Competitive Inhibition Model Research Articles

In vitro analysis of a competitive inhibition model for T7 RNA polymerase biosensors

T7 RNA polymerase (T7 RNAP) biosensors, in which T7 RNAP transcribes some reporter gene or signal in response to external stimuli, have wide applications in synthetic biology and metabolic engineering. We adapted a biochemical reaction network model and used an in vitro transcription assay to determine network parameters for different T7 RNAP constructs. Under conditions where template DNA is limiting, the EC50 values of native and engineered T7 RNAPs ranged from 33 nM (29–37 95 % c.i.) to 570 nM (258–714 95 % c.i.) (wild-type T7 RNAP). The measured EC50 values were largely insensitive to free magnesium, pH, or other buffer conditions. Many biosensor configurations use a split RNAP construct, where the C-terminal (CT7) and N-terminal T7 (NT7) are fused to proximity induced dimerization modules. We used proteolysis and ion exchange chromatography to prepare a CT7 (80 kDa) product. The impact of free CT7 on T7 RNAP transcriptional activity was well described by a competitive inhibition model, with an inhibitory constant KI = 23 nM (18–28 95 % c.i.) of the sensor. These model parameters will be useful for forward modeling and design of T7 RNAP-based genetic circuits.

Rehmannioside A inhibits the activity of CYP3A4, 2C9 and 2D6 in vitro

To assess the effect of Rehmannioside A on CYP450s activity and to estimate its inhibitory properties. The effect of Rehmannioside A on the activity of major CYP450s in human liver microsomes (HLMs) was assessed with the corresponding substrates and marker reactions, and compared with a blank control and the respective inhibitors. Suppression of CYP3A4, 2C9 and 2D6 was assessed by the dose-dependent assay and fitted with non-competitive or competitive inhibition models. The inhibition of CYP3A4 was determined in a time-dependent manner. Rehmannioside A suppressed the activity of CYP3A4, 2C9, and 2D6 with IC50 values of 10.08, 12.62, and 16.43 μM, respectively. Suppression of CYP3A4 was fitted to a non-competitive model with Ki value of 5.08 μM, whereas CYP2C9 and 2D6 were fitted to a competitive model with Ki values of 6.25 and 8.14 μM. Additionally, the inhibitory effect on CYP3A4 was time-dependent with KI value of 8.47 μM−1 and a Kinact of 0.048 min−1. In vitro suppression of CYP3A, 2C9 and 2D6 by Rehmannioside A indicated that Rehmannioside A or its source herbs may interact with drugs metabolised by these CYP450s, which could guide the clinical application.

Negative Regulation of Innate Immune Signaling by Components of the Button Mushroom Agaricus bisporus.

Mushrooms are valued as an edible and medical resource for millennia. As macrofungi, they possess conserved molecular components recognized by innate immune cells like macrophages, yet unlike pathogenic fungi, they do not trigger the immune system in the same way. That these well-tolerated foods both avoid immuno-surveillance and have positive health benefits, highlights the dearth of information on the interactions of mushroom-derived products with the immune system. Using powders produced from the common white button mushroom, Agaricus bisporus, it is observed that pre-treatment of mouse and human macrophages with mushroom powders attenuates innate immune signaling triggered by microbial ligands like LPS and β-glucans, including NFκB activation and pro-inflammatory cytokine production. This effect of mushroom powders is observed at lower doses of TLR ligands, suggesting a model of competitive inhibition whereby mushroom compounds bind and occupy innate immune receptors, precluding activation by microbial stimuli. This effect is preserved following simulated digestion of the powders. Moreover, in vivo delivery of mushroom powders attenuates the development of colitis in a DSS-mouse model. This data highlights an important anti-inflammatory role for powdered A. bisporus mushrooms, which can be further utilized to develop complementary approaches to modulate chronic inflammation and disease.

Effects of Simvastatin on the Metabolism of Vonoprazan in Rats Both in vitro and in vivo.

