Alterations in Plasma Lipid Profile before and after Surgical Removal of Soft Tissue Sarcoma.

Soft tissue sarcoma (STS) is a relatively rare malignancy, accounting for about 1% of all adult cancers. It is known to have more than 70 subtypes. Its rarity, coupled with its various subtypes, makes early diagnosis challenging. The current standard treatment for STS is surgical removal. To identify the prognosis and pathophysiology of STS, we conducted untargeted metabolic profiling on pre-operative and post-operative plasma samples from 24 STS patients who underwent surgical tumor removal. Profiling was conducted using ultra-high-performance liquid chromatography-quadrupole time-of-flight/mass spectrometry. Thirty-nine putative metabolites, including phospholipids and acyl-carnitines were identified, indicating changes in lipid metabolism. Phospholipids exhibited an increase in the post-operative samples, while acyl-carnitines showed a decrease. Notably, the levels of pre-operative lysophosphatidylcholine (LPC) O-18:0 and LPC O-16:2 were significantly lower in patients who experienced recurrence after surgery compared to those who did not. Metabolic profiling may identify aggressive tumors that are susceptible to lipid synthase inhibitors. We believe that these findings could contribute to the elucidation of the pathophysiology of STS and the development of further metabolic studies in this rare malignancy.

Simultaneous isolation and enumeration of virulent Vibrio cholerae and Vibrio vulnificus using an advanced MPN-PCR method.

Vibrio cholerae and Vibrio vulnificus are critical foodborne pathogens that need to be intensively controlled for their infection due to the intake and distribution of seafood, especially raw oysters. For this reason, various methods have already been developed for the detection and enumeration of these bacteria. The most probable number (MPN)-PCR (polymerase chain reaction) method is commonly used with the selective-differential medium for the efficiency and convenience of cell enumeration. One of the most frequently used for detecting Vibrio spp. is thiosulfate-citrate-bile salts-sucrose (TCBS) agar. But this selective-differential medium can fail to distinguish between V. cholerae, V. vulnificus, and Vibrio alginolyticus. For this reason, the conventional MPN-PCR method with TCBS medium for the detection of Vibrio spp. has a problem with processing PCR two times. This study suggests a simple and minimized detection method using one-time PCR and non-NaCl Luria-Bertani (LB-0) medium culture. This detection method is based on the difference in salt requirement between V. cholerae and V. vulnificus. Employing the developed methodology, the simultaneous cell enumeration of V. cholerae and V. vulnificus can be possible at a low cost. Furthermore, this study proposes a new specific primer to detect virulence-related genes from V. cholerae and V. vulnificus. This advanced MPN-PCR method was verified using bioaccumulated pacific oysters (Crassostrea gigas) by V. cholerae and V. vulnificus.

Effectiveness of depuration of Pacific Oyster (Crassostrea gigas): removal of bioaccumulated Vibrio vulnificus by UV-treatment.

The present study aimed to evaluate the efficacy of a depuration system equipped with UV-irradiation to control Vibrio vulnificus infection such as septicemia (or sepsis) using alive oysters. After 6 h of bioaccumulation of V. vulnificus, Pacific oyster Crassostrea gigas were found to be contaminated by > 8.0 log MPN/g of V. vulnificus cells. After 60 h of depuration, the V. vulnificus cell number significantly decreased to < 4.0 log MPN/g. The present depuration process meets the standard effectiveness in reducing V. vulnificus cells by > 3.52 log and < 30 MPN/g as recommended by the National Shellfish Sanitization Procedure Molluscan Shellfish Control guidelines. Furthermore, no significant changes in pH value and glycogen content indicate that the depuration process did not affect the freshness and quality of the oyster samples. The present study could help control any potential infection associated with the consumption of raw oysters without losing their quality.

Streptomycin mediated biofilm inhibition and suppression of virulence properties in Pseudomonas aeruginosa PAO1.

