Validation of a new protocol for a zebrafish MEFL (malformation or embryo-fetal lethality) test method that conforms to the ICH S5 (R3) guideline.

Detecting the toxic effects of chemicals on reproduction and development without using mammalian animal models is crucial in the exploitation of pharmaceuticals for human use. Zebrafish are a promising animal model for investigating pharmacological effects and toxicity during vertebrate development. Several studies have suggested the use of zebrafish embryos for the assessment of malformations or embryo-fetal lethality (MEFL). However, a reproducible protocol as a standard for the zebrafish MEFL test method that fulfills global requests has not been established based on the International Council of Harmonisation (ICH) S5 (R3) guidelines. To establish such a toxicity test method, we developed a new and easy protocol to detect MEFL caused by chemicals, especially those with teratogenic potential, using fertilized zebrafish eggs (embryos) within 5 days of development. Our toxicity test trials using the same protocol in two to four different laboratories corroborated the high inter-laboratory reproducibility. Our test method enabled the detection of 18 out of 22 test compounds that induced rat MEFL. Thus, the prediction rate of our zebrafish test method for MEFL was almost 82% compared with that of rat MEFL. Collectively, our study proposes the establishment of an easy and reproducible protocol for the zebrafish MEFL test method for reproductive and developmental toxicity that meets ICH guideline S5 (R3), which can be further considered in combination with information from other sources for regulatory use.

Vertically aligned MXene bioelectrode prepared by freeze-drying assisted electrophoretic deposition for sensitive electrochemical protein detection.

Two-dimensional (2D) carbides, MXenes, have attracted attention as electrode materials of electrochemical biosensors because of their metallic conductivity, hydrophilicity, and mechanical stability. However, when fabricating electrodes, the nanosheets tend to re-stack and generally align horizontally with respect to the current collector due to the highly anisotropic nature of MXene, resulting in low porosity and poor utilization of the MXene surface. Here we report the electrochemical biosensing of antibody-antigen reactions with a vertically aligned Ti3C2Tx MXene (VA-MXene) electrode prepared by freeze-drying assisted electrophoretic deposition. The macroporous VA-MXene electrode exhibited a better electrochemical response towards the immunoreaction between the allergenic buckwheat protein (BWp16) and the antibody compared to a non-porous, horizontally (in-plane) stacked MXene (HS-MXene) and the sensors reported in the literature. The sensor responsiveness, defined as the ratio of the obtained current density of the electrode to the antigen concentration, was much higher for the VA-MXene electrode (238 µA cm-2 (ng mL-1) -1) than for the HS-MXene electrode. The proposed technique is applicable to other exfoliated nanosheets, and will open a new avenue for porous nanosheet electrodes to improve the sensing characteristics of electrochemical biosensors.

Loss of p53 function promotes DNA damage-induced formation of nuclear actin filaments.

Tumor suppressor p53 plays a central role in response to DNA damage. DNA-damaging agents modulate nuclear actin dynamics, influencing cell behaviors; however, whether p53 affects the formation of nuclear actin filaments remains unclear. In this study, we found that p53 depletion promoted the formation of nuclear actin filaments in response to DNA-damaging agents, such as doxorubicin (DOXO) and etoposide (VP16). Even though the genetic probes used for the detection of nuclear actin filaments exerted a promotive effect on actin polymerization, the detected formation of nuclear actin filaments was highly dependent on both p53 depletion and DNA damage. Whilst active p53 is known to promote caspase-1 expression, the overexpression of caspase-1 reduced DNA damage-induced formation of nuclear actin filaments in p53-depleted cells. In contrast, co-treatment with DOXO and the pan-caspase inhibitor Q-VD-OPh or the caspase-1 inhibitor Z-YVAD-FMK induced the formation of nuclear actin filament formation even in cells bearing wild-type p53. These results suggest that the p53-caspase-1 axis suppresses DNA damage-induced formation of nuclear actin filaments. In addition, we found that the expression of nLifeact-GFP, the filamentous-actin-binding peptide Lifeact fused with the nuclear localization signal (NLS) and GFP, modulated the structure of nuclear actin filaments to be phalloidin-stainable in p53-depleted cells treated with the DNA-damaging agent, altering the chromatin structure and reducing the transcriptional activity. The level of phosphorylated H2AX (γH2AX), a marker of DNA damage, in these cells also reduced upon nLifeact-GFP expression, whilst details of the functional relationship between the formation of nLifeact-GFP-decorated nuclear actin filaments and DNA repair remained to be elucidated. Considering that the loss of p53 is associated with cancer progression, the results of this study raise a possibility that the artificial reinforcement of nuclear actin filaments by nLifeact-GFP may enhance the cytotoxic effect of DNA-damaging agents in aggressive cancer cells through a reduction in gene transcription.

