Biphasic Kinetics Research Articles

Uropathogenic Escherichia coli Shows Antibiotic Tolerance and Growth Heterogeneity in an In Vitro Model of Intracellular Infection.

Uropathogenic Escherichia coli (UPEC), the major causative agent of urinary tract infections, can invade different types of host cells. To compare the pharmacodynamic properties of antibiotics against intra- and extracellular UPEC, an in vitro model of intracellular infection was established in J774 mouse macrophages infected by the UPEC strain CFT073. We tested antibiotics commonly prescribed against urinary tract infections (gentamicin, ampicillin, nitrofurantoin, trimethoprim, sulfamethoxazole, and ciprofloxacin) and the investigational fluoroquinolone finafloxacin. The metabolic activity of individual bacteria was assessed by expressing the fluorescent reporter protein TIMERbac within CFT073. Concentration-response experiments revealed that all tested antibiotics were much less effective against intracellular bacteria than extracellular ones. Most antibiotics, except fluoroquinolones, were unable to reach a bactericidal effect intracellularly at clinically achievable concentrations. Ciprofloxacin and finafloxacin killed 99.9% of extracellular bacteria at concentrations around the MIC, while for intracellular bacteria, concentrations more than 100× over the MIC were required to achieve a bactericidal effect. Time-kill curves showed that finafloxacin was more rapidly bactericidal in acidic medium than at neutral pH, while the reverse observation was made for ciprofloxacin. Intracellularly, kill curves showed biphasic kinetics for both fluoroquinolones, suggesting the presence of drug-tolerant subpopulations. Flow cytometry analysis of TIMERbac fluorescence revealed a marked heterogeneity in intracellular growth of individual bacteria, suggesting that the presence of subpopulations reaching a state of metabolic dormancy was the main reason for increased antibiotic tolerance of intracellular UPEC.

Different SP1 binding dynamics at individual genomic loci in human cells

Using a tamoxifen-inducible time-course ChIP-sequencing (ChIP-seq) approach, we show that the ubiquitous transcription factor SP1 has different binding dynamics at its target sites in the human genome. SP1 very rapidly reaches maximal binding levels at some sites, but binding kinetics at other sites is biphasic, with rapid half-maximal binding followed by a considerably slower increase to maximal binding. While ∼70% of SP1 binding sites are located at promoter regions, loci with slow SP1 binding kinetics are enriched in enhancer and Polycomb-repressed regions. Unexpectedly, SP1 sites with fast binding kinetics tend to have higher quality and more copies of the SP1 sequence motif. Different cobinding factors associate near SP1 binding sites depending on their binding kinetics and on their location at promoters or enhancers. For example, NFY and FOS are preferentially associated near promoter-bound SP1 sites with fast binding kinetics, whereas DNA motifs of ETS and homeodomain proteins are preferentially observed at sites with slow binding kinetics. At promoters but not enhancers, proteins involved in sumoylation and PML bodies associate more strongly with slow SP1 binding sites than with the fast binding sites. The speed of SP1 binding is not associated with nucleosome occupancy, and it is not necessarily coupled to higher transcriptional activity. These results with SP1 are in contrast to those of human TBP, indicating that there is no common mechanism affecting transcription factor binding kinetics. The biphasic kinetics at some SP1 target sites suggest the existence of distinct chromatin states at these loci in different cells within the overall population.

Human Metabolism and Urinary Excretion Kinetics of Nonylphenol in Three Volunteers after a Single Oral Dose.

