Hydrogen Bond Energy Research Articles

An Insight of Cryptocarya Secondary Metabolites as Anticancer P388: Study of Molecular Docking and ADMET Properties

Secondary metabolites isolated from Cryptocarya was known to have various activity especially their cytotoxicity in P388 cell. There were two species of Cryptocarya studied in this research that were Cryptocarya konishii and Cryptocarya lucida. In both species, 8 isolate compounds had bioactivity as anticancer in P388 cells. This study aimed to know the binding affinity and ADMET properties of each isolated compound through P-glycoprotein substrate since this protein was reported to be responsible for the inhibition of P388 cells. Molecular docking was performed using AutoDock4 and AutoDockTools software to know the binding energy and interaction of isolate compounds against the P-glycoprotein substrate. ADMET properties calculation was done using the pkCSM web server for all compounds. Molecular docking results showed that Kurzichalcolactone B (7) isolated from C. lucida had the lowest binding energy. It resulted in the highest total intermolecular energy from the contribution of van der Waals and hydrogen bond energy. The lowest binding energy is indicating the stable interaction of ligand and substrate. Calculation of ADMET properties resulted that some of the isolate compounds fulfilling the minimum standard parameters in ADMET properties.

Degradation of Chinese handmade papers with different fiber raw materials on molecular and supramolecular structures

As the carriers of paper-based cultural relics, ancient handmade papers have greatly promoted the development of human culture, knowledge and civilization. Chinese papermakers wisely selected a broad variety of plants to make papers with better properties and longer lifetime. However, the correlation between the types of fiber raw materials and the durability of handmade papers is still unclear. Herein, six handmade papers classified as bark paper, bamboo paper and grass paper were selected to study their aging behavior and degradation mechanism. Optical microscope and fiber quality analyzer were used to characterize the fiber morphology and length distribution of papers. The degradation kinetics of six handmade papers were systematically studied under accelerated aging condition. On molecular scale, the degradation rate, variation of pH, oxidation degree, chromaticity, and content of reducing carbonyl groups were discussed for bark, bamboo and grass papers. On supramolecular scale, the crystallinity, energy and distance of hydrogen bonds for paper cellulose were quantitatively calculated. For six handmade papers, most of the degradation occurs in the amorphous region of cellulose, leading to the cleavage of glycosidic bonds, the generation of oxidized groups, and the variation of H-bonds arrangement. Owing to the specific molecular and supramolecular structures of cellulose in bast fiber, bark papers have longer lifetime and better durability than bamboo and grass papers. The results of this work will improve the understanding of paper degradation mechanism at microscopic multiscale structures, and provide the scientific foundation for the preparation of handmade papers with better durability.

Molecular docking of monkeypox (mpox) virus proteinase with FDA approved lead molecules

BackgroundMonkeypox virus (mpox) disease is caused by a double-stranded DNA virus from the Poxviridae family. The mpox virus showed structural similarity with smallpox virus disease. The recent outbreak of mpox infection in the rest of African countries causes public health issues of increased pandemic potential. Mpox virus is involved in the viral replication cycle through the biocatalytic reaction of precursor polyproteins cleavage. ObjectivesThe main objective of the study was to analyze the molecular interactions between mpox and FDA-approved drugs. MethodsThe primary and secondary structure of the protein was retrieved and FDA approved drug was screened using AutoDock. The best hit was analyzed and the molecular interactions were studied. Model validation analyzes the peptide, energy of hydrogen bonds, steric conflicts and bond planarity. Z-score was calculated using ProSA-web tool and the score tested the native fold from other alternative folds. ResultsThe confidence level of the submitted amino acids was> 80 % and the maximum confidence score for a single template was 98.2 %. The generated proteinase model was subjected to analyze the distribution of atoms and the using ERRAT server. The overall quality score was 88.535 and this value represents the amino acid percentage with anticipated error value and the value falling below the rejection limit. The Z-score of this study result was within the Z-score range (−4.17) validated for native enzymes. The binding pockets of the enzyme were determined in this study and two binding pockets were predicted using the automatic online tool using the web server. The selected FDA-approved drugs were ordered based on their minimum binding energy to the proteinase. ConclusionsMolecular docking studies revealed the involvement of various hydrophobic interactions between FDA-approved drugs and amino acid residues of monkeypox virus proteinase.

