Cellular Fluorescence Imaging Research Articles

A Versatile Pep-CPDs Nanoprobe for Rapid Detection of mTBI Biomarker in Clinical Instances and Safe Fluorescence Imaging In Vivo for Improved Weight-Drop Mouse Model.

Mild traumatic brain injury (mTBI) is the most common form of traumatic brain injury; however, it is the most difficult to be accurately identified in the early stage because it lacks more reliable biomarkers and detection methods. This study proposes a highly efficient system to detect a molecular biomarker for the early diagnosis of mTBI. The system was prepared by a lower cytotoxic peptide-modified fluorescent nanoprobe based on carbon polymer dots (pep-CPDs) with outstanding imaging capabilities. In vitro and in vivo tests were explored to the efficiency of pep-CPDs, inferring the good performances of cellular fluorescence imaging and in vivo imaging of mice. Moreover, an application of the versatile pep-CPDs on detecting the mTBI biomarker S100-β detection in a novel improved weight-drop mTBI mouse model and human blood samples has been successfully established. Overall, all these results indicate that the pep-CPD system is sensitive, rapid, non-toxic, and reliable for mTBI diagnosis compared with traditional detection methods. It shows a great potential in clinical and translational research and practical applications.

Betel leaf derived multicolor emitting carbon dots as a fluorescent probe for imaging mouse normal fibroblast and human thyroid cancer cells

A facile and eco-friendly approach was adapted for the preparation of multicolor emitting carbon dots (BL-MECDs) by hydrothermal method using betel leaves. BL-MECDs were synthesized at various reaction times-12 h, 24 h, 36 h, and 48 h at 200 °C. The reaction times were varied to find the optimum reaction condition to obtain BL-MECDs with excellent optical- and chemical properties. BL-MECDs were characterized by various analytical techniques. The surface morphology, degree of graphitization, and their composition were examined by field emission scanning electron microscope, Raman spectrum, and attenuated total reflection-Fourier transform infrared spectroscopy, respectively. The optical properties were evaluated via ultraviolet–visible and fluorescence spectroscopy techniques. The result suggests that BL-MECDs prepared at 36 h have enriched functional groups and excellent fluorescence emission with acceptable quantum yield (15%). Thus, BL-MECDs prepared at 36 h were utilized for further studies. X-ray photoelectron spectroscopy confirmed the existence of functional groups such as carboxyl, carbonyl, and hydroxyl groups. Moreover, the obtained BL-MECDs exhibited excellent prolonged stability and photostability even after 150 days of storage in atmospheric condition and by continuous irradiation of about 100 min with 365 nm UV-lamb, respectively. Further, BL-MECDs showed excellent biocompatibility and negligible cytotoxicity and thus explored for cell imaging for normal mouse fibroblast cell line (L929) and human anaplastic thyroid cancer cells line (SW1736). Cellular fluorescence imaging suggesting that BL-MECDs have great potential in bioimaging applications.

Fabrication of protein-coated titanium dioxide nanoparticles for cellular uptake fluorescence imaging and treatment of colorectal cancer

Titanium dioxide (TiO2) coated with different proteins has exhibited exceptional bio-compatibility, leading to various biomedical engineering applications. With the use of green and chemical reduction methods, we fabricated Titanium dioxide nanoparticles that were protein-coated. Bovine serum albumin (BSA), lysozyme proteins, zein, and collagen have been used to coat titanium dioxide-aryl nanoparticles of the form TiO2-NPs. However, in both cases, no catalysts or other stabilizing agents were used. These images of TiO2-NPs fabricated using the green method show high crystallinity. It is a malignant colorectal tumour with dysfunctional cellular processes that cause colorectal cancer cells. It is hoped that studies employing SW1417 cells would give mechanistic ideas on the specifics of the amplification in cancers. This was done by flow cytometry utilizing and laser confocal fluorescence microscopy (LCFM) on the SW1417 colorectal cell line. Of the protein-coated Titanium dioxide nanoparticles fabricated green methods, BSA@TiO2-NPs were the most readily absorbed. Of all TiO2-NPs, lysozyme@TiO2-NPs fabricated by the chemical reduction technique were the most effectively internalized by SW1417 cells out of TiO2-NPs types. However, TiO2-NPs fabricated by the green methodology were coated with zein and lysozyme and tiny. A hydrophobic covering is also on the two nanoparticles. There is a possibility that the variation in hydrophobicity and charge affected the internalization process. Colorectal diagnostic and therapeutic compounds might be synthesized from those coated nanoparticles that were effectively internalized.

