Enhanced effect of autologous EVs delivering paclitaxel in pancreatic cancer.

We engineered human pancreatic cancer cell (PANC-1)-derived extracellular vesicles (EVs) by conjugating the functional ligand RGD and magnetic nanoparticles (MNPs) onto EV surfaces (rmExo), for pancreatic cancer therapy. Paclitaxel (PTX) loaded into rmExo (rmExo-PTX) was intravenously injected into xenograft mice prepared using PANC-1 cells, which showed a significant reduction in tumor size compared to the free PTX-treated and control groups. The enhanced therapeutic effect was attributed to the modification of the surface of EVs using RGD, which has affinity for αvß3 that is highly expressed in pancreatic cancer cells. Moreover, autologous EVs seemed to have more benefits in delivering PTX due to an unknown homing property to parent tumor cells, as exemplified by the reduced therapeutic effect of RGD-modified PANC-1 EVs on HT29 xenograft mice and RGD-modified U937 EVs on PANC-1 xenograft mice. The RGD-modified autologous EV vehicles were effective at penetrating and internalizing tumor cells, and eventually regressing the tumors, by mediating spontaneous removal of α-smooth muscle actin and collagen type 1 in the extracellular matrix of xenografts. Our results also identified an important molecule involved in the home-driving properties of PANC-1 EVs, integrin ß3, which was expressed both on PANC-1 cells and the EVs derived from them. Additional therapeutic effect by permanent magnet near tumor xenograft was not observed in this study.

Numerical learning of deep features from drug-exposed cell images to calculate IC50 without staining.

To facilitate rapid determination of cellular viability caused by the inhibitory effect of drugs, numerical deep learning algorithms was used for unlabeled cell culture images captured by a light microscope as input. In this study, A549, HEK293, and NCI-H1975 cells were cultured, each of which have different molecular shapes and levels of drug responsiveness to doxorubicin (DOX). The microscopic images of these cells following exposure to various concentrations of DOX were trained with the measured value of cell viability using a colorimetric cell proliferation assay. Convolutional neural network (CNN) models for the study cells were constructed using augmented image data; the predicted cell viability using CNN models was compared to the cell viability measured by colorimetric assay. The linear relationship coefficient (r2) between measured and predicted cell viability was determined as 0.94-0.95 for the three cell types. In addition, the measured and predicted IC50 values were not statistically different. When drug responsiveness was estimated using allogenic models that were trained with a different cell type, the correlation coefficient decreased to 0.004085-0.8643. Our models could be applied to label-free cells to conduct rapid and large-scale tests while minimizing cost and labor, such as high-throughput screening for drug responsiveness.

Reactive Oxygen Species-Responsive Miktoarm Amphiphile for Triggered Intracellular Release of Anti-Cancer Therapeutics.

Reactive oxygen species (ROS)-responsive nanocarriers have received considerable research attention as putative cancer treatments because their tumor cell targets have high ROS levels. Here, we synthesized a miktoarm amphiphile of dithioketal-linked ditocopheryl polyethylene glycol (DTTP) by introducing ROS-cleavable thioketal groups as linkers between the hydrophilic and hydrophobic moieties. We used the product as a carrier for the controlled release of doxorubicin (DOX). DTTP has a critical micelle concentration (CMC) as low as 1.55 µg/mL (4.18 × 10-4 mM), encapsulation efficiency as high as 43.6 ± 0.23% and 14.6 nm particle size. The DTTP micelles were very responsive to ROS and released their DOX loads in a controlled manner. The tocopheryl derivates linked to DTTP generated ROS and added to the intracellular ROS in MCF-7 cancer cells but not in HEK-293 normal cells. In vitro cytotoxicity assays demonstrated that DOX-encapsulated DTTP micelles displayed strong antitumor activity but only slightly increased apoptosis in normal cells. This ROS-triggered, self-accelerating drug release device has high therapeutic efficacy and could be a practical new strategy for the clinical application of ROS-responsive drug delivery systems.

Screening of important metabolites and KRAS genotypes in colon cancer using secondary ion mass spectrometry.

