Gold Conjugates Research Articles

On Biodecorated Gold Nanoparticles Distributed within Tissues and Cells

The size and surface charge of gold nanoparticles were reported to be key factors that determine their biological fate. The clearance half times of the biodecorated gold nanoparticles AuNPs from blood were reported to increase with decreasing the particle size. The particle elution time or the length of pathway is inversely proportional to the particle size. The positively charged nanoparticles were most toxic when compared with negatively charged particles and the neutral nanoparticles. Functional (ionic) surfactants (modifiers) have a pronounced effect on the distribution pattern not only by changing the circulation time in the bloodstream but also changing the nature of gold conjugates. The functional tissues present high activity to particle surface which favor their possible irreversible binding to biopolymers. The toxicity of the AuNPs is determined by their ability to irreversibly bind to key biomolecules (DNA and others) and change the functioning of cellular molecular processes.

Flow-Through Assay for Detection of Antibodies Using Protein-A Colloidal Gold Conjugate as a Probe.

Flow-through assay (FTA) is a rapid, simple-to-perform, cost-effective, and user-friendly diagnostic test for monitoring infections in non-laboratory settings. It is mostly applied for antibody detection. FTA employing protein-A colloidal gold conjugate to detect antibodies against porcine cysticerci using cyst fluid and whole cyst antigens of Taenia solium metacestode is described here. Antibodies in the serum are captured by an antigen spotted onto a nitrocellulose membrane mounted on a flow-through device that serves as the antigen capture matrix. The bound antibodies are visualized by the addition of protein-A colloidal gold conjugate, which imparts a pink color. The test can be completed within 3 min at room temperature without any instrumentation. The sensitivity and specificity of the FTA are in agreement with ELISA.

The enhanced longevity and liver targetability of Paclitaxel by hybrid liposomes encapsulating Paclitaxel-conjugated gold nanoparticles

Organic and inorganic drug delivery systems both demonstrate their own advantages and challenges in practical applications. Combining these two drug delivery strategies in one system is expected to solve their current issues and achieve desirable functions. In this paper, gold nanoparticles (GNPs) and liposomes have been chosen as the model systems to construct a hybrid system and investigate its performance for the tumor therapy of Paclitaxel (PTX). The thiol-terminated polyethylene glycol (PEG400)–PTX derivative has been covalently modified on the surface of GNPs, followed by the encapsulation of PTX-conjugated GNPs (PTX–PEG400@GNPs) in liposomes. The hybrid liposomes solve the solubility and stability problems of gold conjugates and show high drug loading capacity. In vitro PTX release from the hybrid system maintains the similar sustained behavior demonstrated in its conjugates. Under the protection of a biocompatible liposome shell, encapsulated PTX shows enhanced circulation longevity and liver targetability compared to Taxol® and PTX–PEG400@GNPs suspension in the pharmacokinetic and biodistribution studies. These indicate that encapsulating drug-conjugated inorganic nanoparticles inside organic carriers maintains the superiority of both vehicles and improves the performance of hybrid systems. Although these attributes of hybrid liposomes lead to a better therapeutic capacity in a murine liver cancer model than that of the comparison groups, it shows no significant difference from Taxol® and conjugate suspension. This result could be due to the delayed and sustained drug release from the system. However, it indicates the promising potential for these hybrid liposomes will allow further construction of a compound preparation with improved performance that is based on their enhanced longevity and liver targetability of Paclitaxel.

Gold nanorod-photosensitizer conjugate with extracellular pH-driven tumor targeting ability for photothermal/photodynamic therapy

