil Red O (adipogenesis); Alizarin Red S and alkaline phosphatase (ALP; osteogenesis); and type II collagen and Safranin O (chondrogenesis) after directional induction. Relative mRNA expression levels of 3 lineage differentiation markers, including PPARc2, C/EBPa, adipsin, collagen 1a, osteopontin, ALP, collagen 11a, collagen 2a, and aggrecan, were significantly higher in AQP5 -/ --differentiating BMMSCs, supporting an increased differentiation capacity of AQP5BMMSCs. Furthermore, a bone-healing process was accelerated in AQP5 -/ -mice in a drill-hole injury model. Mechanistic studies indicated a significantly lower apoptosis rate in AQP5 -/ -than AQP5 + / + BMMSCs. Apoptosis inhibitor Z-VAD-FMK increased the differentiation capacity to a greater extent in AQP5 + / + than AQP5 -/ -BMMSCs. We conclude that AQP5-mediated high plasma membrane water permeability enhances the apoptosis rate of differentiating BMMSCs, thus decreasing their differentiation capacity. These data implicate AQP5 as a novel determinant of differentiation of BMMSCs and therefore a new molecular target for regulating differentiation of BMMSCs during tissue repair and regeneration.

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This part of work was done to explore the basic understanding of the adsorption chromatography by determining the interaction of selected model proteins (n = 5) to monolithic chromatographic materials, with varying densities of butyl and phenyl ligands. Surface energetics approach was applied to study the interaction behavior. The physicochemical properties of the proteins and monolithic chromatographic materials were explored by contact angle and zeta potential values. These values were used to study protein to monolith interaction under various operating conditions. Surface energetics approach allowed the calculation of interaction energy as a function of distance, i.e. energy minimum values. Calculations were performed at various conditions to analyze the effect of major operating parameters on the interaction strength. The interaction strength exposed the hydrophobic nature of the monoliths which increases with increasing ligand density. Further, interaction energy of proteins were higher with monolith with butyl ligand compared to monolith with phenyl ligand. For instance, lactoferrin interaction to monoliths with butyl represents more interaction, i.e. 24.38 kT as compared to monoliths with phenyl i.e. 23.28 kT, keeping lambda as 0.2 nm and salt concentration as 100 mM of ammonium sulphate. Hence, more energy and time will be consumed for elution of proteins immobilized to monoliths with butyl. Similarly, the effect of solid surface for proteins immobilization, effect of ligand density and effect of lambda showed some interesting insights on the interaction behavior. The knowledge generated from the present work will help in the basic understanding as well as development of an efficient, low cost downstream processing design and may mimic the real chromatographic experiments.