Capsaicin combined with stem cells improved mitochondrial dysfunction in PIG3V cells, an immortalized human vitiligo melanocyte cell line, by inhibiting the HSP70/TLR4/mTOR/FAK signaling axis.

BACKGROUND: Vitiligo is a common autoimmune skin disease. Capsaicin has been found to exert a positive effect on vitiligo treatment, and mesenchymal stem cells (MSCs) are also confirmed to be an ideal cell type. This study aimed to explore the influence of capsaicin combined with stem cells on the treatment of vitiligo and to confirm the molecular mechanism of capsaicin combined with stem cells in treating vitiligo. METHODS AND RESULTS: PIG3V cell proliferation and apoptosis were detected using CCK-8 and TUNEL assays, MitoSOX Red fluorescence staining was used to measure the mitochondrial ROS level, and JC-1 staining was used to detect the mitochondrial membrane potential. The expression of related genes and proteins was detected using RT‒qPCR and Western blotting. Coimmunoprecipitation was used to analyze the protein interactions between HSP70 and TLR4 or between TLR4 and mTOR. The results showed higher expression of HSP70 in PIG3V cells than in PIG1 cells. The overexpression of HSP70 reduced the proliferation of PIG3V cells, promoted apoptosis, and aggravated mitochondrial dysfunction and autophagy abnormalities. The expression of HSP70 could be inhibited by capsaicin combined with MSCs, which increased the levels of Tyr, Tyrp1 and DCT, promoted the proliferation of PIG3V cells, inhibited apoptosis, activated autophagy, and improved mitochondrial dysfunction. In addition, capsaicin combined with MSCs regulated the expression of TLR4 through HSP70 and subsequently affected the mTOR/FAK signaling pathway CONCLUSIONS: Capsaicin combined with MSCs inhibits TLR4 through HSP70, and the mTOR/FAK signaling pathway is inhibited to alleviate mitochondrial dysfunction and autophagy abnormalities in PIG3V cells.

A real-world pharmacovigilance study of amivantamab-related cardiovascular adverse events based on the FDA adverse event reporting system (FAERS) database.

Amivantamab is the first dual-specificity antibody targeting EGFR and MET, which is approved for the treatment of locally advanced or metastatic non-small cell lung cancer (NSCLC) with EGFR exon 20 insertion mutations. Cardiovascular toxicities related to amivantamab have not been reported in the CHRYSALIS study. However, the occurrence of cardiovascular events in the real world is unknown. To comprehensively investigate the clinical characteristics, onset times, and outcomes of cardiovascular toxicities associated with amivantamab. The Food and Drug Administration Adverse Event Reporting System (FAERS) database from 1st quarter of 2019 to the 2nd quarter of 2023 was retrospectively queried to extract reports of cardiovascular adverse events (AEs) associated with amivantamab. To perform disproportionality analysis, the reporting odds ratios (RORs) and information components (ICs) were calculated with statistical shrinkage trans-formation formulas and a lower limit of the 95% confidence interval (CI) for ROR (ROR025) > 1 or IC (IC025) > 0 with at least 3 reports was considered statistically significant. A total of 20,270,918 eligible records were identified, among which 98 records were related to cardiovascular events associated with amivantamab. 4 categories of cardiovascular events exhibited positive signals: venous thrombotic diseases, abnormal blood pressure, arrhythmia, and pericardial effusion. Venous thrombotic diseases and abnormal blood pressure were the two most common signals. The median time to onset (TTO) for cardiovascular AEs was 33 days. The cumulative incidence within 90 days was 100% for cardiac failure, 75% for stroke, 63.16% for arrhythmia, 50% for sudden death, and 44.18% for venous thrombotic diseases. Death accounted for 16.3% of all cardiovascular AEs associated with amivantamab. The mortality rates for Major Adverse Cardiovascular Events (MACE) were up to 60%. This pharmacovigilance study systematically explored the cardiovascular adverse events of amivantamab and provided new safety signals based on past safety information. Early and intensified monitoring is crucial, and attention should be directed towards high-risk signals.

Aldo-keto reductase-7A2 protects against atorvastatin-induced hepatotoxicity via Nrf2 activation.