Chlorin e6-pHLIPss-AuNRs, a gold nanorod-photosensitizer conjugate containing a pH (low) insertion peptide (pHLIP) with a disulfide bond which imparts extracellular pH (pHe)-driven tumor targeting ability, has been successfully developed for bimodal photodynamic and photothermal therapy. In this bimodal therapy, chlorin e6 (Ce6), a second-generation photosensitizer (PS), is used for photodynamic therapy (PDT). Gold nanorods (AuNRs) are used as a hyperthermia agent for photothermal therapy (PTT) and also as a nanocarrier and quencher of Ce6. pHLIPss is designed as a pHe-driven targeting probe to enhance accumulation of Ce6 and AuNRs in cancer cells at low pH. In Ce6-pHLIPss-AuNRs, Ce6 is close to and quenched by AuNRs, causing little PDT effect. When exposed to normal physiological pH 7.4, Ce6-pHLIPss-AuNRs loosely associate with the cell membrane. However, once exposed to acidic pH 6.2, pHLIP actively inserts into the cell membrane, and the conjugates are translocated into cells. When this occurs, Ce6 separates from the AuNRs as a result of disulfide bond cleavage caused by intracellular glutathione (GSH), and singlet oxygen is produced for PDT upon light irradiation. In addition, as individual PTT agent, AuNRs can enhance the accumulation of PSs in the tumor by the enhanced permeation and retention (EPR) effect. Therefore, as indicated by our data, when exposed to acidic pH, Ce6-pHLIPss-AuNRs can achieve synergistic PTT/PDT bimodality for cancer treatment.

Highly Stable Positively Charged Dendron-Encapsulated Gold Nanoparticles

We report the development of a novel cationic dendron (TAG1-PCD) and a positively charged gold nanoparticle-dendron conjugate (PCD-AuNP). TAG1-PCD was designed by considering the reactivity, hydrophilicity, and cationic nature that is required to yield a stable gold conjugate in aqueous media. The PCD-AuNPs, nominally 10 nm in size, were synthesized by reduction of chloroauric acid in the presence of TAG1-PCD. The physicochemical properties of PCD-AuNPs were characterized by dynamic light scattering, transmission electron microscopy, UV-vis absorbance, and X-ray photoelectron spectroscopy for investigation of size distribution, shape uniformity, surface plasmon resonance bands, and Au-dendron bonding. Asymmetric-flow field flow fractionation was employed to confirm the in situ size, purity, and surface properties of the PCD-AuNPs. Additionally, the stability of PCD-AuNPs was systematically evaluated with respect to shelf life determination, stability in biological media and a wide range of pH values, chemical resistance against cyanide, redispersibility from lyophilized state, and stability at temperatures relevant to biological systems. Dose dependent cell viability was evaluated in vitro using the human lung epithelial cell line A549 and a monkey kidney Vero cell line. Observations from in vitro studies are discussed. Overall, the investigation confirmed the successful development of stable PCD-AuNPs with excellent stability in biologically relevant test media containing proteins and electrolytes, and with a shelf life exceeding 6 months. The excellent aqueous stability and apparent lack of toxicity for this conjugate enhances its potential use as a test material for investigating interactions between positively charged NPs and biocellular and biomolecular systems, or as a vehicle for drug delivery.

Preparation of Colloidal Gold Immunochromatographic Strip for Detection of Paragonimiasis skrjabini

BackgroundParagonimiasis is a food-borne trematodiasis, a serious public health issue and a neglected tropical disease. Paragonimus skrjabini is a unique species found in China. Unlike paragonimiasis westermani, it is nearly impossible to make a definitive diagnosis for paragonimiasis skrjabini by finding eggs in sputum or feces. Immunodiagnosis is the best choice to detect paragonimiasis skrjabini. There is an urgent need to develop a novel, rapid and simple immunoassay for large-scale screening patients in endemic areas.Methodology/Principal FindingsTo develop a rapid, simple immunodiagnostic assay for paragonimiasis, rabbit anti-human IgG was conjugated to colloidal gold particles and used to detect antibodies in the sera of paragonimiasis patients. The synthesis and identification of colloidal gold particles and antibody-colloidal gold conjugates were performed. The size of colloidal gold particles was examined using a transmission electron microscope (TEM). The average diameter of colloidal gold particles was 17.46 nm with a range of 14.32–21.80 nm according to the TEM images. The formation of antibody-colloidal gold conjugates was monitored by UV/Vis spectroscopy. Excretory-secretory (ES) antigen of Paragonimus skrjabini was coated on nitrocellulose membrane as the capture line. Recombinant Staphylococcus protein A was used to prepare the control line. This rapid gold immunochromatographic strip was assembled in regular sequence through different accessories sticked on PVC board. The relative sensitivity and specificity of the strip was 94.4% (51/54) and 94.1% (32/34) respectively using ELISA as the standard method. Its stability and reproducibility were quite excellent after storage of the strip at 4°C for 6 months.Conclusions/SignificanceImmunochromatographic strip prepared in this study can be used in a rapid one-step immunochromatographic assay, which is instantaneous and convenient.