Improving the photostability of fluorescent dyes by polymer nano‐insulating layer

AbstractFluorescein is a traditional organic fluorescent dye that has the disadvantage of agglomeration‐induced fluorescence quenching and photobleaching due to its large hydrophobic conjugate π‐π system, which makes them have limitations in many fields of application. A method of polymer modification is revealed to address this issue. In this study, fluorescein molecules were modified by polycaprolactone (PCL) to obtain PCL grafting fluorescein (PxF), and subsequently assembled with PEG‐PCL to obtain fluorescent nanoparticles (PPxF). PPxF would realize long term cellular fluorescence imaging. PCL polymerization induces self‐assembly, which effectively avoids fluorescence quenching caused by fluorescent molecules aggregation, and at the same time well inhibit the energy transfer with oxygen to improve photostability. This method is a good way to improve the fluorescence properties of fluorescent dyes, to be suitable for all traditional organic fluorescent dyes to greatly improve their fluorescence and hydrophilic properties and enhance their application fields.

A concise, facile and MCR-GAP chemistry strategy for the synthesis of spiro[benzo[4,5]thiazolo[3,2-a]pyrano[2,3-d]pyrimidine-4,3′-indoline] derivatives as fluorescent cellular imaging agents

In this research, polyheterocycles containing spirooxindole, pyran, and thiazolopyrimidines rings have been synthesized via one-pot three-component reactions of isatins, active methylene compounds and 2-hydroxy-4H-benzo[4,5]thiazolo[3,2-a]pyrimidin-4-ones in the presence of the catalytic amount of NEt3 (2 drops) in ethanol at 70 °C. The photophysical properties of the synthesized fluorophores were investigated against 36 different ions, and compound 4a as a model was successfully used in MCF-7 cell line staining.

3-Hydroxy-7,8,9,10-tetrahydro-6H-benzo[c]chromen-6-one and 3-hydroxy-6H-benzo[c]chromen-6-one act as on-off selective fluorescent sensors for Iron (III) under in vitro and ex vivo conditions.

Regarding the abundant use of metals for different purposes, it becomes more critical from various scientific and technological perspectives to discover novel agents as selective probes for the detection of specific metals. In our previous studies, we have shown that aqueous solutions of natural urolithins (i.e., hydroxyl-substituted benzo[c]chromen-6-one derivatives) are selective Iron (III) sensors in fluorescence assays. In this study, we have extrapolated these findings to another coumarine compound (i.e., 3-Hydroxy-7,8,9,10-tetrahydro-6H-benzo[c]chromen-6-one) and compared the selective metal binding properties with Urolithin B (i.e., 3-Hydroxy-6H-benzo[c]chromen-6-one). Following the synthesis and structure identification studies, the fluorometric studies pointed out that the lactam group in the structure still persists to be the important scaffold for maintaining selective on-off sensor capacity that renders the compound a selective Iron (III) binding probe. Moreover, for the first time, fluorescence cellular imaging studies concomitant to cytotoxicity assays with the title compounds were also performed and the results displayed the cell-penetrative, safe, and fluorescent detectable characteristics of the compounds in neuroblastoma and glioblastoma cells through servings as intracellular Iron (III) on-off sensors.

Dual Functional Hydrogen Peroxide Boosted One Step Solvothermal Synthesis of Highly Uniform Sulfur Quantum Dots at Elevated Temperature and Their Fluorescent Sensing

The dual functional hydrogen peroxide (H2O2) assisted strategy for preparation of homogeneously distributed sulfur quantum dots (SQDs) with tunable size and prominent photoluminescence (PL) property is originally proposed via a one-pot solvothermal method. The synthesis of SQDs was demonstrated by treating sublimated sulfur powder and polyethylene glycol-400 (PEG-400) in the presence of H2O2 at elevated temperature. The H2O2 acts as a dual-functional reagent which could suspend the sulfur powder and etching bulk sulfur to construct uniform SQDs. At elevated temperature, the sulfur powder are easily transformed to molten sulfur which simultaneously acts as solvent. The as obtained SQDs exhibit a uniform size distribution, excellent PL properties with quantum yields (QYs) up to 10.3 %, superior dispersibility, and eminent photo stability. Moreover, morphology and element characterization reveal that the surface of spherical SQDs equipped with abundant oxidized sulphur species, while the diversification in the PL and QYs of SQDs under different reaction time are derived from the difference in their particle size which is proposed by the etching-assemble-etching process. Finally, the resultant SQDs was utilized as a sensitive iron ions (Fe3+) sensor with limit of detection (LOD) up to 10.80 nM as well as their investigation for fluorescent cellular imaging.

