Mechanism and Performance Control Methods of Sulfate Attack on Concrete: A Review.

For concrete structures in marine or groundwater environments, sulfate attack is a major factor contributing to the degradation of concrete performance. This paper analyzes the existing literature on the chemical reactions and physical crystallization effects of sulfate attack on cement-based materials, summarizing the degradation mechanisms of corroded concrete. Experiments have been conducted to study the performance evolution of concrete under sulfate attack, considering both external environmental factors and internal factors of the cement-based materials. External environmental factors, such as the temperature, humidity, concentration, and type of sulfate solutions, wet-dry cycles, freeze-thaw cycles, chloride coupling effects, and stray currents significantly impact sulfate attack on concrete. Internal factors, including internal sources of corrosion, the chemical composition of the cement, water-cement ratio, and the content of C-S-H gel and Ca(OH)2, influence the density and sulfate resistance of the cement-based materials. Additionally, five typical methods for enhancing the sulfate resistance of concrete are summarized. Finally, the paper identifies current challenges in the study of corroded concrete and proposes directions for future research.

sgRNA structure optimization and PTG/Cas9 system synergistically boost gene knockout efficiency in an insect.

Knockouts mediated by CRISPR/Cas9 technology are widely used to study insect gene functions, but the efficiency in Hemiptera is low. New strategies are urgently needed to improve gene knockout efficiency. This study initially explored the impact of modifying the fundamental backbone structure of single guide RNA (sgRNA) on knockout efficiency. The results indicated that both in vitro and in vivo transcription of sgRNA structures (Loop5bp + MT/C type) increased average knockout efficiency by 0.61-fold compared to the original sgRNA. In addition, the PTG/Cas9 system was observed to induce a 0.64-fold increase in average knockout efficiency using the original sgRNA. Notably, an integrated PTG/Cas9 system (iPTG/Cas9 system), the integration of optimized sgRNA structures (Loop5bp + MT/C type) into the conventional PTG/Cas9 system, demonstrated a synergistic effect, resulting in a 1.45-fold increase in average knockout efficiency compared to the original sgRNA structure. The iPTG/Cas9 system was effectively used to simultaneously knockout two different target sites within a single gene and to co-knockout two genes. This study represents the first application of the iPTG/Cas9 system to establish a double knockout system in Hemiptera, offering a promising approach to enhance knockout efficiency in species with low efficiency and improve genetic manipulation tools for pest control.

Functional analysis of Ornithine decarboxylase in manipulating the wing dimorphism in Nilaparvata lugens (Stål) (Hemiptera: Delphacidae).

The brown planthopper (BPH, Nilaparvata lugens), a major insect pest of rice, can make a shift in wing dimorphism to adapt to complex external environments. Our previous study showed that NlODC (Ornithine decarboxylase in N. lugens) was involved in wing dimorphism of the brown planthopper. Here, further experiments were conducted to reveal possible molecular mechanism of NlODC in manipulating the wing dimorphism. We found that the long-winged rate (LWR) of BPH was significantly reduced after RNAi of NlODC or injection of DFMO (D, L-α-Difluoromethylornithine), and LWR of males and females significantly decreased by 21.7% and 34.6%, respectively. Meanwhile, we also examined the contents of three polyamines under DFMO treatment and found that the contents of putrescine and spermidine were significantly lower compared to the control. After 3rd instar nymphs were injected with putrescine and spermidine, LWR was increased significantly in both cases, and putrescine was a little bit more effective, with 5.6% increase in males and 11.4% in females. Three days after injection of dsNlODC, injection of putrescine and spermidine rescued LWR to the normal levels. In the regulation of wing differentiation in BPH, NlODC mutually antagonistic to NlAkt may act through other signaling pathways rather than the classical insulin signaling pathway. This study illuminated a physiological function of an ODC gene involved in wing differentiation in insects, which could be a potential target for pest control.

A novel c-Met/TRK inhibitor 1D228 efficiently inhibits tumor growth by targeting angiogenesis and tumor cell proliferation.

