In Vivo Evaluation of 68Ga-Labeled NOTA-EGFRvIII Aptamer in EGFRvIII-Positive Glioblastoma Xenografted Model.

EGFRvIII is expressed only in tumor cells and strongly in glioblastoma and is considered a promising target in cancer diagnosis and therapy. Aptamers are synthetic single-stranded oligonucleotides that bind to biochemical target molecules with high binding affinity and specificity. This study examined the potential of the 68Ga-NOTA-EGFRvIII aptamer as a nuclear imaging probe for visualizing EGFRvIII-expressing glioblastoma by positron emission tomography (PET). EGFRvIII aptamer was selected using the SELEX technology, and flow cytometry and fluorescence microscopy verified the high binding affinity to EGFRvIII positive U87MG vIII 4.12 glioma cells but not to EGFRvIII negative U87MG cells. The EGFRvIII aptamer was conjugated with a chelator (1,4,7-triazanonane-1,4,7-triyl)triacetic acid (NOTA) for 68Ga-labeling. The 68Ga-NOTA-EGFRvIII aptamer was prepared using the preconcentration-based labeling method with a high radiolabeling yield at room temperature. Ex vivo biodistribution analyses confirmed the significantly higher tumor uptake of the 68Ga-NOTA-EGFRvIII aptamer in EGFRvIII-expressing xenograft tumors than that in EGFRvIII negative tumors, confirming the specific tumor uptake of the 68Ga-NOTA-EGFRvIII aptamer in vivo. PET imaging studies revealed a high retention rate of the 68Ga-NOTA-EGFRvIII aptamer in U87MG vIII 4.12 tumors but only low uptake levels in U87-MG tumors, suggesting that the 68Ga-NOTA-EGFRvIII aptamer may be used as a PET imaging agent for EGFRvIII-expressing glioblastoma.

Pharmacokinetics of collagen dipeptides (Gly-Pro and Pro-Hyp) and tripeptides (Gly-Pro-Hyp) in rats.

Although systemic exposure to peptides, such as Gly-Pro-Hyp, Pro-Hyp, and Gly-Pro, has been reported following administration of collagen hydrolysates from fish scale and porcine skin in vivo, the individual peptide pharmacokinetics remain unknown. We administered the three peptides individually to rats via the intravenous (5 mg/kg) and intragastric (100 mg/kg) routes and then monitored systemic exposure and urinary excretion. The peptides in biological samples were analyzed via liquid chromatography/tandem mass spectrometry. Gly-Pro-Hyp tended to exhibit higher first-pass metabolism than Pro-Hyp; the absolute oral bioavailabilities of Gly-Pro-Hyp and Pro-Hyp were 4.4% and 19.3%, respectively. Gly-Pro levels were very low in the systemic circulation. Pro-Hyp biotransformed from Gly-Pro-Hyp behaved similarly to Pro-Hyp alone when administered orally. Flip-flop kinetics (elimination rate ≫ absorption rate) were evident, probably reflecting transporter-mediated slow absorption. A double-peak phenomenon was observed for Gly-Pro-Hyp and Pro-Hyp when administered orally, and 5.9% ± 2.6% and 1.9% ± 0.3% of each dose were excreted in urine after intravenous administration, respectively. Urinary recovery of Gly-Pro was limited to 0.4% ± 0.5% of the intravenous dose. This work represents the first individual pharmacokinetics of Gly-Pro-Hyp, Pro-Hyp, and Gly-Pro in vivo.

The Point-Of-Care Test to Detect Nephrin from Urine in Preeclampsia.

BACKGROUND: Nephrin is a protein in the glomerular podocyte slit diaphragm; therefore, its presence in urine implies damage to podocytes. This study aimed to determine the usefulness of nephrin as a biomarker in maternal urine to predict preeclampsia (PE). METHODS: This prospective study included pregnant women admitted for delivery at Seoul National University Bundang Hospital from March 2019 to May 2020. Patients who had been diagnosed with PE were included, and patients without a history of underlying diseases were recruited for the control group. Pertinent clinical data were collected. Urine samples were obtained, and nephrin signaling was detected through test strips using a lateral flow assay. The point-of-care test results were compared between patients with PE and without (control group), using the exact concentration of nephrin by enzyme-linked immunosorbent assay. RESULTS: Clinical characteristics - maternal age, parity, proportion of twin pregnancies, height, weight, and cesarean delivery rate - were comparable between the PE and control groups. Nephrin signals were classified into four groups. In the PE group, signals 0, 1, 2, and 3 were found in 18.4% (9/49), 44.9% (22/49), 24.5% (12/49), and 12.2% (6/49) of participants, respectively. Results were significantly different in the control group, in which 84.3% (43/51) were found to have signal 0 (p < 0.001). CONCLUSIONS: Nephrin signaling in maternal urine could be a noninvasive and useful test for early detection of severity of PE.