Time-of-flight secondary ion mass spectrometry (TOF-SIMS) is an imaging-based analytical technique that can characterize the surfaces of biomaterials. We used TOF-SIMS to identify important metabolites and oncogenic KRAS mutation expressed in human colorectal cancer (CRC). We obtained 540 TOF-SIMS spectra from 180 tissue samples by scanning cryo-sections and selected discriminatory molecules using the support vector machine (SVM) algorithm. Each TOF-SIMS spectrum contained nearly 860,000 ion profiles and hundreds of spectra were analyzed; therefore, reducing the dimensionality of the original data was necessary. We performed principal component analysis after preprocessing the spectral data, and the principal components (20) of each spectrum were used as the inputs of the SVM algorithm using the R package. The performance of the algorithm was evaluated using the receiver operating characteristic (ROC) area under the curve (AUC) (0.9297). Spectral peaks (m/z) corresponding to discriminatory molecules used to classify normal and tumor samples were selected according to p-value and were assigned to arginine, α-tocopherol, and fragments of glycerophosphocholine. Pathway analysis using these discriminatory molecules showed that they were involved in gastrointestinal disease and organismal abnormalities. In addition, spectra were classified according to the expression of KRAS somatic mutation, with 0.9921 AUC. Taken together, TOF-SIMS efficiently and simultaneously screened metabolite biomarkers and performed KRAS genotyping. In addition, a machine learning algorithm was provided as a diagnostic tool applied to spectral data acquired from clinical samples prepared as frozen tissue slides, which are commonly used in a variety of biomedical tests.

CD5L as an Extracellular Vesicle-Derived Biomarker for Liquid Biopsy of Lung Cancer.

Cancer screening and diagnosis can be achieved by analyzing specific molecules within serum-derived extracellular vesicles (EVs). This study sought to profile EV-derived proteins to identify potential lung cancer biomarkers. EVs were isolated from 80 serum samples from healthy individuals and cancer patients via polyethylene glycol (PEG)-based precipitation and immunoaffinity separation using antibodies against CD9, CD63, CD81, and EpCAM. Proteomic analysis was performed using 2-D gel electrophoresis and matrix-assisted laser desorption ionization-time-of-flight mass spectrometry (MALDI-TOF MS). The expression of proteins that were differentially upregulated in the EVs or tissue of lung cancer samples was validated by Western blotting. The area under the curve (AUC) was calculated to assess the predictability of each differentially expressed protein (DEP) for lung cancer. A total of 55 upregulated protein spots were selected, seven of which (CD5L, CLEC3B, ITIH4, SERFINF1, SAA4, SERFINC1, and C20ORF3) were found to be expressed at high levels in patient-derived EVs by Western blotting. Meanwhile, only the expression of EV CD5L correlated with that in cancer tissues. CD5L also demonstrated the highest AUC value (0.943) and was found to be the core regulator in a pathway related to cell dysfunction. Cumulatively, these results show that EV-derived CD5L may represent a potential biomarker-detected via a liquid biopsy-for the noninvasive diagnosis of lung cancer.

Work Schedule Characteristics, Missed Nursing Care, and Organizational Commitment Among Hospital Nurses in Korea.

PURPOSE: This study describes Korean nurses' work schedule characteristics and identifies their components to investigate associations of work schedule components with missed nursing care and organizational commitment. DESIGN: This cross-sectional secondary analysis used survey data of 1,057 nurses in 111 units at six hospitals in South Korea. Data were collected between April 2017 and March 2018. METHODS: A self-administered survey, including seven work schedule characteristic items, the Korean version of the MISSCARE Survey, and the Korean version of the Organizational Commitment Questionnaire, was employed. To construct independent components of work schedule characteristics, a principal component analysis was performed. The associations of work schedule components with missed nursing care and organizational commitment were analyzed using multiple linear regression models with generalized estimating equation methods. FINDINGS: The average number of daily work hours was 9.7. Nearly half of the study population worked while sick once or more per month. The two components of nurses' work schedule characteristics were "long work hours" and "lack of rest," and these components showed variations between units. Unhealthy work schedule components were linked to frequently missed nursing care and decreased organizational commitment. CONCLUSIONS: This study showed that proper work hours and adequate rest are important to reduce missed nursing care tasks and enhance organizational commitment, both of which are critical for better patient care and organizational outcomes. CLINICAL RELEVANCE: Healthcare organizations should provide adequate nursing staff and assign reasonable workloads. Furthermore, hospitals should periodically monitor the work schedule characteristics of nurses and actively intervene in cases of scheduling issues to resolve them.