Pseudomonas aeruginosa is known as an opportunistic pathogen whose one of the antibiotic resistance mechanisms includes biofilm formation and virulence factor production. The present study showed that the sub-minimum inhibitory concentration (sub-MIC) of streptomycin inhibited the formation of biofilm and eradicated the established mature biofilm. Streptomycin at sub-MIC was also capable of inhibiting biofilm formation on the urinary catheters. In addition, the sub-MIC of streptomycin attenuated the bacterial virulence properties as confirmed by both phenotypic and gene expression studies. The optimal conditions for streptomycin to perform anti-biofilm and anti-virulence activities were proposed as alkaline TSB media (pH 7.9) at 35 °C. However, sub-MIC of streptomycin also exhibited a comparative anti-biofilm efficacy in LB media at similar pH level and temperature. Furthermore, this condition also improved the biofilm inhibition and eradication properties of streptomycin, tobramycin and tetracycline towards the biofilm formed by a clinical isolate of P. aeruginosa. Findings from the present study provide an important insight for further studies on the mechanisms of biofilm inhibition and dispersion of pre-existing biofilm by streptomycin as well as tobramycin and tetracycline under a specific culture environment.

AERONET-based nonspherical dust optical models and effects on the VIIRS Deep Blue/SOAR over-water aerosol product.

Aerosol Robotic Network (AERONET)-based nonspherical dust optical models are developed and applied to the Satellite Ocean Aerosol Retrieval (SOAR) algorithm as part of the Version 1 Visible Infrared Imaging Radiometer Suite (VIIRS) NASA 'Deep Blue' aerosol data product suite. The optical models are created using Version 2 AERONET inversion data at six distinct sites influenced frequently by dust aerosols from different source regions. The same spheroid shape distribution as used in the AERONET inversion algorithm is assumed to account for the nonspherical characteristics of mineral dust, which ensures the consistency between the bulk scattering properties of the developed optical models with the AERONET-retrieved microphysical and optical properties. For the Version 1 SOAR aerosol product, the dust optical models representative for Capo Verde site are used, considering the strong influence of Saharan dust over the global ocean in terms of amount and spatial coverage. Comparisons of the VIIRS-retrieved aerosol optical properties against AERONET direct-Sun observations at three island/coastal sites suggest that the use of nonspherical dust optical models significantly improves the retrievals of aerosol optical depth (AOD) and Ångström exponent by mitigating the well-known artifact of scattering angle dependence of the variables observed when incorrectly assuming spherical dust. The resulting removal of these artifacts results in a more natural spatial pattern of AOD along the transport path of Saharan dust to the Atlantic Ocean; i.e., AOD decreases with increasing distance transported, whereas the spherical assumption leads to a strong wave pattern due to the spurious scattering angle dependence of AOD.

Real-time monitoring of cisplatin cytotoxicity on three-dimensional spheroid tumor cells.

Three-dimensional (3D) cell cultivation is a powerful technique for monitoring and understanding diverse cellular mechanisms in developmental cancer and neuronal biology, tissue engineering, and drug development. 3D systems could relate better to in vivo models than two-dimensional (2D) cultures. Several factors, such as cell type, survival rate, proliferation rate, and gene and protein expression patterns, determine whether a particular cell line can be adapted to a 3D system. The 3D system may overcome some of the limitations of 2D cultures in terms of cell-cell communication and cell networks, which are essential for understanding differentiation, structural organization, shape, and extended connections with other cells or organs. Here, the effect of the anticancer drug cisplatin, also known as cis-diamminedichloroplatinum (II) or CDDP, on adenosine triphosphate (ATP) generation was investigated using 3D spheroid-forming cells and real-time monitoring for 7 days. First, 12 cell lines were screened for their ability to form 3D spheroids: prostate (DU145), testis (F9), embryonic fibroblast (NIH-3T3), muscle (C2C12), embryonic kidney (293T), neuroblastoma (SH-SY5Y), adenocarcinomic alveolar basal epithelial cell (A549), cervical cancer (HeLa), HeLa contaminant (HEp2), pituitary epithelial-like cell (GH3), embryonic cell (PA317), and osteosarcoma (U-2OS) cells. Of these, eight cell lines were selected: NIH-3T3, C2C12, 293T, SH-SY5Y, A549, HeLa, PA317, and U-2OS; and five underwent real-time monitoring of CDDP cytotoxicity: HeLa, A549, 293T, SH-SY5Y, and U-2OS. ATP generation was blocked 1 day after addition of 50 µM CDDP, but cytotoxicity in HeLa, A549, SH-SY5Y, and U-2OS cells could be visualized only 4 days after treatment. In 293T cells, CDDP failed to kill entirely the culture and ATP generation was only partially blocked after 1 day. This suggests potential CDDP resistance of 293T cells or metabolic clearance of the drug. Real-time monitoring and ATP measurements directly confirmed the cytotoxicity of CDDP, indicating that CDDP may interfere with mitochondrial activity.