Enhancing Rechargeable Persistent Luminescence via Organic Dye Sensitization.

Owing to their unique afterglow ability, long-wavelength light activatable persistent luminescence (PersL) nanoparticles (PLNPs) have been emerging as an important category of imaging probes. Long-wavelength LED light has been shown to be effective in recharging these nanoparticles. However, finding a simple and effective method to amplify such renewable PersL signals under long-wavelength light is still a key challenge. Herein, we discovered that a dye-sensitization strategy was able to effectively boost the renewable PersL signals of the NIR emitting ZnGa2 O4 :Cr3+ (ZGC)) under long-wavelength LED light. Moreover, as a proof-of-principle tumorectomy demonstration, this new class of dye sensitized ZGC enabled simultaneous intraoperative anatomic tumor navigation and effective microscopic detection of tumor cells in pathological diagnosis.

Photosensitive MRI biosensor for BCRP-Targeted uptake and light-induced inhibition of tumor cells

Riboflavin and its derivatives are the most important coenzymes in vivo metabolism, and are closely related to life activities. In this paper, the first photolysis 129Xe biosensor was developed by combining cryptophane-A with riboflavin moiety, which showed photosensitivity recorded by hyperpolarized 129Xe NMR/MRI technology with an obvious chemical shift change of 5.3 ppm in aqueous solution. Cellular fluorescence imaging confirmed that the biosensor could be enriched in MCF-7 cells, and MTT assays confirmed that the cytotoxicity was enhanced after irradiation. Findings suggested that the biosensor has a potential application in tumor targeting and the inhibition of tumor cell proliferation after photodegradation.

Ibuprofen-derived fluorescence inhibitor of COX-2 for breast cancer imaging, prevention and treatment

The integration of precise detection, long-term prevention and effective treatment are beneficial to monitor cancer bioactivity and prevent malignant metastasis for breast cancer patients. Herein, a novel fluorescence probe (IBLN) is designed and synthesized which contains a fluorophore (naphthalimide), a linker and a non-steroidal anti-inflammatory drug (ibuprofen). The specific optical imaging, prevention and treatment effects of breast cancer are relied on the introduction of a powerful cyclooxygenase-2-specific inhibitor, ibuprofen, which makes IBLN possible to successfully differentiate breast cancer cells from normal cells. In addition, fluorescent cellular imaging agent naphthalimide also has anticancer effects via inducing lysosomal membrane permeabilization (LMP). The multi-functional ibuprofen-derived fluorescence inhibitor IBLN realizes the imaging, prevention and treatment for breast cancer, which the selectivity and anticancer effects would be strengthened compared to the single naphthalimide or ibuprofen. Therefore, we envisage that the multi-function strategy in IBLN would be attractive way to explore potential reagent for fluorescence-visible breast cancer therapy in situ.

Structural Investigations, Cellular Imaging, and Radiolabeling of Neutral, Polycationic, and Polyanionic Functional Metalloporphyrin Conjugates.