Interferometric Imaging, and Beam-Formed Study of a Moving Type-IV Radio Burst with LOFAR.

Type-IV radio bursts have been studied for over 50 years. However, the specifics of the radio emission mechanisms is still an open question. In order to provide more information about the emission mechanisms, we studied a moving Type-IV radio burst with fine structures (spike group) by using the high-resolution capability of the Low-Frequency Array (LOFAR) on August 25, 2014. We present a comparison of Nançay Radioheliograph (NRH) and the first LOFAR imaging data of the Type-IV radio burst. The degree of circular polarization (DCP) is calculated at frequencies in the range 20 - 180 MHz using LOFAR data, and it was found that the value of DCP gradually increased during the event, with values of 20 - 30%. LOFAR interferometric data were combined with white-light observations in order to track the propagation of this Type-IV burst. The kinematics shows a westward motion of the radio sources, slower than the CME leading edge. The dynamic spectrum of LOFAR shows a large number of fine structures with durations of less than 1 s and high brightness temperatures ( T B ), i.e., 10 12 - 10 13 K. The gradual increase of DCP supports gyrosynchrotron emission as the most plausible mechanism for the Type IV. However, coherent emissions such as Electron Cyclotron Maser (ECM) instability may be responsible for small-scale fine structures. Countless fine structures altogether were responsible for such high T B . Supplementary Information: The online version contains supplementary material available at 10.1007/s11207-022-02042-0.

Bioavailability of xanthorrhizol following oral administration of a supercritical extract of Java turmeric.

Although xanthorrhizol, a sesquiterpenoid oil obtained from the rhizome of Curcuma xanthorrhiza Roxb., known as Java turmeric, has many pharmacological effects, its pharmacokinetics remain unclear. Therefore, we investigated the pharmacokinetics of xanthorrhizol in mice and rats. Xanthorrhizol was administered intravenously and orally to mice, while xanthorrhizol and a Java turmeric supercritical extract were administered orally to rats. The terminal half-life (t1/2), clearance, and absolute bioavailability (BA) of xanthorrhizol in mice were almost 8 h, 6.5 L/h/kg, and 10.2%, respectively. In comparison, the clearance of xanthorrhizol was 3-fold higher in rats than mice. The absolute BAs of xanthorrhizol in rats were 12.9% and 13.4% after oral administration of xanthorrhizol and a supercritical extract, respectively. Our results regarding the pharmacokinetics of xanthorrhizol could guide the conversion of intravenous and oral doses, and help identify the optimal maintenance doses of xanthorrhizol and the extract for desirable pharmacodynamic effects.

Quantitative determination of ICG-001 in rat plasma using HPLC-MS/MS: A pharmacokinetic study.

Although ICG-001, chemically synthesised from a bicyclic ß-turn peptidomimetic template, represents various pharmacological activities, no validated determination methods in biological samples have been reported. This study was designed to establish a quantitative determination method for ICG-001 in rat plasma using high-performance liquid chromatography coupled with tandem mass spectrometry (HPLC-MS/MS) to validate the analytical method, including stability, and to characterise its pharmacokinetic behaviour in rats. After simple protein precipitation with acetonitrile, ICG-001 was eluted on a reversed-phase column using a mobile phase of water and acetonitrile (3:7 v/v, including 0.1% formic acid). The protonated precursor ion [M+H]+ and the major fragment ion were confirmed at m/z 549.2 and 141.4, respectively, for ICG-001. ICG-001 was stable under bench and storage conditions. The analytical method met the criteria for Food and Drug Administration-validated bioanalytical methods, and was successfully applied to a pharmacokinetic study for the first time following subcutaneous and intravenous administration.

Molecular diagnosis of McArdle disease using whole-exome sequencing.