Multiple tumors are synergistically promoted by c-Met and TRK, and blocking their cross-signalling pathway may give better effects. In this study, we developed a tyrosine kinase inhibitor 1D228, which exhibited excellent anti-tumor activity by targeting c-Met and TRK. Models in vitro, 1D228 showed a significant better inhibition on cancer cell proliferation and migration than the positive drug Tepotinib. Models in vivo, 1D228 showed robust anti-tumor effect on gastric and liver tumor growth with 94.8% and 93.4% of the TGI, respectively, comparing 67.61% and 63.9% of Tepotinib. Importantly, compared with the combination of Larotrectinib and Tepotinib, 1D228 monotherapy in MKN45 xenograft tumor models showed stronger antitumor activity and lower toxicity. Mechanistic studies showed that 1D228 can largely inhibit the phosphorylation of TRKB and c-Met. Interestingly, both kinases, TRKs and c-Met, have been found to be co-expressed at high levels in patients with gastric cancer through IHC. Furthermore, bioinformatics analysis has revealed that both genes are abnormally co-expressed in multiple types of cancer. Cell cycle analysis found that 1D228 induced G0/G1 arrest by inhibiting cyclin D1. Additionally, vascular endothelial cells also showed a pronounced response to 1D228 due to its expression of TRKB and c-Met. 1D228 suppressed the migration and tube formation of endothelial cells, which are the key functions of tumor angiogenesis. Taken together, compound 1D228 may be a promising candidate for the next generation of c-Met and TRK inhibitors for cancer treatment, and offers a novel potential treatment strategy for cancer patients with abnormal expressions of c-Met or NTRK, or simultaneous of them.

HOXD8 suppresses renal cell carcinoma growth by upregulating SHMT1 expression.

Amplification of amino acids synthesis is reported to promote tumorigenesis. The serine/glycine biosynthesis pathway is a reversible conversion of serine and glycine catalyzed by cytoplasmic serine hydroxymethyltransferase (SHMT)1 and mitochondrial SHMT2; however, the role of SHTM1 in renal cell carcinoma (RCC) is still unclear. We found that low SHMT1 expression is correlated with poor survival of RCC patients. The in vitro study showed that overexpression of SHMT1 suppressed RCC proliferation and migration. In the mouse tumor model, SHMT1 significantly retarded RCC tumor growth. Furthermore, by gene network analysis, we found several SHMT1-related genes, among which homeobox D8 (HOXD8) was identified as the SHMT1 regulator. Knockdown of HOXD8 decreased SHMT1 expression, resulting in faster RCC growth, and rescued the SHMT1 overexpression-induced cell migration defects. Additionally, ChIP assay found the binding site of HOXD8 to SHMT1 promoter was at the -456~-254 bp region. Taken together, SHMT1 functions as a tumor suppressor in RCC. The transcription factor HOXD8 can promote SHMT1 expression and suppress RCC cell proliferation and migration, which provides new mechanisms of SHMT1 in RCC tumor growth and might be used as a potential therapeutic target candidate for clinical treatment.

Measurement of Water Absorption of Recycled Aggregate.

Owing to the high absorption capacity of recycled aggregate (RA), it is crucial to accurately measure its saturated-surface-dried water absorption (WSSD), which largely affects an effective water-to-binder ratio of recycled aggregate concrete. In this study, existing measurement methods for the WSSD of RA are extensively reviewed, including Wiping, Slumping, Centrifugation, Infrared, Evaporation, Airflow drying, Conductivity, Pycnometer, Hydrostatic balance, and Extrapolation. In particular, the physical principles and operability of these methods are emphasized. It was determined that the accuracy of all test results was not satisfactory. For example, the water in pores with an open-ended direction that was opposite to the centrifugal force could largely be retained. In Airflow drying, the temperature change was significantly delayed. In addition, in Hydrostatic balance, RA would pre-absorb water before determining the initial reading. Therefore, several suggestions for optimizing these methods are presented, such as the combination of Evaporation and Airflow drying, the liquid selection in Hydrostatic balance, and the addition of a tiny mixer in each centrifuge tube. In summary, this review facilitates the development of an accurate and convenient method for measuring the WSSD of RA.

Modulating Emission of Nonconventional Luminophores from Nonemissive to Fluorescence and Room-Temperature Phosphorescence via Dehydration-Induced Through-Space Conjugation.