Over the past decade, porphyrin derivatives have emerged as invaluable synthetic building blocks and theranostic kits for the delivery of cellular fluorescence imaging and photodynamic therapy. Tetraphenylporphyrin (TPP), its metal complexes, and related derivatives have been investigated for their use as dyes in histology and as components of multimodal imaging probes. The photophysical properties of porphyrin–metal complexes featuring radiometals have been a focus of our attention for the realization of fluorescence imaging probes coupled with radioimaging capabilities and therapeutic potential having “true” theranostic promise. We report hereby on the synthesis, radiochemistry, structural investigations, and preliminary in vitro and in vivo uptake studies on a range of functionalized porphyrin-based derivatives. In pursuit of developing new porphyrin-based probes for multimodality imaging applications, we report new functionalized neutral, polycationic, and polyanionic porphyrins incorporating nitroimidazole and sulfonamide moieties, which were used as targeting groups to improve the notoriously poor pharmacokinetics of porphyrin tags. The resulting functional metalloporphyrin species were stable under serum challenges and the nitroimidazole and sulfonamide derivatives remained fluorescent, allowing in vitro confocal studies and visualization of the lysosomal uptake in a gallium(III) sulfonamide derivative. The molecular structures of selected porphyrin derivatives were determined by single crystal X-ray diffraction using synchrotron radiation. We also investigated the nature of the emission/excitation behavior of model functional porphyrins using in silico approaches such as TD DFT in simple solvation models. The conjugation of porphyrins with the [7-13] and [7-14] fragments of bombesin was also achieved, to provide targeting of the gastrin releasing peptide receptor (GRPR). Depending on the metal, probe conjugates of relevance for single photon emission computed tomography (SPECT) or positron emission tomography (PET) probes have been designed and tested hereby, using TPP and related functional free base porphyrins as the bifunctional chelator synthetic scaffold and 111In[In] or 68Ga[Ga], respectively, as the central metal ions. Interestingly, for simple porphyrin conjugates good radiochemical incorporation was obtained for both radiometals, but the presence of peptides significantly diminished the radio-incorporation yields. Although the gallium-68 radiochemistry of the bombesin conjugates did not show radiochemical incorporation suitable for in vivo studies, likely because the presence of the peptide changed the behavior of the TPP-NH2 synthon taken alone, the optical imaging assays indicated that the conjugated peptide tags do mediate uptake of the porphyrin units into cells.

4-Amino-1,8-naphthalimide-ferrocene conjugates as potential multi-targeted anticancer and fluorescent cellular imaging agents.

Herein we present eight ferrocenyl 4-amino-1,8-naphthalimides. Designed as fluorescent logic gates for acidity and oxidisability, the molecules have been repurposed as anti-proliferation and cellular imaging agents. The compounds were studied in vitro against MCF-7 and K562 cancer cell lines by the MTT method. Compounds with protonable secondary amines tended to exhibit greater cytotoxicity than those with tertiary amines. Compounds with no measurable GI50 values within a 24 hour time window, as well as at shorter exposure times, may be suitable as fluorescent cellular imaging probes.

Transcription Factor RUNX1 Regulates Factor FXIIIA Subunit (F13A1) Expression in Megakaryocytic Cells and Platelet F13A1 Expression is Downregulated in RUNX1 Haplodeficiency