Whole-exome sequencing (WES) analysis has been used recently as a diagnostic tool for finding molecular defects. In the present study, researchers attempted to analyze molecular defects through WES in a 13-year-old female patient who had not been diagnosed through a conventional genetic approach. DNA was extracted and subjected to WES analysis to identify the genetic defect. A total of 106,728 exons and splicing variants were selected, and synonymous single nucleotide variants (SNVs) and general single nucleotide polymorphisms (SNPs) were filtered out. Finally, nonsynonymous SNVs (c.C415T and c.C389T) of the PYGM gene were identified in nine compound heterozygous mutations. PYGM encodes myophosphorylase and degrades glycogen in the muscle to supply energy to muscle cells. The present study revealed that the patient's father had a c.C389T mutation and the mother had a c.C415T mutation, resulting in A130V and R139W missense mutations, respectively. To the best of our knowledge, the A130V variant in PYGM has not been reported in the common variant databases. All variations of the patient's family detected using WES were verified by Sanger sequencing. Because the patient had compound heterozygous mutations in the PYGM gene, the patient was presumed to exhibit markedly decreased muscle phosphorylase activity. To assess the function of myophosphorylase, an ischemic forearm exercise test was performed. The blood ammonia level sharply increased and the lactate level maintained a flat curve shape similar to the typical pattern of McArdle disease. Therefore, the diagnosis of the patient was confirmed to be McArdle disease, a glycogen storage disease. Through WES analysis, accurate and early diagnosis could be made in the present study. This report describes a novel compound heterozygous mutation of the PYGM gene in a Korean patient.

A sensitive analytical method for the determination of SG-SP1 in rat plasma by HPLC-MS/MS and its application to a pharmacokinetic study.

SG-SP1, a newly synthesised gallic acid derivative, blocks histamine release by reducing calcium influx in mast cells and inhibits inflammatory cytokine expression. This derivative has promising anti-allergic potential. Our research was designed to establish a quantitative determination method for SG-SP1 in rat plasma using high-performance liquid chromatography coupled with tandem mass spectrometry (HPLC-MS/MS), to validate the analytical method including stability and to characterise its pharmacokinetic behaviour in rats. After simple protein precipitation with acetonitrile including an internal standard, SG-SP1 was eluted on a reversed-phase column using a mobile phase of water and acetonitrile (2:8 v/v, including 0.1 % formic acid). The protonated precursor ion [M+H]+ and major fragment ion were confirmed at m/z 588.2 and 180.1, respectively. The substance was stable under bench and storage conditions. The analytical method met the criteria of FDA-validated bioanalytical methods and was successfully applied to a pharmacokinetic study for the first time. SG-SP1 decayed in a biphasic pattern with terminal half-life of 5.1 h and clearance of about 3.2 L/h/kg. Double peaks were observed following oral administration, and the absolute oral bioavailability was ∼1 %.

Nanoelectromechanical modulation of a strongly-coupled plasmonic dimer.

The ability of two nearly-touching plasmonic nanoparticles to squeeze light into a nanometer gap has provided a myriad of fundamental insights into light-matter interaction. In this work, we construct a nanoelectromechanical system (NEMS) that capitalizes on the unique, singular behavior that arises at sub-nanometer particle-spacings to create an electro-optical modulator. Using in situ electron energy loss spectroscopy in a transmission electron microscope, we map the spectral and spatial changes in the plasmonic modes as they hybridize and evolve from a weak to a strong coupling regime. In the strongly-coupled regime, we observe a very large mechanical tunability (~250 meV/nm) of the bonding-dipole plasmon resonance of the dimer at ~1 nm gap spacing, right before detrimental quantum effects set in. We leverage our findings to realize a prototype NEMS light-intensity modulator operating at ~10 MHz and with a power consumption of only 4 fJ/bit.

Lipopolysaccharide regulates thymic stromal lymphopoietin expression via TLR4/MAPK/Akt/NF-κB-signaling pathways in nasal fibroblasts: differential inhibitory effects of macrolide and corticosteroid.