A rapid quantitative lateral flow test for the detection of total aflatoxins in maize

IntroductionAflatoxins (B1, B2, G1 and G2) are toxic secondary metabolites of, primarily, the fungi Aspergillus flavus and Aspergillus parasiticus. Aflatoxins are potent carcinogens which cause liver diseases in livestock, domestic animals and humans throughout the world. Regulations for aflatoxins in commodities and food exist in more than 100 countries. A rapid quantitative lateral flow test, namely “Agrastrip”, was developed to measure total aflatoxin levels in maize. The test is developed based on the competitive immuno-chromatography principle. The total assay time for a sample extract is approximately 5 minutes. The results from the test are comparable to the reference HPLC or LC-MS/MS methods. ObjectiveTo validate the quantitative AgraStrip total aflatoxin test in maize samples. MethodsAflatoxins were extracted from ground maize sample using 70% methanol. To start an assay, buffer was added into a microwell to dissolve the pre-coated antibody-colloidal gold conjugate particle in the microwell. This was followed by mixing the dissolved conjugate with sample extract. A test strip was then placed into the mixture in microwell and incubated in a heat block incubator for 3 minutes. The test line zone in the test strip is able to capture the conjugate and form a visible line. The concentration of aflatoxins present in the sample is inversely proportional to the colour intensity of the test line. A line is always visible in positive control zone regardless of the presence of aflatoxins. The test strip is analyzed by the AgraVision® Reader and concentration of aflatoxins is determined. Results & DiscussionThe test was able to measure aflatoxins in the range of 5–100μg/kg. Limit of detection for the test kit on corn sample was 3.6μg/kg. Accuracy and precision of the test on maize sample naturally contaminated with aflatoxins were comparable to the results form reference HPLC or LC-MS/MS methods. The shelf-life of the test kit was 12 months. ConclusionAgrastrip total aflatoxin test is able to screen total aflatoxins in maize samples in its quantitation range.

English

Plasmodium falciparum (Pf) has been found to be the deadliest of all the known species of the parasite capable of infecting humans; this is because it is capable of causing severe cerebral tissue damage. This study was carried out to demonstrate the parasite in the host blood in vitro through immunogold labeling using antibodies against Plasmodium falciparum histidine rich protein 2 (HRP 2); a major metabolite released during the cause of the parasite infection and feeding in the erythrocyte. 12 known Pf positive samples were obtained from across the six geopolitical zones of Nigeria and were further characterized by Geimsa thick and thin film for parasite identification parasite count expressed as parasites/µl of blood. An average of 400 parasites/µl of blood was obtained in each of the samples used for this study. Pf-HRP 2 antibody was conjugated to freshly prepared colloidal gold of particle size 40 nm. The conjugation process was blocked with bovine serum albumin (BSA) and the conjugate itself preserved by 1% glycerol and 0.01% sodium azide. The parasite count was titrated against the Pf-HRP 2 gold conjugate and was analyzed under the light microscope with a fluorescent filter. Reactivity and specificity of Pf-HRP 2 gold conjugate was found to be highly specific and gave direct identification of the erythrocytes infected with the parasite. A good contrast was also obtained between uninfected erythrocytes, parasite and the infected erythrocytes. Keywords : Plasmodium falciparum , malaria, fluorescent microscope, HRP 2, antibody, gold conjugates.

In vivo and in vitro toxicity of nanogold conjugated snake venom protein toxin GNP-NKCT1

Research on nanoparticles has created interest among the biomedical scientists. Nanoparticle conjugation aims to target drug delivery, increase drug efficacy and imaging for better diagnosis. Toxicity profile of the nanoconjugated molecules has not been studied well. In this communication, the toxicity profile of snake venom cytotoxin (NKCT1), an antileukemic protein toxin, was evaluated after its conjugation with gold nanoparticle (GNP-NKCT1). Gold nanoparticle conjugation with NKCT1 was done with NaBH4 reduction method. The conjugated product GNP-NKCT1 was found less toxic than NKCT1 on isolated rat lymphocyte, mice peritoneal macrophage, in culture, which was evident from the MTT/Trypan blue assay. Peritoneal mast cell degranulation was in the order of NKCT1 > GNP-NKCT1. The in vitro cardiotoxicity and neurotoxicity were increased in case of NKCT1 than GNP-NKCT1. On isolated kidney tissue, NKCT1 released significant amount of ALP and γ-GT than GNP-NKCT1. Gold nanoconjugation with NKCT1 also reduced the lethal activity in mice. In vivo acute/sub-chronic toxicity studies in mice showed significant increase in molecular markers due to NKCT1 treatment, which was reduced by gold nanoconjugation. Histopathology study showed decreased toxic effect of NKCT1 in kidney tissue after GNP conjugation. The present study confirmed that GNP conjugation significantly decreased the toxicity profile of NKCT1. Further studies are in progress to establish the molecular mechanism of GNP induced toxicity reduction.

