2016

Permanent URI for this collectionhttps://hdl.handle.net/1807/71210

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Ceruloplasmin-derived peptide is the strongest regulator of oxidative stress and leukotriene synthesis in neutrophils
(Canadian Science Publishing, 2016-12-02) Golenkina, Ekaterina; Livenskyi, Alexey; Viryasova, Galina; Romanova, Yulia; Sud'ina, Galina; Sokolov, Alexey
Ceruloplasmin, an acute-phase protein, can affect the activity of leukocytes through its various enzymatic activities and protein-protein interactions (with lactoferrin, myeloperoxidase, eosinophil peroxidase, serprocidins, and 5-lipoxygenase (5-LOX), among others). However, the molecular mechanisms of ceruloplasmin activity are not clearly understood. In this study, we tested the ability of two synthetic peptides, RPYLKVFNPR (883â 892) (P1) and RRPYLKVFNPRR (882â 893) (P2), corresponding to the indicated fragments of the ceruloplasmin sequence, to affect neutrophil activation. Leukotriene (LT) B4 is the primary eicosanoid product of polymorphonuclear leukocytes (PMNLs, neutrophils).We studied leukotriene synthesis in PMNLs upon interaction with Salmonella enterica serovar Typhimurium. Priming of neutrophils with phorbol 12-myristate 13-acetate (PMA) elicited the strong regulatory function of P2 peptide as a superoxide formation inducer and leukotriene synthesis inhibitor. Ceruloplasmin-derived P2 peptide appeared to be a strong inhibitor of 5-LOX product synthesis under conditions of oxidative stress.
Potential lncRNA regulatory mechanisms in diabetes and its complications
(Canadian Science Publishing, 2016-10-27) Feng, Shuidong; YANG, Ji-Hua; Yao, Chao Hua; YANG , Si-Si; ZHU, Ze-Mei; WU , Di; Ling, Hongyan; Zhang, Liang
Long noncoding RNAs (lncRNA) are transcripts longer than 200 nucleotides without protein-coding potential. Though these molecules were initially considered as “junk-products” of transcription without biological meaning, recent research advancements have shown that lncRNA plays an important role not only in cellular processes such as proliferation, differentiation and metabolism, but also in pathological processes of cancers，diabetes, and neurodegenerative diseases．In this review, we focus on the potential regulatory roles of lncRNA in diabetes and diabetic complications.
Functional assessment of MeCP2 in Rett syndrome and cancers of breast, colon and prostate
(Canadian Science Publishing, 2016-09-23) PANDEY, SOMNATH; PRUITT, KEVIN
Ever since the first report that mutations in methyl-CpG-binding protein 2 (MeCP2) causes Rett syndrome (RTT), a severe neurological disorder in females world-wide, there has been a keen interest to gain a comprehensive understanding of this protein. While the classical model associated with MeCP2 function suggests its role in gene suppression via recruitment of co-repressor complexes and histone deacetylases to methylated CpG-sites, recent discoveries have brought to light its role in transcription activation, modulation of RNA splicing and chromatin compaction. Various post-translational modifications (PTMs) of MeCP2 further increase its functional versatility. Involvement of MeCP2 in pathologies other than RTT, such as tumorigenesis however, remains poorly explored and understood. This review provides a survey of the literature implicating MeCP2 in breast, colon and prostate cancer.
Chronic intermittent hypoxia disturbs insulin secretion and causes pancreatic injury via MAPK signaling pathway
(Canadian Science Publishing, 2016-11-23) Wang, Yeying; Hai, Bing; Niu, Xiaoqun; Ai, Li; Cao, Yu; Li, Ran; Li, Yongxia
Obstructive sleep apnea (OSA) is a breath disorder during sleep with a most prominent character of chronic intermittent hypoxia (CIH), inducing the generation of reactive oxygen species (ROS) to damage multiple tissues and cause metabolism disorders. In this study, we established rat varying OSA model by graded concentration of CIH (12.5% O2, 10% O2, 7.5%O2 and 5% O2) for 12 weeks, and found that CIH stimulated insulin secretion, reduced the insulin/proinsulin ratio in the pancreatic tissue, and caused pancreatic tissue lesions and cells apoptosis in a dose-dependent manner. Moreover, CIH promoted the production of tumor necrosis factor (TNF)-Îą, Interleukin (IL)-1Î˛ and IL-6, and activated mitogen-activated protein kinase (MAPK) family members, extracellular regulated protein kinase (ERK), c-Jun N-terminal kinase (JNK) and P38 relying on the O2 concentration. In summary, CIH disturbed insulin secretion, caused inflammation, lesion and cell apoptosis in pancreatic tissue via MAPK signaling pathway, which may be of great significance for clinical treatment of OSA and type 2 diabetes mellitus (T2DM).