Evaluating the Height of Biomass Burning Smoke Aerosols Retrieved from Synergistic Use of Multiple Satellite Sensors over Southeast Asia.

This study evaluates the height of biomass burning smoke aerosols retrieved from a combined use of Visible Infrared Imaging Radiometer Suite (VIIRS), Ozone Mapping and Profiler Suite (OMPS), and Cloud-Aerosol Lidar with Orthogonal Polarization (CALIOP) observations. The retrieved heights are compared against spaceborne and ground-based lidar measurements during the peak biomass burning season (March and April) over Southeast Asia from 2013 to 2015. Based on the comparison against CALIOP, a quality assurance (QA) procedure is developed. It is found that 74% (81-84%) of the retrieved heights fall within 1 km of CALIOP observations for unfiltered (QA-filtered) data, with root-mean-square error (RMSE) of 1.1 km (0.8-1.0 km). Eliminating the requirement for CALIOP observations from the retrieval process significantly increases the temporal coverage with only a slight decrease in the retrieval accuracy; for best QA data, 64% of data fall within 1 km of CALIOP observations with RMSE of 1.1 km. When compared with Micro-Pulse Lidar Network (MPLNET) measurements deployed at Doi Ang Khang, Thailand, the retrieved heights show RMSE of 1.7 km (1.1 km) for unfiltered (QA-filtered) data for the complete algorithm, and 0.9 km (0.8 km) for the simplified algorithm.

The isolation and characterization of simultaneous saccharification and fermentation microorganisms for Laminaria japonica utilization.

Brown seaweed contains various carbohydrates, such as alginate, laminaran, and mannitol, therefore ethanol fermentation was attempted with Nuruk and a mixed culture that included Laminaria japonica. Nuruk is used to make Korean traditional alcohol. In the research, four microorganisms that produced ethanol and had the ability to achieve alginate degradation were obtained on the L. japonica medium. Nuruk 4 was found to produce a better result than the other tested microorganisms, and the optimal substrate for ethanol production was found to be mannitol (2.59 g/L at 96 h). Nuruk 4 was more than three times better compared with Candida tropicalis in regards to ethanol production. When alginate lyase activity occurred, it appeared as a clear zone around Nuruk 3. The maximal ethanol production yield conditions were comprised of Nuruk 3 and 4 on the anaerobic culture. In this case, 2.0 g/L of ethanol were efficiently produced under the same conditions.

Factors Associated with Depressive Symptom in Chronic Obstructive Pulmonary Disease Based on the Third (2005) and Fourth (2008) Korea National Health and Nutritional Examination Survey (KNHANES III, IV).

BACKGROUND: Depressive symptoms accompanied by chronic obstructive pulmonary disease (COPD) can be influenced by socioeconomic status, associated chronic diseases and the current smoking status. This study was conducted to assess factors that are associated with depressive symptoms accompanied by COPD, using the data obtained from the Korea National Health and Nutritional Survey (KNHANES) conducted in 2005 and 2008. METHODS: From the third (2005) and the fourth (2008) KNHANES, 407 (0.9%) with physician-diagnosed COPD were selected. Of the 407 subjects, only 180 (0.4%) who reported having depressive symptoms were included in this study. The associations of depressive symptoms with socioeconomic status, associated chronic diseases and smoking status were investigated. RESULTS: Of the total 180 subjects, 45 (25%) had depressive symptoms. There were 102 males (55%) and 78 females (45%) with a slight predilection for males. In multivariate analysis, significant predictors of depressive symptoms were dependent activities of daily living (odds ratio [OR], 2.42; 95% confidence interval [CI], 2.06 to 2.84) and association with number of chronic diseases (OR of one, two, and three, 1.40, 1.72, 2.60; 95% CI of one, two, and three, 1.20 to 1.63,1.41 to 2.10,1.99 to 3.39). CONCLUSION: This study provides the basis for managing COPD patients in a clinical setting by understanding the number and characteristics of COPD patients with depressive symptoms. The results of this study suggest that primary physicians should manage COPD patients with consideration of risk factors for depressive symptom.