BACKGROUND: Chronic rhinosinusitis (CRS) is an inflammatory disease of the sinonasal mucosa. Thymic stromal lymphopoietin (TSLP) is associated with T-helper 2 (Th2) response and induced by pathogen, allergen, toll-like receptor (TLR) ligands, and cytokines. Fibroblasts are known to be modulators of wound-healing, from inflammation to tissue remodeling. We examined effect of lipopolysaccharide (LPS) on TSLP production and the underlying mechanisms. We aimed to determine whether the effects of commonly used medications in CRS, namely corticosteroids, and macrolides, are related to LPS-induced TSLP in nasal fibroblasts. METHODS: Fibroblasts were isolated from inferior turbinate tissues of CRS patients. TSLP and TLR4 expressions were determined by reverse transcript-polymerase chain reaction (RT-PCR), Western blot, enzyme-linked immunoassay, and immunofluorescence staining. Mitogen-activated protein kinase (MAPK), protein kinase B (Akt), and nuclear factor-kappaB (NF-κB) phosphorylation was determined by Western blot and/or luciferase assay. RESULTS: LPS increased TSLP expression in a dose- and time-dependent manner. LPS antagonist and corticosteroids inhibited TLR4 expression in LPS-stimulated fibroblasts. LPS-RS, macrolides, corticosteroids, and specific inhibitors suppressed LPS-induced alterations. Ex vivo culture showed similar results. CONCLUSION: LPS induces TSLP production via the TLR4, MAPK, Akt, and NF-κB pathways. The effects of corticosteroids and macrolides are related to LPS-induced TSLP expression. We explored new treatment modalities targeting LPS-induced TSLP production that could replace the currently used corticosteroid and macrolides for treatment of CRS.

TGF-ß1 Activates Nasal Fibroblasts through the Induction of Endoplasmic Reticulum Stress.

(1) Background: Tissue remodeling and extracellular matrix (ECM) accumulation contribute to the development of chronic inflammatory diseases of the upper airway. Endoplasmic reticulum (ER) stress is considered to be the key signal for triggering tissue remodeling in pathological conditions. The present study aimed to investigate the role of ER-stress in TGF-ß1-stimulated nasal fibroblasts and inferior turbinate organ cultures; (2) Methods: Fibroblasts and organ cultures were pretreated with 4-phenylbutyric acid (PBA) and stimulated with TGF-ß1 or thapsigargin (TG). Expression of ER-stress markers, myofibroblast marker, and ECM components was measured by Western blotting and real-time PCR. Reactive oxygen species (ROS) were quantified using 2',7'-dichlorofluorescein diacetate. Cell migration was evaluated using Transwell assays. Contractile activity was measured by collagen contraction assay; (3) Results: 4-PBA inhibited TGF-ß1 or TG-induced ER-stress marker expression, phenotypic changes, and ECM. Pre-treatment with ROS scavengers inhibited the expression of TGF-ß1-induced ER-stress markers. Migration and collagen contraction of TGF-ß1 or TG-stimulated fibroblasts were ameliorated by 4-PBA treatment. These findings were confirmed in ex vivo organ cultures; (4) Conclusions: 4-PBA downregulates TGF-ß1-induced ER-stress marker expression, migration, and collagen contraction via ROS in fibroblasts and organ cultures. These results suggest that ER-stress may play an important role in progression of chronic upper airway inflammatory diseases by aiding pathological tissue remodeling.

Dynamic thermal emission control with InAs-based plasmonic metasurfaces.

Thermal emission from objects tends to be spectrally broadband, unpolarized, and temporally invariant. These common notions are now challenged with the emergence of new nanophotonic structures and concepts that afford on-demand, active manipulation of the thermal emission process. This opens a myriad of new applications in chemistry, health care, thermal management, imaging, sensing, and spectroscopy. Here, we theoretically propose and experimentally demonstrate a new approach to actively tailor thermal emission with a reflective, plasmonic metasurface in which the active material and reflector element are epitaxially grown, high-carrier-mobility InAs layers. Electrical gating induces changes in the charge carrier density of the active InAs layer that are translated into large changes in the optical absorption and thermal emission from metasurface. We demonstrate polarization-dependent and electrically controlled emissivity changes of 3.6%P (6.5% in relative scale) in the mid-infrared spectral range.

Thermoplasmonic Ignition of Metal Nanoparticles.