Method Comparison for Analyzing Wound Healing Rates
(Canadian Science Publishing, 2016-11-23) Dhillon, Prabhpreet K.; Li, Xinyin; Sanes, Jurgen T.; Akintola, Oluwafemi S.; Sun, Bingyun
Wound healing scratch assay is a frequently used method to characterize cell migration, which is an important biological process in the course of development, tissue repair, and immune response for example. The measurement of wound healing rate, however, varies among different studies. Here we summarized these measurements into three types: I) Direct Rate Average; II) Regression Rate Average; and III) Average Distance Regression Rate. Using Chinese Hamster Ovary (CHO) cells as a model, we compared the three types of analyses on quantifying the wound closing rate, and discovered that type I III measurements are more resistant to outliers and type II analysis is more sensitive to outliers. We hope this study can help researchers to better use this simple yet effective assay.
Temporo-spacial micoranatomical distribution of the murine sodium-dependent ascorbic acid transporters Slc23a1 and Slc23a2 in the kidney throughout development
(Canadian Science Publishing, 2016-11-24) Eck, Peter Karl; Corpe, Christopher; Levine, Mark Alan
The two membrane transporters Slc23a1 and Slc23a2 mediate ascorbic acid uptake into cells. We recently determined the key role of Slc23a1 in renal re-absorption of ascorbic acid in a knockout mouse model. However, the renal spatial and temporal expression patterns of murine Slc23a1 and Slc23a2 are not defined. This study utilizes database evidence combined with experimental confirmation via in situ hybridization to define spatial and temporal expression of Slc23a1 in the murine kidney. Slc23a1 is expressed in the early proximal tubule, but not in its precursors during embryonic development, and exclusive proximal tubular expression persists throughout the animalâ s lifetime. In contrast, Slc23a2 is uniformly expressed in metabolic cell types such as stromal cells. The expression patterns appear to be conserved from rodent lineages to humans.
Cellular stress associated with Plasmodium berghei ookinete differentiation
(Canadian Science Publishing, 2016-07-01) Duran-Bedolla, Josefina; Tellez-Sosa, Juan; Valdovinos-Torres, Humberto; Pavon, Natalia; Buelna-Chontal, Mabel; Tello-Lopez, Angel; Argotte-Ramos, Rocio; Rodriguez, Mario Henry; Rodriguez, Maria Carmen
For malaria transmission, Plasmodium parasites must develop in the mosquito vector. Oxidative stress in the insect midgut, triggered by environmental changes (e.g., pH and temperature), influences the cellular signaling of parasites involved in differentiation from gametocytes to mobil ookinetes. It activates the homeostatic response to stress characterized by the phosphorylation eIF2ď Ą, the attenuation of protein synthesis and the transcription of genes participating in the unfolded protein response and antioxidant processes, forming a part of an integrated stress response (ISR). We hypothesized that ISR operates during the differentiation of gametocytes to ookinetes to assure Plasmodium survival. Using in vitro conditions resembling the mosquito midgut conditions, we cultured Plasmodium berghei gametocytes to ookinetes, and evaluated the redox balance by detecting ROS and SOD activity. Additionally, we evaluated the phosphorylation of eIF2Îą, the attenuation of the global protein synthesis and the gene expression of cellular stress markers (e.g., ER chaperones and antioxidant molecules, measured by RT-qPCR), finding that these processes were all taking place, probably to improve survival during Plasmodium berghei ookinete differentiation.