Targeted removal of leukemia cells from the circulating system by whole-body magnetic hyperthermia in mice.

Until now, magnetic hyperthermia was used to remove solid tumors by targeting magnetic nanoparticles (MNPs) to tumor sites. In this study, leukemia cells in the bloodstream were directly removed by whole-body hyperthermia, using leukemia cell-specific MNPs. An epithelial cellular adhesion molecule (EpCAM) antibody was immobilized on the surface of MNPs (EpCAM-MNPs) to introduce the specificity of MNPs to leukemia cells. The viability of THP1 cells (human monocytic leukemia cells) was decreased to 40.8% of that in control samples by hyperthermia using EpCAM-MNPs. In AKR mice, an animal model of lymphoblastic leukemia, the number of leukemia cells was measured following the intravenous injection of EpCAM-MNPs and subsequent whole-body hyperthermia treatment. The result showed that the leukemia cell number was also decreased to 43.8% of that without the treatment of hyperthermia, determined by Leishman staining of leukemia cells. To support the results, simulation analysis of heat transfer from MNPs to leukemia cells was performed using COMSOL Multiphysics simulation software. The surface temperature of leukemia cells adhered to EpCAM-MNPs was predicted to be increased to 82 °C, whereas the temperature of free cells without adhered MNPs was predicted to be 38 °C. Taken together, leukemia cells were selectively removed by magnetic hyperthermia from the bloodstream, because EpCAM-modified magnetic particles were specifically attached to leukemia cell surfaces. This approach has the potential to remove metastatic cancer cells, and pathogenic bacteria and viruses floating in the bloodstream.

Immunochromatographic assay to detect α-tubulin in urine for the diagnosis of kidney injury.

BACKGROUNDS: Shortening of primary cilia in kidney epithelial cells is associated with kidney injury and involved with the induced level of α-tubulin in urine. Therefore, rapid detection and quantification of α-tubulin in the urine samples could be used to the preliminary diagnosis of kidney injury. METHODS: Cellulose-based nanobeads modified with α-tubulin were used for the detection probe of competitive immunochromatographic (IC) assay. The concentration of α-tubulin in the urine samples was determined by IC assay and compared with the amount determined by Western blotting analysis. RESULTS: The relationship between α-tubulin concentration and the colorimetric intensity resulted from IC assay was determined by logistic regression, and the correlation coefficient (R2 ) was 0.9948. When compared to the amount determined by Western blotting analysis, there was a linear relationship between the α-tubulin concentrations measured by the two methods and the R2 value was 0.823. CONCLUSIONS: This method is simple, rapid, and adequately sensitive to detect α-tubulin in patient urine samples, which could be used for the clinical diagnosis of kidney injury.

In vivo removal of radioactive cesium compound using Prussian blue-deposited iron oxide nanoparticles.

Aim: To mitigate the side effects of medical treatment by Prussian blue (PB), a well-known adsorbent for radioactive cesium (Cs), PB-deposited magnetic nanoparticles (MNPs), were prepared and analyzed on the adsorbent capacity for Cs removal. Materials & methods: The PB-deposited MNPs were prepared by photo-deposition method and investigated for their Cs adsorption properties in vitro and in vivo. The distribution of the adsorbents was also evaluated in C57BL/6 mice. Results: PB-deposited MNPs provided an improved adsorbent capacity for Cs removal and reduced toxicity to blood cells compared with those of bulk PB. Conclusion: PB-deposited MNPs could be considered as an alternative of PB-based medicine to reduce the possible hazards caused by high dose of PB intake.

RGD-containing elastin-like polypeptide improves islet transplantation outcomes in diabetic mice.

