Mole Decrease Research Articles

Chemical inhibitors of hexokinase-2 enzyme reduce lactate accumulation, alter glycosylation processing, and produce altered glycoforms in CHO cell cultures.

Chinese hamster ovary (CHO) cells, predominant hosts for recombinant biotherapeutics production, generate lactate as a major glycolysis by-product. High lactate levels adversely impact cell growth and productivity. The goal of this study was to reduce lactate in CHO cell cultures by adding chemical inhibitors to hexokinase-2 (HK2),the enzyme catalyzing the conversion of glucose to glucose 6-phosphate, and examine their impact on lactate accumulation, cell growth, protein titers, and N-glycosylation. Five inhibitors of HK2 enzyme at different concentrations were evaluated, of which 2-deoxy- d-glucose (2DG) and 5-thio- d-glucose (5TG) successfully reduced lactate accumulation with only limited impacts on CHO cell growth. Individual 2DG and 5TG supplementation led to a 35%-45% decrease in peak lactate, while their combined supplementation resulted in a 60% decrease in peak lactate. Inhibitor supplementation led to at least 50% decrease in moles of lactate produced per mol of glucose consumed. Recombinant EPO-Fctiters peaked earlier relative to the end of culture duration in supplemented cultures leading to at least 11% and as high as 32% increase in final EPO-Fctiters. Asparagine, pyruvate, and serine consumption rates also increased in the exponential growth phase in 2DG and 5TG treated cultures, thus, rewiring central carbon metabolism due to low glycolytic fluxes. N-glycan analysis of EPO-Fcrevealed an increase in high mannose glycans from 5% in control cultures to 25% and 37% in 2DG and 5TG-supplementedcultures, respectively. Inhibitor supplementation also led to a decrease in bi-, tri-, and tetra-antennary structures and up to 50% lower EPO-Fcsialylation. Interestingly, addition of 2DG led to the incorporation of 2-deoxy-hexose (2DH) on EPO-Fc N-glycans and addition of 5TG resulted in the first-ever observed N-glycan incorporation of 5-thio-hexose (5TH). Six percent to23% of N-glycans included 5TH moieties, most likely 5-thio-mannose and/or 5-thio-galactose and/or possibly 5-thio-N-acetylglucosamine, and 14%-33% of N-glycans included 2DH moieties, most likely 2-deoxy-mannose and/or 2-deoxy-galactose, for cultures treated with different concentrations of 5TG and 2DG, respectively. Our study is the first to evaluate the impact of these glucose analogs on CHO cell growth, protein production, cell metabolism, N-glycosylation processing, and formation of alternative glycoforms.

Desalination of produced water via CO2 + C3H8 hydrate formation

Recent years have seen increased interest in gas hydrate production as a viable saline water desalination technique. Produced water is the liquid that is extracted from oil and gas fields and is typically salty. The management of produced water is a significant aspect of the petroleum industry because of its potential to serve as a source of fresh water for oil-producing nations with limited access to clean water, growing environmental concerns, and the strict laws governing the discharge of produced water into the environment. In this study, the effect of mixed gas (CO2 + C3H8 (70:30)) hydrate formation in treating produced water and kinetic studies on induction time, moles of gas consumed, rate, water recovery, and water-to-hydrate conversion were experimentally investigated. An aqueous solution with the same ion concentrations was prepared and experiments were performed at 275.15 K/277.15 K, 2.0 MPa, and 450 rpm. The findings unveiled that with an increase in salinity, the water recovery decreased showing the hindering effect of salts on mixed hydrate formation by reducing the solubility of mixed gas due to the strong electrostatic force of attraction between salt ions and water. A maximum of 67.3% water recovery was observed in 2.8 wt% produced water solution at 275.15 K, 2.0 MPa, and 450 rpm. Furthermore, the induction time increased by 52.64% in treating 2.8 wt% and a decrease in moles of gas consumed, water recovery, and water-to-hydrate conversion by 6.99%, 2.08%, and 9.69% compared to the deionized water system at 275.15 K. A removal efficiency of 47%-63% is achieved by using CO2 + C3H8 and the sequence of metal cation removal is in the order K+ > Na+ > Mg2+ > Ca2+ and anions SO42− > Cl− at 275.15 K. The results also showed that with a rise in the experimental temperature to 277.15 K the removal efficiency increased though the amount of hydrate formed is less. This study illustrates that the formation of mixed gas (CO2 + C3H8) hydrate can be used for treating produced water based on the water recovery, removal efficiency, electrical conductivity, and total dissolved solids results with lower energy consumption.

