Prospecting the therapeutic edge of a novel compound (B12) over berberine in the selective targeting of Retinoid X Receptor in colon cancer.

The Retinoid X Receptor (RXR) is an attractive target in the treatment of colon cancer. Different therapeutic binders with high potency have been used to specifically target RXR. Among these compounds is a novel analogue of berberine, B12. We provided structural and molecular insights into the therapeutic activity properties of B12 relative to its parent compound, berberine, using force field estimations and thermodynamic calculations. Upon binding of B12 to RXR, the high instability elicited by RXR was markedly reduced; similar observation was seen in the berberine-bound RXR. However, our analysis revealed that B12 could have a more stabilizing effect on RXR when compared to berberine. Interestingly, the mechanistic behaviour of B12 in the active site of RXR opposed its impact on RXR protein. This disparity could be due to the bond formation and breaking elicited between B12/berberine and the active site residues. We observed that B12 and berberine could induce a disparate conformational change in regions Gly250-Asp258 located on the His-RXRα/LBD domain. Comparatively, the high agonistic and activation potential reported for B12 compared to berberine might be due to its superior binding affinity as evidenced in the thermodynamic estimations. The total affinity for B12 (-25.76 kcal/mol) was contributed by electrostatic interactions from Glu243 and Glu239. Also, Arg371, which plays a crucial role in the activity of RXR, formed a strong hydrogen bond with B12; however, a weak interaction was elicited between Arg371 and berberine. Taken together, our study has shown the RXRα activating potential of B12, and findings from this study could provide a framework in the future design of RXRα binders specifically tailored in the selective treatment of colon cancer.

Discovery of Potential Chemical Probe as Inhibitors of CXCL12 Using Ligand-Based Virtual Screening and Molecular Dynamic Simulation.

CXCL12 are small pro-inflammatory chemo-attractant cytokines that bind to a specific receptor CXCR4 with a role in angiogenesis, tumor progression, metastasis, and cell survival. Globally, cancer metastasis is a major cause of morbidity and mortality. In this study, we targeted CXCL12 rather than the chemokine receptor (CXCR4) because most of the drugs failed in clinical trials due to unmanageable toxicities. Until now, no FDA approved medication has been available against CXCL12. Therefore, we aimed to find new inhibitors for CXCL12 through virtual screening followed by molecular dynamics simulation. For virtual screening, active compounds against CXCL12 were taken as potent inhibitors and utilized in the generation of a pharmacophore model, followed by validation against different datasets. Ligand based virtual screening was performed on the ChEMBL and in-house databases, which resulted in successive elimination through the steps of pharmacophore-based and score-based screenings, and finally, sixteen compounds of various interactions with significant crucial amino acid residues were selected as virtual hits. Furthermore, the binding mode of these compounds were refined through molecular dynamic simulations. Moreover, the stability of protein complexes, Root Mean Square Deviation (RMSD), Root Mean Square Fluctuation (RMSF), and radius of gyration were analyzed, which led to the identification of three potent inhibitors of CXCL12 that may be pursued in the drug discovery process against cancer metastasis.

Characterization, antioxidant activities, and inhibition on α-glucosidase activity of corn silk polysaccharides obtained by different extraction methods.

The polysaccharides (CSPw, CSPc, CSPa, and CSPu) were prepared by hot water extraction, acid-assisted extraction, alkaline-assisted extraction, and ultrasound-assisted extraction from corn silk, respectively. High performance gel permeation chromatography (HPGPC), fourier-transform infrared (FT-IR) spectroscopy, and scanning electron microscopy (SEM) results indicated that the extraction methods had an obvious impact on the molecular weight, structure, and morphology of the CSPs. Among the four polysaccharides, CSPu showed the highest inhibitory α-glucosidase activity, which might be related to its smaller molecular weight. Furthermore, kinetics analyses revealed that CSPu had significant inhibition of α-glucosidase in a non-reversible and competitive manner. Fluorescence quenching analysis illustrated that the interaction mechanism of CSPu and α-glucosidase was claimed as a static quenching mechanism. Isothermal titration calorimetry (ITC) analysis showed that the main driving forces for the interaction of CSPu with α-glucosidase was hydrogen bonding and the binding interactions of them occurred spontaneously.

Preparation of stabilized submicron fenofibrate crystals on niacin as a hydrophilic hydrotropic carrier.