Explosives, propellants, and pyrotechnics are energetic materials that can store and quickly release tremendous amounts of chemical energy. Aluminum (Al) is a particularly important fuel in many applications because of its high energy density, which can be released in a highly exothermic oxidation process. The diffusive oxidation mechanism (DOM) and melt-dispersion mechanism (MDM) explain the ways powders of Al nanoparticles (NPs) can burn, but little is known about the possible use of plasmonic resonances in NPs to manipulate photoignition. This is complicated by the inhomogeneous nature of powders and very fast heating and burning rates. Here, we generate Al NPs with well-defined sizes, shapes, and spacings by electron beam lithography and demonstrate that their plasmonic resonances can be exploited to heat and ignite them with a laser. By combining simulations with thermal-emission, electron-, and optical-microscopy studies, we reveal how an improved control over NP ignition can be attained.

Anti-Hermitian photodetector facilitating efficient subwavelength photon sorting.

The ability to split an incident light beam into separate wavelength bands is central to a diverse set of optical applications, including imaging, biosensing, communication, photocatalysis, and photovoltaics. Entirely new opportunities are currently emerging with the recently demonstrated possibility to spectrally split light at a subwavelength scale with optical antennas. Unfortunately, such small structures offer limited spectral control and are hard to exploit in optoelectronic devices. Here, we overcome both challenges and demonstrate how within a single-layer metafilm one can laterally sort photons of different wavelengths below the free-space diffraction limit and extract a useful photocurrent. This chipscale demonstration of anti-Hermitian coupling between resonant photodetector elements also facilitates near-unity photon-sorting efficiencies, near-unity absorption, and a narrow spectral response (∼ 30 nm) for the different wavelength channels. This work opens up entirely new design paradigms for image sensors and energy harvesting systems in which the active elements both sort and detect photons.

Chemical Chaperone of Endoplasmic Reticulum Stress Inhibits Epithelial-Mesenchymal Transition Induced by TGF-ß1 in Airway Epithelium via the c-Src Pathway.

Epithelial-mesenchymal transition (EMT) is a biological process that allows epithelial cells to assume a mesenchymal cell phenotype. EMT is considered as a therapeutic target for several persistent inflammatory airway diseases related to tissue remodeling. Herein, we investigated the role of endoplasmic reticulum (ER) stress and c-Src in TGF-ß1-induced EMT. A549 cells, primary nasal epithelial cells (PNECs), and inferior nasal turbinate organ cultures were exposed to 4-phenylbutylic acid (4PBA) or PP2 and then stimulated with TGF-ß1. We found that E-cadherin, vimentin, fibronectin, and α-SMA expression was increased in nasal polyps compared to inferior turbinates. TGF-ß1 increased the expression of EMT markers such as E-cadherin, fibronectin, vimentin, and α-SMA and ER stress markers (XBP-1s and GRP78), an effect that was blocked by PBA or PP2 treatment. 4-PBA and PP2 also blocked the effect of TGF-ß1 on migration of A549 cells and suppressed TGF-ß1-induced expression of EMT markers in PNECs and organ cultures of inferior turbinate. In conclusion, we demonstrated that 4PBA inhibits TGF-ß1-induced EMT via the c-Src pathway in A549 cells, PNECs, and inferior turbinate organ cultures. These results suggest an important role for ER stress and a diverse role for TGF-ß1 in upper airway chronic inflammatory disease such as CRS.

Electrical tuning of a quantum plasmonic resonance.

Surface plasmon (SP) excitations in metals facilitate confinement of light into deep-subwavelength volumes and can induce strong light-matter interaction. Generally, the SP resonances supported by noble metal nanostructures are explained well by classical models, at least until the nanostructure size is decreased to a few nanometres, approaching the Fermi wavelength λF of the electrons. Although there is a long history of reports on quantum size effects in the plasmonic response of nanometre-sized metal particles, systematic experimental studies have been hindered by inhomogeneous broadening in ensemble measurements, as well as imperfect control over size, shape, faceting, surface reconstructions, contamination, charging effects and surface roughness in single-particle measurements. In particular, observation of the quantum size effect in metallic films and its tuning with thickness has been challenging as they only confine carriers in one direction. Here, we show active tuning of quantum size effects in SP resonances supported by a 20-nm-thick metallic film of indium tin oxide (ITO), a plasmonic material serving as a low-carrier-density Drude metal. An ionic liquid (IL) is used to electrically gate and partially deplete the ITO layer. The experiment shows a controllable and reversible blue-shift in the SP resonance above a critical voltage. A quantum-mechanical model including the quantum size effect reproduces the experimental results, whereas a classical model only predicts a red shift.