Plasma Factor XIII, a transglutaminase, is a complex of two A (gene F13A1) chains with catalytic activity and two B chains (gene F13B). FXIII induces clot stabilization and retraction via cross-linking of fibrin monomers and other proteins. Factor XIII deficiency in plasma causes a major bleeding disorder with impaired clot retraction. FXIIIA is synthesized in megakaryocytes, and is abundant in platelets. It is expressed on activated platelets and cross-links proteins on platelet surface. Little is known regarding the regulation of F13A1 in megakaryocytes and platelets. RUNX1 is a major hematopoietic transcription factor and regulates expression of numerous genes in megakaryocytes and platelets. Patients with RUNX1 haplodeficiency (RHD) with heterozygous mutations have thrombocytopenia, platelet dysfunction with impaired responses to activation, and predisposition to myeloid malignancies. Platelet expression profiling using Affymetrix microarrays of a patient (P1) with a heterozygous RUNX1 mutation (c.969-323G>T) revealed decreased F13A expression, which encodes for subunit A of FXIII: fold-change 0.30; p=0.006; patient profiled twice 10 months apart and compared to 6 healthy subjects. These findings were confirmed by qPCR With RT-PCR platelet F13A mRNA expression was decreased to ~ 20% of mean expression in 4 subjects. Platelet F13A protein expression was decreased by ~50% as assessed by corrected total cellular fluorescence imaging. Platelet F13A mRNA expression was also decreased (to less than 20% of expression in 5 controls) in two siblings (P2 and P3 ages 8 and 3 years) from an unrelated family with a heterozygous RUNX1 mutation 508+1G>A). Plasma FXIII was normal in the patient P1. We studied the regulation of F13A by RUNX1 in human erythroleukemia (HEL) cells treated with phorbol myristate acetate (PMA) to induce megakaryocytic transformation. In silico analyses of the 5' upstream region of F13A showed 7 RUNX1 consensus sites within 545bp from ATG. Chromatin immunoprecipitation studies performed using HEL cells and RUNX1 antibody showed that that RUNX1 binds in the region encompassing site 4 and sites 5-7. In parallel, electrophoretic mobility shift assay (EMSA) studies showed binding of nuclear protein to labeled DNA probes with sites 1, 4, 5, 6, and 7. RUNX1 antibody produced a "supershift" with addition suggesting RUNX1 binding. Site-directed mutagenesis of the RUNX1 binding sites in F13A1 promoter decreased promoter activity in luciferase reporter studies in HEL cells, providing evidence that the binding sites are functional. Further, overexpression of RUNX1 in HEL cells increased F13A1 promoter activity and protein while siRNA RUNX1 knockdown reduced F13A1 protein. We assessed clot retraction over 90 min at 37oC in the patient P1 and his daughter (also with RUNX1 mutation) in citrated whole blood following addition of tissue factor (1 pM) and CaCl2 (1 mM), and in washed platelets resuspended in platelet poor plasma from a healthy subject supplemented with added fibrinogen (0.5 mg/mL) and stimulated with 0.5 U/mL thrombin in the presence of CaCl2. These preliminary studies suggested that clot retraction was slower in the patient P1 compared to the controls, particularly in studies with platelet suspensions. Conclusions: Overall, these studies provide the first evidence that hematopoietic transcription factor RUNX1 regulates expression of F13A1 in megakaryocytic cells and that platelet expression of F13A1 is decreased in platelets from patients with RUNX1 haplodeficiency. The decreased expression of F13A1 in RHD may contribute to the platelet dysfunction in RHD. These findings are particularly interesting because they reflect regulation of a coagulation protein (FXIIIA) by hematopoietic transcription factor RUNX1. Disclosures Lambert: Dova: Consultancy, Membership on an entity's Board of Directors or advisory committees; Principia: Consultancy, Membership on an entity's Board of Directors or advisory committees; Novartis: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Shionogi: Consultancy; Educational Concepts in Medicine: Consultancy; Octapharma: Consultancy, Research Funding; Bayer: Consultancy; Platelet Disorder Support Association (PDSA): Consultancy; 22qSociety: Consultancy; ITP Australia: Consultancy; CdLS Foundation: Consultancy; RDMD ITP study: Consultancy; Sysmex: Research Funding; AstraZeneca: Research Funding; Argenix: Consultancy; ClinGen: Honoraria.

Apelin-13 regulates angiotensin ii-induced Cx43 downregulation and autophagy via the AMPK/mTOR signaling pathway in HL-1 cells.

Atrial fibrillation is associated with atrial remodeling, in which connexin 43 (Cx43) and cell hypertrophy play important roles. In this study, apelin-13, an aliphatic peptide, was used to explore the protective effects of the adenosine monophosphate-activated protein kinase (AMPK)/mTOR signaling pathway on Cx43 expression and autophagy, using murine atrial HL-1 cells. The expression of Cx43, AMPK, B-type natriuretic peptide (BNP) and pathway-related proteins was detected by Western blot analysis. Cellular fluorescence imaging was used to visualize Cx43 distribution and the cytoskeleton. Our results showed that the Cx43 expression was significantly decreased in HL-1 cells treated with angiotensin II but increased in cells additionally treated with apelin-13. Meanwhile, apelin-13 decreased BNP expression and increased AMPK expression. However, the expression of Cx43 and LC3 increased by apelin-13 was inhibited by treatment with compound C, an AMPK inhibitor. In addition, rapamycin, an mTOR inhibitor, promoted the development of autophagy, further inhibited the protective effect on Cx43 expression and increased cell hypertrophy. Thus, apelin-13 enhances Cx43 expression and autophagy via the AMPK/mTOR signaling pathway, and serving as a potential therapeutic target for atrial fibrillation.

Gold embedded chitosan nanoparticles with cell membrane mimetic polymer coating for pH-sensitive controlled drug release and cellular fluorescence imaging.