Platinum nanosheets synthesized via topotactic reduction of single-layer platinum oxide nanosheets for electrocatalysis.

Increasing the performance of Pt-based electrocatalysts for the oxygen reduction reaction (ORR) is essential for the widespread commercialization of polymer electrolyte fuel cells. Here we show the synthesis of double-layer Pt nanosheets with a thickness of 0.5 nm via the topotactic reduction of 0.9 nm-thick single-layer PtOx nanosheets, which are exfoliated from a layered platinic acid (HyPtOx). The ORR activity of the Pt nanosheets is two times greater than that of conventionally used state-of-the-art 3 nm-sized Pt nanoparticles, which is attributed to their large electrochemically active surface area (124 m2 g-1). These Pt nanosheets show excellent potential in reducing the amount of Pt used by enhancing its ORR activity. Our results unveil strategies for designing advanced catalysts that are considerably superior to traditional nanoparticle systems, allowing Pt catalysts to operate at their full potential in areas such as fuel cells, rechargeable metal-air batteries, and fine chemical production.

Symmetric Breakage-Induced Semimetallic State: Polymorphism in Ruthenate Nanosheets.

Atomic arrangements and their symmetries govern the physical properties of materials, including nanosheets that are low-dimensional nanomaterials. Although they have the same composition, symmetric changes associated with atomic displacements sometimes induce unexpected physical properties. Herein, we report that symmetric breakage induces a semimetallic state in chemically exfoliated ruthenate nanosheets. The atomic arrangements and symmetries are determined by a pair distribution function (PDF); subsequently, the physical properties are discussed using ab initio calculations and resistivity measurements. Ruthenate nanosheets can adopt an electronic structure similar to that of graphene owing to symmetric breakage. We experimentally confirmed the polymorphism in ruthenate nanosheets that highlights the importance of symmetric analysis, even in low-dimensional materials.

Zero-Overpotential Redox Reactions of Quinone-Based Molecules Confined in Carbon Micropores.

Quinone-based aromatic compounds have been studied as electrode materials for various energy-storage devices. However, the relatively large activation barrier of the charge-transfer process of these redox-active molecules causes sluggish reactions and a decrease in energy efficiency. To lower the activation barrier, aromatic compounds must be strongly adsorbed on the electrode surface, preferably via π-π stacking interactions. Molecules in slit-shaped micropores strongly adsorb on the graphitic walls, thus experiencing unique micropore-confinement properties. In this study, the micropore-confinement effect is extended to the adsorption of quinone-based redox-active molecules in 0.8 nm slit-shaped micropores of activated carbon, which produces a drastic reduction in the activation barrier of the charge-transfer process and creates a zero-overpotential redox reaction. The property originates from the short distance (approximately 0.3 nm) between the quinone molecules and the graphitic wall due to the strong adsorption of the aromatic compound. Our results provide the first demonstration that the micropore-confinement effect can reduce and nearly eliminate the activation barrier of an electrochemical reaction. We also demonstrate the applicability of this approach via the charge/discharge performance of a two-electrode cell. Cells comprising the aromatic compound/activated carbon material as positive and negative electrodes exhibit a greater retention capacity than those without activated carbon. The technique described herein can guide the development of high-performance, rapid charging/discharging electrodes for energy-storage devices such as batteries, supercapacitors, and hybrid devices using organic materials.

Enhancement in the Charge-Transfer Kinetics of Pseudocapacitive Iridium-Doped Layered Manganese Oxide.

