Development of Molecularly Imprinted Polymer in Porous Film Format for Binding of Phenol and Alkylphenols from Water

Molecularly imprinted polymers (MIPs) were fabricated on glass slides with a “sandwich” technique giving ~20 µm thick films. Methanol/water as a solvent, and polyethyleneglycol and polyvinylacetate as solvent modifiers, were used to give a porous morphology, which was studied with scanning electron microscopy and gravimetric analysis. Various MIPs were synthesized through non-covalent imprinting with phenol as the template; itaconic acid, 4-vinylpyridine, and styrene as monomers; ethylene glycol dimethacrylate, triethylene glycol dimethacrylate, and pentaerythritol triacrylate (PETA) as cross-linkers. Binding and imprinting properties of the MIPs were evaluated based on phenol adsorption isotherms. Since phenol has only one weakly acidic hydroxyl group and lacks unique structural characteristics necessary for binding specificity, the preparation of selective MIPs was challenging. The recognition of phenol via hydrogen bonding is suppressed in water, while hydrophobic interactions, though promoted, are not specific enough for highly-selective phenol recognition. Nevertheless, the styrene-PETA MIP gave modest imprinting effects, which were higher at lower concentrations (Imprinting Factor (IF) = 1.16 at 0.5 mg·L−1). The isotherm was of a Freundlich type over 0.1–40 mg·L−1 and there was broad cross-reactivity towards other structurally similar phenols. This shows that phenol MIPs or simple adsorbents can be developed based on styrene for hydrophobic binding, and PETA to form a tighter, hydrophilic network

Kissing loop interaction in adenine riboswitch: insights from umbrella sampling simulations

Introduction: Riboswitches are cis-acting regulatory RNA elements prevalently located in the leader sequences of bacterial mRNA. An adenine sensing riboswitch cis-regulates adeninosine deaminase gene (add) in Vibrio vulnificus. The structural mechanism regulating its conformational changes upon ligand binding mostly remains to be elucidated. In this open framework it has been suggested that the ligand stabilizes the interaction of the distal "kissing loop" complex. Using accurate full-atom molecular dynamics with explicit solvent in combination with enhanced sampling techniques and advanced analysis methods it could be possible to provide a more detailed perspective on the formation of these tertiary contacts. Methods In this work, we used umbrella sampling simulations to study the thermodynamics of the kissing loop complex in the presence and in the absence of the cognate ligand. We enforced the breaking/formation of the loop-loop interaction restraining the distance between the two loops. We also assessed the convergence of the results by using two alternative initialization protocols. A structural analysis was performed using a novel approach to analyze base contacts. Results Contacts between the two loops were progressively lost when larger inter-loop distances were enforced. Inter-loop Watson-Crick contacts survived at larger separation when compared with non-canonical pairing and stacking interactions. Intra-loop stacking contacts remained formed upon loop undocking. Our simulations qualitatively indicated that the ligand could stabilize the kissing loop complex. We also compared with previously published simulation studies. Discussion and Conclusions Kissing complex stabilization given by the ligand was compatible with available experimental data. However, the dependence of its value on the initialization protocol of the umbrella sampling simulations posed some questions on the quantitative interpretation of the results and called for better converged enhanced sampling simulations

Towards de novo RNA 3D Structure Prediction

RNA is a fundamental class of biomolecules that mediate a large variety of molecular processes within the cell. Computational algorithms can be of great help in the understanding of RNA structure-function relationship. One of the main challenges in this field is the development of structure-prediction algorithms, which aim at the prediction of the three-dimensional (3D) native fold from the sole knowledge of the sequence. In a recent paper, we have introduced a scoring function for RNA structure prediction. Here, we analyze in detail the performance of the method, we underline strengths and shortcomings, and we discuss the results with respect to state-of-the-art techniques. These observations provide a starting point for improving current methodologies, thus paving the way to the advances of more accurate approaches for RNA 3D structure prediction

A Homogenization Approach for Turbulent Channel Flows over Porous Substrates: Formulation and Implementation of Effective Boundary Conditions

The turbulent flow through a plane channel bounded by a single permeable wall is considered; this is a problem of interest since a carefully chosen distribution of grains and voids in the porous medium can result in skin friction reduction for the flow in the channel. In the homogenization approach followed here, the flow is not resolved in the porous layer, but an effective velocity boundary condition is developed (and later enforced) at a virtual interface between the porous bed and the channel flow. The condition is valid up to order two in terms of a small gauge factor, the ratio of microscopic to macroscopic length scales; it contains slip coefficients, plus surface and bulk permeability coefficients, which arise from the solution of microscale problems solved in a representative elementary volume. Using the effective boundary conditions, free of empirical parameters, direct numerical simulations are then performed in the channel, considering a few different porous substrates. The results, examined in terms of mean values and turbulence statistics, demonstrate the drag-reducing effects of porous substrates with streamwise-preferential alignment of the solid grains

Development and application of a thin-film molecularly imprinted polymer for the measurement of mycophenolic acid in human plasma