PurposeTo study the potential drug–drug interactions between simvastatin and vonoprazan and to provide the scientific basis for rational use of them in clinical practice.MethodsAn incubation system was established with rat liver microsomes, and the main metabolite of vonoprazan M-I was detected by UPLC-MS/MS. The IC50 value of simvastatin was then calculated and its inhibitory mechanism against vonoprazan was also analyzed. Twelve SD rats were randomly divided into 2 groups, then they were given simvastatin or saline for 2 weeks continuously. On the day of the experiment, both groups were intragastrically administered with vonoprazan once, followed by the collection of blood at different time points. Then the plasma concentration of vonoprazan and M-I in rats were detected by UPLC-MS/MS.ResultsIn vitro experiments revealed that simvastatin could inhibit the metabolism of vonoprazan, and its inhibition type belonged to the mixed non-competitive and competitive inhibition model. In vivo experiments in rats demonstrated that the area under concentration time curve (AUC) of vonoprazan was decreased but the clearance (CLz/F) of it was increased in the simvastatin administrated group, as compared to those of the control group. However, M-I in simvastatin treated group exhibited the higher AUC and lower CLz/F values compared to those in the control group. These data indicated that multiple doses of simvastatin administration could reduce the plasma concentration of vonoprazan and accelerate its metabolic rate in rats.ConclusionSimvastatin could inhibit the metabolism of vonoprazan in vitro but multiple doses of simvastatin exhibited the opposite effect In vivo. Altogether, our data indicated that an interaction existed between simvastatin and vonoprazan and additional cares might be taken when they were co-administrated in clinic.

In vitro evaluation of the inhibition potential of echinacoside on human cytochrome P450 isozymes

BackgroundEchinacoside (ECH) possesses a wide range of biological activity. This present study analyzes the effect of ECH on cytochrome P450 isozymes (CYPs) activities of human liver microsomes.MethodsThe effect of ECH on CYPs enzyme activities were studied using the enzyme-selective substrates phenacetin (1A2), chlorzoxazone (2E1), S-mephenytoin (2C19), testosterone (3A4), coumarin (2A6), diclofenac (2C9), paclitaxel (2C8), and dextromethorphan (2D6). The IC50 values for CYP1A2, CYP2E1, CYP2C19, and CYP3A4 isoforms were examined to express the strength of inhibition. Further, the inhibition of CYPs was checked for time-dependent or not, and then fitted with competitive or non-competitive inhibition models. The corresponding parameters were also obtained.ResultsECH caused inhibitions on CYP1A2, CYP2E1, CYP2C19 and CYP3A4 enzyme activities in HLMs with IC50 of 21.23, 19.15, 8.70 and 55.42 μM, respectively. The obtained results showed that the inhibition of ECH on CYP3A4 was time-dependent with the KI/Kinact value of 6.63/0.066 min− 1·μM− 1. Moreover, ECH inhibited the activity of CYP1A2 and CYP2E1 via non-competitive manners (Ki = 10.90 μM and Ki = 14.40 μM, respectively), while ECH attenuated the CYP2C19 activity via a competitive manner (Ki = 4.41 μM).ConclusionsThe results of this study indicate that ECH inhibits CYP1A2, CYP2E1, CYP2C19 and CYP3A4 activities in vitro. In vivo and clinical studies are warranted to verify the relevance of these interactions.

Inhibitory effects of Thai herbal extracts on the cytochrome P450 3A-mediated the metabolism of gefitinib, lapatinib and sorafenib