Atorvastatin (ATO), as a cholesterol-lowering drug, was the world's best-selling drug in the early 2000s. However, ATO overdose-induced liver or muscle injury is a threat to many patients, which restricts its application. Previous studies suggest that ATO overdose is accompanied with ROS accumulation and increased lipid peroxidation, which are the leading causes of ATO-induced liver damage. This study is, therefore, carried out to investigate the roles of anti-oxidant pathways and enzymes in protection against ATO-induced hepatotoxicity. Here we show that in ATO-challenged HepG2 cells, the expression levels of transcription factor NFE2L2/Nrf2 (nuclear factor erythroid 2 p45-related factor 2) are significantly upregulated. When Nrf2 is pharmacologically inhibited or genetically inactivated, ATO-induced cytotoxicity is significantly aggravated. Aldo-keto reductase-7A (AKR7A) enzymes, transcriptionally regulated by Nrf2, are important for bioactivation and biodetoxification. Here, we reveal that in response to ATO exposure, mRNA levels of human AKR7A2 are significantly upregulated in HepG2 cells. Furthermore, knockdown of AKR7A2 exacerbates ATO-induced hepatotoxicity, suggesting that AKR7A2 is essential for cellular adaptive response to ATO-induced cell damage. In addition, overexpression of AKR7A2 in HepG2 cells can significantly mitigate ATO-induced cytotoxicity and this process is Nrf2-dependent. Taken together, these findings indicate that Nrf2-mediated AKR7A2 is responsive to high concentrations of ATO and contributes to protection against ATO-induced hepatotoxicity, making it a good candidate for mitigating ATO-induced side effects.

Conventional and pulsed hybrid triboelectric nanogenerator with tunable output time and wider impedance matching range.

Sliding grating-structured triboelectric nanogenerators (SG-TENGs) can multiply transferred charge, reduce open-circuit voltage, and increase short-circuit current, which have wide application prospects in self-powered systems. However, conventional SG-TENGs have an ultrahigh internal equivalent impedance, which reduces the output voltage and energy under low load resistances (<10 MΩ). The Pulsed SG-TENGs can reduce the equivalent impedance to near zero by introducing a synchronously triggered mechanical switch (STMS), but its limited output time causes the incomplete charge transfer under high load resistances (>1 GΩ). In this paper, a conventional and pulsed hybrid SG-TENG (CPH-SG-TENG) is developed through rational designing STMS with tunable width and output time. The matching relationship among grid electrode width, contactor width of STMS, sliding speed, and load resistance has been studied, which provides a feasible solution for simultaneous realization of high output energy under small load resistances and high output voltage under high load resistances. The impedance matching range is extended from zero to at least 10 GΩ. The output performance of CPH-SG-TENG under low and high load resistances are demonstrated by passive power management circuit and arc discharge, respectively. The general strategy using tunable STMS combines the advantages of conventional and pulsed TENGs, which has broad application prospects in the fields of TENGs and self-powered systems.

Targeted versus Empiric Embolization for Delayed Postpancreatectomy Hemorrhage: A Retrospective Study of 312 Patients.

PURPOSE: To assess the safety and clinical effectiveness of empiric embolization (EE) compared with targeted embolization (TE) in the treatment of delayed postpancreatectomy hemorrhage (PPH). MATERIALS AND METHODS: The data of patients with delayed PPH between January 2012 and August 2022 were analyzed retrospectively. In total, 312 consecutive patients (59.6 years ± 10.8; 239 men) were included. The group was stratified into 3 cohorts according to angiographic results and treatment strategies: TE group, EE group, and no embolization (NE) group. The χ2 or Fisher exact test was implemented for comparing the clinical success and 30-day mortality. The variables related to clinical failure and 30-day mortality were identified by univariable and multivariable analyses. RESULTS: Clinical success of transcatheter arterial embolization was achieved in 70.0% (170/243) of patients who underwent embolization. There was no statistical difference in clinical success and 30-day mortality between the EE and TE groups. Multivariate analyses demonstrated that malignant disease (odds ratio [OR] = 5.76), Grade C pancreatic fistula (OR = 7.59), intra-abdominal infection (OR = 2.54), and concurrent extraluminal and intraluminal hemorrhage (OR = 2.52) were risk factors for clinical failure. Moreover, 33 patients (13.6%) died within 30 days after embolization. Advanced age (OR = 2.59) and intra-abdominal infection (OR = 5.55) were identified as risk factors for 30-day mortality. CONCLUSIONS: EE is safe and as effective as TE in preventing rebleeding and mortality in patients with angiographically negative delayed PPH.