Processing nonconventional luminophores into ultralong room-temperature phosphorescence (RTP) materials with bright emission is extremely difficult but highly desired because of their intrinsic advantages together with the relatively weak spin-orbit coupling and rapid nonradiative decay in comparison to traditional aromatic compounds. Here, a straightforward heat treatment method was developed to promote the intersystem crossing efficiency and to suppress nonradiative pathways. A "dehydration-induced through-space conjugation" mechanism was proposed for explaining the activating of fluorescence and RTP of nonconventional luminophores. RTP materials with a phosphorescence quantum yield of 23.8% and emission lifetime of 1.3 s are developed. In addition, the emission color and lifetimes can be modulated by tuning the structure of ligands, which allows their applications in multilevel information encryption. These results open the door for designing highly efficient ultralong RTP materials, which also provides a clue to clarify the detailed emission profiles of RTP materials.

Photoluminescence investigations of sulfur quantum dots synthesized by a bubbling-assisted strategy.

Sulfur quantum dots (S-dots) emerge as promising luminescent materials owing to their remarkable optical properties. However, the mechanisms of their formation and photoluminescence remain concealed. We reveal these mechanisms by the bubbling-assisted synthesis and spectroscopic study of S-dots formed from sulfur ions produced by the alkaline oxidation of bulk sulfur under the passivation of PEG. The emission colour of the S-dots depends on the size, explained by the quantum confinement effect. The dots' luminescent quantum efficiency is strongly affected by the surface sulfur species, which is optimized by the proper surface oxidation. The simple synthesis, excellent luminescence properties, and metal-free nature attract S-dots to optoelectronic and electroluminescence applications.

Accommodation is unrelated to myopia progression in Chinese myopic children.

This study shows accommodative accuracy and distance accommodation facility in myopic children do not play a role in myopia progression. In 144 subjects, the monocular distance accommodative facility (DAF) and continuous accommodative stimulus-response curves (ASRCs) were measured at the enrolment. Retrospective and prospective refraction with regard to the enrolment visit were obtained from the outpatient database system based on noncycloplegic subjective spherical equivalent refraction (SER). The rate of myopic progression at enrolment was the first derivative of the Gompertz function, which was fitted with each subject's longitudinal refractive error data, including at least four records of SER with an interval of more than 6 months between each visit. A mixed linear model for multilevel repeated-measures data was used to explore the associations between the rate of myopia progression and accommodative parameters. The mean rate of myopia progression at enrolment was -0.61 ± 0.31 D/y with a mean age of 12.27 ± 1.61 years. By adjusting for age and SER, it was shown that the myopic progression rate was not associated with the accommodative lag (F = 0.269, P = 0.604), accommodative lag area (F = 0.086, P = 0.354), slope of ASRC (F = 0.711, P = 0.399), and DAF (F = 0.619, P = 0.432).

Two-Step Oxidation Synthesis of Sulfur with a Red Aggregation-Induced Emission.

Sulfur is not normally considered a light-emitting material, even though there have been reports of a dim luminescence of this compound in the blue-to-green spectral region. Now, it is shown how to make red-emissive sulfur by a two-step oxidation approach using elemental sulfur and Na2 S as starting materials, with a high photoluminescence quantum yield of 7.2 %. Polysulfide is formed first and is partially transformed into Na2 S2 O3 in the first step, and then turns back to elemental S in the second step. The elevated temperature and relatively oxygen-deficient environment during the second step transforms Na2 S2 O3 into Na2 SO3 incorporated with oxygen vacancies, thus resulting in the formation of a solid-state powder consisting of elemental S embedded in Na2 SO3 . It shows aggregation-induced emission properties, attributed to the influence of oxygen vacancies on the emission dynamics of sulfur by providing additional lower energy states that facilitate the radiative relaxation of excitons.

A MXene of type Ti3C2Tx functionalized with copper nanoclusters for the fluorometric determination of glutathione.

Luminescent copper nanoclusters (Cu NCs) are chosen to functionalize Ti3C2Tx MXene flakes to form a new kind of nanohybrid. It was applied to the determination of glutathione (GSH) via photoluminescence (PL). The Cu NCs and MXene flakes are in close contact, and the blue PL of the Cu NCs (with excitation/emission peaks at 380/425 nm) is quenched. The addition of GSH triggers the separation of the nanohybrid. This results in the recovery of PL. GSH also promotes the PL of Cu NCs via host-guest interactions. Thus, target recognition, corresponding signal output and further magnification are accomplished in a single step. Under optimum conditions, the nanohybrid can detect GSH in the 5.0 to 100 µM concentration range and with a 3.0 µM detection limit. The assay is very specific and shows high selectivity towards metal ions, small biomolecules, amino acids, and thiol containing molecules. Graphical abstractLuminescent copper nanoclusters are used to functionalize Ti3C2Tx MXene flakes, forming a nanohybrid, which is applied to detect glutathione. Target recognition, signal output and magnification are accomplished in a single step, resulting in high selectivity.