Herbal products are widely used in cancer patients via co−administration with chemotherapy. Previous studies have demonstrated that pharmacokinetic interactions between herbs and anticancer drugs exist due to inhibition of drug−metabolizing enzymes, particularly cytochrome P450s (CYPs). The aim of this study was to determine the inhibitory effects of Andrographis paniculata, Curcuma zedoaria, Ganoderma lucidum, Murdannia loriformis and Ventilago denticulata extracts on the metabolism of gefitinib, lapatinib and sorafenib. The activities of CYP3A in human liver microsome on the metabolism of gefitinib, lapatinib and sorafenib in the absence and presence of Thai herbal extracts were assayed using high-performance liquid chromatography analysis. Curcuma zedoaria extract potently inhibited CYP3A−mediated lapatinib and sorafenib metabolism with IC50 values of 4.18 ± 3.20 and 7.59 ± 1.23 μg/mL, respectively, while the metabolism of gefitinib was strongly inhibited by Murdannia loriformis and Ventilago denticulata extracts with IC50 values of 7.53 ± 2.87 and 7.06 ± 1.23 μg/mL, respectively. Andrographis paniculata and Ganoderma lucidum extracts had less effect on the metabolism of the tested anticancers (IC50 values >10 μg/mL). In addition, kinetic analysis of the ability of Curcuma zedoaria extract to inhibit CYP3A-mediated metabolism of anticancer drugs was best described by the noncompetitive and competitive inhibition models with Ki values of 20.08 and 11.55 μg/mL for the metabolism of gefitinib and sorafenib, respectively. The present study demonstrated that there were potential pharmacokinetic interactions between tyrosine kinase inhibitors and herbal extracts.

In vitro study on the effect of leonurine hydrochloride on the enzyme activity of cytochrome P450 enzymes in human liver microsomes

Leonurine hydrochloride (LH) is derived from an ingredient of Leonurus japonicus Houtt which is widely used for diseases in women. The influence of LH on the activity of cytochrome P450 (CYPs) enzymes was investigated in this study. The effect of LH on CYPs enzyme activities were studied using the enzyme-selective substrates phenacetin (1A2), coumarin (2A6), diclofenac (2C9), S-mephenytoin (2C19), paclitaxel (2C8), dextromethorphan (2D6), chlorzoxazone (2E1) and testosterone (3A4). The IC50 value was calculated to express the strength of inhibition. The inhibition of CYPs was fitted with competitive or non-competitive inhibition models and corresponding parameters were also obtained. LH exerted inhibitory effects on the activity of CYP1A2, 2D6, and 3A4 with the IC50 values of 18.05, 15.13, and 20.09 μM, respectively. The obtained results showed that LH inhibited the activity of CYP1A2 and CYP2D6 via competitive manners (Ki = 8.667 μM and Ki = 7.805 μM, respectively), while LH attenuated the CYP3A4 activity via a non-competitive manner (Ki = 9.507 μM). Moreover, LH showed time-dependent inhibition on CYP3A4 with the KI/Kinact value of 4.31/0.044 min−1·μM−1. The inhibition of CYP1A2, CYP2D6, and CYP3A4 by LH, demonstrated in vitro, indicated the potential herb–drug interaction. Therefore, pharmacokinetic interactions involving LH and CYP1A2 or CYP2D6 or CYP1A2 substrates are likely to occur.

Encapsulated NOLA™ Fit 5500 Lactase—An Economically Beneficial Way to Obtain Lactose-Free Milk at Low Temperature

The current requirements of industrial biocatalysis are related to economically beneficial and environmentally friendly processes. Such a strategy engages low-temperature reactions. The presented approach is essential, especially in food processes, where temperature affects the quality and nutritional value foodstuffs. The subject of the study is the hydrolysis of lactose with the commercial lactase NOLA™ Fit 5500 (NOLA). The complete decomposition of lactose into two monosaccharides gives a sweeter product, recommended for lactose intolerant people and those controlling a product’s caloric content. The hydrolysis reaction was performed at 15 °C, which is related to milk transportation and storage temperature. The enzyme showed activity over the entire range of substrate concentrations (up to 55 g/L lactose). For reusability and easy isolation, the enzyme was encapsulated in a sodium alginate network. Its stability allows carrying out six cycles of the complete hydrolysis of lactose to monosaccharides, lasting from two to four hours. During the study, the kinetic description of native and encapsulated NOLA was conducted. As a result, the model of competitive galactose inhibition and glucose mixed influence (competitive inhibition and activation) was proposed. The capsule size does not influence the reaction rate; thus, the substrate diffusion into capsules can be omitted from the process description. The prepared 4 mm capsules are easy to separate between cycles, e.g., using sieves.