Heterologous expression of an alginate lyase from Streptomyces sp. ALG-5 in Escherichia coli and its use for preparation of the magnetic nanoparticle-immobilized enzymes.

The marine alginate lyase from Streptomyces sp. ALG-5, which specifically degrades poly-G block of alginate, was functionally expressed as a His-tagged form with an Escherichia coli expression system. The recombinant alginate lyase expressed with pColdI at 15 °C exhibited the highest alginate-degrading activity. The recombinant alginate lyase was efficiently immobilized onto two types of magnetic nanoparticles, superparamagnetic iron oxide nanoparticle, and hybrid magnetic silica nanoparticle, based on the affinity between His-tag and Ni(2+) that displayed on the surfaces of nanoparticles. An alginate oligosaccharide mixture consisting of dimer and trimer was prepared by the immobilized alginate lyase. The immobilized enzymes were re-used repeatedly more than 10 times after magnetic separation.

Serotype distribution and beta-lactam resistance in Haemophilus influenzae isolated from patients with respiratory infections in Korea.

Haemophilus influenzae is a frequent causative bacterial pathogen of respiratory tract infections. Resistance to beta-lactam antibiotics has been a significant clinical problem in treatment for H. influenzae respiratory infections. This study describes the serotype, antibiotic resistance and distribution of TEM-1 or ROB-1 beta-lactamase in H. influenzae isolates from local private hospitals from 2002 to 2004. Among the 100 H. influenzae respiratory isolates, only 7% were identified as serotypes a, b, e, and f, with the remaining 93% being nontypeable. Resistance to ampicillin, cefaclor, and tetracycline was 57%, 46%, and 16%, respectively. All strains were susceptible to azithromycin and ciprofloxacin, whereas amoxicillin/clavulanate, cefotaxime, and imipenem exhibited reduced susceptibilities of 99%, 99%, and 91%, respectively. All 57 ampicillin-resistant strains (minimum inhibitory concentration, MIC>or=4 microg/ml) were beta-lactamase-positive and possessed the TEM-1 type beta-lactamase. One beta-lactamase-positive amoxicillin/clavulanate-resistant isolate that was resistant to ampicillin (MIC>128 microg/ml) had the TEM-1 type beta-lactamase and not susceptible to cefaclor and cefotaxime. Analysis of penicillin binding protein 3 revealed six residues (Asp-350, Met-377, Ala-502, Asn-526, Val-547, and Asn-569) that were substituted by Asn, Ile, Val, Lys, Ile, and Ser, respectively.

Antimicrobial resistance in Haemophilus influenzae respiratory tract isolates in Korea: results of a nationwide acute respiratory infections surveillance.

Antimicrobial susceptibility patterns and beta-lactam resistance mechanisms of 544 Haemophilus influenzae isolates through the nationwide Acute Respiratory Infections Surveillance (ARIS) network in Korea during 2005 and 2006 were determined. Resistance to ampicillin was 58.5%, followed by resistance to cefuroxime (23.3%), clarithromycin (18.7%), cefaclor (17.0%), amoxicillin-clavulanate (10.4%), and chloramphenicol (8.1%). Levofloxacin and cefotaxime were the most active agents tested in this study. beta-Lactamase production (52.4%) was the main mechanism of ampicillin resistance, affecting 96.1% of TEM-1-type beta-lactamase. According to their beta-lactam resistance mechanisms, all isolates were classified into the following groups: beta-lactamase-negative, ampicillin-sensitive (BLNAS) strains (n = 224; 41.5%); beta-lactamase-positive, ampicillin-resistant (BLPAR) strains (n = 255; 47.2%); beta-lactamase-negative, ampicillin-resistant (BLNAR) strains (n = 33; 6.1%); and beta-lactamase-positive, amoxicillin-clavulanate-resistant (BLPACR) strains (n = 28; 5.2%). Among the BLNAR and BLPACR strains, there were various patterns of multiple-amino-acid substitutions in penicillin-binding protein 3. Particularly, among BLNAR, group III isolates, which had three simultaneous substitutions (Met377Ile, Ser385Thr, and Leu389Phe), were identified for the first time in Korea. Three group III strains displayed the highest MIC of cefotaxime (1 to 2 mug/ml). The results indicate the importance of monitoring a changing situation pertaining to the increase and spread of BLNAR and BLPACR strains of H. influenzae for appropriate antibiotic therapy for patients with respiratory tract infections in Korea.