A deletion variant partially complements a porin-less strain of Neurospora crassa
(Canadian Science Publishing, 2016-10-16) Ferens, Fraser G.; Spicer, Victor; Krokhin, Oleg V; Motnenko, Anna; Summers, William A.T.; Court, Deborah A
Mitochondrial porin, the voltage-dependent anion channel, plays an important role in metabolism and other cellular functions within eukaryotic cells. To further the understanding of porin structure and function, Neurospora crassa wild-type porin was replaced with a deletion variant lacking residues 238-242 (238porin). 238porin was assembled in the mitochondrial outer membrane, but the steady state levels were only about 3% of those of the wild-type protein. The strain harbouring 238porin displayed cytochrome deficiencies and expressed alternative oxidase. Nonetheless, it exhibited an almost normal linear growth rate. Analysis of mitochondrial proteomes from a wild-type strain FGSC 9718, a strain lacking porin (Î Por-1) and that expressing 238porin revealed that the major differences between the variant strains were in the levels of subunits of the NADH:ubiquinone oxidoreductase (complex I) of the electron transport chain, which were reduced only in Î Por-1 strain. These, and other proteins related to electron flow and mitochondrial biogenesis, are differentially affected by relative porin levels.
The discovery and development of the CRISPR system in applications in genome manipulation
(Canadian Science Publishing, 2016-09-06) Lau, Veronica; Davie, James R.
The Clustered regularly interspaced short palindromic repeat (CRISPR) CRISPR associated 9 (Cas9) system is a microbial adaptive immune system that has been recently developed for genomic engineering. From the moment the CRISPR system was discovered in Escherichia coli, the drive to understand the mechanism prevailed, leading to rapid advancement in the knowledge and applications of the CRISPR system. With the ability to characterize and understand the function of the Cas9 endonuclease came the ability to adapt the CRISPR-Cas9 system for use in a variety of applications and disciplines ranging from agriculture to biomedicine. This review will provide a brief overview of the discovery and development of the CRISPR-Cas9 system in applications such as genome regulation and epigenome engineering, as well as the challenges faced.
Frizzled Gene Expression and Negative Regulation of Canonical WNT-β-catenin Signaling in Mouse F9 Teratocarcinoma Cells
(Canadian Science Publishing, 2016-08-31) Golenia, Gregory; Gatie, Mohamed I; Kelly, Gregory M
Mouse F9 cells differentiate into primitive endoderm (PrE) following the activation of the canonical WNT/β-catenin pathway. The upregulation of Wnt6 and activation of β-catenin-TCF/LEF-dependent transcription is known to accompany differentiation, but the Frizzled (FZD) receptor responsible for transducing the WNT6 signal is not known. Eight of the ten Fzd genes were found to be expressed in F9 cells, with Fzd7 being the most highly expressed, and chosen for further analysis. To alter steady-state Fzd7 levels and test the effect this has on differentiation, siRNA and overexpression approaches were used to knockdown and ectopically express the Fzd7 message, respectively. siRNA knock down of Fzd7 resulted in reduced DAB2 levels, and the overexpression activated a TCF-LEF reporter, but neither approach affected differentiation. Our focus turned to how canonical WNT6 signaling was attenuated to allow PrE cells to form PE. Dkk1, encoding a WNT antagonist, was examined and results showed that its expression increased in F9 cells treated with RA or overexpressing Wnt6. F9 cells overexpressing human DKK1 or treated with DKK1-conditioned medium and then treated with RA failed to differentiate, indicating that a negative feedback loop involving WNT6 and DKK1 attenuates canonical WNT/β-catenin signaling, thereby allowing PE cells to differentiate.
Proteomics Analysis of Latex from Hevea brasiliensis (Clone RRIM 600)
(Canadian Science Publishing, 2016-08-26) Habib, Mohd Afiq Hazlami; Gan, Chee Yuen; Othman, Fazilah; Zainudin, Nurul Nabilah; Abdul Latiff, Aishah; Ismail, Mohd Nazri
The natural rubber latex extracted from the bark of Hevea brasiliensis plays various important roles in today's modern society. Following ultracentrifugation, the latex can be separated into 3 layers; C-serum, lutoids, and rubber particle. Previous studies showed that a large number of proteins were present inside these three layers. However, a complete proteome for this important plant is still unavailable. Protein sequences have been recently translated from the completed draft genome database of H. brasiliensis leading to the creation of annotated protein databases of H. brasiliensis biosynthetic pathways; photosynthesis, latex allergens, rubberwood formation, latex biosynthesis, and disease resistance. This research was conducted to identify the proteins contained within the latex by way of de novo sequencing from mass spectric data obtained from the 3 layers of the latex. Peptides from these proteins were fragmented using collision-induced dissociation (CID), higher-energy collisional dissociation (HCD) and electron-transfer dissociation (ETD) activation methods. A large percentage of proteins from the biosynthetic pathways were successfully identified, ranging from 63% to 100%. In addition, a total of 1839 unique proteins were identified from the whole translated draft genome database (AnnHBM).