Profile of the inhibitory effects of gefitinib on CYP2D6 variants in vitro.

CYP2D6 is a highly polymorphic metabolic enzyme with more than 100 genetic variants, some of which are associated with significantly altered enzyme activity, such as CYP2D6.2 (Arg296Cys, Ser486Thr), CYP2D6.10 (Pro34Ser, Ser486Thr), and CYP2D6.39 (Ser486Thr). Gefitinib is a tyrosine kinase inhibitor of the epidermal growth factor receptor (EGFR) and is used to treat non-small-cell lung cancer with EGFR mutations. Gefitinib is known to competitively inhibit CYP2D6 activity. The aim of this study was to quantitatively compare the inhibitory effects of gefitinib on CYP2D6 enzyme kinetics among CYP2D6 variants. The enzymatic activity of several CYP2D6 genetic variants; i.e., CYP2D6.1 (wild type), CYP2D6.2, CYP2D6.10, and CYP2D6.39, was assessed by examining the O-demethylation of dextromethorphan. The intrinsic clearance of dextromethorphan (Vmax/Km) for CYP2D6.2, CYP2D6.10, and CYP2D6.39 were 0.565-, 0.0376-, and 0.470-fold of that for wild type, respectively. For all variants, the mixed inhibition model was better at explaining the nature of the inhibitory effects of gefitinib than the competitive inhibition model. However, the inhibitory potency of gefitinib varied among the CYP2D6 genetic variants. The Ki values for CYP2D6.2, CYP2D6.10, and CYP2D6.39 were 1.4-, 2.5- and 1.5-fold higher than that for wild type, respectively, implying that these variants are less susceptible to the inhibition by gefitinib. The genetic variations in CYP2D6 might be one of the factors responsible for inter-individual differences in the strength of CYP2D6-mediated drug interactions involving gefitinib.

The Effect of Pb2+ Short Term Stress on the Anammox Biomass—A Batch Test Experiment

The anaerobic ammonium oxidation process for treating industrial wastewater is widely preferred since Anammox bacteria has been discovered, due to energy savings and cost reduction comparing to classical biological methods. However high concentration of heavy metals contained in such type of wastewater is well known inhibitor for biological processes and may limit the bacteria activity, as well as worsen the effectiveness of wastewater purification. The present state of the art shows that high concentration of certain heavy metals (Pb2+) can be found in municipal waste landfill leachates, therefore their negative effect may occur. The main goals of this research were to: (a) to study the negative effect of Pb2+ on the Anammox process in short term exposures. The inhibition of lead salt on the Anammox bacteria activity and nitrogen removal rate were determined by batch test, based on gas measurements procedure. Inhibition effects were monitored over 80 min experimental runs. Also, the kinetic parameters of the inhibition were investigated. A non competitive inhibition model successfully described the relationship between microbial inhibition and metal concentration. Data has shown that Pb2+ is toxic for Anammox bacteria biomass. The results showed that lead caused 16.2% of the Anammox activity decrease in highest tested concentration of 10 mg L–1 of Pb2+. However, the 50% inhibition has not been reached the IC50 has been calculated at 39.94 mg L. It was also suggested that long term exposure on the heavy metal presence might be more toxic. Thus, the soluble concentration of lead should be monitored on wastewater treatment plants not only before applying Anammox process to the treatment plants, but also during their operation to prevent negative changes in the processes. Also, further investigation should be introduced to understand the matter of the toxicity and the possibility to carry out the process in the heavy metal presence.

