Factors controlling the drop evaporation constant

In this paper, we discuss the factors affecting drop evaporation. We found that the droplet morphology at a specific temperature was controlled by the physical properties of the liquid itself, such as the molecular weight, density, diffusion coefficient in air, and heat of vaporization. Two processes are included in drop evaporation: diffusion of liquid molecules into the air (diffusion part) and flow of the liquid molecules from inside the drop to the free outer shell liquid layer within the liquid-vapor interface (evaporation part). The diffusion part remained steady during drying and was not sensitive to the variation of temperature. The evaporation part, however, was an active factor and determined the differences in drop evaporation behaviors.     

Highly controlled electrofusion of individually selected cells in dielectrophoretic field cages

The prospect of novel therapeutic approaches has renewed the current interest in the fusion of rare cells, like stem cells or primary immune cells. While conventional techniques are only capable of mass fusion, lab-on-a-chip systems often still lack an acceptable method for making the cells available after processing. Here, we present a microfluidic approach for electrofusion on the single-cell level that offers high control over the cells both before and after fusion. For cell pairing and fusion, we employed dielectrophoresis and AC voltage pulses, respectively. Each cell has been characterized and selected before they were paired, fused and released from the fluidic system for subsequent analysis and cultivation. The successful experimental evaluation of our system was further corroborated by numerical simulations. We obtained fusion efficiencies of more than 30% for individual pairs of mouse myeloma and B cell blasts and showed the proliferating ability of the hybrid cells 3 d after fusion. Since aggregates of more than two cells can be fused, the technique could also be developed further for generating giant cells for low-noise electrophysiology in the context of semi-automated pharmaceutical screening procedures.     

Factors controlling the mobility of photosynthetic proteins

Protein diffusion in and around the photosynthetic membrane must play a crucial role in photosynthetic functions including electron transport, regulation of light-harvesting, and biogenesis, turnover and repair of membrane components. Protein mobility is controlled by a complex web of specific interactions, plus the viscosity of the environment and the extent of macromolecular crowding. I discuss the techniques that can be used to measure protein mobility in photosynthetic membranes. I then summarize what we know about the constraints on protein mobility imposed by macromolecular aggregation and crowding in and around the thylakoid membranes of green plants and cyanobacteria, with particular reference to the fluidity of the thylakoid membrane and the aqueous phases on either side of the membrane (the stroma/cytoplasm and the thylakoid lumen). Current indications are that the stroma/cytoplasm is a relatively fluid environment, whereas protein mobility in the lumen may be extremely restricted. The thylakoid membrane itself has an intermediate fluidity: some protein complexes are virtually immobile, probably due to their incorporation into large, stable macromolecular aggregates. However, there is sufficient free space to allow the long-range diffusion of some complexes. Finally, I discuss some future directions for research in this area.     

Factors controlling the reactivity of zinc finger cores

Although the Zn(2+) cation in Zn·Cys(4), Zn·Cys(3)His, Zn·Cys(2)His(2), and Zn(2)Cys(6) cores of zinc finger (Zf) proteins typically plays a structural role, the Zn-bound thiolates in some Zf cores are reactive. Such labile Zf cores can serve as drug targets for retroviral or cancer therapies. Previous studies showed that the reactivity of a Zn-bound thiolate toward electrophiles is significantly reduced if it forms S---NH hydrogen bonds with the backbone amide. However, we found several well-known inactive Zf cores containing Cys ligands with no H-bonding interactions. Here, we show that H bonds from the peptide backbone or bonds from a second Zn cation to Zn-bound S atoms suppress the reactivity not only of these S atoms, but also of Zn-bound S* atoms with no interactions. Indeed, two or more indirect NH---S hydrogen bonds raise the free energy barrier for methylation of a Zn-bound S* in a Cys(4) core more than a direct NH---S* hydrogen bond. These findings help to elucidate why several well-known Zf cores have Cys ligands with no H bonds, but are unreactive. They also help to provide guidelines for distinguishing labile Cys-rich Zn sites from structural ones, which in turn help to identify novel potential Zf drug targets.     