The formation mechanism of the precursor film in high temperature molten metal systems: insight into structural disjoining pressure.

A precursor film is a unique microfluidic entity that arises at the liquid/solid interface. The formation mechanism of this entity in high-temperature systems is yet to be explained, mainly due to the limitations posed by the increased reaction at the solid/liquid interface. In this study, we investigate the formation process of the precursor film in high-temperature molten metal systems (Ag/Ni, Au/Ni, and Cu/Ni) using molecular dynamics simulations. The alloying energies for different alloying pairs were determined to extract the excess energy, which was found to be distributed from the interface to the upper liquid. The pattern of this energy distribution determines the shape of the near-surface liquid, including the precursor film. This relationship is further reflected by the structural disjoining pressure, which is the excess pressure exerted by the ordered microstructures within the wedge-shaped area of the droplet. Strong nonlinearity has been found in the structural disjoining pressure of Ag/Ni and Au/Ni systems, which is considered to be the main reason for the formation of the precursor film. The fluctuation of the dissolution rate is also reflected in the disjoining pressure, and the inhibition of dissolution on the precursor film formation is phenomenally clarified.

An update on the bridging factors connecting autophagy and Nrf2 antioxidant pathway.

Macroautophagy/autophagy is a lysosome-dependent catabolic pathway for the degradation of intracellular proteins and organelles. Autophagy dysfunction is related to many diseases, including lysosomal storage diseases, cancer, neurodegenerative diseases, cardiomyopathy, and chronic metabolic diseases, in which increased reactive oxygen species (ROS) levels are also observed. ROS can randomly oxidize proteins, lipids, and DNA, causing oxidative stress and damage. Cells have developed various antioxidant pathways to reduce excessive ROS and maintain redox homeostasis. Treatment targeting only one aspect of diseases with autophagy dysfunction and oxidative stress shows very limited effects. Herein, identifying the bridging factors that can regulate both autophagy and antioxidant pathways is beneficial for dual-target therapies. This review intends to provide insights into the current identified bridging factors that connect autophagy and Nrf2 antioxidant pathway, as well as their tight interconnection with each other. These factors could be potential dual-purpose targets for the treatment of diseases implicated in both autophagy dysfunction and oxidative stress.

Template-free scalable growth of vertically-aligned MoS2 nanowire array meta-structural films towards robust superlubricity.

Two-dimensional (2D) molybdenum disulfide exhibits a variety of intriguing behaviors depending on its orientation layers. Therefore, developing a template-free atomic layer orientation controllable growth approach is of great importance. Here, we demonstrate scalable, template-free, well-ordered vertically-oriented MoS2 nanowire arrays (VO-MoS2 NWAs) embedded in an Ag-MoS2 matrix, directly grown on various substrates (Si, Al, and stainless steel) via one-step sputtering. In the meta-structured film, vertically-standing few-layered MoS2 NWAs of almost micron length (∼720 nm) throughout the entire film bulk. While near the surface, MoS2 lamellae are oriented in parallel, which are beneficial for caging the bonds dangling from the basal planes. Owing to the unique T-type topological characteristics, chemically inert Ag@MoS2 nano-scrolls (NSCs) and nano-crystalline Ag (nc-Ag) nanoparticles (NPs) are in situ formed under the sliding shear force. Thus, incommensurate contact between (002) basal planes and nc-Ag NPs is observed. As a result, robust superlubricity (friction coefficient µ = 0.0039) under humid ambient conditions is reached. This study offers an unprecedented strategy for controlling the basal plane orientation of 2D transition metal dichalcogenides (TMDCs) via substrate independence, using a one-step solution-free easily scalable process without the need for a template, which promotes the potential applications of 2D TMDCs in solid superlubricity.

A novel NADH-dependent leucine dehydrogenase for multi-step cascade synthesis of L-phosphinothricin.