Precision Genome Editing in the CRISPR Era
(Canadian Science Publishing, 2016-09-13) Salsman, Jayme; Dellaire, Graham
With the introduction of precision genome editing using CRISPR/Cas9 technology, we have entered a new era of genetic engineering and gene therapy. With RNA-guided endonucleases, such as Cas9, it is possible to engineer DNA double strand breaks (DSB) at specific genomic loci. DSB repair by the error-prone non-homologous end joining (NHEJ) pathway can disrupt a target gene by generating insertions and deletions. Alternatively, Cas9-mediated DSBs can be repaired by homology directed repair (HDR) using a homologous DNA repair template, thus allowing precise gene editing by incorporating genetic changes into the repair template. HDR can introduce gene sequences for protein epitope tags, delete genes, make point mutations or alter enhancer and promoter activities. In anticipation of adapting this technology for gene therapy in human somatic cells, much focus has been placed on increasing the fidelity of CRISPR/Cas9 and increasing HDR efficiency to improve precision genome editing. In this review, we will discuss applications of CRISPR technology for gene inactivation and genome editing with a focus on approaches to enhancing CRISPR/Cas9-mediated HDR for the generation of cell and animal models, and conclude with a discussion of recent advances and challenges towards the application of this technology for gene therapy in humans.
Linker histones: Novel insights into structure-specific recognition of the nucleosome
(Canadian Science Publishing, 2016-06-15) Cutter, Amber R.; Hayes, Jeffrey J.
Linker histones (H1s) are a primary component of metazoan chromatin, fulfilling numerous functions, both in vitro and in vivo, including stabilizing wrapping of DNA around the nucleosome, promoting folding and assembly of higher order chromatin structures influencing nucleosome spacing on DNA and regulating specific gene expression. However, many molecular details of how H1 binds to nucleosomes and recognizes unique structural features on the nucleosome surface remain undefined. Numerous, confounding studies are complicated not only by experimental limitations, but the use of different linker histone isoforms and nucleosome constructions. This review summarizes the decades of research that has resulted in several models of H1 association with nucleosomes, with a focus on recent advances that suggest multiple modes of H1 interaction in chromatin, while also highlighting remaining questions.
MgF3- and AlF4- Transition State Analogue complexes of Yeast Phosphoglycerate Kinase
(Canadian Science Publishing, 2016-07-11) McCormick, Nicole E; Forget, Stephanie M; Syvitski, Raymond T; Jakeman, David L.
The phospho-transfer mechanism of yeast phosphoglycerate kinase (PGK) has been probed through formation of trifluoromagnesate (MgF3-) and tetrafluoroaluminate (AlF4-) transition state analogue complexes and analyzed using 19F, 1H waterLOGSY and 1H chemical shift perturbation NMR spectroscopy. We observed the first 19F NMR spectroscopic evidence for the formation of metal fluoride transition state analogues of yeast PGK and also observed significant changes to proton chemical shifts of PGK in the presence, but not in the absence, of fluoride upon titration of ligands, providing indirect evidence of the formation of a closed ternary transition state. WaterLOGSY NMR spectroscopy experiments using an uncompetitive model were used in an attempt to measure ligand binding affinities within the transition state analogue complexes.
Characterization of a Salmonella sugar kinase essential for utilization of fructose-asparagine
(Canadian Science Publishing, 2016-08-03) Biswas, Pradip K; Behrman, Edward J; Gopalan, Venkat
Salmonella can utilize fructose-asparagine (F-Asn), a naturally occurring Amadori product, as its sole carbon and nitrogen source. Conversion of F-Asn to the common intermediates glucose-6-phosphate, aspartate, and ammonia was predicted to involve the sequential action of an asparaginase, a kinase and a deglycase. Mutants lacking the deglycase are highly attenuated in mouse models of intestinal inflammation due to the toxic build-up of the deglycase substrate. The limited distribution of this metabolic pathway in the animal gut microbiome raises the prospects for antibacterial discovery. We report the biochemical characterization of the kinase that was expected to transform fructose-aspartate to 6-phosphofructose-aspartate during F-Asn utilization. In addition to confirming its anticipated function, we determined through studies of fructose-aspartate analogues that this kinase exhibits a substrate-specificity with greater tolerance to changes to the amino acid (including the D-isomer of aspartate) than to the sugar.