Factors controlling the efficiencies of photoinduced electron-transfer reactions

The overall efficiencies of photoinduced electron transfer reactions in polar solvents are usually determined by the efficiency with which separated radical ions are formed from the initially formed geminate radical-ion pairs. These separation efficiencies are determined by the competition between retum electron transfer and separation within the geminate pairs. A method is described for determining whether variations in the quantum yields for formation of separated radical ions are due to changes in the reorganization parameters for the return electron transfer reactions, or to other factors. The use of the method is illustrated in studies of the effects of varying steric bulk and molecular size of the donors, and also in studies of the effect of using a charged sensitizer.     

Optimization of bulk cell electrofusion in vitro for production of human-mouse heterohybridoma cells

Cell electrofusion is a phenomenon that occurs, when cells are in close contact and exposed to short high-voltage electric pulses. The consequence of exposure to pulses is transient and nonselective permeabilization of cell membranes. Cell electrofusion and permeabilization depend on the values of electric field parameters including amplitude, duration and number of electric pulses and direction of the electric field. In our study, we first investigated the influence of the direction of the electric field on cell fusion in two cell lines. In both cell lines, applications of pulses in two directions perpendicular to each other were the most successful. Cell electrofusion was finally used for production of human-mouse heterohybridoma cells with modified Koehler and Milstein hybridoma technology, which was not done previously. The results, obtained by cell electrofusion, are comparable to usually used polyethylene glycol mediated fusion on the same type of cells.     

Factors controlling the addition of carbon-centered radicals to alkenes

The addition rate constants of radicals to alkenes are strongly substituent dependent because of enthalpic, polar and steric effects. Recent absolute experimental and high level ab initio data for many prototype additions of small radicals are analyzed with the aid of the state correlation diagram. This leads to a unifying rationalization of the various effects and allows the prediction of rate constants to one order of magnitude or better. Propagation rate coefficients of homo- and copolymerizations and penultimate effects are also discussed.     

On chip electrofusion of single human B cells and mouse myeloma cells for efficient hybridoma generation

This article describes the development and full characterization of a microfluidic chip for electrofusion of human peripheral blood B-cells and mouse myeloma (NS-1) cells to generate hybridomas. The chip consists of an array of 783 traps, with dimensions that were optimized to obtain a final cell pairing efficiency of 33±6%. B cells were stained with a cytoplasmic stain CFDA to assess the different stages of cell fusion, i.e. dye transfer to NS-1 cells (initiating fusion) and membrane reorganization (advanced fusion). Six DC pulses of 100 μs (2.5 kV/cm) combined with an AC field (30 s, 2 MHz, 500 V/cm) and pronase treatment resulted in the highest electrofusion efficiency of paired cells (51±11%). Hybridoma formation, with a yield of 0.33 and 1.2%, was observed after culturing the fused cells for 14 days in conditioned medium. This work provides valuable leads to improve the current electrofusion protocols for the production of human antibodies for diagnostic and therapeutic applications.     

Factors controlling the mechanism of NAD(+) non-redox reactions

β-Nicotinamide adenine dinucleotide (NAD(+)) is an indispensable coenzyme or substrate for enzymes involved in catalyzing redox and non-redox reactions. ADP-ribosylating enzymes catalyze cleavage of the nicotinamide-glycosyl bond of NAD(+) and addition of a nucleophilic group from their substrate proteins to the N-ribose anomeric carbon of NAD(+). Although the role of the nicotinamide-ribose fragment in the mechanism of NAD(+) hydrolysis has been examined, the role of the doubly negatively charged, flexible, and chemically reactive NAD(+) diphosphate moiety in the reaction process has largely been neglected. Thus, the participation of the pyrophosphate group in stabilizing intra- and intermolecular interactions in the ground state and transition state has not been explored. Furthermore, the roles of other factors such as the type/nucleophilicity of the attacking nucleophile and the medium in influencing the reaction pathway have not been systematically evaluated. In this study, we endeavor to fill in these gaps and elucidate the role of these factors in controlling the NAD(+) nicotinamide-glycosyl bond cleavage. Using density functional theory combined with continuum dielectric methods, we modeled both S(N)1 and S(N)2 reaction pathways and assessed the role of the diphosphate group in stabilizing the (i) NAD(+) ground state, (ii) oxocarbocation intermediate, (iii) reaction product, and (iv) nucleophile. We also assessed the chemical nature of the attacking nucleophile and the role of the protein matrix in affecting the reaction mechanism. Our results reveal an intricate interplay among various factors in controlling the reaction pathway, which in turn suggests ways in which the enzyme can accelerate the reaction.     