Bond energies and hydrogen density distributions for hydrogen bond

Abstract The three-body problem for hydrogen bond (X–Y–X; Y=H or D) is discussed assuming symmetrical double minimum of the Morse potentials (X–Y). Two types for hydrogen bond energies are estimated by deriving the confocal elliptic differential equation, and the lowest energy for hydrogen bond may be estimated using the trial function of the linear combination of the lowest Morse wave functions by variation method. The repulsive potentials for X2 are determined by the expectation values E | z p | E $\langle E\vert {z}^{p}\vert E\rangle $ in continuum state for the Morse potential for X2. Figures of the potential curve for O–H–O and the hydrogen density distribution for O–D–O are displayed. The hydrogen bond energies for O–H–O, O–D–O, N–H–N, and N–D–N are obtained.

Revisiting the hydrogen atom transfer reactions through a simple and accurate theoretical model: Role of hydrogen bond energy in polyphenolic antioxidants

The importance of hydrogen atom transfer (HAT) reaction has been realized in various fields, ranging from atmospheric chemistry to biological processes. HAT is noteworthy in exploring phenolic antioxidants as these molecules scavenge harmful reactive free radicals through this popular pathway. The determining index of the HAT reaction is the bond dissociation enthalpy (BDE). This article proposes a theoretical model for estimating the OH BDE, a key concept for assessing HAT reactions. The model is shown to have high accuracy, and is computationally cheaper than the conventional methods of computing BDE. It has been tested with 35 polyphenolic systems having antioxidant activity involved through the HAT mechanism. Further, topological parameters within the QTAIM are computed along with the hydrogen bond energies to understand the intramolecular hydrogen bonding in such polyphenolic systems undergoing HAT reactions.

Mobility of Li+, Na+, and Cs+ cations in Nafion membrane, as studied by NMR techniques

The local mobility and diffusion of Li+, Na+, and Cs+ cations in Nafion 117 membrane were explored by 7Li, 23Na, and 133Cs spin relaxation and pulsed field gradient NMR techniques. It was shown that the macroscopic mass transfer of cations is controlled by ion motion near sulfonate groups. Lithium and sodium cations, whose hydrated energy is higher than the water hydrogen bond energy, are moving together with water molecules, but cesium cations possessing a low hydrated energy are jumping directly between the neighboring sulfonate groups.

Quantum chemical study of tautomeric equilibriums, intramolecular hydrogen bonds, and π-electron delocalization in the first singlet and triplet excited states of 2-selenoformyl-3-thioxo-propionaldehyde

In the present study, the tautomeric process, intramolecular hydrogen bonding (IMHB), and π-electron delocalization (π-ED) of 2-selenoformyl-3-thioxo-propionaldehyde (STP) in the first singlet and triplet excited states were investigated by CIS and TD-DFT methods. The relative energies of hydrogen-bonded tautomers in both excited states indicate that the thiol/enol conformers are the most/least stable forms. In this regard, a detailed analysis of various tautomeric equilibriums, different types of hydrogen bonds, and π-electron delocalization was performed. The electronic energies of different tautomers indicate the thermodynamic preference of thiol with respect to the other forms. Furthermore, the low activation energy barriers of thione⇄thiol equilibrium also show the kinetic preference of thiol. On the other hand, the estimation of different hydrogen bond energies emphasizes the stronger IMHB of enol. Moreover, the evaluation of π-ED by the structural parameter of Gilli (λ) represents the significance of electron mobility in the enol conformers. Consequently, the duality between the IMHB and π-ED with the thermodynamic stability order of tautomers indicates that the tautomeric phenomenons play a dominant role in determining the stability of the benchmark structures in both singlet and triplet excited states.

Thermally Crosslinked Hydrogen-Bonded Organic Framework Membranes for Highly Selective Ion Separation.