Fenofibrate is antihyperlipidemic which has low and variable oral bioavailability due to erratic dissolution characteristics. Niacin showed a potential atheroprotective effects suggesting possible co-administration with fenofibrate with a potential for development of fixed dose combination. The chemical structure of both drugs highlights the opportunity for interaction upon co-processing due to the existence of complementary hydrogen bonding sites. Accordingly, fenofibrate and niacin were co-processed by wet co-grinding and the resulting product was assessed using scanning electron microscopy, FTIR, thermal analysis and X-ray diffraction in addition to dissolution studies. The instrumental analysis indicated the development of submicron fenofibrate crystals stabilized over the surface of niacin crystals. The developed submicron crystals showed fast dissolution of fenofibrate depending on the relative proportions of fenofibrate to niacin. Co-processing of both drugs at dose ratio which contained higher proportion of niacin resulted in further enhancement in the dissolution rate. This further enhancement was attributed to the hydrotropic effect of niacin which was proved by solubility study in addition to size reduction. This supposition was confirmed from the inferior dissolution of fenofibrate from the physical mixture. The study introduces fenofibrate/niacin as potential fixed dose combination for augmented dissolution rate and pharmacological effects.

Development of active packaging based on chitosan-gelatin blend films functionalized with Chinese hawthorn (Crataegus pinnatifida) fruit extract.

The fruits of Chinese hawthorn (Crataegus pinnatifida) have been used as the functional food and folk medicine due to potent antioxidant activity. In this study, polyphenols were extracted from the fruits of Chinese hawthorn and further added into chitosan-gelatin blend films to develop active packaging. The microstructure, physical, mechanical, barrier and antioxidant properties of the films were investigated in details. Results showed epicatechin, chlorogenic acid and procyanidin B2 were the main polyphenols in the extract of hawthorn fruits. The inner microstructure of chitosan-gelatin blend films became more compact when the extract was incorporated. The intermolecular interactions between film matrix and the extract were through hydrogen bonding and electrostatic interactions. The incorporation of the extract remarkably increased the thickness, tensile strength and elongation at break of chitosan-gelatin blend films. However, the moisture content, water vapor permeability and light transmittance of chitosan-gelatin blend films were significantly reduced by the addition of the extract. Moreover, chitosan-gelatin blend films containing the extract exhibited potent free radical scavenging ability. Our results suggest Chinese hawthorn fruit extract can be used as a natural antioxidant to improve the mechanical, barrier and antioxidant properties of chitosan-gelatin blend films.

A Comprehensive Study on the Biological Activity of Elderberry Extract and Cyanidin 3-O-Glucoside and Their Interactions with Membranes and Human Serum Albumin.

In our research we used the extract from dietary supplement of elderberry (EE) and its dominant anthocyanin-cyanidin 3-O-glucoside (Cy 3-gluc). By interacting with a model membrane that reflects the main lipid composition of tumor membranes, the extract components, including Cy 3-gluc, caused an increase in packing order, mainly in the hydrophilic region of the membrane. It can thus be stated that EE caused a rigidifying effect, which is fundamental for understanding its anticancer and antioxidant activity. This study represents the first attempt to unravel the mechanism of interaction of elderberry extract with membranes. The results of the interaction with human serum albumin (HSA) proved that the studied substance quenches the fluorescence of HSA through a static mechanism in which the main interaction forces are Van der Waals and hydrogen bonding. The antioxidant activity of EE and Cy 3-gluc on liposomal membranes, antiradical properties and ability to inhibited the activity of the enzymes cyclooxygenase-1 and cyclooxygenase-2 were also demonstrated. Moreover, the anticancer activity of EE and Cy 3-gluc on human breast adenocarcinoma cell line were investigated. In addition, EE also exhibited the ability to form lipid aggregates in the form of liposomal capsules that can be applied as carriers of active biological substances, and the highest efficacy of EE encapsulation was obtained for multilayered liposome formulations.

Deglycosylation of wogonoside enhances its anticancer potential.