Factors controlling charge recombination under dark and light conditions in dye sensitised solar cells

A simple and powerful approach for assessing the recombination losses in dye sensitised solar cells (DSSCs) across the current voltage curve (j-V) as a function of TiO(2) electron concentration (n) is demonstrated. The total flux of electrons recombining with iodine species in the electrolyte and oxidised dye molecules can be thought of as a recombination current density, defined as j(rec) = j(inj)-j where j(inj) is the current of electrons injected from optically excited dye states and j is the current density collected at cell voltage (V). The electron concentration at any given operating conditions is determined by charge extraction. This allows comparison of factors influencing electron recombination rates at matched n. We show that j(rec) is typically 2-3 times higher under 1 sun equivalent illumination (j(inj) > 0) relative to dark (j(inj) = 0) conditions. This difference was increased by increasing light intensity, electrolyte iodine concentration and electrolyte solvent viscosity. The difference was reduced by increasing the electrolyte iodide concentration and increasing the temperature. These results allowed us to verify a numerical model of complete operational cells (Barnes et al., Phys. Chem. Chem. Phys., DOI: 10.1039/c0cp01554g) and to relate the differences in j(rec) to physical processes in the devices. The difference between j(rec) in the light and dark can be explained by two factors: (1) an increase in the concentration of electron acceptor species (I(3)(-) and/or I(2)) when current is flowing under illumination relative to dark conditions where the current is flowing in the opposite direction, and (2) a non-trivial contribution from electron recombination to oxidised dye molecules under light conditions. More generally, the technique helps to assign the observed relationship between the components, processing and performance of DSSCs to more fundamental physical processes.     

Influence of additives on the protoplasts electrofusion.

Various neutral or charged surface active substances were used for testing the influence of additives on the electrofusion of barley protoplasts. It was found that neutral surface active agents DX, TAGB, Span-80 and AEO-9 could promote the electrofusion. The positively charged surface active agents Bardac 2080, Bardac 2280 and amphoteric surface active agents dodecyl-propyl betaine and CAB betaine also promote the electrofusion, but at high concentration the electrofusion efficiency will reduce. The negatively charged polymer agents Cibacron blue DX, Fluoresceinylthiocarbamoyl DX, and active surface substances K12 and Carsonol TLS- presented negative effect. These phenomena were discussed from the view of adsorption of additives on the membrane and the interactions between protoplasts.     

Factors controlling the barriers to degenerate hydrogen atom transfers

High-level electronic structure calculations have been used to study the factors contributing to the barriers to degenerate hydrogen-atom transfer (HAT) reactions. Understanding of these reactions is a prerequisite to the development of any more general theory of HAT reactions, and yet, the existing models for such reactions perform quite poorly when applied to even simple self-exchanges. The reasons behind these failures are elucidated in the present work. They include a near cancellation of bond-strength effects between reactant and transition state, as well as a strong dependence of the geometry of the transition state on the nature of the heavy atoms.     

On the role of intermembrane contact in cell electrofusion

The question: is cell electrofusion mediated by a long-lived fusogenic state or does the electric field fuse the membranes directly, was investigated by a new centrifugal approach. Mouse L-cells were brought into contact by a special centrifuge device allowing high voltage pulses to be applied upon the cell pellet during centrifugation. Both stages, membrane contact and electrical breakdown of cell membranes, were controlled. The degree of cell-to-cell compression and corresponding intermembrane contact area were estimated by measuring the low-voltage resistance R of the cell pellet which grows sharply with the increase of centripetal acceleration G. The extent of electrical membrane poration and critical pulse parameters were detected by recording the breakdown current. Supercritical pulse delivery to a cell pellet compressed by intensive centrifugation (400–600 g) leads to polycaryon mass formation. The pulse amplitude required for efficient fusion (2–3 kV/cm, 20–50 μs: fusion index F ∼ 25–35%) was found to be several times higher than the amplitude sufficient to induce noticeable breakdown (300 V/cm). The shapes of the F(G) and R(G) dependences were similar, which revealed a correlation between the area of intermembrane contact and fusion probability. Fusion was negligible if the moment of pulsation and the period of intensive centrifugation were separated in time. The data obtained allow us to conclude that in the case of fusion of L-cells the action of the electric field is not mediated by any long-lived fusogenic state. The process of the common membrane surface formation driven directly by the electric field is discussed.     