L-Phosphinothricin (L-PPT) is the effective constituent in racemic PPT (a high-efficiency and broad-spectrum herbicide), and the exploitation of green and sustainable synthesis route for L-PPT has always been the focus in pesticide industry. In recent years, "one-pot, two-step" enzyme-mediated cascade strategy is a mainstream pathway to obtain L-PPT. Herein, RgDAAO and BsLeuDH were applied to expand "one-pot, two-step" process. Notably, a NADH-dependent leucine dehydrogenase from Bacillus subtilis (BsLeuDH) was firstly characterized and attempted to generate L-PPT, achieving an excellent enantioselectivity (99.9% ee). Meanwhile, a formate dehydrogenase from Pichia pastoris (PpFDH) was utilized to implement NADH cofactor regeneration and only CO2 was by-product. Sufficient amount of the corresponding keto acid precursor PPO was obtained by oxidation of D-PPT relying on a D-amino acid oxidase from Rhodotorula gracilis (RgDAAO) with content conversion (46.1%). L-PPT was ultimately prepared from racemized PPT via oxidative deamination catalyzed by RgDAAO and reductive amination catalyzed by BsLeuDH, achieving 80.3% overall yield and > 99.9% ee value.

DNA repair byproduct 8-oxoguanine base promotes myoblast differentiation.

Muscle contraction increases the level of reactive oxygen species (ROS), which has been acknowledged as key signaling entities in muscle remodeling and to underlie the healthy adaptation of skeletal muscle. ROS inevitably endows damage to various cellular molecules including DNA. DNA damage ought to be repaired to ensure genome integrity; yet, how DNA repair byproducts affect muscle adaptation remains elusive. Here, we showed that exercise elicited the generation of 8-oxo-7,8-dihydroguanine (8-oxoG), that was primarily found in mitochondrial genome of myofibers. Upon exercise, TA muscle's 8-oxoG excision capacity markedly enhanced, and in the interstitial fluid of TA muscle from the post-exercise mice, the level of free 8-oxoG base was significantly increased. Addition of 8-oxoG to myoblasts triggered myogenic differentiation via activating Ras-MEK-MyoD signal axis. 8-Oxoguanine DNA glycosylase1 (OGG1) silencing from cells or Ogg1 KO from mice decreased Ras activation, ERK phosphorylation, MyoD transcriptional activation, myogenic regulatory factors gene (MRFs) expression. In reconstruction experiments, exogenously added 8-oxoG base enhanced the expression of MRFs and accelerated the recovery of the injured skeletal muscle. Collectively, these data not only suggest that DNA repair metabolite 8-oxoG function as a signal entity for muscle remodeling and contribute to exercise-induced adaptation of skeletal muscle, but also raised the potential for utilizing 8-oxoG in clinical treatment to skeletal muscle damage-related disorders.

Case Report: A long-term survival case of diffuse large B-cell lymphoma with left ventricular infiltration and spinal cord compression.

Background: Diffuse large B-cell lymphoma (DLBCL) is the most common subtype of non-Hodgkin lymphoma and may occur with lymph node and/or extranodal involvement. However, DLBCL with intracardiac mass is exceedingly rare. In the reported literature, the intracardiac infiltration of DLBCL mostly involves the right ventricle. Lymphoma that invades the heart has an aggressive nature, with symptoms that are easily ignored initially and can lead to multiple complications in severe cases, resulting in a poor prognosis. Early screening and diagnosis may significantly improve the survival rate. Early diagnosis may significantly improve outcomes. Case summary: We presented a 68-year-old woman with back pain. PET/CT suggested increased FDG metabolism in the left ventricle, right adrenal gland, right erector spinae intramuscularis, multiple bones and multiple lymph nodes. Contrast-enhanced ultrasound showed a left ventricular apical mass with ventricular septum thickening. Cardiac MRI suggested a 1.6*1.1*2.1 cm mass in the apical-central portion of the left ventricle. Biopsy of the right neck mass confirmed the pathologic diagnosis of diffuse large B-cell lymphoma. However, before the pathologic diagnosis was confirmed, the patient was paralyzed due to spinal cord compression caused by the progression of bone metastases. Subsequently, pathology confirmed the diagnosis of diffuse large B-cell lymphoma, and rituximab plus cyclophosphamide, doxorubicin, vincristine, and prednisone (R-CHOP) was treated immediately as first-line therapy. In addition, glucocorticoids and mannitol dehydration were administered to relieve the symptoms of spinal cord compression. After 8 cycles of R-CHOP, the tumor at all sites had almost complete regression. The patient was able to walk normally and had no tumor-related symptoms. Conclusions: We present a case of DLBCL with a very high tumor load that involved multiple organs, including the left ventricle, but exhibited no cardiac-related symptoms. The combination of various imaging modalities is valuable for the diagnosis of cardiac infiltration. The mass in the left ventricle almost completely regressed after R-CHOP treatment, and no recurrence has occurred in the 5 years of follow-up so far.