Role of Monovalent and Divalent Ions in Low-Salinity Water Flood in Carbonate Reservoirs: An Integrated Analysis through Zeta Potentiometric and Simulation Studies

The presence of principal ions in the water injected is essential for enhanced oil recovery by formation of water-wet state in carbonates. This study reaffirms this and presents an evaluation of the positive influence of both divalent as wells as monovalent ions on wettability alteration mechanisms during low salinity waterflooding using brines of varying ionic composition, referred to as “smart brines”. Zeta potentiometric analysis and reservoir simulation studies were conducted with diluted and smart brines that were prepared by varying the composition of principal ions. Surface charge of oil-saturated whole core samples of rock in the presence of various diluted and smart brines was estimated by zeta potential measurements. A comprehensive analysis of zeta potentiometric and reservoir simulation studies was done to establish and investigate the linkage between the recovery mechanism and the incremental recovery achieved. It is noted that zeta potential increases with the increasing level of dilution and it can be attributed to electric double-layer mechanism. On the contrary, simulation studies implied a different mechanism where an increase in effluent’s pH and Ca2+ mole fraction along with decrease in moles of minerals and saturation index implied rock dissolution was dominant mechanism. Moreover, the effect of mineral dissolution beyond the injection block is highly doubtful. This study demonstrates that an integrated approach from both zeta potentiometric and simulation studies can be used to provide insights into the underlying science of interactions at pore scale during a low salinity waterflood using smart brines. With the aid of an adequately designed upscaling procedure and protocol, the laboratory results can be further used towards developing field-scale models to obtain with realistic recovery factors with optimized brine composition and salinity.

Membrane alterations as causes of impaired signal transduction in Alzheimer's disease and aging

Changes in cell-membrane composition in normal aging and in Alzheimer's and other age-related diseases appear to result in impaired neurotransmitter-triggered signal transduction. The impaired signal transduction seems to be related to dysfunctions in the coupling of G proteins to their receptors and effectors. Direct demonstration of altered physiochemical properties of brain tissue of patients with Alzheimer's disease has been achieved by small-angle X-ray diffraction. In this disease, thinner membranes correlate with a 30% decrease in moles of cholesterol: phospholipid. Such changes can affect directly the coupling and uncoupling properties of G proteins, and can account for signal transduction deficits. These findings offer a complementary alternative to the β-amyloid hypothesis, and an opportunity to consider new types of therapeutic interventions.

Photosensitized dissociation of di-tert-butyl peroxide. Energy transfer to a repulsive excited state

Energy transfer from a variety of aromatic hydrocarbons and ketones to di-tert-butyl peroxide has been examined by using nanosecond laser flash photolysis techniques. Triplet energy transfer to the peroxide leads to its efficient cleavage into two tert-butoxy radicals. Representative rate constants for triplet quenching in benzene at 25/sup 0/C are 7.9 x 10/sup 6/, 3.4 x 10/sup 6/, and 7.0 x 10/sup 4/M/sup -1/s/sup -1/ for p-methoxypropiophenone, benzophenone, and benz(a)anthracene, respectively. The rate of transfer for p-methoxypropiophenone (E/sub T/ = 72.5 kcal/mol) is approximately temperature independent; for lower energy sensitizers ca. 0.17 kcal/mol activation energy is required for each kilocalorie per mole decrease in triplet energy. No evidence indicating exciplex intermediacy was found. A model for energy transfer to a repulsive state of the peroxide is proposed in which no activation energy is required if the sensitizer meets the energy requirements at the 0-0 equilibrium distance. For sensitizers of lower triplet energy, energy transfer to a repulsive state is proposed to occur from a thermally activated ground state having a greater than equilibrium oxygen-oxygen bond length. The same mechanism may apply in other systems where the acceptor lacks low-lying excited states. A few rate constants for the quenching of singletmore » sensitizers have also been determined by using fluorescence techniques.« less