Succinic acid inhibits the activity of cytochrome P450 (CYP450) enzymes

Context Succinic acid, extracted from amber, is widely used in cardiovascular therapy. Objective The effect of succinic acid on the activity of cytochrome P450 (CYP450) enzymes was investigated in this study. Materials and methods The effect of succinic acid (100 μM) on the activity of eight isoforms of CYP450 (i.e., 1A2, 3A4, 2A6, 2E1, 2D6, 2C9, 2C19 and 2C8) was investigated compared to the specific inhibitor and blank controls in pooled human liver microsomes in vitro. The inhibition of CYPs was fitted with competitive or non-competitive inhibition models and corresponding parameters were also obtained. Results Succinic acid exerted inhibitory effect on the activity of CYP3A4, 2D6, and 2C9 with the IC50 values of 12.82, 14.53, and 19.60 μM, respectively. Succinic acid inhibited the activity of CYP3A4 in a non-competitive manner with the Ki value of 6.18 μM, and inhibited CYP2D6 and 2C9 competitively with Ki values of 7.40 and 9.48 μM, respectively. Furthermore, the inhibition of CYP3A4 was found to be time-dependent with the KI/Kinact value of 6.52/0.051 min−1·μM−1. Discussion and conclusions Succinic acid showed in vitro inhibitory effects on the activity of CYP3A4, 2D6, and 2C9, which indicated the potential drug-drug interactions. Succinic acid should be carefully co-administrated with the drugs metabolized by CYP3A4, 2D6, and 2C9.

Biotransformation of Polycyclic Aromatic Hydrocarbons by Trout Liver S9 Fractions: Evaluation of Competitive Inhibition Using a Substrate Depletion Approach.

Environmental contaminants frequently occur as part of a chemical mixture, potentially resulting in competitive inhibition among multiple substrates metabolized by the same enzyme. Trout liver S9 fractions were used to evaluate the biotransformation of 3 polycyclic aromatic hydrocarbons (PAHs): phenanthrene, pyrene, and benzo[a]pyrene, tested as binary mixtures. Initial rates of biotransformation were determined using a substrate-depletion approach. The resulting data were then fitted by simultaneous nonlinear regression to a competitive inhibition model. In each case, the PAH possessing the lower Michaelis-Menten affinity constant (KM ) competitively inhibited biotransformation of the other compound. Inhibition constants determined for the lower-KM compound were generally close to previously determined KM values, consistent with the suggestion that phase I biotransformation of PAHs is largely catalyzed by one or a small number of cytochrome P450 enzymes. The use of a substrate-depletion approach to perform enzyme-inhibition studies imposes practical limitations on experimental design and complicates the interpretation of derived kinetic constants. Nevertheless, the resulting information may have utility for chemical hazard assessments as well as the design and interpretation of controlled laboratory studies. Depletion experiments informed by measured chemical concentrations in tissues may also provide a means of determining whether enzyme inhibition occurs under relevant environmental conditions. Environ Toxicol Chem 2019;38:2729-2739. Published 2019 Wiley Periodicals, Inc. on behalf of SETAC. This article is a US government work, and as such, is in the public domain in the United States of America.

Aromatic rosane diterpenoids from the roots of Euphorbia ebracteolata and their inhibitory effects against lipase.

The bioactive chemical constituents of Euphorbia ebracteolata have been investigated in the present work using various techniques. On the basis of chromatographic methods, such as silica gel, RP C-18 column chromatography, five novel rosane type diterpenoids with an aromatic A-ring (1-5) have been isolated from the roots of Euphorbia ebracteolata. Their structures were elucidated by widely spectroscopic data, including HRESI-MS, 1D and 2D NMR. Additionally, the inhibitory effects on lipase of these isolated diterpenoids were evaluated in vitro. Compound 1 as a new diterpenoid displayed significant inhibitory effect on lipase (IC50 = 1.0 μM). And, the inhibitory kinetics has been studied fully, which determined a competitive inhibition model for compound 1 on the enzymatic activity of lipase (Ki = 1.8 μM).