INTRODUCTION: Scutellaria baicalensis is commonly used in Asia as an herbal medicine to treat a variety of ailments, including cancer. Wogonoside, one major constituent of S. baicalensis, can be primarily converted to wogonin through deglycosylation via enteric microbiome metabolism. MATERIALS AND METHODS: The antiproliferative effects of the glycoside (wogonoside) and its deglycosylated compound (wogonin) on a panel of human cancer cell lines from the most common solid tumors were evaluated using the MTS colorimetric assay. Cell cycle and apoptosis were determined using flow cytometry. Enzymatic activities of caspases were measured, and the interactions of wogonin and caspases were explored by a docking analysis. RESULTS: Wogonoside did not have obvious antiproliferative effects on the cancer cells. In contrast, wogonin showed significant antiproliferative activities on all the tested cancer cells. Wogonin arrested the cells in the G1 phase and significantly induced cell apoptosis. The compound also activated the expression of caspases 3 and 9. The docking results suggest that the compound forms hydrogen bonds with Phe250 and Ser251, and π-π interactions with Phe256 in caspase 3, and with Asp228 in caspase 9. CONCLUSIONS: After wogonoside deglycosylation, wogonin significantly enhanced its anticancer potential as a potent anticancer compound derived from S. baicalensis.

Hippeastrum reticulatum (Amaryllidaceae): Alkaloid Profiling, Biological Activities and Molecular Docking.

The Amaryllidaceae family has proven to be a rich source of active compounds, which are characterized by unique skeleton arrangements and a broad spectrum of biological activities. The aim of this work was to perform the first detailed study of the alkaloid constituents of Hippeastrum reticulatum (Amaryllidaceae) and to determine the anti-parasitological and cholinesterase (AChE and BuChE) inhibitory activities of the epimers (6α-hydroxymaritidine and 6ß-hydroxymaritidine). Twelve alkaloids were identified in H. reticulatum: eight known alkaloids by GC-MS and four unknown (6α-hydroxymaritidine, 6ß-hydroxymaritidine, reticulinine and isoreticulinine) by NMR. The epimer mixture (6α-hydroxymaritidine and 6ß-hydroxymaritidine) showed low activity against all protozoan parasites tested and weak AChE-inhibitory activity. Finally, a molecular docking analysis of AChE and BuChE proteins showed that isoreticulinine may be classified as a potential inhibitory molecule since it can be stabilized in the active site through hydrogen bonds, π-π stacking and hydrophobic interactions.

Arctigenin inhibits STAT3 and exhibits anticancer potential in human triple-negative breast cancer therapy.

Triple-negative breast cancers (TNBCs) are the most aggressive and hard-to-treat breast tumors with poor prognosis, and exploration for novel therapeutic drugs is impending. Arctigenin (Atn), a bioactive lignan isolated from seeds of Arctium lappa L, has been reported to inhibit many cancer types; however, the effect of Atn on TNBC remains unclear. In this study, we demonstrated that Atn decreased proliferation, and induced apoptosis in TNBC cells. Furthermore, we explored the underlying mechanism of Atn inhibition on TNBC cells. Computational docking and affinity assay showed that Atn bound to the SH2 domain of STAT3. Atn inhibited STAT3 binding to genomic DNA by disrupting hydrogen bond linking between DNA and STAT3. In addition, Atn augmented Taxotere®-induced TNBC cell cytotoxicity. TNBC xenograft tests also confirmed the antitumor effect of Atn in vivo. These characteristics render Atn as a promising candidate drug for further development and for designing new effective STAT3 inhibitors.

Synthesis of porous starch xerogels modified with mercaptosuccinic acid to remove hazardous gardenia yellow.

Mercaptosuccinic acid (MSA) molecules were inserted into potato starch, leading to the breaking of intrinsic H-bonds within macromolecular chains of starch and the formation of intermolecular H-bonds between MSA and starch, which could be verified by Fourier transform infrared spectroscopy (FT-TR). MSA modified porous starch xerogels (PSX/MSA) were obtained after freeze-drying the MSA modified starch, and they were characterized by field emission scanning electron microscopy (FESEM), exhibiting the intriguing porous structure due to the separation of starch chains by MSA molecules. The PSX/MSA were then used as the adsorbents to remove gardenia yellow (GY), a natural colorant with genotoxicity. Due to the porous structure of PSX and the introduced carboxyl groups from MSA, the adsorption capacity of the PSX/MSA was much higher than that of the starch xerogels alone (SX). The adsorption behaviors of GY by the PSX/MSA fitted both the Freundlich isotherm model and the pseudo-second-order kinetic model, and the efficient adsorption of GY suggested that the PSX/MSA might be potential adsorbents for the removal of dyes from contaminated aquatic systems.