Nonylphenol (NP) is an endocrine-disrupting anthropogenic chemical that is ubiquitous in the environment. Human biomonitoring data and knowledge on internal NP exposure are still sparse, and its human metabolism is largely unknown. Therefore, in this study, we investigated human metabolism and urinary excretion of NP. Three male volunteers received a single oral dose of 1 mg 13C6-labeled NP (10.6-11.7 μg/kg body weight). Consecutive full urine voids were collected for 48 h. A metabolite screening identified nine ring- and/or side chain-oxidized metabolites. We chose the most promising hits, the alkyl chain-oxidized metabolites hydroxy-NP (OH-NP) and oxo-NP, for quantitative investigation next to the parent NP. For this purpose, we newly synthesized specific n - 1-oxidized monoisomeric analytical standards. Quantification of the polyisomeric metabolites was performed via online-solid phase extraction-LC-MS/MS with stable isotope dilution using a previously published consensus method. Alkyl chain hydroxylation (OH-NP) constituted the major metabolism pathway representing 43.7 or 62.2% (depending on the mass transition used for quantification) of the NP dose excreted in urine. The urinary excretion fraction (FUE) for oxo-NP was 6.0 or 9.3%. The parent NP, quantified via an analogous isomeric 13C6-NP standard, represented 6.6%. All target analytes were excreted predominately as glucuronic acid conjugates. Excretion was rather quick, with concentration maxima in urine 2.3-3.4 h after dosing and biphasic elimination kinetics (elimination half-times first phase: 1.0-1.5 h and second phase: 5.2-6.8 h). Due to its high FUE and insusceptibility to external contamination (contrary to parent NP), OH-NP represents a robust and sensitive novel exposure biomarker for NP. The novel FUEs enable to robustly back-calculate the overall NP intakes from urinary metabolite levels in population samples for a well-informed cumulative exposure and risk assessment.

Immune Synapse Residency of Orai1 Alters Ca2+ Response of T Cells.

CRAC, which plays important role in Ca2+-dependent T-lymphocyte activation, is composed of the ER-resident STIM1 and the plasma membrane Orai1 pore-forming subunit. Both accumulate at the immunological synapse (IS) between a T cell and an antigen-presenting cell (APC). We hypothesized that adapter/interacting proteins regulate Orai1 residence in the IS. We could show that mGFP-tagged Orai1-Full channels expressed in Jurkat cells had a biphasic IS-accumulation kinetics peaked at 15 min. To understand the background of Orai1 IS-redistribution we knocked down STIM1 and SAP97 (adaptor protein with a short IS-residency (15 min) and ability to bind Orai1 N-terminus): the mGFP-Orai1-Full channels kept on accumulating in the IS up to the 60th minute in the STIM1- and SAP97-lacking Jurkat cells. Deletion of Orai1 N terminus (mGFP-Orai1-Δ72) resulted in the same time course as described for STIM1/SAP97 knock-down cells. Ca2+-imaging of IS-engaged T-cells revealed that of Orai1 residency modifies the Ca2+-response: cells expressing mGFP-Orai1-Δ72 construct or mGFP-Orai1-Full in SAP-97 knock-down cells showed higher number of Ca2+-oscillation up to the 90th minute after IS formation. Overall, these data suggest that SAP97 may contribute to the short-lived IS-residency of Orai1 and binding of STIM1 to Orai1 N-terminus is necessary for SAP97-Orai1 interaction.

Reverse Ordered Sequential Mechanism for Lactoperoxidase with Inhibition by Hydrogen Peroxide.

Lactoperoxidase (LPO, FeIII in its resting state in the absence of substrates)—an enzyme secreted from human mammary, salivary, and other mucosal glands—catalyzes the oxidation of thiocyanate (SCN−) by hydrogen peroxide (H2O2) to produce hypothiocyanite (OSCN−), which functions as an antimicrobial agent. The accepted catalytic mechanism, called the halogen cycle, comprises a two-electron oxidation of LPO by H2O2 to produce oxoiron(IV) radicals, followed by O-atom transfer to SCN−. However, the mechanism does not explain biphasic kinetics and inhibition by H2O2 at low concentration of reducing substrate, conditions that may be biologically relevant. We propose an ordered sequential mechanism in which the order of substrate binding is reversed, first SCN− and then H2O2. The sequence of substrate binding that is described by the halogen cycle mechanism is actually inhibitory.

Singlet and Triplet Excited-State Dynamics of a Nonfullerene Electron Acceptor Y6

Understanding the excited-state dynamics of nonfullerene electron acceptors is essential for further improvement of organic solar cells. Herein, we investigated the singlet and triplet excited-state dynamics in Y6, a novel nonfullerene acceptor, using transient absorption spectroscopy. We found that pristine Y6 films show biphasic singlet exciton decay kinetics with decay constants of ~220 ps and ~1200 ps, which is the origin of the large discrepancies in the previously reported exciton lifetimes in the solid state. The majority of the Y6 singlet excitons decayed with the faster (~220 ps) component, whereas a clear photoluminescence with the slower (~1200 ps) component was observed. Y6 singlet excitons undergo fast diffusion in the crystalline domains, resulting in fast singlet–singlet exciton annihilation, after which ultrafast triplet formation, assigned to singlet fission from higher excited singlet states, is observed.