Birnessite manganese oxide is a promising candidate as an electrode material for aqueous supercapacitors owing to its pseudocapacitance associated with fast redox processes. While manganese oxides are semiconductive, the conductivity is much lower than that of typical materials used for capacitive electrodes such as activated carbon or ruthenium oxide. In an attempt to increase the electronic conductivity of birnessite, a new solid solution phase, Ky(Mn1-xIrx)O2, was synthesized, and the electrochemical charge storage capability of Ir-doped birnessite was studied in aqueous Li2SO4. Structural characterization revealed that the single-phase Ky(Mn1-xIrx)O2 could be synthesized up to x = 0.1. An increase in the pseudocapacitive charge was observed with the increase in Ir content. In addition to the increase in the pseudocapacitive charge, an unusual change in the peak potential was observed. The peak-to-peak difference for the Mn4+/Mn3+ redox decreased with increasing Ir content, indicating an increase in the reversibility of the pseudocapacitive process. The decrease in peak-to-peak difference was observed only by Ir substitution and was not observed for physical mixtures of K0.28MnO2 and IrO2, suggesting a strong electronic interaction between the host Mn ion and the substituting Ir ion.

Intramolecular G-quadruplex-hairpin loop structure competition of a GC-rich exon region in the TMPRSS2 gene.

We identified cytosine-rich regions adjacent to guanine-rich regions in protease genes. A typical GC-rich sequence derived from the TMPRSS2 gene showed structural competition between a G-quadruplex and a hairpin loop, and this competition significantly affected transcription efficiency. These results suggest an impact of neighboring sequences on the gene expression of guanine-rich sequences.

Detection of Intracellular Reactive Oxidative Species Using the Fluorescent Probe Hydroxyphenyl Fluorescein.

Various fluorescent probes for the detection of intracellular reactive oxidative species (ROS) have been developed because ROS levels are closely associated with cellular states. Here, we describe a method for detection of intracellular ROS in living cells using the fluorescent probe, hydroxyphenyl fluorescein (HPF), which detects hydroxyl radicals and peroxynitrite. NIH3T3 cells and p53 knockout (p53-/-) mouse embryonic fibroblasts (MEFs) were transformed by expressing oncogenic RAS using a retrovirus system. The cells were treated with HPF at 37 °C for 30 min, and subsequently, images were acquired using a confocal fluorescence microscope at an excitation wavelength of 488 nm after washing with PBS.

The effect of rectal gas on dose distribution during prostate cancer treatment using full arc and partial arc Volumetric Modulated Arc Therapy (VMAT) treatment plans.

BACKGROUND/AIM: In this study, we investigated the effect of rectal gas on the dose distribution of prostate cancer using a volumetric modulated arc therapy (VMAT) treatment planning. MATERIALS AND METHODS: The first is the original structure set, clinical target volume (CTV), the rectum, and the bladder used clinically. The second is a structure set (simulated gas structure set) in which the overlapping part of the rectum and PTV is overwritten with Hounsfield Unit -950 as gas. Full arc and limited gantry rotation angle with VMAT were the two arcs. The VMAT of the full arc was 181°-179° in the clockwise (CW) direction and 179°-181° in the counterclockwise (CCW) direction. Three partial arcs with a limited gantry rotation angle were created: 200°-160 °CW and 160°-200 °CCW; 220°-140 °CW and 140°-220 °CCW; and finally, 240°-120 °CW and 120°-240 °CCW. The evaluation items were dose difference, distance to agreement, and gamma analysis. RESULT: In the CTV, the full arc was the treatment planning technique with the least effect of rectal gas. In the rectum, when the gantry rotation angle range was short, the pass rate tended to reduce for all evaluation indices. The bladder showed no characteristic change between the treatment planning techniques in any of the evaluation indices. CONCLUSIONS: The VMAT treatment planning with the least effect on dose distribution caused by rectal gas was shown to be a full arc.

Determination of reproducibility of end-exhaled breath-holding in stereotactic body radiation therapy.