Absolute configuration of podophyllotoxone and its inhibitory activity against human prostate cancer cells.

Podophyllotoxone (1) was isolated from the roots of Dysosma versipellis. The structure was determined by spectroscopic analysis in combination with single-crystal X-ray analysis. The absolute configuration of compound 1 was assigned based on the Flack parameter. It showed significant inhibitory activities against human prostate cancer cells PC3 and DU145 with IC50 values being 14.7 and 20.6 µmol·L(-1), respectively. It also arrested the cells at G2/M phase. Tubulin polymerization assay showed that it inhibited the tubulin polymerization in a dose-dependent manner, and molecular docking analysis revealed a different binding mode with tubulin as compared with those known tubulin inhibitors.

Treatment of cardiovascular disease by traditional Chinese medicine against pregnane X receptor.

Recently, cardiovascular disease, also known as loop circulatory system diseases or disorders, is one of the serious diseases including heart disease, stroke, atherosclerosis, myocardial infarction, hypertension, hypotension, and thrombosis. Human pregnane X receptor, PXR, plays a crucial role in exogenous and endobiotic metabolism for rabbit, rat, mouse, and human. The PXR activation can protect the blood vessels from damage of hazardous substances. In this study we aim to investigate the potent lead compounds as PXR receptor agonist against cardiovascular disease. To improve drug development of TCM compounds, we aim to investigate the potent lead compounds as PXR agonists from the TCM compounds in TCM Database@Taiwan. The top three TCM compounds, bis(4-hydroxybenzyl) ether mono-ß-D-glucopyranoside (BEMG), ixerisoside, and tangshenoside II, have displayed higher potent binding affinities than the positive control, PNU-142721, in the docking simulation. After MD simulations, which can optimize the result of docking simulation and validate the stability of H-bonds between each ligand and PXR protein under dynamic conditions, top TCM compounds, BEMG and tangshenoside II, maintain most of interactions with PXR protein, which keep the ligand binding stable in the binding domain. Hence, we propose BEMG and tangshenoside II as potential lead compounds for further study in drug development process with the PXR protein.

Investigation of potent lead for acquired immunodeficiency syndrome from traditional Chinese medicine.

Acquired immunodeficiency syndrome (AIDS), caused by human immunodeficiency virus (HIV), has become, because of the rapid spread of the disease, a serious global problem and cannot be treated. Recent studies indicate that VIF is a protein of HIV to prevent all of human immunity to attack HIV. Molecular compounds of traditional Chinese medicine (TCM) database filtered through molecular docking and molecular dynamics simulations to inhibit VIF can protect against HIV. Glutamic acid, plantagoguanidinic acid, and Aurantiamide acetate based docking score higher with other TCM compounds selected. Molecular dynamics are useful for analysis and detection ligand interactions. According to the docking position, hydrophobic interactions, hydrogen bonding changes, and structure variation, the study try to select the efficacy of traditional Chinese medicine compound Aurantiamide acetate is better than the other for protein-ligand interactions to maintain the protein composition, based on changes in the structure.

Mechanistic insights into mode of action of potent natural antagonists of BACE-1 for checking Alzheimer's plaque pathology.

Alzheimer's is a neurodegenerative disorder resulting in memory loss and decline in cognitive abilities. Accumulation of extracellular beta amyloidal plaques is one of the major pathology associated with this disease. ß-Secretase or BACE-1 performs the initial and rate limiting step of amyloidic pathway in which 37-43 amino acid long peptides are generated which aggregate to form plaques. Inhibition of this enzyme offers a viable prospect to check the growth of these plaques. Numerous efforts have been made in recent years for the generation of BACE-1 inhibitors but many of them failed during the preclinical or clinical trials due to drug related or drug induced toxicity. In the present work, we have used computational methods to screen a large dataset of natural compounds to search for small molecules having BACE-1 inhibitory activity with low toxicity to normal cells. Molecular dynamics simulations were performed to analyze molecular interactions between the screened compounds and the active residues of the enzyme. Herein, we report two natural compounds of inhibitory nature active against ß-secretase enzyme of amyloidic pathway and are potent lead molecules against Alzheimer's disease.