Ultrasonication-promoted synthesis of luminescent sulfur nano-dots for cellular imaging applications.

An ultrasonication-promoted strategy was proposed to synthesize luminescent S-dots, which reduced the synthesis time from the commonly used 5 days to several hours. The as-synthesized S-dots show a high photostability and low cytotoxicity, and are then successfully applied for cellular imaging.

Static and dynamical isomerization of Cu38 cluster.

The lowest-energy geometrical and electronic structures of Cu38 cluster are investigated by density-functional calculations combined with a genetic algorithm based on a many body semi-empirical interatomic potential, the traditional FCC-truncated Octahedron (OH) and an incomplete-Mackay icosahedron (IMI) are recognized as the two lowest energy structures (energetically degenerate isomers) but with different electronic structures: a semiconductor-type with the energy-gap of 0.356 eV for the IMI and a metallic-type with negligible gap for the OH, which is in good agreement with the experimental results. The electron affinity and ionization potential of Cu38 are also discussed and compared with the observations of the ultraviolet photoelectron spectroscopy experiments. The dynamical isomerization of the OH-like and IMI-like structures of Cu38 is revealed to dominate the pre-melting stage through the investigation by the molecular dynamics annealing simulations.

Boys, Girls, and Grandparents: The Impact of the Sex of Preschool-Aged Children on Family Living Arrangements and Maternal Labor Supply.

In this study, we consider household decision-making on living arrangements and maternal labor supply in extended families with young children. In such a context, decision-making is driven by the concerns that the companionship of children is a household public good and that family members share childcare and related domestic duties. The incentive to share children's companionship is affected by son preference, whereas the economic motive of labor division hinges on the potential wage rate of the mother. Both channels play important roles in households with mothers whose wage rates are high, while sharing the companionship of (grand) sons is the main driving force in households with mothers whose wage rates are low. Using China Health and Nutrition Survey (CHNS) data, we find that among less-educated mothers, the incidence of a family coresiding with the paternal grandmother is at least 8.6 percentage points higher if the firstborn is a boy. At the same time, maternal labor supply increases by 2.9 days per month. By contrast, for educated mothers, the propensity for coresidence is higher, the working hours are longer, and the impact of the child's sex is not significant. Our study not only provides a better understanding of the demographic and economic factors determining coresidence and intrahousehold time allocations but also lends empirical support to policies aiming to increase female labor supply and improve the well-being of girls.

Repeatability of two subjective accommodative amplitude measurements and agreement with an objective method.

BACKGROUND: The aim was to compare the repeatability of subjective accommodative amplitude (AA) measurements obtained using the modified push-up and minus-lens methods and determine the agreement with objective AA by an autorefractor. METHODS: The right eyes of 47 adults aged 22-30 years (mean 25.1 ± 1.9) were measured by subjective AA using the modified push-up and minus-lens methods. Subjective AAs with first sustained blur and unreadable criteria were obtained with an average of three consecutive measurements. Measurements of the objective AA were simulated by the minus lens using an open-field autorefractor. The repeatability of two subjective AA methods and the agreement among the different methods was assessed. RESULTS: The AA value was dependent on the measuring method (F = 139.68, p < 0.001). The AA of the minus-lens method with first sustained blur criterion (10.10 ± 1.71 D) was closest to the objective AA (9.01 ± 1.49 D). The minus-lens method with unreadable criterion had the best repeatability. The agreement between the minus-lens method and the objective methods was better than that between the modified push-up and the objective methods based on the difference and 95 per cent limits of agreement. The agreement was highest between the minus-lens method with the first sustained blur criterion and the objective method, which had the lowest average difference and a narrow 95 per cent limits of agreement. CONCLUSIONS: The subjective minus-lens method was more accurate for assessing the subjective AA in adults than the modified push-up method. The minus-lens method with the first sustained blur criterion was the optimal method for subjective AA. The minus-lens method with unreadable criterion can also be applied to clinical measurements, although it was approximately 0.95 D larger than the minus-lens method with the first sustained blur criterion.