The weak bonding energy and flexibility of hydrogen bonds can hinder the long-term use of hydrogen-bonded organic framework (HOF) materials under harsh conditions. Here we invented a thermal-crosslinking method to form polymer materials based on a diamino triazine (DAT) HOF (FDU-HOF-1), containing high-density hydrogen bonding of N-H⋯N. With the increase of temperature to 648 K, the formation of -NH- bonds between neighboring HOF tectons by releasing NH3 was observed based on the disappearance of the characteristic peaks of amino groups on FDU-HOF-1 in the Fourier transform infrared (FTIR) and solid-state nuclear magnetic resonance (ss-NMR). The variable temperature PXRD indicated the formation of a new peak at 13.2° in addition to the preservation of the original diffraction peaks of FDU-HOF-1. The water adsorption, acid-base stability (12 M HCl to 20 M NaOH) and solubility experiments concluded that the thermally crosslinked HOFs (TC-HOFs) are highly stable. The membranes fabricated by TC-HOF demonstrate the permeation rate of K+ ions as high as 270 mmol m-2 h-1 as well as high selectivity of K+/Mg2+ (50) and Na+/Mg2+ (40), which was comparable to Nafion membranes. This study provides guidance for the future design of highly stable crystalline polymer materials based on HOFs.

The combination of polyphenols and phospholipids as an efficient platform for delivery of natural products

Although nature is a rich source of potential drugs and drug leads, the widespread application of natural products (NPs) is limited due to their poor absorption when administered orally. A strategy of using phytosome has emerged as a promising technique to increase the bioavailability of NPs. Here, a comprehensive computational investigation is performed to explore the nature of interactions in the formation of phytosomes between phosphatidylcholine (PC) and a series of polyphenols (PP), including epigallocatechin-3-gallate (Eg), luteolin (Lu), quercetin (Qu), and resveratrol (Re). Our quantum mechanical calculation revealed that the intermolecular hydrogen bonds (HBs) of phosphate and glycerol parts of PC with the polyphenol compounds are the main driving force in the formation of phytosomes. The strongest HB (with energy HB = − 108.718 kJ/mol) is formed between the Eg molecule and PC. This hydrogen bond results from the flexible structure of the drug which along with several van der Waals (vdW) interactions, makes Eg-PC the most stable complex (adsorption energy = − 164.93 kJ/mol). Energy decomposition analysis confirms that the electrostatic interactions (hydrogen bond and dipole-diploe interactions) have a major contribution to the stabilization of the studied complexes. The obtained results from the molecular dynamics simulation revealed that the formation of phytosomes varies depending on the type of polyphenol. It is found that the intermolecular hydrogen bonds between PP and PC are a key factor in the behavior of the PP-PC complex in the self-aggregation of phytosome. In Eg-PC, Lu-PC, and Qu-PC systems, the formation of strong hydrogen bonds (HBCP < 0 and ∇2ρBCP > 0) between PP and PC protects the PP-PC complexes from degradation. The steered molecular dynamics simulation results have a good agreement with experimental data and confirm that the phytosome platform facilitates the penetration of PP compounds into the membrane cells.

History-dependent hydrogen bonds energy distributions in NaCl aqueous solutions undergoing osmosis and diffusion through a ceramic barrier

History-dependent hydrogen bonds energy distributions in NaCl aqueous solutions undergoing osmosis and diffusion through a ceramic barrier

Electronic and magnetic properties of TATA-DNA sequence driven by chemical functionalization.

The TATA box is a promoter sequence able to interact directly with the components of the basal transcription initiation machinery. We investigate the changes in the electronic and magnetic properties of a TATA-DNA sequence when functionalized with different chemical groups; using the first-principles density functional theory specifically, the TATA-DNA sequences were functionalized with methyl groups (CH3 , methylation), amino groups (NH2 , amination), imine groups (NH, imination), chloroamine groups (NCl2 , chloramination), H-adatom (hydrogenation), and Cl-adatom (chlorination). The functional groups were anchored at nitrogen atoms from adenine and oxygen atoms from thymine at sites pointed as reactive regions. We demonstrated that chemical functionalization induces significant changes in charge transfer, hydrogen bond distance, and hydrogen bond energy. The hydrogenation and imination increased the hydrogen bond energy. Results also revealed that the chemical functionalization of DNA molecules exhibit a ferromagnetic ground state, reaching magnetization up to 4.665 μB and complex magnetic ordering. We further demonstrated that the functionalization could induce tautomerism (proton migration in the base pair systems). The present study provides a theoretical basis for understanding the functionalization further into DNA molecules and visualizing possible future applications.

Two-Step ONIOM Method for the Accurate Estimation of Individual Hydrogen Bond Energy in Large Molecular Clusters.