Human papilloma virus 16 E6 protein as a target for curcuminoids, curcumin conjugates and congeners for chemoprevention of oral and cervical cancers.

Curcumin (diferuloyl methane) and its naturally occurring analogs viz. demethoxy, bisdemethoxy and cyclocurcumin, present in rhizomes of curcuma species turmeric, have been shown to inhibit the proliferation of a wide variety of tumor cells. Target nuclear protein HPV 16 E6 (PDB ID: 2fk4) is the major protein actively participating in oral and cervical cancers. In silico studies indicate that curcumin and its natural analogs have effective binding with different active sites on HPV 16 E6 protein, ideal target for restoring the tumor suppressor function of p53 and thus allowing the apoptosis of infected cells. The main limitation in the use of curcuminoids as therapeutic agents is their low bioavailability. Since piperine is known to enhance curcumin bioavailability to more than two thousand times by inhibiting its efflux, a conjugate of curcumin-piperic acid was also used. Although curcumin has been found to have strongest binding with this target and the two curcuminoids, demethoxy and bisdemethoxy curcumin have lower but comparable affinity, chlorogenic acid amongst the naturally occurring analogs has been found to have best binding affinity amongst all the analogs. Although curcumin-piperoyl conjugate does not show very encouraging results, it is likely to have potential activity in vitro and in vivo. These results throw light on the SAR of curcuminoids, leading to future designing of potent, non-toxic drugs for oral and cervical cancers.

Structural changes of oil palm empty fruit bunch (OPEFB) after fungal and phosphoric acid pretreatment.

Oil palm empty fruit bunch (OPEFB) was pretreated using white-rot fungus Pleurotus floridanus, phosphoric acid or their combination, and the results were evaluated based on the biomass components, and its structural and morphological changes. The carbohydrate losses after fungal, phosphoric acid, and fungal followed by phosphoric acid pretreatments were 7.89%, 35.65%, and 33.77%, respectively. The pretreatments changed the hydrogen bonds of cellulose and linkages between lignin and carbohydrate, which is associated with crystallinity of cellulose of OPEFB. Lateral Order Index (LOI) of OPEFB with no pretreatment, with fungal, phosphoric acid, and fungal followed by phosphoric acid pretreatments were 2.77, 1.42, 0.67, and 0.60, respectively. Phosphoric acid pretreatment showed morphological changes of OPEFB, indicated by the damage of fibre structure into smaller particle size. The fungal-, phosphoric acid-, and fungal followed by phosphoric acid pretreatments have improved the digestibility of OPEFB's cellulose by 4, 6.3, and 7.4 folds, respectively.

Probing the structure of Leishmania major DHFR TS and structure based virtual screening of peptide library for the identification of anti-leishmanial leads.

Leishmaniasis, a multi-faceted ethereal disease is considered to be one of the World's major communicable diseases that demands exhaustive research and control measures. The substantial data on these protozoan parasites has not been utilized completely to develop potential therapeutic strategies against Leishmaniasis. Dihydrofolate reductase thymidylate synthase (DHFR-TS) plays a major role in the infective state of the parasite and hence the DHFR-TS based drugs remains of much interest to researchers working on Leishmaniasis. Although, crystal structures of DHFR-TS from different species including Plasmodium falciparum and Trypanosoma cruzi are available, the experimentally determined structure of the Leishmania major DHFR-TS has not yet been reported in the Protein Data Bank. A high quality three dimensional structure of L.major DHFR-TS has been modeled through the homology modeling approach. Carefully refined and the energy minimized structure of the modeled protein was validated using a number of structure validation programs to confirm its structure quality. The modeled protein structure was used in the process of structure based virtual screening to figure out a potential lead structure against DHFR TS. The lead molecule identified has a binding affinity of 0.51 nM and clearly follows drug like properties.

Theoretical investigation of differences in nitroreduction of aristolochic acid I by cytochromes P450 1A1, 1A2 and 1B1.