Residue Kinetics of Ethofumesate in Texturally Diverse Soils of Sugar Beet Crop under Field Conditions

Indiscriminate use of pesticides and growing awareness of environmental and health problems had led to monitoring their residues in soil and crops. Ethofumesate is one of the widely used herbicides for controlling weeds in sugar beet. Dissipation kinetics and terminal residues of ethofumesate were investigated in two diverse soils under subtropical field conditions. Ethofumesate dissipated slowly after application and follows biphasic first-order kinetics in soils. The average half-life for initial and later phases in sandy loam soil, respectively, was 14.54 and 20.42 and 51.83 and 65.21 days, while for silty clay loam, it was 10.09 and 13.00 and 71.42 and 73.10 days, respectively. Recoveries in soil, leaves, and beetroot ranged from 78.15 to 88.05, 77.01 to 88.58, and 76.25 to 84.50%, respectively. The quantitation limit for soil, roots, and leaves was 0.002 μg g−1. At harvest, no residues were detected in soils, leaves, and sugar beetroots. Residues were below the maximum residue limits in sugar beetroots and leaves as set by EU (0.2 ppm). Ethofumesate is safe from weed control and environmental aspects as it does not persist for a long duration in soils and does not appear to pose any adverse effect on human/animal health under subtropical field conditions.

Human metabolism and kinetics of the UV absorber 2-(2H-benzotriazol-2-yl)-4,6-di-tert-pentylphenol (UV 328) after oral administration

2-(2H-Benzotriazol-2-yl)-4,6-di-tert-pentylphenol (UV 328; CAS: 25973-55-1) is an ultraviolet light (UV) absorber which belongs to the class of hydroxy phenol benzotriazoles. Therefore, UV 328 is added to plastics and other polymers due to its photostability to prevent discoloration and prolong product stability which may result in an exposure of consumers. However, information about the toxic effects on humans and the human metabolism are still lacking. In the present study, human metabolism pathways of UV 328 and its elimination kinetics were explored. For that purpose, three healthy volunteers were orally exposed to a single dose of 0.3 mg UV 328/kg bodyweight. UV 328 and its metabolites were investigated in blood and urine samples collected until 48 and 72 h after exposure, respectively. Thereby, previously published analytical procedures were applied for the sample analysis using dispersive liquid–liquid microextraction and subsequent measurement via gas chromatography coupled to tandem mass spectrometry with advanced electron ionization. UV 328 was found to be oxidized at its alkyl side chains leading to the formation of hydroxy and/or oxo function with maximum blood concentrations at 8–10 h after exposure for UV 328-6/3-OH, UV 328-4/3-OH and UV 328-4/3-CO. In contrast, a plateau for UV 328-4/3-CO-6/3-OH levels was reached around 10 h post-dosage. The highest blood levels were found for native UV 328 at 8 h after ingestion. Furthermore, biphasic elimination kinetics in blood were revealed for almost all detected metabolites. UV 328 and its metabolites did not occur in blood as conjugates. The renal elimination kinetics were very similar with the kinetics in blood. However, the prominence of the metabolites in urine was somewhat different compared to blood. In contrast, mostly conjugated metabolites occurred for renal elimination. In urine, UV 328-4/3-CO-6/3-OH was found to be the most dominant urinary biomarker followed by UV 328-6/3-OH and UV 328-4/3-OH. In total, approximately 0.1% of the orally administered dose was recovered in urine within 72 h. Although high levels of UV 328 in blood proved good resorption and high systemic availability of the substance in the human body, the urine results revealed a rather low quantitative metabolism and urinary excretion rate. Consequently, biliary excretion as part of the enterohepatic cycle and elimination via feces are assumed to be the preferred pathways instead of renal elimination.