Co-metabolic degradation of naphthalene and pyrene by acclimated strain and competitive inhibition kinetics

A dominant strain named Ochrobactrum sp. was isolated from soils contaminated with coal tar. The batch experiments were carried out to study the co-metabolic degradation of pyrene by Ochrobactrum MB-2 with naphthalene as the main substrate and the effects of several significant parameters such as naphthalene concentration, pH and temperature on removal efficiency were explored. The results showed that Ochrobactrum MB-2 effectively degraded naphthalene and that the addition of naphthalene favored the degradation of pyrene. The maximum elimination efficiency of naphthalene (10 mg L−1) and pyrene (1 mg L−1) was achieved at pH 7 and 25 °C, and the corresponding values were 99 and 41%, respectively. A competitive inhibition model based on the Michaelis–Menten equation was used to characterize the inhibitory effect of pyrene on naphthalene degradation. The values of the half-saturation coefficient for naphthalene (KS) and dissociation constant of enzyme-inhibitor complex (KC) were determined to be 4.93 and 1.38 mg L−1, respectively.

Inhibition effect of zinc, cadmium, and nickel ions in microalgal growth and nutrient uptake from water: An experimental approach

The effect of heavy metals (Cd, Ni, and Zn) on the growth of Chlorella vulgaris and nutrient uptake was assessed. C. vulgaris was cultivated in a synthetic solution with a single or ternary metal system. Biomass growth was well predicted considering a competitive inhibition model. The higher specific growth rate (0.34 d−1), biomass productivity (98.1 mg·L−1·d−1), heavy metals uptake efficiency (85%) was obtained for the cultures with Cd and Zn ions. The higher nutrient removal was observed in the presence of Cd ions (91% nitrogen and 99% phosphorus). The highest inhibition on biomass growth (>88%) was observed in the presence of nickel ions. In the ternary system, microalgae growth inhibition was similar to that observed in the presence of only nickel ions. Although, wastewaters containing nutrients and residual concentrations of zinc and cadmium ions can be used for C. vulgaris production, in the presence of residual concentrations of nickel ions, biomass growth is negligible.

Efficacy of a novel sequential enzymatic hydrolysis of lignocellulosic biomass and inhibition characteristics of monosugars.

Efficient production of sugar monomers from lignocellulose is often hampered by serious bottle-necks in biomass hydrolysis. The present study reveals that ultra-sonication assisted pretreatment following autoclaving, termed as combined pretreatment, can lead to more efficient delignification of lignocellulosic biomass and an open, deformed polysaccharide matrix, found favorable for subsequent enzymatic hydrolysis, is formed. The pattern of inhibition for the enzymatic hydrolysis reaction on combined-pretreated saw dust is identified. Two main inhibition models (competitive and noncompetitive) are proposed and a better fit of experimental values with the theoretical values for the competitive inhibition model validates the proposition that in the present experiment, glucose inhibits the enzymes competitively. Additionally, accuracy of the inhibitory kinetics based models is estimated over a series of enzyme and substrate concentrations.

Cholinesterase and phenyl valerate-esterase activities sensitive to organophosphorus compounds in membranes of chicken brain

Some effects of organophosphorus compounds (OPs) esters cannot be explained by action on currently recognized targets acetylcholinesterase or neuropathy target esterase (NTE). In previous studies, in membrane chicken brain fractions, four components (EPα, EPβ, EPγ and EPδ) of phenyl valerate esterase activity (PVase) had been kinetically discriminated combining data of several inhibitors (paraoxon, mipafox, PMSF). EPγ is belonging to NTE. The relationship of PVase components and acetylcholine-hydrolyzing activity (cholinesterase activity) is studied herein. Only EPα PVase activity showed inhibition in the presence of acetylthiocholine, similarly to a non-competitive model. EPα is highly sensitive to mipafox and paraoxon, but is resistant to PMSF, and is spontaneously reactivated when inhibited with paraoxon. In this papers we shows that cholinesterase activities showed inhibition kinetic by PV, which does not fit with a competitive inhibition model when tested for the same experimental conditions used to discriminate the PVase components. Four enzymatic components (CP1, CP2, CP3 and CP4) were discriminated in cholinesterase activity in the membrane fraction according to their sensitivity to irreversible inhibitors mipafox, paraoxon, PMSF and iso-OMPA. Components CP1 and CP2 could be related to EPα as they showed interactions between substrates and similar inhibitory kinetic properties to the tested inhibitors.