Effect of doxycycline on epithelial-mesenchymal transition via the p38/Smad pathway in respiratory epithelial cells.

PURPOSE: Doxycycline has antibacterial and anti-inflammatory effects, and it also suppresses collagen biosynthesis. This study aimed to confirm the effects and mechanism of doxycycline on transforming growth factor (TGF) beta 1 induced epithelial-mesenchymal transition and cell migration in A549 and primary nasal epithelial cells. METHODS: A 3-(4,5-dimethylthiazol-2yl)-2,5-diphenyl-tetrazolium bromide assay and phalloidin-fluorescein isothiocyanate staining were used to evaluate cytotoxicity and cellular morphologic changes. Western blot and immunofluorescence staining were used to determine the expression levels of E-cadherin, vimentin, alpha-smooth muscle actin, fibronectin, phosphorylated Smad2/3, and mitogen-activated protein kinases. Scratch and transwell migration assays were used to assess cellular migration ability. RESULTS: Doxycycline (0-10 µg/mL) had no significant cytotoxic effects in A549 and primary nasal epithelial cells. Increased expression of mesenchymal markers, including vimentin, alpha-smooth muscle actin, and fibronectin in TGF beta 1 induced A549 cells were downregulated by doxycycline treatment. In contrast, E-cadherin expression was upregulated in TGF beta 1 induced A549 cells. An in vitro cell migration assay showed that doxycycline also inhibited the ability of TGF beta 1 induced migration. Doxycycline treatment suppressed the activation of Smad2/3 and p38, whereas its inhibitory effects were similar to each element-specific inhibitor in A549 and primary nasal epithelial cells. CONCLUSION: Doxycycline inhibited TGF beta 1 induced epithelial-to-mesenchymal transition and migration by targeting Smad2/3 and p38 signal pathways in respiratory epithelial cells.

Switching of Photonic Crystal Lasers by Graphene.

Unique features of graphene have motivated the development of graphene-integrated photonic devices. In particular, the electrical tunability of graphene loss enables high-speed modulation of light and tuning of cavity resonances in graphene-integrated waveguides and cavities. However, efficient control of light emission such as lasing, using graphene, remains a challenge. In this work, we demonstrate on/off switching of single- and double-cavity photonic crystal lasers by electrical gating of a monolayer graphene sheet on top of photonic crystal cavities. The optical loss of graphene was controlled by varying the gate voltage Vg, with the ion gel atop the graphene sheet. First, the fundamental properties of graphene were investigated through the transmittance measurement and numerical simulations. Next, optically pumped lasing was demonstrated for a graphene-integrated single photonic crystal cavity at Vg below -0.6 V, exhibiting a low lasing threshold of ∼480 µW, whereas lasing was not observed at Vg above -0.6 V owing to the intrinsic optical loss of graphene. Changing quality factor of the graphene-integrated photonic crystal cavity enables or disables the lasing operation. Moreover, in the double-cavity photonic crystal lasers with graphene, switching of individual cavities with separate graphene sheets was achieved, and these two lasing actions were controlled independently despite the close distance of ∼2.2 µm between adjacent cavities. We believe that our simple and practical approach for switching in graphene-integrated active photonic devices will pave the way toward designing high-contrast and ultracompact photonic integrated circuits.

Dynamic Reflection Phase and Polarization Control in Metasurfaces.

Optical metasurfaces are two-dimensional optical elements composed of dense arrays of subwavelength optical antennas and afford on-demand manipulation of the basic properties of light waves. Following the pioneering works on active metasurfaces capable of modulating wave amplitude, there is now a growing interest to dynamically control other fundamental properties of light. Here, we present metasurfaces that facilitate electrical tuning of the reflection phase and polarization properties. To realize these devices, we leverage the properties of actively controlled plasmonic antennas and fundamental insights provided by coupled mode theory. Indium-tin-oxide is embedded into gap-plasmon resonator-antennas as it offers electrically tunable optical properties. By judiciously controlling the resonant properties of the antennas from under- to overcoupling regimes, we experimentally demonstrate tuning of the reflection phase over 180°. This work opens up new design strategies for active metasurfaces for displacement measurements and tunable waveplates.