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Production of monoclonal antibodies specific for the recombinant viral coat protein of Apple stem grooving virus-citrus isolate and their application for a simple, rapid diagnosis by an immunochromatographic assay

A simple and rapid immunochromatographic assay (ICA) for the diagnosis of Apple stem grooving virus (ASGV) in citrus was developed. Nine lines of monoclonal antibodies (mAbs) were produced by immunizing with a recombinant viral coat protein of ASGV as the antigen. According to the competitive-binding ELISA results, the 9 mAbs comprised 2 paratope groups, A and B. After screening for the most effective combination of mAbs, the two lines from different paratope groups (4A12 from group A and 6N31 from group B) were used to create a colloidal gold conjugate and for the test line, respectively, in ICA test plate preparation. The ICA detection using this test plate was accurate for positive and negative samples, and ASGV was detectable to a dilution of 1:2430 for the infected citrus sample. Furthermore, ICA was more sensitive than ELISA for the detection of ASGV isolates in citrus. The simple and sensitive ICA for ASGV provides a straightforward method for diagnosis by non-experts, including nursery workers and growers.

The performance of thiol-terminated PEG-paclitaxel-conjugated gold nanoparticles

A series of thiol-terminated polyethylene glycol (PEG)-paclitaxel (PTX) derivatives are designed and synthesized to fabricate PTX-conjugated gold nanoparticles (PTX@GNPs) and improve their overall performance. By extending the molecular weight of PEG from 400 to 1000 Da, the optimized water solubility of the conjugate reaches 184 mg/mL, equal to 4.6 × 105 times that of PTX alone (0.4 μg/mL). High drug loading is obtained by eliminating the steric hindrance between PTX molecules on the surface of GNPs. The gold conjugate shows double simultaneous stimulation-induced drug release behavior in the presence of both esterase and high concentrations of glutathione. The synergic release characteristics of this conjugate results in significant performance improvements, including prolonged circulation due to high stability in vivo, targeted release of PTX inside tumor cells, and increased tumor cell killing efficiency. Improving the in vitro properties of the conjugate not only significantly enhances its therapeutic efficacy in a murine liver cancer model, but also allows drug-conjugated gold nanoparticles to be used as a promising nanoprodrug system in the cancer therapeutics.

Development of a Liver‐Targeting Gold–PEG–Galactose Nanoparticle Platform and a Structure–Function Study

For the specific liver parenchymal cell delivery, a series of short heterobifunctional poly(ethylene glycol) (PEG) derivatives containing dimercapto and galactose (Gal) terminals is synthesized for the preparation of gold conjugates. The Gal density on the surface of all gold conjugates can be well controlled and the prepared gold conjugates are stable in various media, even in the presence of serum. For the liver targeting and reflectance imaging applications, the structure–function relationships of this platform, including the influence of the PEG molecular weight and the Gal ligand coverage of hybrid particles on the cytotoxicity and cellular recognition of tumor cells in vitro and on their liver‐targeting ability in small animals, are studied. Biocompatibility results show that HepG2 cells are more sensitive than HeLa cells to gold conjugates. Cellular uptake studies demonstrate that a lower PEG molecular weight, a higher Gal density, or a higher gold concentration can increase the cellular uptake efficiency of these hybrid particles in HepG2 cells when the other parameters are constant. The results reveal the importance of parameter modulation for the design and control of nanoprobes and the gold conjugates with short PEG chains and a high Gal density are a potential vector for active‐targeting therapy.