New 2-Aryl-9-methyl-\u03b2-carbolinium salts as Potential Acetylcholinesterase Inhibitor agents: Synthesis, Bioactivity and Structure\u2013Activity Relationship

A series of 2-aryl-9-methyl-β-carbolinium bromides (B) were synthesized and explored for anti-acetylcholinesterase (AChE) activities in vitro, action mechanism and structure-activity relationship. All the compounds B along with their respective 3,4-dihydro intermediates (A) presented anti-AChE activity at 10 μM. Thirteen compounds B showed the excellent activity with IC50 values of 0.11–0.76 μM and high selectivity toward AChE relative to butyrylcholinesterase (BChE), superior to galantamine (IC50 = 0.79 μM), a selective AChE inhibitor drug. Kinetic analysis showed that the action mechanisms of both compounds B and A are a competitive inhibition model. Structure-activity relationship analyses showed that the C = N+ moiety is a determinant for the activity. Substituents at 6, 7 or 4′ site, the indole-N-alkyl and the aromatization of the C-ring can significantly improve the activity. Molecular docking studies showed that the compounds could combine with the active site of AChE by the π-π or cation-π action between the carboline ring and the phenyl rings of the residues, and the β-carboline moiety is embedded in a cavity surrounded by four aromatic residues of Trp86, Tyr337, Trp439 and Tyr449. The present results strongly suggest that the para-position of the D-ring should be a preferred modification site for further structural optimization design. Thus, 2-aryl-9-methyl-β-carboliniums emerged as novel and promising tool compounds for the development of new AChE inhibitor agents.

Role of Cell-Mediated Enzymatic Degradation and Cytoskeletal Tension on Dynamic Changes in the Rheology of the Pericellular Region Prior to Human Mesenchymal Stem Cell Motility.

Human mesenchymal stem cells (hMSCs) are encapsulated in synthetic matrix metalloproteinase (MMP) degradable poly(ethylene glycol)-peptide hydrogels to characterize cell-mediated degradation of the pericellular region using multiple particle tracking microrheology. The hydrogel scaffold is degraded by cell-secreted enzymes and cytoskeletal tension. We determine that cell-secreted enzymatic degradation is the main contributor to changes in the pericellular region, with cytoskeletal tension playing a minimal role. Measured degradation profiles for untreated and myosin II inhibited hMSCs have the highest cross-link density around the cell. We hypothesize that cells are also secreting tissue inhibitor of metalloproteinases (TIMPs) to inhibit MMPs, which allow cell spreading and attachment prior to motility. We develop a Michaelis-Menten competitive enzymatic inhibition model which accurately describes the degradation profile due to MMP-TIMP unbinding.

DNA residence time is a regulatory factor of transcription repression.

Transcription comprises a highly regulated sequence of intrinsically stochastic processes, resulting in bursts of transcription intermitted by quiescence. In transcription activation or repression, a transcription factor binds dynamically to DNA, with a residence time unique to each factor. Whether the DNA residence time is important in the transcription process is unclear. Here, we designed a series of transcription repressors differing in their DNA residence time by utilizing the modular DNA binding domain of transcription activator-like effectors (TALEs) and varying the number of nucleotide-recognizing repeat domains. We characterized the DNA residence times of our repressors in living cells using single molecule tracking. The residence times depended non-linearly on the number of repeat domains and differed by more than a factor of six. The factors provoked a residence time-dependent decrease in transcript level of the glucocorticoid receptor-activated gene SGK1. Down regulation of transcription was due to a lower burst frequency in the presence of long binding repressors and is in accordance with a model of competitive inhibition of endogenous activator binding. Our single molecule experiments reveal transcription factor DNA residence time as a regulatory factor controlling transcription repression and establish TALE-DNA binding domains as tools for the temporal dissection of transcription regulation.