OBJECTIVES: The herbal drug aristolochic acid (AA) derived from Aristolochia species has been shown to be the cause of aristolochic acid nephropathy (AAN), Balkan endemic nephropathy (BEN) and their urothelial malignancies. One of the common features of AAN and BEN is that not all individuals exposed to AA suffer from nephropathy and tumor development. One cause for these different responses may be individual differences in the activities of the enzymes catalyzing the biotransformation of AA. Thus, the identification of enzymes principally involved in the metabolism of AAI, the major toxic component of AA, and detailed knowledge of their catalytic specificities is of major importance. Human cytochrome P450 (CYP) 1A1 and 1A2 enzymes were found to be responsible for the AAI reductive activation to form AAI-DNA adducts, while its structurally related analogue, CYP1B1 is almost without such activity. However, knowledge of the differences in mechanistic details of CYP1A1-, 1A2-, and 1B1- mediated reduction is still lacking. Therefore, this feature is the aim of the present study. METHODS: Molecular modeling capable of evaluating interactions of AAI with the active site of human CYP1A1, 1A2 and 1B1 under the reductive conditions was used. In silico docking, employing soft-soft (flexible) docking procedure was used to study the interactions of AAI with the active sites of these human enzymes. RESULTS: The predicted binding free energies and distances between an AAI ligand and a heme cofactor are similar for all CYPs evaluated. AAI also binds to the active sites of CYP1A1, 1A2 and 1B1 in similar orientations. The carboxylic group of AAI is in the binding position situated directly above heme iron. This ligand orientation is in CYP1A1/1A2 further stabilized by two hydrogen bonds; one between an oxygen atom of the AAI nitro-group and the hydroxyl group of Ser122/Thr124; and the second bond between an oxygen atom of dioxolane ring of AAI and the hydroxyl group of Thr497/Thr498. For the CYP1B1:AAI complex, however, any hydrogen bonding of the nitro-group of AAI is prevented as Ser122/Thr124 residues are in CYP1B1 protein replaced by hydrophobic residue Ala133. CONCLUSION: The experimental observations indicate that CYP1B1 is more than 10× less efficient in reductive activation of AAI than CYP1A2. The docking simulation however predicts the binding pose and binding energy of AAI in the CYP1B1 pocket to be analogous to that found in CYP1A1/2. We believe that the hydroxyl group of S122/T124 residue, with its polar hydrogen placed close to the nitro group of the substrate (AAI), is mechanistically important, for example it could provide a proton required for the stepwise reduction process. The absence of a suitable proton donor in the AAI-CYP1B1 binary complex could be the key difference, as the nitro group is in this complex surrounded only by the hydrophobic residues with potential hydrogen donors not closer than 5 Å.

Discovery of potential M2 channel inhibitors based on the amantadine scaffold via virtual screening and pharmacophore modeling.

The M2 channel protein on the influenza A virus membrane has become the main target of the anti-flu drugs amantadine and rimantadine. The structure of the M2 channel proteins of the H3N2 (PDB code 2RLF) and 2009-H1N1 (Genbank accession number GQ385383) viruses may help researchers to solve the drug-resistant problem of these two adamantane-based drugs and develop more powerful new drugs against influenza A virus. In the present study, we searched for new M2 channel inhibitors through a combination of different computational methodologies, including virtual screening with docking and pharmacophore modeling. Virtual screening was performed to calculate the free energies of binding between receptor M2 channel proteins and 200 new designed ligands. After that, pharmacophore analysis was used to identify the important M2 protein-inhibitor interactions and common features of top binding compounds with M2 channel proteins. Finally, the two most potential compounds were determined as novel leads to inhibit M2 channel proteins in both H3N2 and 2009-H1N1 influenza A virus.

Trans-sodium crocetinate for treating hypoxia/ischemia.

BACKGROUND: Trans-sodium crocetinate (TSC) is a novel compound that offers promise as a treatment for conditions caused by hypoxia or ischemia. TSC was originally developed at the University of Virginia for hemorrhagic shock, as part of the battlefield casualty program sponsored by the US Office of Naval Research. Animal toxicology studies have demonstrated that high levels of TSC are well-tolerated, and a Phase I clinical study has shown that TSC is also safe in humans. OBJECTIVE: The drug acts via a mechanism that has not been previously exploited in a pharmaceutical. TSC increases the rate of oxygen diffusion between the erythrocytes and the tissues by altering the 'structure' of water in blood plasma. It does this by causing additional hydrogen bonds to form among the water molecules. CONCLUSION: Further development of TSC for hemorrhagic shock and other hypoxic/ischemic conditions is being done by Diffusion Pharmaceuticals LLC.