In this work, gold embedded chitosan nanoparticles (Au@CS NPs) were fabricated by a one-pot method. The benzaldehyde-terminated poly[(2-methacryloyloxy) ethyl phosphorylcholine] (PMPC) was applied to modification of the gold doped chitosan nanoparticles. The obtained Au@CS-PMPC NPs had the diameter of 135 nm with a narrow distribution. The size of the Au@CS-PMPC NPs, as well as the size of the embedded gold NPs, might be well-controlled by adjusting the feeding ratio between chitosan and HAuCl4. Furthermore, the Au@CS-PMPC NPs showed increased colloidal stability, high drug loading content, pH-responsive drug release, excellent biocompatibility and bright fluorescence emission. The results demonstrated that Au@CS-PMPC NPs showed a great potential for tumor therapy via the combination advantages of pH-sensitive controlled drug release and cellular fluorescence imaging.

Manganese–Fluorouracil Metallodrug Nanotheranostic for MRI-Correlated Drug Release and Enhanced Chemoradiotherapy

For cancer therapy, drug delivery systems are often limited by insufficient drug loading capacity, which usually results in systemic toxicity and heavy metabolic burden to excrete the carriers. Her...

Facile preparation of FITC-modified silicon nanodots for ratiometric pH sensing and imaging.

A ratiometric fluorescent pH sensor was facilely constructed by covalent modification of amino-terminated silicon nanodots (SiND) with pH-sensitive fluorescein isothiocyanate (FITC). After optimization, the SiND-FITC(40:1) material with a SiND:FITC initial mass ratio of 40:1 was selected for the sensing of hydrogen ions. It was observed that the material inherits the unique features of SiND and FITC, and there is significant improvement of SiND acid-base stability, which is a favorable factor in terms of providing fluorescence reference signal. The SiND-FITC(40:1) material displays not only high pH sensitivity, but also good stability and anti-interference ability, and the response process is highly reversible. Deploying the SiND-FITC(40:1) material, we have made available a simple, sensitive, and precise approach for pH sensing. In aqueous solutions, the I517/I466 fluorescence intensity ratio of SiND-FITC(40:1) increases linearly in the pH range of 5.40-7.76. This dual emission nanosensor was successfully applied for pH sensing and cellular fluorescence imaging.

Ultra-fast Cycling for Multiplexed Cellular Fluorescence Imaging.

Rapid analysis of single and scant cell populations is essential in modern diagnostics, yet existing methods are often limited and slow. Herein, we describe an ultra-fast, highly efficient cycling method for the analysis of single cells based on unique linkers for tetrazine (Tz)/trans-cyclooctene (TCO)-mediated quenching. Surprisingly, the quenching reaction rates were more than 3 orders of magnitude faster (t1/2 <1 s) than predicted. This allowed multi-cycle staining and immune cell profiling within an hour, leveraging the accelerated kinetics to open new diagnostic possibilities for rapid cellular analyses.

Ultra-fast cycling for multiplexed cellular fluorescence imaging.

Rapid analysis of single and scant cell populations is essential in modern diagnostics, yet existing methods are often limited and slow. Here we describe an ultra-fast, highly efficient cycling method for the analysis of single cells based on unique linkers for tetrazine (Tz) / trans-cyclooctene (TCO) mediated quenching. Surprisingly, the quenching reaction rates were more than 3 orders of magnitude faster (t1/2 < 1 sec) than predicted. This allowed multi-cycle staining and immune cell profiling within an hour, leveraging the accelerated kinetics to open new diagnostic possibilities for rapid cellular analyses.

A fluorescent turn-on probe for detection of hypochlorus acid and its bioimaging in living cells

Hypochlorous acid has played several functions in the biological system. However, excess HOCl can cause damage to biomolecules and result in some diseases. Accordingly, a new fluorescent probe, BSP, has been developed for fast recognition of HOCl through the HOCl-induced oxidation of methyl phenyl sulfide to sulfoxide. The reaction of BSP with HOCl caused a 22-fold fluorescence enhancement (quantum yield increase from 0.006 to 0.133). The detection limit of HOCl is found to be 30nM (S/N=3). The fluorescence enhancement is due to the suppression of the photo-induced electron transfer from the methyl phenyl sulfide moiety to BODIPY. Eventually, the cellular fluorescence imaging experiment showed that BSP could be effectively used for monitoring HOCl in living cells.