Fusicoccin (FC)-Induced Rapid Growth, Proton Extrusion and Membrane Potential Changes in Maize (Zea mays L.) Coleoptile Cells: Comparison to Auxin Responses

The fungal toxin fusicoccin (FC) induces rapid cell elongation, proton extrusion and plasma membrane hyperpolarization in maize coleoptile cells. Here, these three parameters were simultaneously measured using non-abraded and non-peeled segments with the incubation medium having access to their lumen. The dose–response curve for the FC-induced growth was sigmoidal shaped with the maximum at 10−6 M over 10 h. The amplitudes of the rapid growth and proton extrusion were significantly higher for FC than those for indole-3-acetic acid (IAA). The differences between the membrane potential changes that were observed in the presence of FC and IAA relate to the permanent membrane hyperpolarization for FC and transient hyperpolarization for IAA. It was also found that the lag times of the rapid growth, proton extrusion and membrane hyperpolarization were shorter for FC compared to IAA. At 30 °C, the biphasic kinetics of the IAA-induced growth rate could be changed into a monophasic (parabolic) one, which is characteristic for FC-induced rapid growth. It has been suggested that the rates of the initial phase of the FC- and IAA-induced growth involve two common mechanisms that consist of the proton pumps and potassium channels whose contribution to the action of both effectors on the rapid growth is different.

Optimization of a fluorescent-mRNA based real-time assay for precise kinetic measurements of ribosomal translocation

ABSTRACT Kinetic characterization of ribosomal translocation is important for understanding the mechanism of elongation in protein synthesis. Here we have optimized a popular fluorescent-mRNA based translocation assay conducted in stopped-flow, by calibrating it with the functional tripeptide formation assay in quench-flow. We found that a fluorescently labelled mRNA, ten bases long from position +1 (mRNA+10), is best suited for both assays as it forms tripeptide at a fast rate equivalent to the longer mRNAs, and yet produces a large fluorescence change upon mRNA movement. Next, we compared the commonly used peptidyl tRNA analog, N-acetyl-Phe-tRNAPhe, with the natural dipeptidyl fMet-Phe-tRNAPhe in the stopped-flow assay. This analog translocates about two times slower than the natural dipeptidyl tRNA and produces biphasic kinetics. The rates reduce further at lower temperatures and with higher Mg2+ concentration, but improve with higher elongation factor G (EF-G) concentration, which increase both rate and amplitude of the fast phase significantly. In summary, we present here an improved real time assay for monitoring mRNA-translocation with the natural- and an N-Ac-analog of dipeptidyl tRNA.

Lifetime Accumulation of Microplastic in Children and Adults.

Human exposure to microplastic is recognized as a global problem, but the uncertainty, variability, and lifetime accumulation are unresolved. We provide a probabilistic lifetime exposure model for children and adults, which accounts for intake via eight food types and inhalation, intestinal absorption, biliary excretion, and plastic-associated chemical exposure via a physiologically based pharmacokinetic submodel. The model probabilistically simulates microplastic concentrations in the gut, body tissue, and stool, the latter allowing validation against empirical data. Rescaling methods were used to ensure comparability between microplastic abundance data. Microplastic (1–5000 μm) median intake rates are 553 particles/capita/day (184 ng/capita/day) and 883 particles/capita/day (583 ng/capita/day) for children and adults, respectively. This intake can irreversibly accumulate to 8.32 × 103 (90% CI, 7.08 × 102–1.91 × 106) particles/capita or 6.4 (90% CI, 0.1–2.31 × 103) ng/capita for children until age 18, and up to 5.01 × 104 (90% CI, 5.25 × 103–9.33 × 106) particles/capita or 40.7 (90% CI, 0.8–9.85 × 103) ng/capita for adults until age 70 in the body tissue for 1–10 μm particles. Simulated microplastic concentrations in stool agree with empirical data. Chemical absorption from food and ingested microplastic of the nine intake media based on biphasic, reversible, and size-specific sorption kinetics, reveals that the contribution of microplastics to total chemical intake is small. The as-yet-unknown contributions of other food types are discussed in light of future research needs.

Preclinical Pharmacokinetics and Biodistribution of Anticancer Dinuclear Palladium(II)-Spermine Complex (Pd2Spm) in Mice.