Methods to evaluate the positional reproducibility of breath-hold irradiation mostly require manual operation. The purpose of this study is to propose a method to determine the reproducibility of breath-hold irradiation of lung tumors between fractions using non-artificial methods. This study included 13 patients who underwent terminal exhaled breath-hold irradiation for primary and metastatic lung cancer. All subjects received a prescribed dose of 60 Gy/8 fractions. The contours of the gross tumor volume (GTV) were extracted by threshold processing using treatment-planning computed tomography (CT) and cone-beam CT (CBCT), which was done just before the beginning of the treatment. The method proposed in this study evaluates the dice similarity coefficient (DSC) and Hausdorff distance (HD) by comparing two volumes, the GTVCTS (GTV obtained from treatment-planning CT) and GTVCBCT (GTV obtained from CBCT). The reference contours for DSC and HD are represented by GTVCTS. The results demonstrated good visual agreement for cases with a DSC of ~0.7. However, apparent misalignment occurred when the DSC was <0.5. HD was >2 mm in 3 out of 13 cases, and when the DSC was ~0.7, the HD was ~1 mm. In addition, cases with greater HD also demonstrated more significant variability. It was found that the DSC and HD evaluation methods for the positional reproducibility of breath-hold irradiation proposed in this study are straightforward and can be performed without the involvement of humans. Our study is of extreme significance in the field of radiation studies.

Simplification of head and neck volumetric modulated arc therapy patient-specific quality assurance, using a Delta4 PT.

BACKGROUND/AIM: In many facilities, intensity-modulated radiation therapy (IMRT), and volumetric modulated arc therapy (VMAT) use intensity-modulated beams, formed by a multi-leaf collimator (MLC). In IMRT and VMAT, MLC and linear accelerator errors (both geometric and dose), can significantly affect the doses administered to patients. Therefore, IMRT and VMAT treatment plans must include the use of patient-specific quality assurance (QA) before treatment to confirm dose accuracy. MATERIALS AND METHODS: In this study, we compared and analyzed the results of dose verification using a multi-dimensional dose verification system Delta4 PT, an ionization chamber dosimeter, and gafchromic film, using data from 52 patients undergoing head and neck VMAT as the test material. RESULT: Based on the results of the absolute dose verification for the ionization chamber dosimeter and Delta4 PT, taking an axial view, the upper limit of the 95% confidence interval was 3.13%, and the lower limit was -3.67%, indicating good agreement. These results mean that as long as absolute dose verification for the axial view does not deviate from this range, Delta4 PT can be used as an alternative to an ionization chamber dosimeter for absolute dose verification. When we then reviewed dose distribution verification, the pass rate for Delta4 PT was acceptable, and was less varied than that of gafchromic film. CONCLUSION: This results in that provided the pass rate result for Delta4 PT does not fall below 96%, it can be used as a substitute for gafchromic film in dose distribution verification. These results indicate that patient-specific QA could be simplified.

Scalable Design of Two-Dimensional Oxide Nanosheets for Construction of Ultrathin Multilayer Nanocapacitor.

Large size of capacitors is the main hurdle in miniaturization of current electronic devices. Herein, a scalable solution-based layer-by-layer engineering of metallic and high-κ dielectric nanosheets into multilayer nanosheet capacitors (MNCs) with overall thickness of ≈20 nm is presented. The MNCs are built through neat tiling of 2D metallic Ru0.95 O2 0.2- and high-κ dielectric Ca2 NaNb4 O13 - nanosheets via the Langmuir-Blodgett (LB) approach at room temperature which is verified by cross-sectional high-resolution transmission electron microscopy (HRTEM). The resultant MNCs demonstrate a high capacitance of 40-52 µF cm-2 and low leakage currents down to 10-5 -10-6 A cm-2 . Such MNCs also possess complimentary in situ robust dielectric properties under high-temperature measurements up to 250 °C. Based on capacitance normalized by the thickness, the developed MNC outperforms state-of-the-art multilayer ceramic capacitors (MLCC, ≈22 µF cm-2 /5 × 104  nm) present in the market. The strategy is effective due to the advantages of facile, economical, and ambient temperature solution assembly.

MMP24 as a Target of YAP is a Potential Prognostic Factor in Cancer Patients.

The extracellular matrix (ECM) surrounding cancer cells becomes stiffer during tumor progression, which influences cancer cell behaviors such as invasion and proliferation through modulation of gene expression as well as remodeling of the actin cytoskeleton. In this study, we show that MMP24 encoding matrix metalloproteinase (MMP)-24 is a novel target gene of Yes-associated protein (YAP), a transcription coactivator known as a mechanotransducer. We first examined the effect of substrate stiffness on MMP24 expression in MCF-7 human breast cancer cells and showed that the expression of MMP24 was significantly higher in cells grown on stiff substrates than that on soft substrates. The MMP24 expression was significantly reduced by knockdown of YAP. In contrast, the expression of constitutively active YAP increased MMP24 promoter activity. In addition, binding of YAP to the MMP24 promoter was confirmed by the chromatin immunoprecipitation (ChIP) assay. These results show that ECM stiffening promotes YAP activation, thereby inducing MMP24 expression. Based on the Human Protein Atlas database, breast cancer patients with lower MMP24 expression exhibit the worse survival rates overall. Thus, MMP24 may negatively regulate the aggressiveness of cancer cells under the stiff ECM environment during tumor progression.