Comparison of three monocular methods for measuring accommodative stimulus-response curves.

PURPOSE: The aim was to evaluate the repeatability of dynamic measurement of the accommodative stimulus-response curve (ASRC) at three different dioptric speeds using a modified instrument and its agreement with two other methods. METHODS: Twenty-nine adults (23.5 ± 2.0 years) were enrolled in the study. ASRC was measured monocularly using three methods: dynamic and static measurement using a motorised Badal system mounted on an open-field auto-refractor (WAM-5500, Grand Seiko Co., Ltd, Japan) and the minus lens technique. Dynamic measurements were conducted at three dioptric stimulus speeds to simulate continuous stimuli for ASRC (0.25, 0.40 and 0.55 D/s), with three repetitions for each speed. All three types of ASRCs were fitted with third-degree polynomial equations. The slope and objective accommodative amplitude of the ASRC were analysed. RESULTS: The repeatability of objective accommodative amplitude worsened as the speed of the stimuli increased. The repeatability of the slope was best at a speed of 0.40 D/s and worst at 0.55 D/s. The measurement method significantly influenced the objective accommodative amplitude values and slope (both, p < 0.001). The minus lens technique yielded the highest amplitude of accommodation (6.21 ± 0.84 D) and steepest slope (1.11 ± 0.14), followed by the static Badal method (5.60 ± 0.83 D and 0.89 ± 0.09 D). The objective accommodative amplitude decreased with increasing speed during dynamic measurements. There was no difference between the slopes at 0.25 D and 0.40 D/s (p > 0.05) and the slope was lowest at 0.55 D/s. CONCLUSION: The accommodative stimulus-response curve values are method-dependent and the significant differences between three methods used to determine the ASRC based on slope and accommodative amplitude indicate that these methods are non-interchangeable. Using dynamic measurements, accommodative behaviour varies with the speed of dioptric-change of the stimulus. A speed of 0.40 D/s appears to be the best compromise in terms of time, results and repeatability for dynamic ASRC measurement.

Health Insurance and Health Care among the Mid-Aged and Older Chinese: Evidence from the National Baseline Survey of CHARLS.

We document the recent profile of health insurance and health care among mid-aged and older Chinese using data from the China Health and Retirement Longitudinal Study conducted in 2011. Overall health insurance coverage is about 93%. Multivariate regressions show that respondents with lower income as measured by per capita expenditure have a lower chance of being insured, as do the less-educated, older, and divorced/widowed women and rural-registered people. Premiums and reimbursement rates of health insurance vary significantly by schemes. Inpatient reimbursement rates for urban people increase with total cost to a plateau of 60%; rural people receive much less. Demographic characteristics such as age, education, marriage status, per capita expenditure, and self-reported health status are not significantly associated with share of out-of-pocket cost after controlling community effects. For health service use, we find large gaps that vary across health insurance plans, especially for inpatient service. People with access to urban health insurance plans are more likely to use health services. In general, Chinese people have easy access to median low-level medical facilities. It is also not difficult to access general hospitals or specialized hospitals, but there exists better access to healthcare facilities in urban areas. Copyright © 2016 John Wiley & Sons, Ltd.

Concentration-dependent cytotoxicity of copper ions on mouse fibroblasts in vitro: effects of copper ion release from TCu380A vs TCu220C intra-uterine devices.