Palladium-based compounds are regarded as potential analogs to platinum anticancer drugs with improved properties. The present study assessed the pharmacokinetics and biodistribution of a dinuclear palladium(II)-spermine chelate (Pd2Spm), which has previously been shown to possess promising in vitro activity against several therapy-resistant cancers. Using inductively coupled plasma-mass spectrometry, the kinetic profiles of palladium/platinum in serum, serum ultrafiltrate and tissues (kidney, liver, brain, heart, lungs, ovaries, adipose tissue and mammary glands) were studied in healthy female Balb/c mice after a single intraperitoneal bolus injection of Pd2Spm (3 mg/kg bw) or cisplatin (3.5 mg/kg bw) between 0.5 and 48 h post-injection. Palladium in serum exhibited biphasic kinetics with a terminal half-life of 20.7 h, while the free palladium in serum ultrafiltrate showed a higher terminal half-life than platinum (35.5 versus 31.5 h). Palladium was distributed throughout most of the tissues except for the brain, with the highest values in the kidney, followed by the liver, lungs, ovaries, adipose tissue and mammary glands. The in vitro cellular accumulation was also evaluated in breast cancer cells, evidencing a passive diffusion as a mechanism of Pd2Spm’s cellular entry. This study reports, for the first time, the favorable pharmacokinetics and biodistribution of Pd2Spm, which may become a promising pharmacological agent for cancer treatment.

Controlled drug release: On the evolution of physically entrapped drug inside the electrospun poly(lactic-co-glycolic acid) matrix

The drug loading and releasing properties of poly(lactic-co-glycolic acid) (PLGA) were approached with the application of neutron techniques. The neutron reflection (NR) study on the response of PLGA material to vapor and to bulk water revealed that the hydration of PLGA origins from the molecular compatibility between water and PLGA. Hydration is reversible with regard to the change in humidity and temperature. Capecitabine as drug was embedded in the electrospun PLGA fibers. Small angle neutron scattering (SANS) was able to disclose the domain of entrapped drug inside the fibers and trace its evolution over time when the electrospun membrane was incubated in D2O buffer solution. The evolution of drug domains is discussed in terms of the concentration dependence, the temperature dependence, and the relevance between the drug diffusion inside the polymer matrix and the drug release out to the medium. It was observed that, at 20 °C the drug-related domains are relatively small (~ 100 Å) and relax extremely slow while at 37 °C the drug-related domains are relatively larger (~ 200 Å) and relax faster. These behaviors can be related to the glassy property of structural material. The transportation of drug through the polymer matrix relies on the global relaxation of PLGA chains. The variation of fiber diameter vs. incubation time was followed by ultra-small angle neutron scattering (USANS). The bi-phasic or tri-phasic release kinetics from a series of fibers with different drug loading (2%, 5%, 10%, 20%, 30%, 40%, 50%) were discussed based on the SANS and USANS discovery.

Degradation of imazethapyr in soil: impact of application rate, soil physicochemical properties and temperature

A dissipation study of pesticides in soil is essential as it is a major sink for bulk of globally used pesticides. Soil properties, temperature and application rate play important role in its degradation. Laboratory studies were carried out in order to assess the degradation dynamics of imazethapyr under subtropical conditions. In all soils, at all studied temperatures dissipation followed single first-order kinetics at lower application rates (0.0375 and 0.187 µg g−1) with half-life varying from 30.15 to 52.16 days. However, at higher application rate (0.8 and 1.5 µg g−1), dissipation followed biphasic first-order kinetics and half-life varied from 6.57 to 7.79 days (p > 0.05) during initial phase and 93.74–152.83 days (p < 0.05) during final phase. Dissipation rate was fastest in alkaline soils (pH = 8.0–8.8) followed by neutral soil (pH = 7.4) and acidic soil (pH = 5.0). Amendment of soil with farmyard manure and increase in temperature enhanced the dissipation of imazethapyr. Application of imazethapyr at low rate has nonsignificant effect on dehydrogenase and alkaline phosphates activity, whereas short and momentary toxic effect was observed at 0.8 and 1.5 µg g−1. These findings indicate that soil physicochemical parameters especially pH, OM content and temperature should be considered in combination along with the application rate of imazethapyr for achieving satisfactory weed control and reducing environmental risk associated with the use of imazethapyr in different crops.