Competitive immunochromatographic assay for the detection of the organophosphorus pesticide EPN

An immunochromatographic assay (ICA) based on competitive antigen-coated format and colloidal gold label was developed for the detection of the organophosphorus insecticide O-ethyl O-4-nitrophenyl phenylphosphonothioate (EPN). The ICA test strip consisted of a membrane with a detection zone, a sample pad and an absorbent pad. The membrane was separately coated with EPN hapten-OVA conjugate (test line) and anti-mouse IgG (control line). As the competition is between the migrating analyte and the immobilised analyte hapten for the binding sites of the migrating antibody–colloidal gold (Ab–CG) conjugate, this study suggests that the relative migration speed between the two migrating substances is a critically important factor for the sensitive detection by competitive ICA. Based on this criterion, a nitrocellulose (NC) membrane was chosen as the most suitable for EPN ICA. The detection limit of the ICA for EPN standard and EPN spiked into agricultural samples were 10−2 and 5×10−2 µg mL−1, respectively.

Dot immunogold filtration assay (DIGFA) for the rapid detection of specific antibodies against the rat lungwormAngiostrongylus cantonensis(Nematoda: Metastrongyloidea) using purified 31-kDa antigen

A rapid dot immunogold filtration assay (DIGFA) was adopted for specific immunodiagnosis of human cerebral angiostrongyliasis, using purified 31-kDa glycoprotein specific to Angiostrongylus cantonensis as diagnostic antigen and protein A colloidal gold conjugate as antigen-antibody detector. A total of 59 serum samples were assayed - 11 samples from clinically diagnosed patients with detectable A. cantonensis-specific antibody in immunoblotting; 23 samples from patients with other related parasitic diseases, i.e. gnathostomiasis (n=8), cysticercosis (n=5), toxocariasis (n=2), filariasis (n=4), paragonimiasis (n=2) and malaria (n=2); and 25 samples from normal healthy subjects. The sensitivity and specificity of DIGFA to detect anti-A. cantonensis specific antibodies in serologically confirmed angiostrongyliasis cases, were both 100%. No positive DIGFA was observed in cases with other parasitic diseases, and the healthy control subjects. The 3-min DIGFA is as sensitive and specific as the 3-h immunoblot test in angiostrongyliasis confirmed cases that revealed a 31-kDa reactive band. The gold-based DIGFA is more rapid and easier to perform than the traditional enzyme-linked immunosorbent assay (ELISA). The test utilizing purified A. cantonensis antigen is reliable and reproducible for specific immunodiagnosis of human infection with A. cantonensis - thus can be applied as an additional routine test for clinical diagnostic support. Large-scale sero-epidemiological studies in endemic communities in north-east Thailand are under way to evaluate its usefulness under field conditions.

Synthesis and Evaluation of Polymeric Gold Glyco-Conjugates as Anti-Cancer Agents

The antitumor activity of organo-gold compounds is a focus of research from the past two decades. A variety of gold stabilizing ligands such as vitamins and xanthanes have been prepared and explored for their 'chelating effect' as well as for their antitumor activity. Dithiocarbamates (DTC) compounds and their metallic conjugates have been well explored for their antiproliferative activities. In this study, glycopolymer based DTC-conjugates are prepared by reversible addition-fragmentation chain transfer polymerization (RAFT) and subsequently modified with gold(I) phosphine. These polymer-DTC derivatives and their gold compounds are tested for their in vitro toxicity in both normal and cancer cell lines. The Au(I) phosphine conjugated cationic glycopolymers of 10 kDa and 30 kDa are evaluated for their cytotoxicity profiles using MTT assay. Au(I) compounds are well-known for their mitochondrial toxicity, hence hypoxic cell lines bearing unusually enlarged mitochondria are subjected to these anticancer compounds. It is concluded that these polymeric DTC derivatives and their gold conjugates indeed show higher accumulation as well as cytotoxicity to cancer cells under hypoxic conditions in comparison to the normoxic ones. Hypoxic MCF-7 cells showed significant sensitivity toward the low molecular weight (10 kDa) glycopolymer-Au(I) complexes.