The study of molecular clusters to understand the properties of condensed systems has been the subject of immense interest. To get insight into these properties, the knowledge of various noncovalent interactions present in these molecular clusters is indispensable. Our recently developed molecular tailoring approach-based (MTA-based) method for the estimation of the individual hydrogen bond (HB) energy in molecular clusters is useful for this purpose. However, the direct application of this MTA-based method becomes progressively difficult with the increase in the size of the cluster. This is because of the difficulty in the evaluation of single-point energy at the correlated level of theory. To overcome this caveat, herein, we propose a two-step method within the our own N-layer integrated molecular orbital molecular mechanics (ONIOM) framework. In this method, the HB energy evaluated by the MTA-based method employing the actual molecular cluster at a low Hartree-Fock (HF) level of theory is added to the difference in the HB energies evaluated by the MTA-based method, employing an appropriate small model system, called the shell-1 model, calculated at high (MP2) and low (HF) levels of theory. The shell-1 model of a large molecular cluster is made up of only a few molecules that are in direct contact (by a single HB) with the two molecules involved in the formation of an HB under consideration. We tested this proposed two-step ONIOM method to estimate the individual HB energies in various molecular clusters, viz., water (Wn, n = 10-16, 18 and 20), (H2O2)12, (H2O3)8, (NH3)n and strongly interacting (HF)15 and (HF)m(W)n clusters. Furthermore, these estimated individual HB energies by the ONIOM method are compared with those calculated by the MTA-based method using actual molecular clusters. The estimated individual HB energies by the ONIOM method, in all these clusters, are in excellent linear one-to-one agreement (R2 = 0.9996) with those calculated by the MTA-based method using actual molecular clusters. Furthermore, the small values of root-mean-square deviation (0.06), mean absolute error (0.04), |ΔEmax| (0.21) and Sε (0.06) suggest that this two-step ONIOM method is a pragmatic approach to provide accurate estimates of individual HB energies in large molecular clusters.

Effect of oxygen-containing functional groups on the wettability of coal through DFT and MD simulation

To investigate the wettability of different oxygen-containing functional group (OFG) surfaces, graphite substrates were used as a model for coal adsorbents. The substrates were modified with COOH, OH, CO, and OCH3. The adsorption-diffusion behavior of H2O molecules/water droplets on different OFG surfaces was investigated using molecular dynamics (MD) simulations with frontier orbital energy difference as a metric for different surface wettability degrees in quantum chemical analysis. The results indicated that the frontier orbital energy difference of the H2O molecule was 3.480, 3.491, 3.631, and 3.680 eV for PhCOOH, PhOH, PhCO, and PhOCH3, respectively. In addition, the equilibrium contact angle, interaction energy, and number of hydrogen bonds after the adsorption equilibrium of water droplets for COOH, OH, CO, and OCH3 surfaces were 22.34°, −5.03 kcal/mol, and 652; –23.72°, −4.19 kcal/mol, and 450; 68.01°, −0.79 kcal/ mol, and 61; 90.51°, −0.50 kcal/mol, and 28, respectively. The smaller the energy difference between the frontier orbitals of the H2O molecule and the OFG, the smaller the equilibrium contact angle between the water droplet and the OFG surface, the more hydrogen bonds were formed, and the larger the absolute value of the interaction energy, the better the wettability of the surface of the OFG. The order of wettability of the different OFG surfaces was COOH > OH > CO > OCH3, which is consistent with the radial distribution function and the analysis results for the extended area, etc. The results of density functional theory (DFT) calculations and MD simulations exhibited identical patterns, indicating the reasonableness of the simulations. This study may serve as a reference for the suppression of hydrophilicity in low-order coal and the enhancement of the flotation effect.

Molecular Dynamics Study on the Diffusion Behavior of Water Molecules and the Dielectric Constant of Vegetable/Mineral Oil Blends.