Enzyme Kinetics of Oxidative Metabolism-Cytochromes P450.

The cytochrome P450 enzymes (CYPs) are the most important enzymes in the oxidative metabolism of hydrophobic drugs and other foreign compounds (xenobiotics). The versatility of these enzymes results in some unusual kinetic properties, stemming from the simultaneous interaction of multiple substrates with the CYP active site. Often, the CYPs display kinetics that deviate from standard hyperbolic saturation or inhibition kinetics. Non-Michaelis-Menten or "atypical" saturation kinetics include sigmoidal, biphasic, and substrate inhibition kinetics (see Chapter 2 ). Interactions between substrates include competitive inhibition, noncompetitive inhibition, mixed inhibition, partial inhibition, activation, and activation followed by inhibition (see Chapters 4 and 6 ). Models and equations that can result in these kinetic profiles will be presented and discussed.

Repair Kinetics of DNA Double Strand Breaks Induced by Simulated Space Radiation.

Radiation is unavoidable in space. Energetic particles in space radiation are reported to induce cluster DNA damage that is difficult to repair. In this study, normal human fibroblasts were irradiated with components of space radiation such as proton, helium, or carbon ion beams. Immunostaining for γ-H2AX and 53BP1 was performed over time to evaluate the kinetics of DNA damage repair. Our data clearly show that the repair kinetics of DNA double strand breaks (DSBs) induced by carbon ion irradiation, which has a high linear energy transfer (LET), are significantly slower than those of proton and helium ion irradiation. Mixed irradiation with carbon ions, followed by helium ions, did not have an additive effect on the DSB repair kinetics. Interestingly, the mean γ-H2AX focus size was shown to increase with LET, suggesting that the delay in repair kinetics was due to damage that is more complex. Further, the 53BP1 focus size also increased in an LET-dependent manner. Repair of DSBs, characterized by large 53BP1 foci, was a slow process within the biphasic kinetics of DSB repair, suggesting non-homologous end joining with error-prone end resection. Our data suggest that the biological effects of space radiation may be significantly influenced by the dose as well as the type of radiation exposure.

Rat liver glutathione S-transferase-catalyzed conjugation of glutathione to the endogenous epoxides of oleic acid and cholesterol

cis-9,10-Epoxy-octadecanoic acid (oleic acid epoxide, OAE) and 5α,6α-epoxy-cholesterol (ChE) are endogenous epoxides. Unlike other epoxides, the oxirane groups of OAE and ChE are relatively stable against nucleophiles. OAE lacks toxicity and mutagenicity, while ChE is considered harmful, mutagenic and cancerogenic to animals. In humans, ChE is associated with cancer. The metabolism of OAE and ChE includes hydrolysis by cytosolic and microsomal hydrolases to their diols and glutathione (GSH) conjugation by GSH S-transferases (GST) to form the GSH conjugates (R-SG; R, residue). The GST-catalyzed GSH conjugation of OAE and ChE is poorly investigated. This article reports on the GSH conjugation of OAE, its methyl ester (OAEMe) and of ChE by rat liver homogenate GST. The GSH conjugates of OAE, OAEMe and ChE, i.e., OAE-SG, OAEMe-SG and ChE-SG, respectively, were determined by pre-column derivatization with o-phthaldialdehyde (OPA)/2-mercaptoethanol, high-performance liquid chromatography (HPLC) and fluorescence detection. Complex biphasic kinetics were observed with substrate inhibition of GST activity by OAE, OAEMe and ChE, an optimum pH of about 8.3 for OAE, and no measurable chemical GSH conjugation, underlying the importance of GST for the biotransformation of these epoxides. The results confirm the substrate concentration-dependent kinetic mechanism of GST isoforms first reported by William B. Jakoby (J. Biol. Chem. 1974) for exogenous electrophiles including the epoxide 1,2-epoxy-3-(p-nitrophenoxy)propane and the organic nitrates. This mechanism allows for maximal GST activity that can be achieved under given concentrations of GSH, epoxides and other electrophiles.

Site-Directed Mutagenesis at the Molybdenum Pterin Cofactor Site of the Human Aldehyde Oxidase: Interrogating the Kinetic Differences Between Human and Cynomolgus Monkey.