Triboelectric Plasma CO2 Reduction Reaching a Mechanical Energy Conversion Efficiency of 2.3.

Mechanical energy-induced CO2 reduction is a promising strategy for reducing greenhouse gas emissions and simultaneously harvesting mechanical energy. Unfortunately, the low energy conversion efficiency is still an open challenge. Here, multiple-pulse, flow-type triboelectric plasma with dual functions of harvesting mechanical energy and driving chemical reactions is introduced to efficiently reduce CO2 . CO selectivity of 92.4% is achieved under normal temperature and pressure, and the CO and O2 evolution rates reach 12.4 and 6.7 µmol h-1 , respectively. The maximum energy conversion efficiencies of 2.3% from mechanical to chemical energy and 31.9% from electrical to chemical energy are reached. The low average electron energy in triboelectric plasma and vibrational excitation dissociation of CO2 with low barrier is revealed by optical emission spectra and plasma simulations, which enable the high energy conversion efficiency. The approach of triboelectric plasma reduction reported here provides a promising strategy for efficient utilization of renewable and dispersed mechanical energy.

Efficient synthesis of an apremilast precursor and chiral ß-hydroxy sulfones via ketoreductase-catalyzed asymmetric reduction.

Ketoreductase (KRED)-catalyzed asymmetric reduction of prochiral ketones is an attractive method to synthesize chiral alcohols. Herein, two KREDs LfSDR1-V186A/E141I and CgKR1-F92I with complementary stereopreference were identified towards reduction of apremilast prochiral ketone intermediate 1a. LfSDR1-V186A/E141I exhibited >99% conversion and 99.2% ee yielding an apremilast chiral alcohol intermediate ((R)-2a) at 50 g L-1 substrate loading. Furthermore, we investigated the substrate scope of ß-keto sulfones by using LfSDR1-V186A/E141I and CgKR1-F92I to produce both enantiomers of the corresponding ß-hydroxy sulfones, with good-to-excellent conversion (up to >99%) and enantioselectivity (up to 99.9% ee) being obtained in most cases. Finally, the gram-scale synthesis of (R)-2a was performed by employing the crude enzyme of LfSDR1-V186A/E141I and BsGDH to afford the desired enantiomer with >99% conversion, 85.9% isolated yield and 99.2% ee. This study presents a biocatalytic strategy to synthesize chiral ß-hydroxy sulfones.

Identification and Characterization of Enzymes Catalyzing Early Steps in Miharamycin and Amipurimycin Biosynthesis.

The biochemical elucidation of the early biosynthetic pathways of miharamycins and amipurimycin revealed the roles of several enzymes, which include GMP hydrolase, represented by MihD/ApmD, and hypothetical proteins, MihI/ApmI, unexpectedly exhibiting the dual function of the guanylglucuronic acid assembly and GMP cleavage. In addition, MihE, a carbonyl reductase that functions on the C2 branch of high-carbon sugars, and MihF, a rare guanine O-methyltransferase, were also functionally verified.

The water droplet with huge charge density excited by triboelectric nanogenerator for water sterilization.

Water is one of the most essential resources for the survival of human beings and all other living things. For the point of daily use, water sterilization has enormous social and economic significance, especially for remote and undeveloped areas. Here, we developed a self-powered water sterilization device, which consists of a rotating-disk freestanding triboelectric-layer mode triboelectric nanogenerator (RF-TENG), a voltage-multiplying circuit, and a water droplet control system. The output voltage of the RF-TENG is boosted by a voltage-multiplying circuit and then utilized to charge water droplet. When the rotation rate of the RF-TENG is 300 rpm, the output voltage of a six-fold voltage-multiplying circuit can reach 9319 V, and a 62.50µl water droplet can be positively charged to 6320 nC at the flow rate of 0.31 ml min-1. The charge density and electric filed of the water droplet can reach 101.12 nCµl-1and 11.28 kV cm-1, respectively. The charged water droplet can killE. coliandS. aureusquickly and efficiently through electroporation mechanism. With the advantages of low cost, simple in fabrication and usage, portability, and etc, the self-powered water sterilization device has wide application prospects in remote and undeveloped areas.