Factors controlling the alkyne prins cyclization: the stability of dihydropyranyl cations

The relative stability of the intermediates involved in the alkyne Prins cyclization and the competitive 2-oxonia-[3,3]-sigmatropic rearrangement was studied. This rearrangement was shown to occur slowly under typical alkyne Prins cyclization conditions when the allenyl oxocarbenium ion that results from the rearrangement is similar to or higher in energy than the starting alkynyl oxocarbenium ion. The formal 2-oxonia-[3,3]-sigmatropic rearrangement may be disfavored by destabilizing the resultant allenyl oxocarbenium ion or by stabilizing an intermediate dihydropyranyl cation. The trimethylsilyl group as a substituent at the alkyne and electron-withdrawing groups (CH2Cl and CH2CN) located at the alpha-position to the carbinol center are shown to be effective. DFT calculations suggest that these substituents stabilize the dihydropyranyl cations, thus leading to a more uniform reorganization of the electronic density in the ring, and do not have a direct effect on the formally positively charged carbon atom.     

Factors controlling extremely strong AAA-DDD triply hydrogen-bonded complexes

Triply hydrogen-bonded complexes of the form AAA-DDD are shown to have the strongest interaction when the complex is substituted with electron withdrawing groups on the donor molecule (DDD) and electron donating groups on the acceptor molecule (AAA). In particular, the largest effects are observed when the withdrawing groups act through resonance. This serves to flatten the entire system resulting in more linear, and consequently stronger, hydrogen bonds. Furthermore, the present calculations show that the binding energy correlates with the electron density at the bond critical points and inversely with the hydrogen bond lengths.     

Factors controlling sulfur dioxide solubilities in organic solvents

Sulfur dioxide solubility in a liquid phase was studied as affected by the donor-acceptor interactions of the system’s components, these interactions being dependent on the basicity of organic solvents. Other factors of influence were also studied in order for fitting solubility data by means of linear multiparameter equations, primarily, the nonspecific solvation ability of solvents and their cohesion energy, which counteracts the incorporation of gas molecules into the liquid phase.     

Microfabricated embryonic stem cell divider for large-scale propagation of human embryonic stem cells

We have developed a novel method for fabricating an embryonic stem cell divider (ESCD) constructed from a poly(dimethylsiloxane) (PDMS) replica with a square or hexagonal pattern, and have proposed a new dissociation method for human embryonic stem cells (ESCs). An aspect ratio of the device as high as 2 was perfectly replicated in the cutting line. Using the ESCD, human ESC colonies can be easily and efficiently dissociated into regular-sized ESC clumps without enzymatic treatment. The regularity of the ESC clumps dissociated by the ESCD was compared to that dissociated by a conventional mechanical method. Its quality and reliability were confirmed by maintaining undifferentiated ESCs up to the 15th passage. The ESCD will contribute to the advance quality control of in vitro ESC cultures and allow large-scale production of qualified ESCs with tremendous time- and work-saving.     

Reconstructing the differentiation niche of embryonic stem cells using biomaterials

The biochemical cues and topographical architecture of the extracellular environment extensively influence ES cell fate. The microenvironment surrounding the developing embryo presents these instructive cues in a complex and interactive manner in order to guide cell fate decisions. Current stem cell research aims to reconstruct this multifaceted embryonic niche to recapitulate development in vitro. This review focuses on 2D and 3D differentiation niches created from natural and synthetic biomaterials to guide the differentiation of ES cells toward specific lineages. Biomaterials engineered to present specific physical constraints are also reviewed for their role in differentiation.