Long-term stability of a three-dimensional dose verification system.

Recently, the number of patients undergoing intensity-modulated radiation therapy and volumetric modulated arc therapy has increased with the expansion of the adaptation site. However, it is necessary to improve the efficiency of time-consuming dose verification. Therefore, patient-specific quality assurance is expected to shift from dose verification using a conventional ionization chamber dosimeter and film to a three-dimensional dose verification system. However, caution is required when using a three-dimensional dose verification system, especially when it comes to the calibration of the detector. Calibration is performed regularly, but not all necessary verifications are done routinely. There are many uncertainties on how the sensitivity of the three-dimensional dose verification system changes over time. In this study, on the same day, when dose verification using a three-dimensional system for one head and neck case was performed, dose verification using a conventional ionization chamber dosimeter and film was also performed once every two months, for 2 years. From the results of the absolute dose and dose distribution verification using the ionization chamber dosimeter and Gafchromic film, the output of the linear accelerator, mechanical accuracy and precision were secured. From the results of the three-dimensional dose verification system, when the distance to agreement was evaluated at 2 mm and 3 mm, and gamma analysis was performed at 2 mm/2% and 3 mm/3%, the passing rate was almost 100%, and a sensitivity change in 2 years was not observed.

DMPK is a New Candidate Mediator of Tumor Suppressor p53-Dependent Cell Death.

Tumor suppressor p53 plays an integral role in DNA-damage induced apoptosis, a biological process that protects against tumor progression. Cell shape dramatically changes when cells undergo apoptosis, which is associated with actomyosin contraction; however, it remains entirely elusive how p53 regulates actomyosin contraction in response to DNA-damaging agents. To identify a novel p53 regulating gene encoding the modulator of myosin, we conducted DNA microarray analysis. We found that, in response to DNA-damaging agent doxorubicin, expression of myotonic dystrophy protein kinase (DMPK), which is known to upregulate actomyosin contraction, was increased in a p53-dependent manner. The promoter region of DMPK gene contained potential p53-binding sequences and its promoter activity was increased by overexpression of the p53 family protein p73, but, unexpectedly, not of p53. Furthermore, we found that doxorubicin treatment induced p73 expression, which was significantly attenuated by downregulation of p53. These data suggest that p53 induces expression of DMPK through upregulating p73 expression. Overexpression of DMPK promotes contraction of the actomyosin cortex, which leads to formation of membrane blebs, loss of cell adhesion, and concomitant caspase activation. Taken together, our results suggest the existence of p53-p73-DMPK axis which mediates DNA-damage induced actomyosin contraction at the cortex and concomitant cell death.

Dopamine D2L Receptor Deficiency Causes Stress Vulnerability through 5-HT1A Receptor Dysfunction in Serotonergic Neurons.

Mental disorders are caused by genetic and environmental factors. We here show that deficiency of an isoform of dopamine D2 receptor (D2R), D2LR, causes stress vulnerability in mouse. This occurs through dysfunction of serotonin [5-hydroxytryptamine (5-HT)] 1A receptor (5-HT1AR) on serotonergic neurons in the mouse brain. Exposure to forced swim stress significantly increased anxiety- and depressive-like behaviors in D2LR knock-out (KO) male mice compared with wild-type mice. Treatment with 8-OH-DPAT, a 5-HT1AR agonist, failed to alleviate the stress-induced behaviors in D2LR-KO mice. In forced swim-stressed D2LR-KO mice, 5-HT efflux in the medial prefrontal cortex was elevated and the expression of genes related to 5-HT levels was upregulated by the transcription factor PET1 in the dorsal raphe nucleus. Notably, D2LR formed a heteromer with 5-HT1AR in serotonergic neurons, thereby suppressing 5-HT1AR-activated G-protein-activated inwardly rectifying potassium conductance in D2LR-KO serotonergic neurons. Finally, D2LR overexpression in serotonergic neurons in the dorsal raphe nucleus alleviated stress vulnerability observed in D2LR-KO mice. Together, we conclude that disruption of the negative feedback regulation by the D2LR/5-HT1A heteromer causes stress vulnerability.SIGNIFICANCE STATEMENT Etiologies of mental disorders are multifactorial, e.g., interactions between genetic and environmental factors. In this study, using a mouse model, we showed that genetic depletion of an isoform of dopamine D2 receptor, D2LR, causes stress vulnerability associated with dysfunction of serotonin 1A receptor, 5-HT1AR in serotonergic neurons. The D2LR/5-HT1AR inhibitory G-protein-coupled heteromer may function as a negative feedback regulator to suppress psychosocial stress.