Enzymatically inactive OGG1 binds to DNA and steers base excision repair toward gene transcription.

8-Oxoguanine DNA glycosylase1 (OGG1)-initiated base excision repair (BER) is the primary pathway to remove the pre-mutagenic 8-oxo-7,8-dihydroguanine (8-oxoG) from DNA. Recent studies documented 8-oxoG serves as an epigenetic-like mark and OGG1 modulates gene expression in oxidatively stressed cells. For this new role of OGG1, two distinct mechanisms have been proposed: one is coupled to base excision, while the other only requires substrate binding of OGG1--both resulting in conformational adjustment in the adjacent DNA sequences providing access for transcription factors to their cis-elements. The present study aimed to examine if BER activity of OGG1 is required for pro-inflammatory gene expression. To this end, Ogg1/OGG1 knockout/depleted cells were transfected with constructs expressing wild-type (wt) and repair-deficient mutants of OGG1. OGG1's promoter enrichment, oxidative state, and gene expression were examined. Results showed that TNFα exposure increased levels of oxidatively modified cysteine(s) of wt OGG1 without impairing its association with promoter and facilitated gene expression. The excision deficient K249Q mutant was even a more potent activator of gene expression; whereas, mutant OGG1 with impaired substrate recognition/binding was not. These data suggested the interaction of OGG1 with its substrate at regulatory regions followed by conformational adjustment in the adjacent DNA is the primary mode to modulate inflammatory gene expression.

Characterization of Miharamycin Biosynthesis Reveals a Hybrid NRPS-PKS to Synthesize High-Carbon Sugar from a Complex Nucleoside.

Miharamycins are peptidyl nucleoside antibiotics with a unique branched C9 pyranosyl amino acid core and a rare 2-aminopurine moiety. Inactivation of 19 genes in the biosynthetic gene cluster and identification of several unexpected intermediates suggest an alternative biosynthetic pathway, which is further supported by feeding experiments and in vitro characterization of an unusual adenylation domain recognizing a complex nucleoside derivative as the substrate. These results thereby provide an unprecedented biosynthetic route of high-carbon sugar catalyzed by atypical hybrid nonribosomal peptide synthetase-polyketide synthase.

Key sites insight on the stereoselectivity of four mined aldo-keto reductases toward α-keto esters and halogen-substituted acetophenones.

Biocatalytic reduction catalyzed by aldo-keto reductases (AKRs) is a valuable approach for asymmetric synthesis of chiral alcohols. In this study, four novel aldo-keto reductases with significant activity and stereoselectivity toward a variety of α-keto esters and halogen-substituted acetophenones were identified by genome mining. Through analysis of the crystal structure and multiple-sequence alignment of the starting AKR YvgN from Bacillus subtilis, residues F25 and W113 were proposed as the key positions that might control the stereoselectivity of YvgN. F25S and F25S/W113F variants of YvgN were able to improve its activity and stereoselectivity toward some α-keto ester compounds and halogen-substituted acetophenone derivatives. In addition, similar enhancement of catalytic activity and stereoselectivity was also found in the other three AKRs with corresponding mutations of starting YvgN.

Enantioselective bioreduction of benzo-fused cyclic ketones with engineered Candida glabrata ketoreductase 1 - a promising synthetic route to ladostigil (TV3326).

Biocatalysis has been recently emerging as a promising alternative to traditional chemical synthesis because of its "green" characteristics and comparable selectivities, which accord with the concept of sustainable development and demand for asymmetric synthesis. In this study, whole-cell biocatalysts containing glucose dehydrogenase (GDH) and Candida glabrata ketoreductase 1 (CgKR1) variants were constructed. These biocatalysts were applied to the reduction of benzo-fused cyclic ketones and showed good to high activities and enantioselectivities. Particularly, CgKR1 variants displayed high activities (90.6%-98.4% conversions) and enantioselectivities (>99.9% ee) towards 5a, a key intermediate of ladostigil (TV3326). Based on these results, a chemoenzymatic synthesis of (S)-5b was developed by using biocatalytic asymmetric reduction as a key step, giving the product with a total yield of 34.0% and 99.9% ee.