Effective purification of ginsenosides from cultured wild ginseng roots,red ginseng, and white ginseng with macroporous resins.

This study was aimed (i) to develop an effective method for the purification of ginsenosides for industrial use and (ii) to compare the distribution of ginsenosides in cultured wild ginseng roots (adventitious root culture of Panax ginseng) with those of red ginseng (steamed ginseng) and white ginseng (air-dried ginseng). The crude extracts of cultured wild ginseng roots, red ginseng, and white ginseng were obtained by using a 75% ethanol extraction combined with ultrasonication. This was followed sequentially by AB-8 macroporous adsorption chromatography, Amberlite IRA 900 Cl anion-exchange chromatography, and Amberlite XAD16 adsorption chromatography for further purification. The contents of total ginsenosides were increased from 4.1%, 12.1%, and 11.3% in the crude extracts of cultured wild ginseng roots, red ginseng, and white ginseng to 79.4%, 71.7%, and 72.5% in the final products, respectively. HPLC analysis demonstrated that ginsenosides in cultured wild ginseng roots were distributed in a different ratio compared with red ginseng and white ginseng.

Cloning, expression, and characterization of a glycoside hydrolase family 50 beta-agarase from a marine Agarivorans isolate.

The gene for a thermostable beta-agarase from Agarivorans sp. JA-1 was cloned and sequenced. It comprised an open reading frame of 2,988 base pairs, which encode a protein of 109,450 daltons consisting of 995 amino acid residues. A comparison of the entire sequence showed that the enzyme has 98.8% sequence similarities to beta-agarase from Vibrio sp. JT1070, indicating that it belongs to the family glycoside hydrolase (GH)-50. The gene corresponding to a mature protein of 976 amino acids was inserted and expressed in Escherichia coli. The recombinant beta-agarase was purified to homogeneity. It had maximal activity at 40 degrees C and pH 8.0 in the presence of 1 mM NaCl and 1 mM CaCl(2). The enzyme hydrolyzed agarose as well as neoagarohexaose and neoagarotetraose to yield neoagarobiose as the main product. Thus, the enzyme would be useful for the industrial production of neoagarobiose.

Protective effect of puerariae radix on oxidative stress induced by hydrogen peroxide and streptozotocin.

This study evaluated the protective effect of Puerariae radix against the oxidative stress induced by hydrogen peroxide (H2O2) and streptozotocin in vitro and in vivo, respectively. The ethanol extract scavenged intracellular reactive oxygen species (ROS), and 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical, and prevented lipid peroxidation. This radical scavenging activity of the ethanol extract protected the cell viability of Chinese hamster lung fibroblast (V79-4) cells exposed to H2O2. Furthermore, this extract reduced the formation of apoptotic cells induced by H2O2, which was demonstrated by the decreased number of sub G(1) hypo-diploid cells and apoptotic cell body formation. The extract increased the activities of the cellular antioxidant enzymes, superoxide dismutase (SOD) and catalase (CAT). Administration of the extract to the streptozotocin induced diabetic rats decreased the blood glucose levels. The diabetic rats showed low activities of superoxide dismutase and catalase in the liver, and the ethanol extract increased the CAT activity. The increased level of lipid peroxidation in the diabetic rats reverted to near normal levels after being treated with the extract. This study showed that Puerariae radix was effective in the amelioration of diabetes, which may be a consequence of its antioxidant potential.

Establishment of in vitro test system for the evaluation of the estrogenic activities of natural products.