Conductive Nanosized Magnéli-Phase Ti4O7 with a Core@Shell Structure.

Magnéli-phase Ti4O7, known for its high electrical conductivity and corrosion resistance, is typically prepared by hydrogen reduction at high temperatures (∼1000 °C), leading to large particles. Nanosized Ti4O7 have been explored for application toward high specific surface area electrode materials and electrocatalyst supports; nonetheless, the particle size of Ti4O7 is still insufficient for utilization as a support. In this study, we have pursued a novel synthetic approach for nanosized Ti4O7 platelets with a length of 10-80 nm and thickness of 3-10 nm even under high-temperature conditions. We herein describe the use of SiO2 beads as a core to obtain a SiO2 core coated with multilayers of TiO2 nanosheets exfoliated from layered H2Ti4O7 which is subsequently subjected to high-temperature reduction to prepare a SiO2-core@Ti4O7-shell structure. The pair distribution function technique has proven that the shell is transformed to single-phase Ti4O7. The electrical double layer capacitance of SiO2-core@Ti4O7-shell was much larger than that of conventionally synthesized Ti4O7 particles with a micrometer size. The results show the beneficial effects of the SiO2-core@Ti4O7-shell structure, and it is the first example of the synthesis for conductive Ti4O7 with a high specific surface area even under conditions of high-temperature synthesis.

Electrochemical and Spectroscopic Analysis of the Ionogel-Electrode Interface.

Ionogels, pseudo-solid-state electrolytes consisting of an ionic liquid electrolyte confined in a mesoporous inorganic matrix, have attracted interest recently due to their high ionic conductivity and physicochemical stability. These traits, coupled with their inherent solution processability, make them a viable solid electrolyte for solid-state battery systems. Despite the promising properties of ionogels, there have been very few investigations of the electrode-ionogel interface. In the present study, X-ray photoelectron spectroscopy, Raman spectroscopy, and electrochemical measurements were utilized to probe the surface reactions occurring at the electrode-ionogel interface for several electrode materials. Our results indicate that the sol acidity initiates breakdown of the organic constituents of the sol and reduction of the transition metals present in the electrode materials. This chemical attack forms an organic surface layer and affects the electrode composition, both of which can impede Li+ access. By modifying the silica sol-gel reaction via a two-step acid-base catalysis, these interfacial reactions can be avoided. Results are shown for a LiCoO2 electrode in which a high Li-ion capacity and stable cycling were achieved.

Mitochondrial protein E2F3d, a distinctive E2F3 product, mediates hypoxia-induced mitophagy in cancer cells.

Mitochondrial damage is caused by changes in the micro-environmental conditions during tumor progression. Cancer cells require mechanisms for mitochondrial quality control during this process; however, how mitochondrial integrity is maintained is unclear. Here we show that E2F3d, a previously unidentified E2F3 isoform, mediates hypoxia-induced mitophagy in cancer cells. Aberrant activity and expression of the E2F3 transcription factor is frequently observed in many cancer cells. Loss of retinoblastoma (Rb) protein family function increases the expression of E2F3d and E2F3a. E2F3d localizes to the outer mitochondrial membrane and its cytosolic domain contains an LC3-interacting region motif. Overexpression of E2F3d induces mitochondrial fragmentation and mitophagy, suggesting that E2F3d plays an important role in mitophagy. Furthermore, depletion of E2F3s attenuates hypoxia-induced mitophagy and increases intracellular levels of reactive oxygen species, which is reversed by the reintroduction of E2F3d. This study presents another key player that regulates mitochondrial quality control in cancer cells.