Background: Mycophenolic acid (MPA) is used to suppress the immune response following organ transplantation; however, complex pharmacokinetic behavior and a large interpersonal variability necessitate therapeutic drug monitoring. To overcome the limitations of current sample preparation techniques, we present a novel thin-film molecularly imprinted polymer (TF-MIP) extraction device as part of a simple, sensitive, and fast method for analysis of MPA from human plasma. Methods: Mycophenolic acid is extracted from plasma using a tailor-made TF-MIP that is subsequently desorbed into an organic solvent system compatible with mass spectrometry. The MIP yielded higher recovery of MPA relative to a corresponding non-imprinted polymer. The method allows for the determination of MPA in 45 min including analysis time and can be scaled for high throughput to process as many as 96 samples per hour. Results: The method gave an LOD of 0.3 ng mL-1 and was linear from 5 to 250 ng mL-1 . Patient plasma samples (35 μL) were diluted using charcoal-stripped pooled plasma to a final extraction volume of 700 μL; when MPA in patient plasma is high, this ratio can easily be adjusted to ensure samples are within the method linear range. Intra- and inter-day variability were 13.8% and 4.3% (at 15 ng mL-1 ) and 13.5% and 11.0% (at 85 ng mL-1 ), respectively (n = 3); inter-device variability was 9.6% (n = 10). Conclusions: Low inter-device variability makes these devices suitable for single use in a clinical setting, and the fast and robust method is suitable for therapeutic drug monitoring, where throughput and time-to-result are critical

Fitting Corrections to an RNA Force Field Using Experimental Data

Empirical force fields for biomolecular systems are usually derived from quantum chemistry calculations and validated against experimental data. We here show how it is possible to refine the full dihedral-angle potential of the Amber RNA force field by using solution NMR data as well as stability of known structural motifs. The procedure can be used to mix multiple systems and heterogeneous experimental information and crucially depends on a regularization term chosen with a cross-validation procedure. By fitting corrections to the dihedral angles on the order of less than 1 kJ/mol per angle, it is possible to increase the stability of difficult-to-fold RNA tetraloops by more than 1 order of magnitude

MicroRNA as Possible Mediators of the Synergistic Effect of Celecoxib and Glucosamine Sulfate in Human Osteoarthritic Chondrocyte Exposed to IL-1β

This study investigated the role of a pattern of microRNA (miRNA) as possible mediators of celecoxib and prescription-grade glucosamine sulfate (GS) effects in human osteoarthritis (OA) chondrocytes. Chondrocytes were treated with celecoxib (1.85 µM) and GS (9 µM), alone or in combination, for 24 h, with or without interleukin (IL)-1β (10 ng/mL). Cell viability was determined using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, apoptosis and reactive oxygen species (ROS) by cytometry, nitric oxide (NO) by Griess method. Gene levels of miRNA, antioxidant enzymes, nuclear factor erythroid (NRF)2, and B-cell lymphoma (BCL)2 expressions were analyzed by quantitative real time polymerase chain reaction (real time PCR). Protein expression of NRF2 and BCL2 was also detected at immunofluorescence and western blot. Celecoxib and GS, alone or in combination, significantly increased viability, reduced apoptosis, ROS and NO production and the gene expression of miR-34a, -146a, -181a, -210, in comparison to baseline and to IL-1β. The transfection with miRNA specific inhibitors significantly counteracted the IL-1β activity and potentiated the properties of celecoxib and GS on viability, apoptosis and oxidant system, through nuclear factor (NF)-κB regulation. The observed effects were enhanced when the drugs were tested in combination. Our data confirmed the synergistic anti-inflammatory and chondroprotective properties of celecoxib and GS, suggesting microRNA as possible mediators

Maackia amurensis Seed Lectin (MASL) Increases Movement Velocity of Mice with TNFα Induced Rheumatoid Arthritis

Up to 70 million people around the world suffer from rheumatoid arthritis. Current treatment options have varied efficacy and can cause unwanted side effects. New approaches are needed to treat this condition. Sialic acid modifications on chondrocyte receptors have been associated with arthritic inflammation and joint destruction. The transmembrane mucin receptor protein podoplanin (PDPN) has been identified as a functionally relevant receptor that presents extracellular sialic acid motifs. PDPN signaling promotes inflammation and invasion associated with arthritis and, therefore, has emerged as a target that can be used to inhibit arthritic inflammation. Maackia amurensis seed lectin (MASL) can target PDPN on chondrocytes to decrease inflammatory signaling cascades and reduce cartilage destruction in a lipopolysaccharide induced osteoarthritis mouse model. Here, we investigated the effects of MASL on rheumatoid arthritis progression in a TNFα transgenic (TNF-Tg) mouse model. Results from this study indicate that MASL can be administered orally to ameliorate joint malformation and increase velocity of movement exhibited by these TNF-Tg mice. These data support the consideration of MASL as a potential treatment for rheumatoid arthritis

Heart Rate Turbulence Predicts Survival Independently From Severity of Liver Dysfunction in Patients With Cirrhosis

Background: Reduced heart rate variability (HRV) is an independent predictor of mortality in patients with cirrhosis. However, conventional HRV indices can only be interpreted in individuals with normal sinus rhythm. In patients with recurrent premature ventricular complexes (PVCs), the predictive capacity of conventional HRV indices is compromised. Heart Rate Turbulence (HRT) represents the biphasic change of the heart rate after PVCs. This study was aimed to define whether HRT parameters could predict mortality in cirrhotic patients. Materials and Methods: 24 h electrocardiogram recordings were collected from 40 cirrhotic patients. Turbulence Onset was calculated as HRT indices. The enrolled patients were followed up for 12 months after the recruitment in relation to survival and/or transplantation. Results: During the follow-up period, 21 patients (52.5%) survived, 12 patients (30%) died and 7 patients (17.5%) had liver transplantation. Turbulence Onset was found to be strongly linked with mortality on Cox regression (Hazard ratio = 1.351, p < 0.05). Moreover, Turbulence Onset predicted mortality independently of MELD and Child-Pugh's Score. Conclusion: This study provides further evidence of autonomic dysfunction in cirrhosis and suggests that HRT is reliable alternative to HRV in patients with PVCs