High Concentration Calcitriol Induces Endoplasmic Reticulum Stress Related Gene Profile in Breast Cancer Cells
(Canadian Science Publishing, 2016-05-19) Ozkaya, Ali Burak; Ak, Handan; Aydin, Hikmet Hakan
Calcitriol, the active form of vitamin D, is known for its anti-cancer properties including induction of apoptosis, inhibition of angiogenesis and metastasis. Understanding the mechanism of action for calcitriol will help development of novel treatment strategies. Since vitamin D exerts its cellular actions via binding to its receptor and by altering expressions of a set of genes, we aimed to evaluate the effect of calcitriol on transcriptomic profile of breast cancer cells. We previously demonstrated that calcitriol alters ER stress markers, therefore in this study we have focused on ER-stress related genes to reveal calcitriols action on these genes in particular. We have treated breast cancer cell lines MCF-7 and MDA-MB-231 with previously determined IC50 concentrations of calcitriol and evaluated transcriptomic alterations via microarray. During analysis only genes altered by at least 2 fold with a p value less than 0.05 were taken into consideration. Our findings revealed an ER stress associated transcriptomic profile induced by calcitriol. Induced genes include genes with pro-survival function (NUPR1, DNAJB9, HMOX1, LCN2 and LAMP3) and with pro-death function (CHOP, DDIT4, NDGR1, NOXA and CLGN). These results suggest that calcitriol induces an ER-stress like response inducing both pro-survival and pro-death transcripts of the process.
Gastroprotective and antielastase effect of protein inhibitors from Erythrina velutina seeds in an experimental ulcer model
(Canadian Science Publishing, 2016-07-01) Lima, Vanessa Cristina Oliveira de; Machado, Richele Janaína de Araújo; Monteiro, Norberto; Camillo, Christina da Silva; Serquiz, Alexandre Coelho; Oliveira, Adeliana Silva de; Rufino, Fabiola; Maciel, Bruna Leal Lima; Uchôa, Adriana Ferreira; Santos, Elizeu Antunes; Morais, Ana Heloneida de Araújo
Trypsin and chymotrypsin inhibitors from Erythrina velutina seeds have been previously isolated by our group. In previous studies using a sepsis model, we demonstrated the anti-tumor and anti-inflammatory action of these compounds. This study aimed to evaluate the gastroprotective and antielastase effect of protein inhibitors from Erythrina velutina seeds in an experimental stress induced ulcer model. Two protein isolates from E. velutina seeds, with antitrypsin (PIAT) and anti-chymotrypsin (PIAQ) activities were tested. Both protein isolates showed a high affinity and inhibitory effect against HNE, with 84% and 85% of inhibition, respectively. Gastric ulcer was induced using ethanol (99%) in six groups of animals (female Wistar rats, n = 6). Before ulcer induction, these animals received during 5 days one of the following: 1- PIAT (0.2 mg/kg), 2- PIAT (0.4 mg/kg), 3- PIAQ (0.035 mg/kg), 4- Ranitidine hydrochloride (50 mg/kg), 5- Saline solution (0.9%), 6- No intervention (Sham). Both PIAT and PIAQ protected gastric mucosa, preventing hemorrhagic lesions, edema and mucus loss. No histologic toxic effects of PIAT or PIAQ were seen in liver and pancreatic cells. Our results show that protein isolates from E. velutina seeds have potential gastroprotective effects, placing these compounds as natural candidates for gastric ulcer prevention.