In order to evaluate estrogenic compounds in natural products, an in vitro detection system was established. For this system, the human breast cancer cell line MCF7 was stably transfected using an estrogen responsive chloramphenicol acetyltransferase (CAT) reporter plasmid yielding MCF7/pDsCAT-ERE119-Ad2MLP cells. To test the estrogenic responsiveness of this in vitro assay system, MCF7/pDsCAT-ERE119-Ad2MLP cells were treated with various concentrations of 17beta-estradiol. Treatments of 10(-8) to 10(-12) M 17beta-estradiol revealed significant concentration dependent estrogenic activities compared with ethanol. We used in vitro assay system to detect estrogenic effects in Puerariae radix and Ginseng radix Rubra extracts. Treatment of 500 and 50 microg/ml of Puerariae radix extracts increased the transcriptional activity approximately 4- and 1.5-fold, respectively, compared with the ethanol treatment. Treatment of 500, 50, and 5 microg/ml of Ginseng radix Rubra extracts increased the transcriptional activity approximately 3.2-, 2.7-, and 1.4-fold, respectively, compared with the ethanol treatment. These observations suggest that Puerariae radix and Ginseng radix Rubra extracts have effective estrogenic actions and that they could be developed as estrogenic supplements.

The preventive inhibition of chondroitin sulfate against the CCl4-induced oxidative stress of subcellular level.

Our work in this study was made in the microsomal fraction to evaluate the lipid peroxidation by measuring superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx), and malondialdehyde (MDA) and to elucidate the preventive role of CS in the CCl4-induced oxidative stress. The excessive lipid peroxidation by free radicals derived from CCl4 leads to the condition of oxidative stress which results in the accumulation of MDA. MDA is one of the end-products in the lipid peroxidation process and oxidative stress. MDA, lipid peroxide, produced in this oxidative stress causes various diseases related to aging and hepatotoxicity, etc. Normal cells have a number of enzymatic and nonenzymatic endogenous defense systems to protect themselves from reactive species. The enzymes in the defense systems, for example, are SOD, CAT, and GPx. They quickly eliminate reactive oxygen species (ROS) such as superoxide anion free radical *O2(-), hydrogen peroxide H2O2 and hydroxyl free radical *OH. CS inhibited the accumulation of MDA and the deactivation of SOD, CAT and GPx in the dose-dependent and preventive manner. Our study suggests that CS might be a potential scavenger of free radicals in the oxidative stress originated from the lipid peroxidation of the liver cells of CCl4-treated rats.

Production and secretion of biologically active human granulocyte-macrophage colony stimulating factor in transgenic tomato suspension cultures.

A complementary DNA encoding human granulocyte-macrophage colony stimulating factor (hGM-CSF) was cloned and introduced into tomato (Lycopersicon esculentum cv. Seokwang) using Agrobacterium-mediated transformation. Genomic PCR and Northern blot analysis demonstrated the integration of the construction into the plant nuclear genome and expression of the hGM-CSF in transgenic tomato. The cell suspension culture was established from leaf-derived calli of the transgenic tomato plants transformed with the hGM-CSF gene. Recombinant hGM-CSF was synthesized by the transgenic cell culture and secreted into the growth medium at 45 microg l(-1) after 10 d' cultivation.

Human act and AR1 sequences differentially regulate murine and human D1A dopamine receptor promoters.

The D1A dopamine receptor gene underlies complex transcriptional regulation in order to achieve the tissue-specific expression. Transcription in the D1A genes proceeds from two distinct promoters utilized for the tissue-specific regulation of these genes. Furthermore, analysis of the human D1A dopamine receptor gene has revealed that the region between nucleotides -1173 and -1136 (ActAR1) of the gene might be important for its neural cell-specific expression. To investigate the function of D1A dopamine receptor promoters in the brain cell-specific expression of transgenes, we analyzed the regulatory patterns of two distinct protein-binding regions of ActAR1, i.e., an Act sequence (-1174/-1154) and an AR1 sequence (-1154/-1136), toward murine and human D1A promoters. Transient expression analyses using various chloramphenicol acetyltransferase constructs revealed that Act could not activate murine or human D1A promoters, and that AR1 could effectively stimulate these promoters in a cell type-non-specific manner. Only ActAR1, a combination of Act and AR1, could activate murine and human D1A promoters in a prominent cell type-specific manner. Abundant protein binding to Act was detected by gel mobility shift assay using nuclear extracts from SK-N-MC, NS20Y, OK, and C6 but faint protein binding using nuclear extracts from HepG2. Furthermore, strong protein binding to AR1 was detected using nuclear extracts from SK-N-MC, NS20Y, HepG2 but faint protein binding from C6 extracts and no detectable protein binding from OK extracts. These observations suggest that the tissue-specific expression of the D1A gene is due, at least in part, to the differential expression of these activator proteins that bind to Act and AR1.