Insulating oil plays a crucial role in internal insulation of oil-impregnated transformers. It has been demonstrated in a variety of experimental studies that mineral oil (MO) and vegetable oil (VO) can be blended in different ratios to improve insulation properties; however, the mechanisms underlying this phenomenon remain unclear. In this study, a molecular dynamics (MD) simulation approach was used to investigate diffusion of water molecules in VO/MO blends and dielectric constants of a mixture. The results show that the diffusion coefficient of water molecules is negatively correlated with the proportion of VO; thus, addition of VO helps to improve the insulation properties of a mixture. Due to introduction of strong polar functional groups, a decrease in the diffusion behavior of water molecules can be attributed to an increase in the interaction energy and formation of hydrogen bonds between water molecules and the mixed oil system. There is a direct correlation between the dielectric constant of a mixture and VO content; however, it is very sensitive to water content. The presence of strong polar water molecules or functional groups in a mixture leads to an increase in the dielectric constant, which results in a reduction in insulating properties. Accordingly, presence of polar groups plays an important role in determining the insulating properties of a mixture. To increase the insulation performance of a mixture, it is important to consider the diffusion-inhibiting and dielectric effects of the stronger polar groups in vegetable oil compared to those in mineral oil.

Revealing the Reasons for Degeneration of Resonance-Assisted Hydrogen Bond on the Aromatic Platform: Calculations of Ortho-, Meta-, Para-Disubstituted Benzenes, and (Z)-(E)-Olefins

The energies of the O-H∙∙∙O=C intramolecular hydrogen bonds were compared quantitatively for the series of ortho-disubstituted benzenes and Z-isomers of olefins via a molecular tailoring approach. It was established that the hydrogen bond energy in the former series is significantly less than that in the latter one. The reason for lowering the hydrogen bond energy in the ortho-disubstituted benzenes compared to the Z-isomers of olefins is the decrease in the π-contribution to the total energy of the complex interaction, in which the hydrogen bond per se is enhanced by the resonance effect. By the example of the para- and meta-disubstituted benzenes, as well as E-isomers of olefins, it was explicitly shown that the aromatic ring is a much poorer conductor of the resonance effect compared to the double bond. The hydrogen bond in the ortho-disubstituted benzenes has a lower energy than a typical resonance-assisted hydrogen bond because the aromatic moiety cannot properly assist the hydrogen bond with a resonance effect. Thus, a hydrogen bond on an aromatic platform should fall into a special category, namely an aromaticity-assisted hydrogen bond, which is closer by nature to a simple hydrogen bond rather than to a resonance-assisted one.

COCRYSTAL PREDICTION OF THE SALICYLIC ACIDNICOTINAMIDE

Salicylic acid-nicotinamide was used as a prediction model in the hydrogen bond interactions dominating a cocrystal structure. The combined conformation of the two molecules was analyzed at the B3LYP-D3BJ and WB97M-D3BJ theoretical level. The existing hydrogen bond dimer conformations were compared with putative conformations to determine the bond energy and stability. Some parameters were assessed for predictive descriptors, such as geometric parameters, total energy, interaction energy, single hydrogen bond energy, HOMO-LUMO gap, Laplacian Bond Order, and Natural Bond Orbital. An existing conformation, SACNIC1, produced intermolecular hydrogen bond energy which was quite higher than the other conformations, including the pure homomolecular conformations of either salicylic acid or nicotinamide. The cocrystal packing formation seems to be initiated with SACNIC1 as the basic structure conformation. Furthermore, the presence of the strongest intermolecular hydrogen bond energy in SACNIC1 suggests its potential utility as a predictive descriptor.

A Self-Matting Waterborne Polyurethane Coating for PVC Artificial Leather

A type of polyester-based self-matting waterborne polyurethane (ESMWPU) with an excellent matte effect of 0.8° and transmittance of 78.5% for PVC artificial leather was synthesized. The influence of synthesis parameters, including R value, crosslinking and hydrophilic group content, on coating gloss and transmittance was investigated. Meanwhile, the properties necessary for applying water-based resins to plasticized PVC were detailed. The results demonstrated that R value, crosslinking degree and hydrophilic group content synergistically decided the morphological changes of latex particles during their film-forming process in three aspects: particle stiffness, three-dimensional structure and particle size, respectively. With optimized parameters of R = 1.9, TMP = 2 wt% and DMPA = 1.75 wt%, ESMWPU latex particles stabilized their spherical shapes without collapsing in the film-forming process and created a rough surface, resulting in a matte effect. In terms of application performance, good wetting and adhesion for ESMWPU to a plasticized PVC surface was achieved with a 2 wt% leveling agent load. Moreover, due to the high cohesion energy of ester bonds and intermolecular hydrogen bonds, this type of polyester-based ESMWPU also depicted admirable thermal adhesion resistance. All aforementioned results distinctly demonstrate a feasible yet promising paradigm for applying ESMWPU on PVC artificial leather.