Successful islet transplantation critically depends on the isolation of healthy islets. However, the islet isolation procedure itself contributes to islet death due to the destruction of intra- and peri-islet extracellular matrices (ECMs) during digestion. We investigated whether an RGD-containing elastin-like polypeptide (REP) could function as a self-assembling matrix to replenish ECMs and protects islets from cell death. Immediately following isolation, islets were coated with REP coacervate particles via isothermal adsorption of an REP solution followed by thermal gelation. REP-coated islets displayed increased viability and insulin secretory capacity in pretransplant culture compared to untreated islets. Co-transplantation of REP-treated islets and REP beneath the renal sub-capsule in streptozotocin-induced diabetic mice restored normoglycemia and serum insulin levels. Mice that received co-transplants maintained normoglycemia for a longer period of time than those receiving untreated islets without REP. Moreover, co-transplantation sites exhibited enhanced ß-cell proliferation and vascularization. Thus, the REP-based coacervation strategy improve the survival, function and therapeutic potential of transplanted islets. STATEMENT OF SIGNIFICANCE: 1). An artificial matrix polypeptide comprised of thermoresponsive elastin-like peptides and integrin-stimulatory RGD ligands (REP) to reconstitute damaged or lost matrices. 2). Through body temperature-induced coacervation, REP reconstitutes intra-islet environment and enhances islet viability and insulin secretion by activating the pro-survival and insulin signaling pathways. 3). REP-coated islets were transplanted together with the matrix polypeptide under the kidney sub-capsule of mice, it develops a new peri-insular environment, which protects the islet grafts from immune rejection thus extending islet longevity. 4). Our data suggest that in situ self-assembly of biomimetic islet environments become a new platform allowing for improved islet transplantation at extrahepatic sites.

Dual-responsive Gemini Micelles for Efficient Delivery of Anticancer Therapeutics.

Polymeric micelles as drug delivery vehicles are popular owing to several advantages. In this study, a gemini amphiphile (gemini mPEG-Cys-PMT) consisting of hydrophilic poly(ethylene glycol) and hydrophobic poly(methionine) with cystine disulfide spacer was synthesized and its micellar properties for thiol- or reactive oxygen species (ROS)-dependent intracellular drug delivery were described. The cleavage of cystine linkage in a redox environment or the oxidation of methionine units in a ROS environment caused the destabilization of micelles. Such redox- or ROS-triggered micellar destabilization led to enhanced release of encapsulated doxorubicin (DOX) to induce cytotoxicity against cancer cells. Further, the therapeutic effects of the DOX-loaded micelles were demonstrated using the KB cell line. This study shows that thiol and ROS dual-responsive gemini micelles are promising platforms for nano-drug delivery in various cancer therapies.

Effect of young exosomes injected in aged mice.

INTRODUCTION: Exosomes, nanosized extracellular vesicles, are known to circulate through the blood stream to transfer molecular signals from tissue to tissue. METHODS: To determine whether exosomes affect aging in animals, we primarily identified the changes in exosomal miRNA contents during the aging process. In exosomes from 12-month-old mice, mmu-miR-126-5p and mmu-miR-466c-5p levels were decreased and mmu-miR-184-3p and mmu-miR-200b-5p levels were increased significantly compared with those of 3-month-old mice. Their levels in exosomes were partially correlated with those in tissues: levels of only mmu-miR-126-5p and mmu-miR-466c-5p in lungs and/or liver were decreased, but those of mmu-miR-184-3p and mmu-miR-200b-5p in tissues did not coincide with those of exosomes. RESULTS AND DISCUSSION: In the aged tissues injected with young exosomes isolated from serum, mmu-miR-126b-5p levels were reversed in the lungs and liver. Expression changes in aging-associated molecules in young exosome-injected mice were obvious: p16Ink4A, MTOR, and IGF1R were significantly downregulated in the lungs and/or liver of old mice. In addition, telomerase-related genes such as Men1, Mre11a, Tep1, Terf2, Tert, and Tnks were significantly upregulated in the liver of old mice after injection of young exosomes. CONCLUSION: These results indicate that exosomes from young mice could reverse the expression pattern of aging-associated molecules in aged mice. Eventually, exosomes may be used as a novel approach for the treatment and diagnosis of aging animals.

Enhanced Therapeutic Treatment of Colorectal Cancer Using Surface-Modified Nanoporous Acupuncture Needles.

Acupuncture originated within the auspices of Oriental medicine, and today is used as an alternative method for treating various diseases and symptoms. The physiological mechanisms of acupuncture appear to involve the release of endogenous opiates and neurotransmitters, with the signals mediating through electrical stimulation of the central nervous system (CNS). Earlier we reported a nanoporous stainless steel acupuncture needle with enhanced therapeutic properties, evaluated by electrophysiological and behavioral responses in Sprague-Dawley (SD) rats. Herein, we investigate molecular changes in colorectal cancer (CRC) rats by acupuncture treatment using the nanoporous needles. Treatment at acupoint HT7 is found most effective at reducing average tumor size, ß-catenin expression levels, and the number of aberrant crypt foci in the colon endothelium. Surface modification of acupuncture needles further enhances the therapeutic effects of acupuncture treatment in CRC rats.