Aerosolized bovine lactoferrin reduces neutrophils and pro-inflammatory cytokines in mouse models of Pseudomonas aeruginosa lung infections
(Canadian Science Publishing, 2016-07-01) Valenti, Piera; Frioni, Alessandra; Rossi, Alice; Ranucci, Serena; De Fino, Ida; Cutone, Antimo; Rosa, Luigi; Bragonzi, Alessandra; Berlutti, Francesca
Lactoferrin (Lf), an iron-chelating glycoprotein of the innate immunity, produced by exocrine glands and neutrophils in infection/inflammation sites, is one of the most abundant defence molecules in airway secretions. Lf, a pleiotropic molecule, exerts antibacterial and anti-inflammatory functions. These properties may play a relevant role in airway infections characterized by exaggerate inflammatory response as in Pseudomonas aeruginosa lung infection in cystic fibrosis (CF) subjects. To verify the Lf role in Pseudomonas aeruginosa lung infection, here we evaluated the efficacy of aerosolized bovine Lf (bLf) in mouse models of P. aeruginosa acute and chronic lung infections. C57BL/6NCrl mice were challenged with 106 CFUs of P. aeruginosa PAO1 (acute infection) or MDR-RP73 strain (chronic infection) by intra-tracheal administration. In both acute and chronic infections, aerosolized bLf determined not-significant reduction of bacterial load but significant decrease of the neutrophil recruitment and pro-inflammatory cytokine levels. Moreover, in chronic infection the bLf-treated mice recovered the body weight faster and at higher extent than the control mice. These findings add new insights into the benefits of bLf as a mediator of general health and its potential therapeutic applications.
Anticancer activities of bovine and human lactoferricin-derived peptides
(Canadian Science Publishing, 2016-10-03) Arias, Mauricio; Hilchie, Ashley; Haney, Evan F.; Bolscher, Jan G.M.; Hyndman, Matthew Eric; Hancock, Robert E.W.; Vogel, Hans J.
Lactoferrin (LF) is a mammalian host defense glycoprotein with diverse biological activities. Peptides derived from the cationic region of LF possess cytotoxic activity against cancer cells in vitro and in vivo. Bovine lactoferricin (LFcinB), a peptide derived from bovine LF (bLF), exhibits broad-spectrum anticancer activity, while a similar peptide derived from human LF (hLF) is not as active. In this work, several peptides derived from the N-terminal regions of bLF and hLF were studied for their anticancer activities against leukamia and breast cancer cells, as well as normal peripheral blood mononuclear cells. The cyclized LFcinB-CLICK peptide, which possesses a stable triazole linkage, showed improved anticancer activity, while short peptides hLF11 and bLF10 were not cytotoxic to cancer cells. Interestingly, hLF11 can act as a cell penetrating peptide (CPP), and when combined with the antimicrobial core sequence of LFcinB (RRWQWR) through either a Pro or Gly-Gly linker, toxicity to Jurkat cells increased. Together, our work extends the library of LF-derived peptides tested for anticancer activity, and identified new chimeric peptides with high cytotoxicity towards cancerous cells. Additionally, these results support the notion that short CPPs and antimicrobial peptides can be combined to create new adducts with increased potency.
Bovine lactoferrin and Crohn's Disease: A case study
(Canadian Science Publishing, 2016-10-03) Alexander, David Bedell; Iigou, Masaaki; Abdelgied, Mohamed Ahmed Mahmoud; Ozeki, Keiji; Tanida, Satoshi; Tatashi, Joh; Takahashi, Satoru; Tsuda, Hiroyuki
A 22 year old male suffering from abdominal pain, repeated diarrhea, and weight loss visited the Digestive Disease Department of Nagoya City University Hospital on Dec. 19, 2011. He was hospitalized and diagnosed with Crohnâ s colitis. His Crohn's Disease Activity Index (CDAI) was 415. Treatment by granulocyte apheresis, mesalazine, and adalimumab was started. His CDAI was 314 on Dec 30 and 215 on Jan. 5. A colonoscopic examination on Jan. 19 showed almost complete remission in the transverse colon and marked remission in the rectum. Mesalazine therapy stopped on Feb 28, and the patient was instructed to self-inject 40 mg adalimumab every other week. His CDAI was 50 on April 10 indicating clinical remission. His last self-injection of adalimumab was April 24, 2012, and he started taking 1 g bovine lactoferrin (bLF) daily. His CDAI was 35 on Jan 08, 2013. He continued taking 1 g bLF daily without any other treatment for Crohn's disease. Laboratory blood tests on Sept 07, 2015, showed no sign of disease recurrence, and a colonoscopic examination on Oct 23, 2015, showed almost complete mucosal healing. This case indicates that ingestion of bLF to maintain Crohn's disease in a remissive state should be further explored.

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