Hydrogen bonds energy distribution and information-theoretic analysis of blood serum from hamsters with experimental Graffi tumor

The aim of the present study was to estimate the feasibility of blood serum hydrogen bonds energy distribution, and information-theoretic characteristics, as diagnostic markers for tumor disease in Graffi tumor bearing hamsters. Golden Syrian hamsters were randomly divided into two groups: a healthy control and an experimental one, challenged with Graffi tumor cells. The distributions of hydrogen bonds energies (-E) in the blood serum samples were investigated with a nonequilibrium process of droplets evaporation and sequential measurements of their wetting angle (Non-equilibrium Energy Spectrum (NES)). The measurements show significantly lower average hydrogen bond energy of the experimental samples than the controls. Shifts in NES toward depletion of (-E) at energy levels 0.0937, 0.1187, 0.1212, 0.1337, and 0.1387 eV, and an additional population at 0.0987, 0.1012, 0.1137, and 0.1237 eV energy levels were observed in the experimental samples compared to the controls. Such a pattern points to the possibility of utilizing the f(E) shifts at these energy levels as diagnostic markers of Graffi tumor in hamsters. Information-theoretic measures of Shannon entropy, Variation of information and Transformational information entropy were used to investigate possible differences between the experimental and control spectrum. Although they differ significantly in definitions, a comparison with the three information-theoretic measures did not reveal a statistically significant difference.

Adsorption mechanism of ammonia nitrogen and phenol on lignite surface: Molecular dynamics simulations and quantum chemical calculations

To elucidate the adsorption mechanisms between the lignite and typical fractions from the wastewater, the microscopic adsorption behavior of ammonia nitrogen and phenol on the lignite surface was investigated by molecular dynamics simulation. Meanwhile, the interaction strength and properties of lignite structural units with ammonia nitrogen and phenol were examined by combining quantum chemical calculations and wave function analysis. The results showed that a small amount of NH4+ was adsorbed in the lignite structure, but most ions remained in the aqueous phase. Compared with NH4+, phenol was adsorbed more easily on the lignite surface. In addition to O···H hydrogen bond, there were cation-π interactions between NH4+ and lignite structural units. The main interactions between phenol and lignite structural units include π-π stacking and hydrogen bonding, as well as a small amount of van der Waals (vdW) interactions. The total hydrogen bond energy of the lignite structural unit to the four conformations of NH4+ as a percentage of the total binding energy was 72.34, 82.64, 51.00, and 51.02 %, respectively, while that for phenol was 40.94, 53.42, 62.18 and 73.31 %, respectively. And the electrostatic effects played a major role in the binding interaction of the NH4+ and lignite structural units, while the dispersion effects and electrostatic effects dominated the binding interaction of the phenol and lignite structural units. In the gas phase, the maximum binding energy between NH4+ and lignite structural units was higher than that between phenol and lignite. However, the binding energy between phenol and lignite structural units was higher in aqueous solution, which was consistent with the result that phenol was more easily adsorbed by lignite in molecular dynamics simulations.

Improved hydroxyl radical production by electric-field-induced catalysis in O3/H2O2 process: A reactive molecular dynamics perspective

O3/H2O2 process, as a well-used ozone advanced oxidation technology, has been considered to be suitable for treating a variety of organic wastewater. However, the low solubility of ozone in hydrogen peroxide aqueous solution makes the catalytic effect to ozone reaction unsatisfactory. In this study, a novel method of external electric field (EEF) catalyzing O3/H2O2 process was proposed. The chemical reaction in O3/H2O2 process under EEF were investigated by reaction molecular dynamics simulation. The mechanism of the catalytic effect of EEF on chemical reactions in the O3/H2O2 system was revealed by analyzing the system energy, hydrogen bond energy, reaction rate and degree of reaction of the reactants, and the amount of reaction products under EEF with different strength. The results show that EEF can significantly promote the ionization reaction of H2O2 to produce more HO– 2, which further promote the reaction of O3, thus ultimately leads to the increase of •OH production and generation rate. In addition, the catalytic effect of EEF is proportional to the field strength. When the amount of ozone is constant, the concentration of hydrogen peroxide will affect the catalytic effect of EEF.