Reverse Expression of Aging-Associated Molecules through Transfection of miRNAs to Aged Mice.

Molecular changes during aging have been studied to understand the mechanism of aging progress. Herein, changes in microRNA (miRNA) expression in the whole blood of mice were studied to systemically reverse aging and propose them as non-invasive biomarkers. Through next-generation sequencing analysis, we selected 27 differentially expressed miRNAs during aging. The most recognized function involved was liver steatosis, a type of non-alcoholic fatty liver disease (NAFLD). Among 27 miRNAs, six were predicted to be involved in NAFLD, miR-16-5p, miR-17-5p, miR-21a-5p, miR-30c-5p, miR-103-3p, and miR-130a-3p; alterations in their blood and liver levels were confirmed by real-time qPCR. The expression of the genes associated in the network of these miRNAs, Bcl2, Ppara, E2f1, E2f2, Akt, Ccnd1, and Smad2/3, also was altered in the liver of aged mice. Following transfection of these miRNAs into 18-month-old mice, levels of miR-21a-5p, miR-103-3p, and miR-30c-5p increased, and their related genes exhibited a reversed expression in the liver. Expression of Mre11a, p16INK4a, and Mtor, reported to be aging-associated molecules, also was reversed in the livers of miRNA-transfected mice. These miRNAs could be non-invasive biomarkers for aging, and they might induce a reverse regulation of aging-associated pathways. This study provides preliminary data on reverse aging, which could be applied further for treatments of adult diseases.

Transthyretin as a new transporter of nanoparticles for receptor-mediated transcytosis in rat brain microvessels.

Many drugs are unable to breach the blood-brain barrier (BBB). Protein-directed transport of nanomedicine by receptor-mediated transcytosis (RMT) has been investigated as a means to overcome this problem. In this study, we screened transporters using an in vitro transcytosis assay system in rat serum to identify candidates that could guide nanoparticles through the BBB by RMT. The proteins that showed over 5-fold decreases in RMT when treated with chloropromazine, an inhibitor of clathrin-dependent endocytosis, were selected and identified by Maldi-TOF mass spectroscopy. Eleven proteins, including transthyretin (Ttr), and creatine kinase-muscle type (CKM), were identified as being capable of penetrating the endothelial cell layer by RMT. Among them, 10 proteins have not yet been used to transport nanomaterials across the BBB. To validate their activity as nanoparticle transporters in vivo, Ttr and CKM were conjugated to the surface of quantum dot (QD) nanoparticles and administrated intravenously. After 8h, the distribution of Ttr-QDs and CKM-QDs in brain tissue was analyzed. The results showed transcytosis of Ttr-QD conjugates across the BBB in rats as well as in in vitro assays, which was in contrast to the results observed for bare QDs and CKM-QDs. Taken together, these results indicate that Ttr is a new putative transporter for nanomedicines across the BBB.

Carcinogenic activity of PbS quantum dots screened using exosomal biomarkers secreted from HEK293 cells.

Lead sulfide (PbS) quantum dots (QDs) have been applied in the biomedical area because they offer an excellent platform for theragnostic applications. In order to comprehensively evaluate the biocompatibility of PbS QDs in human cells, we analyzed the exosomes secreted from cells because exosomes are released during cellular stress to convey signals to other cells and serve as a reservoir of enriched biomarkers. PbS QDs were synthesized and coated with 3-mercaptopropionic acid (MPA) to allow the particles to disperse in water. Exosomes were isolated from HEK293 cells treated with PbS-MPA at concentrations of 0 µg/mL, 5 µg/mL, and 50 µg/mL, and the exosomal expression levels of miRNAs and proteins were analyzed. As a result, five miRNAs and two proteins were proposed as specific exosomal biomarkers for the exposure of HEK293 cells to PbS-MPA. Based on the pathway analysis, the molecular signature of the exosomes suggested that PbS-MPA QDs had carcinogenic activity. The comet assay and expression of molecular markers, such as p53, interleukin (IL)-8, and C-X-C motif chemokine 5, indicated that DNA damage occurred in HEK293 cells following PbS-MPA exposure, which supported the carcinogenic activity of the particles. In addition, there was obvious intensification of miRNA expression signals in the exosomes compared with that of the parent cells, which suggested that exosomal biomarkers could be detected more sensitively than those of whole cellular extracts.