Colloidal Dye Immunobinding Assay for Detection of Trypanosoma evansi Antibodies in Animals

Colloidal dye immunobinding assay (CDIA) was developed for detection of circulatory antibodies of Trypanosoma evansi in the sera of domestic animals. Specific antibodies in the serum were captured by T. evansi antigen spotted on to nitro cellulose membrane mounted on a flow through device. The bound antibodies were visualised by the addition of protein A colloidal gold conjugate which imparts pink colour to the membrane. The optimum concentrations of antigen, test serum and colloidal dye protein A conjugate in the assay were standardized. The efficacy of CDIA was determined by comparing with the results of indirect ELISA. CDIA could detect T. evansi antibodies in 261 ﴾cattle (n=92), buffaloes (n=132) and sheep (n=37) ﴿ serum samples out of 290 ﴾cattle (n=100), buffaloes (n=140) and sheep (n=50) ﴿ indirect ELISA positive samples. All the 22 samples found positive for T. evansi infection by Wet blood film (WBF) examination were also found to be positive by CDIA. The diagnostic sensitivity and diagnostic specificity of CDIA were recorded as 90.00 and 100 per cent, respectively. The CDIA developed in the present study for detection of T. evansi infection in domestic animals is simple to perform, rapid, could potentially applied in all host species, economical and suitable for a wide variety of field applications without any sophisticated instrumentation.

Production of antibody labeled gold nanoparticles for influenza virus H5N1 diagnosis kit development

Preparation of colloidal gold conjugated antibodies specific for influenza A/H5N1 and its use in developing a virus A/H5N1 rapid diagnostic kit is presented. Colloidal gold nanoparticles (AuNPs) were prepared through citrate reduction. Single chain antibodies specific to H5N1 (scFv7 and scFv24) were produced using pTI2 + vector and E. coli strain HB2151. These antibodies were purified by affinity chromatography technique employing HiTrap Chelating HP columns pre-charged with Ni2 + . The method for preparation of antibody–colloidal gold conjugate was based on electrostatic force binding antibody with colloidal gold. The effect of factors such as pH and concentration of antibody has been quantitatively analyzed using spectroscopic methods after adding 1 wt% NaCl which induced AuNP aggregation. The morphological study by scanning electron microscopy (SEM) showed that the average size of the spherical AuNPs was 23 nm with uniform sizes. The spectroscopic properties of colloidal AuNPs showed the typical surface plasmon resonance band at 523 nm in UV-visible spectrum. The optimal pH of conjugated colloidal gold was found between 8.0 and 10.0. The activity of synthesized antibody labeled AuNPs for detection of H5N1 flu virus was checked by dot blot immunological method. The results confirmed the ability in detection of the A/H5N1 virus of the prepared antibody labeled gold particles and opened up the possibility of using them in manufacturing rapid detection kit for this virus.

Immuno electron microscopy – tracking the sub‐cellular localization of antigens in high resolution

AbstractPurpose Any antigen to which an antibody can be generated can be localized, at sub‐cellular level and viewed under very high magnification by immuno‐electron microscopy (immune‐EM). The applications of EM immunocytochemistry are very wide ranging.Methods A particular antigen is localized by primary antibody (monoclonal or polyclonal) within a tissue. The primary antibody is visualized using a secondary antibody, the gold conjugates of 5 or 10nm size. The gold particles are opaque and can be seen under an electron microscope. The antibody can be used on pre‐embedded or post embedded tissue. In the pre‐embedding method, a fresh thin piece of tissue is treated with primary and secondary antibodies. In the post‐embedding method, primary and secondary antibodies are used on ultrathin sections. The immunocytochemistry is also carried out on cryosection. Double immune labeling is also carried out by some authors.Results A number of extracellular matrix proteins and non‐collagenous glycoproteins, such as collagen, elastin, fibronectin, laminin, keratan sulphate proteoglycan, BKS1, chondroitin sulphate, keratoepithilin have been demonstrated by the immuno‐EM procedure. A number of cellular proteins such as keratoepithelin and actin were also shown by immuno‐EM.Conclusion Immuno‐EM is a very powerful technique since it combines the specificity and flexibility of immunocytochemistry with the resolution at high magnification of electron microscopy. Acknowledgement: Supported by National Plan for Science and Technology, KSU, Riyadh.

DNA–Gold Conjugates: Formation of 1D and 2D Gold Nanoparticle Arrays by Divalent DNA–Gold Nanoparticle Conjugates (Small 15/2012)

DNA–Gold Conjugates: Formation of 1D and 2D Gold Nanoparticle Arrays by Divalent DNA–Gold Nanoparticle Conjugates (Small 15/2012)