The estimation of the drug clearance by aldehyde oxidase (AO) has been complicated because of this enzyme's atypical kinetics and species and substrate specificity. Since human AO (hAO) and cynomolgus monkey AO (mAO) have a 95.1% sequence identity, cynomolgus monkeys may be the best species for estimating AO clearance in humans. Here, O6-benzylguanine (O6BG) and dantrolene were used under anaerobic conditions, as oxidative and reductive substrates of AO, respectively, to compare and contrast the kinetics of these two species through numerical modeling. Whereas dantrolene reduction followed the same linear kinetics in both species, the oxidation rate of O6BG was also linear in mAO and did not follow the already established biphasic kinetics of hAO. In an attempt to determine why hAO and mAO are kinetically distinct, we have altered the hAO V811 and F885 amino acids at the oxidation site adjacent to the molybdenum pterin cofactor to the corresponding alanine and leucine in mAO, respectively. Although some shift to a more monkey-like kinetics was observed for the V811A mutant, five more mutations around the AO cofactors still need to be investigated for this purpose. In comparing the oxidative and reductive rates of metabolism under anaerobic conditions, we have come to the conclusion that despite having similar rates of reduction (4-fold difference), the oxidation rate in mAO is more than 50-fold slower than hAO. This finding implies that the presence of nonlinearity in AO kinetics is dependent upon the degree of imbalance between the rates of oxidation and reduction in this enzyme. SIGNIFICANCE STATEMENT: Although they have as much as 95.1% sequence identity, human and cynomolgus monkey aldehyde oxidase are kinetically distinct. Therefore, monkeys may not be good estimators of drug clearance in humans.

A Comparison of Chitosan, Mesoporous Silica and Poly(lactic-co-glycolic) Acid Nanocarriers for Optimising Intestinal Uptake of Oral Protein Therapeutics

Efficacious oral delivery of therapeutic proteins remains challenging and nanoparticulate approaches are gaining interest for enhancing their permeability. In this study, we explore the ability for three comparably sized nanocarriers, with diverse physicochemical properties [i.e., chitosan (CSNP), mesoporous silica nanoparticles (MSNP) and poly(lactic-co-glycolic) acid (PLGA-NP)], to successfully facilitate epithelial uptake of a model protein, ovalbumin (OVA). We report the effect of nanoparticle surface chemistry and nanostructure on protein release, cell toxicity and the uptake mechanism in a Madin Darby Canine Kidney (MDCK) cell model of the intestinal epithelium. All nanocarriers exhibited bi-phasic OVA release kinetics with sustained and incomplete release after 4 days, and more pronounced release from MSNP than either polymeric nanocarriers. CSNP and MSNP displayed the highest cellular uptake, however CSNP was prone to significant dose-dependent toxicity attributed to the cationic surface charge. Approximately 25% of MSNP uptake was governed by a clathrin-independent endocytic mechanism, while CSNP and PLGA-NP uptake was not controlled via any endocytic mechanisms investigated herein. Furthermore, endosomal localisation was observed for CSNP and MSNP, but not for PLGA-NP. These findings may assist in the optimal choice and engineering of nanocarriers for specific intestinal permeation enhancement for oral protein delivery.

The Mechanism of Vesicle Solubilization by the Detergent Sodium Dodecyl Sulfate.

Membrane solubilization by sodium dodecyl sulfate (SDS) is indispensable for many established biotechnological applications, including viral inactivation and protein extraction. Although the ensemble thermodynamics have been thoroughly explored, the underlying molecular dynamics have remained inaccessible, owing to major limitations of traditional measurement tools. Here, we integrate multiple advanced biophysical approaches to gain multiangle insight into the time-dependence and fundamental kinetic steps associated with the solubilization of single submicron sized vesicles in response to SDS. We find that the accumulation of SDS molecules on intact vesicles triggers biphasic solubilization kinetics comprising an initial vesicle expansion event followed by rapid lipid loss and micellization. Our findings support a general mechanism of detergent-induced membrane solubilization, and we expect that the framework of correlative biophysical technologies presented here will form a general platform for elucidating the complex kinetics of membrane perturbation induced by a wide variety of surfactants and disrupting agents.