Evaluation of the National Train-the-Trainer Program for Hospice and Palliative Care in Korea.

BACKGROUND: To evaluate the effectiveness of the National Train-the-Trainers Program for Hospice and Palliative Care Experts (TTHPC) sponsored by the National Cancer Center of Korea between 2009 and 2012. This program was developed to improve the teaching skills of those in the field of hospice and palliative care (HPC). MATERIALS AND METHODS: Training was offered in eight 1-day sessions between 2009 and 2012. The effect of the program was measured using Kirkpatrick's model of educational outcomes. First, levels 1 and 2 were evaluated immediately after the 1-day program (n=120). In 2012, the level-3 evaluation test was administered to trainers who offered at least one HPC training (n=78) as well as to their trainees (n=537). RESULTS: The level-1 evaluation addressed participant reactions to and satisfaction with the program. Participants (n=120) were generally satisfied with the content, the method, and the overall course (mean range: 3.94-4.46 on a five-point Likert scale). The level-2 evaluation (learning) showed that participants gained knowledge and confidence related to teaching HPC (4.24 vs. 4.00). The level-3 evaluation (behavioral), which assessed trainers' application of teaching skills to HPC, showed that trainees rated the teaching methods of trainers (mean range: 4.03-4.08) more positively than did trainers (p<0.05). Female trainers were more likely than were male trainers to plan sessions in consideration of their trainees' characteristics (4.11 vs. 3.58; p<0.05), and nurse trainers were more likely than physician trainers to use a variety of instructional methods (4.05 vs. 3.36; p<0.05) CONCLUSIONS: We conducted systematic evaluations based on Kirkpatrick's model to assess the effectiveness of our train-the- trainers program. Our educational program was practical, effective, and followed by our HPC experts, who needed guidance to learn and improve their clinical teaching skills.

Photothermal cancer therapy using graphitic carbon-coated magnetic particles prepared by one-pot synthesis.

We describe here a simple synthetic strategy for the fabrication of carbon-coated Fe3O4 (Fe3O4@C) particles using a single-component precursor, iron (III) diethylenetriaminepentaacetic acid complex. Physicochemical analyses revealed that the core of the synthesized particles consists of ferromagnetic Fe3O4 material ranging several hundred nanometers, embedded in nitrogen-doped graphitic carbon with a thickness of ~120 nm. Because of their photothermal activity (absorption of near-infrared [NIR] light), the Fe3O4@C particles have been investigated for photothermal therapeutic applications. An example of one such application would be the use of Fe3O4@C particles in human adenocarcinoma A549 cells by means of NIR-triggered cell death. In this system, the Fe3O4@C can rapidly generate heat, causing >98% cell death within 10 minutes under 808 nm NIR laser irradiation (2.3 W cm(-2)). These Fe3O4@C particles provided a superior photothermal therapeutic effect by intratumoral delivery and NIR irradiation of tumor xenografts. These results demonstrate that one-pot synthesis of carbon-coated magnetic particles could provide promising materials for future clinical applications and encourage further investigation of this simple method.

Rapid detection of 6×-histidine-labeled recombinant proteins by immunochromatography using dye-labeled cellulose nanobeads.

A rapid and easy immunochromatography assay using dye-labeled cellulose nanobeads (CNBs) was developed to detect proteins with hexa-histidine tag (His-tag) to characterize recombinant proteins during purification. Recombinant ATG8 protein was used as a His-tagged protein, and ATG8-conjugated CNBs (A-CNBs) were prepared. The original ATG8 in the sample solution competed with A-CNBs for anti-His-tag antibodies spotted on to the strip resulting in an inverse relationship between ATG8 concentration and the colorimetric signal. The usefulness of this method was shown by adding ATG8 to a 1% Escherichia coli extract. In addition, this assay can be used to detect other His-tagged proteins without protein-specific antibodies. Because the identification of fractions containing His-tagged proteins by western blotting or ELISA is labor-intensive and expensive, our method provides an efficient and cheaper alternative.