Sustained release of copper (Cu) ions from Cu-containing intrauterine devices (CuIUD) is quite efficient for contraception. However, the tissue surrounding the CuIUD is exposed to toxic Cu ion levels. The objective for this study was to quantify the concentration dependent cytotoxic effects of Cu ions and correlate the toxicity due to Cu ion burst release for two popular T-shaped IUDs - TCu380A and TCu220C on L929 mouse fibroblasts. Fibroblasts were cultured in 98 well tissue culture plates and 3-(4,5-dimethylthiazol- 2-yl)-2,5-diphehyltetrazolium bromide (MTT) assay was used to determine their viability and proliferation as a function of time. For cell seeding numbers ranging from 10,000 to 100,000, a maximum culture time of 48 h was identified for fibroblasts without significant reduction in cell proliferation due to contact inhibition. Thus, for Cu cytotoxicity assays, a cell seeding density of 50,000 and a maximum culture time of 48 h in 96 well plates were used. 24 h after cell seeding, culture media were replaced with Cu ion containing media solutions of different concentrations, including 24 and 72 h extracts from TCuIUDs and incubated for a further 24 h. Cell viability decreased with increasing Cu ion concentration, with 30 % and 100 % reduction for 40 µg/ml and 100 µg/ml respectively at 24 h. The cytotoxic effects were further evaluated using light microscopy, apoptosis and cell cycle analysis assays. Fibroblasts became rounded and eventually detached from TCP surface due to Cu ion toxicity. A linear increase in apoptotic cell population with increasing Cu ion concentration was observed in the tested range of 0 to 50 µg/ml. Cell cycle analysis indicated the arrest of cell division for the tested 25 to 50 µg/ml Cu ion treatments. Among the TCuIUDs, TCu220C having 265 mm(2) Cu surface area released 9.08 ± 0.16 and 26.02 ± 0.25 µg/ml, while TCu380A having 400 mm(2) released 96.7 ± 0.11 and 159.3 ± 0.15 µg/ml respectively following 24 and 72 h extractions. The effects of TCuIUD extracts on viability, morphology, apoptosis and cell cycle assay on L929 mouse fibroblasts cells, were appropriate for their respective Cu ion concentrations. Thus, a concentration of about 46 µg/ml (~29 µM) was identified as the LD50 dose for L929 mouse fibroblasts when exposed for 24 h based on our MTT cell viability assay. The burst release of lethal concentration of Cu ions from TCu380A, especially at the implant site, is a cause of concern, and it is advisable to use TCuIUD designs that release Cu ions within cytotoxic limits yet therapeutic, similar to TCu220C.

Bioequivalence of Two Formulations of a Single Oral Dose of 500-mg Azithromycin Granules: A Randomized, Open-Label, Two-Period Crossover Study in Healthy Han Chinese Volunteers.

BACKGROUND: In recent years, the use of generic drugs has been increasing due to their effectiveness and to the increasing variety of drugs that are now available in generic formulations. Although several generic oral formulations of azithromycin are available in China, information concerning the bioavailability of these formulations in the Chinese population is unavailable. OBJECTIVE: The purpose of this study was to compare the bioequivalence and tolerability of a single oral dose of 2 commercial brands of 500-mg azithromycin granules in healthy Han Chinese volunteers. METHODS: In a randomized, open-label, 2-period crossover study, the bioequivalence and tolerability of 2 commercial formulations of azithromycin granules (test: Dayin Ocean Biochemical Company Ltd., Shandong, China; reference: Taiyang Drug Company Ltd., Beijing, China) were compared in healthy adult Han Chinese volunteers. Both the test and the reference formulations were administered to each subject. The 2 treatment phases were separated by a 3-week washout period. Liquid chromatography-tandem mass spectrometry was used to determine plasma drug concentrations. The formulations were considered bioequivalent if the natural log-transformed ratios of Cmax and AUC were within the predetermined equivalence range of 70% to 143% and 80% to 125%, respectively, and if P ≤ 0.05 for the 90% CIs. RESULTS: Twenty-four male Han Chinese volunteers (mean [SD] age, 21.0 [2.0] years [range, 18-25 years]; mean [SD] weight, 67.6 [5.6] kg [range, 56-81 kg]; mean [SD] height, 176.0 [5.0] cm [range, 165-188 cm]) were enrolled. Twenty-two subjects completed the study, with 2 withdrawing for personal reasons. No period or sequence effect was observed. The 90% CIs for the corresponding ratios of Cmax, AUC from hour 0 to time t, and AUC from hour 0 to any time point were 85.9 to 103.9, 83.6 to 106.0, and 84.7 to 105.9 (in the 2 one-sided t tests; all, P < 0.05), respectively. Similar results were found in data without a logarithmic transformation. There were no significant differences in the plasma concentration-time curves of the test and reference formulations. No adverse events were reported by the subjects or revealed by clinical or laboratory tests. CONCLUSIONS: Single oral doses of 2 commercial brands of azithromycin granules (500 mg) were equivalent with regard to the rate and extent of absorption among these healthy Han Chinese volunteers. Both formulations were well tolerated.