The auxin-inducible degron 2 technology provides sharp degradation control in yeast, mammalian cells, and mice

Protein knockdown using the auxin-inducible degron (AID) technology is useful to study protein function in living cells because it induces rapid depletion, which makes it possible to observe an immediate phenotype. However, the current AID system has two major drawbacks: leaky degradation and the requirement for a high dose of auxin. These negative features make it difficult to control precisely the expression level of a protein of interest in living cells and to apply this method to mice. Here, we overcome these problems by taking advantage of a bump-and-hole approach to establish the AID version 2 (AID2) system. AID2, which employs an OsTIR1(F74G) mutant and a ligand, 5-Ph-IAA, shows no detectable leaky degradation, requires a 670-times lower ligand concentration, and achieves even quicker degradation than the conventional AID. We demonstrate successful generation of human cell mutants for genes that were previously difficult to deal with, and show that AID2 achieves rapid target depletion not only in yeast and mammalian cells, but also in mice.

Lead Complexation by Humic Acids and Their Analogs: A Voltammetric Study

Differential pulse polarography (DPP) was used to assess the interaction of Pb2+ with various humic acid analogs and several humic acids. DPP analysis demonstrated that the reduction peak maximum (Ep) for Pb2+ shifted to more negative values in the presence of humic acids and humic acid analogs. The observed Ep for Pb2+ in the presence of humic acids and humic acid analogs is influenced by ligand concentration, solution pH and Pb2+ concentration. Shifts in the Ep for Pb2+ are related to the reduction potential and can be rationalized using the Lingane equation.

Concentration Sensing in Crowded Environments

ABSTRACTSignal transduction within crowded cellular compartments is essential for the physiological function of cells and organisms. While the accuracy with which receptors can probe the concentration of ligands has been thoroughly investigated in dilute systems, the effect of macromolecular crowding on the inference of concentration remains unknown. In this work we develop a novel algorithm to simulate reversible reactions between reacting Brownian particles. This facilitates the calculation of reaction rates and correlation times for ligand-receptor systems in the presence of macromolecular crowding. Using this method, we show that it is possible for crowding to increase the accuracy of estimated ligand concentration based on receptor occupancy. In particular, we find that crowding can enhance the effective association rates between small ligands and receptors to a large enough degree to overcome the increased chance of rebinding due to caging by crowding molecules. For larger ligands, crowding decreases the accuracy of the receptor’s estimate primarily by decreasing the microscopic association and dissociation rates.SIGNIFICANCEDeveloping an understanding of how cells effectively transmit signals within or between compartments under physical constraints is an important challenge for biophysics. This work investigates the effect that macromolecular crowding can have on the accuracy of a simple ligand-receptor signaling system. We show that the accuracy of an inferred ligand concentration based on the occupancy of the receptor can be enhanced by crowding under certain circumstances. Additionally, we develop a simulation algorithm that speeds the calculation of reaction rates in crowded environments and can be readily applied to other, more complex systems.

Interaction of Ampicillin and Amoxicillin with Mn2+: A Speciation Study in Aqueous Solution

A potentiometric and UV spectrophotometric investigation on Mn2+-ampicillin and Mn2+-amoxicillin systems in NaCl aqueous solution is reported. The potentiometric measurements were carried out under different conditions of temperature (15 ≤ t/°C ≤ 37). The obtained speciation pattern includes two species for both the investigated systems. More in detail, for system containing ampicillin MLH and ML species, for that containing amoxicillin, MLH2 and MLH ones. The spectrophotometric findings have fully confirmed the results obtained by potentiometry for both the systems, in terms of speciation models as well as the stability constants of the formed species. Enthalpy change values were calculated via the dependence of formation constants of the species on temperature. The sequestering ability of ampicillin and amoxicillin towards Mn2+ was also evaluated under different conditions of pH and temperature via pL0.5 empirical parameter (i.e., cologarithm of the ligand concentration required to sequester 50% of the metal ion present in traces).

Single-Molecule Mechanics in Ligand Concentration Gradient

Single-molecule experiments provide unique insights into the mechanisms of biomolecular phenomena. However, because varying the concentration of a solute usually requires the exchange of the entire solution around the molecule, ligand-concentration-dependent measurements on the same molecule pose a challenge. In the present work we exploited the fact that a diffusion-dependent concentration gradient arises in a laminar-flow microfluidic device, which may be utilized for controlling the concentration of the ligand that the mechanically manipulated single molecule is exposed to. We tested this experimental approach by exposing a λ-phage dsDNA molecule, held with a double-trap optical tweezers instrument, to diffusionally-controlled concentrations of SYTOX Orange (SxO) and tetrakis(4-N-methyl)pyridyl-porphyrin (TMPYP). We demonstrate that the experimental design allows access to transient-kinetic, equilibrium and ligand-concentration-dependent mechanical experiments on the very same single molecule.

Solvent Extraction of Copper (II) Ions Using Unmodified and Aromatic Amine Modified Red Onion Skin Extract

This study investigated the use of unmodified red onion skin extract (UROSE), aniline modified red onion skin extract (AmROSE) and 2-aminophenol modified red onion skin extract (APmROSE) for the extraction of copper (II) ions from aqueous media. The effect of pH, agitation time, ligand concentration and metal ion concentration on the percentage extraction were explored. The stoichiometric coefficients of the metal ions and the ligands (UROSE, AmROSE and APmROSE) in each extraction experiment were determined using slope analysis. The results revealed that the percentage extraction of copper (II) ions increased with increasing ligand concentration and agitation time and decreased with increasing initial concentration of copper (II) ions. The optimum pH for the extraction of copper (II) was found to be 6.77, 6.10 and 2.57 for UROSE, AmROSE and APmROSE respectively, while slope analysis showed that UROSE, AmROSE, and APmROSE ligands reacted with the metal ion in 1:1 molar ratio.