Low caveolin-1 levels and symptomatic intracranial haemorrhage risk in large-vessel occlusive stroke patients after endovascular thrombectomy.

BACKGROUND AND PURPOSE: Caveolin-1 (Cav-1) is reported to mediate blood-brain barrier integrity after ischaemic stroke. Our purpose was to assess the role of circulating Cav-1 levels in predicting symptomatic intracranial haemorrhage (sICH) amongst ischaemic stroke patients after endovascular thrombectomy (EVT). METHODS: Patients with large-vessel occlusive stroke after EVT from two stroke centres were prospectively included. Serum Cav-1 level was tested after admission. sICH was diagnosed according to the Heidelberg Bleeding Classification. RESULTS: Of 325 patients (mean age 68.6 years; 207 men) included, 47 (14.5%) were diagnosed with sICH. Compared with patients without sICH, those with sICH had a lower concentration of Cav-1. After adjusting for potential confounders, multivariate regression analysis demonstrated that the increased Cav-1 level was associated with a lower sICH risk (odds ratio 0.055; 95% confidence interval 0.005-0.669; p = 0.038). Similar results were obtained when Cav-1 levels were analysed as a categorical variable. Using a logistic regression model with restricted cubic splines, a linear and negative association of Cav-1 concentration was found with sICH risk (p = 0.001 for linearity). Furthermore, the performance of the conventional risk factors model in predicting sICH was substantially improved after addition of the Cav-1 levels (integrated discrimination index 2.7%, p = 0.002; net reclassification improvement 39.7%, p = 0.007). CONCLUSIONS: Our data demonstrate that decreased Cav-1 levels are related to sICH after EVT. Incorporation of Cav-1 into clinical decision-making may help to identify patients at a high risk of sICH and warrants further consideration.

Periprocedural intravenous heparin in patients with acute ischemic stroke treated with endovascular thrombectomy after intravenous thrombolysis.

OBJECTIVE: The benefit-to-risk ratio of periprocedural heparin in patients treated with endovascular thrombectomy (EVT) after intravenous thrombolysis (IVT) remains unclear. This study aimed to evaluate the potential effects of periprocedural heparin on clinical outcomes of EVT after IVT. METHODS: The authors retrospectively analyzed patients from multicenter studies treated with EVT after IVT in the anterior circulation. The endpoints were unfavorable outcome (defined as modified Rankin Scale score ≥ 3 at 90 days), 90-day mortality, symptomatic intracranial hemorrhage (SICH), successful recanalization, and early neurological deterioration. Patients were divided into two groups based on whether they were treated with heparin (heparin-treated group) or not (untreated group), and the efficacy and safety outcomes were compared using multivariable logistic regression models and propensity score-matching methods. RESULTS: Among the 322 included patients (mean age 67.4 years, 54.3% male), 32% of patients received periprocedural heparin. In multivariable analyses, the administration of periprocedural heparin was a significant predictor for unfavorable outcome (OR 2.821, 95% CI 1.15-7.326; p = 0.027), SICH (OR 24.925, 95% CI 2.363-780.262; p = 0.025), and early neurological deterioration (OR 5.344, 95% CI 1.299-28.040; p = 0.029). Regarding successful recanalization and death, no significant differences between the groups were found after propensity score matching. CONCLUSIONS: The results showed that periprocedural heparin is associated with an increased risk of unfavorable outcomes and SICH in patients treated with EVT after IVT. Further studies are warranted to evaluate the utility and safety of periprocedural heparin.

Trajectories of stroke severity and functional outcomes after endovascular treatment in ischemic stroke: A post hoc analysis of a randomized controlled trial.

BACKGROUND: The trajectory of early neurological changes in patients with acute ischemic stroke has been understudied. This study aimed to investigate the association between longitudinal trajectories of stroke severity and 90-day functional outcomes in patients with acute ischemic stroke receiving endovascular treatment. METHODS: We enrolled patients from a prospective, multicenter, randomized controlled trial. The stroke severity was assessed with the National Institute of Health Stroke Scale at the pre-procedure, 24 hours, and seven days after the procedure. Group-based trajectory modeling (GBTM) was used to identify trajectories of stroke severity. Multivariable logistic regression was performed to explore the association between stroke severity markers and 90-day functional outcomes. RESULTS: Of 218 enrolled patients, 127 (58.3%) had poor functional outcomes at 90 days. We identified three trajectories of stroke severity in the GBTM: stable symptom (38.1%), symptom deterioration (17.0%), and symptom improvement (44.9%). In multivariable analyses, trajectories of stroke severity were associated with an increased risk of poor functional outcomes (symptom improvement versus symptom deterioration: odds ratio, 0.007; 95% confidence interval, 0.001-0.040; P <0.001). Reclassification indexes revealed that trajectories of stroke severity would increase the predictive ability for poor functional outcomes at 90 days. CONCLUSION: After endovascular treatment, patients would follow one of three distinct trajectories of stroke severity. Symptom deterioration trajectory was associated with an increased risk of poor functional outcomes at 90 days. TRIAL REGISTRATION NUMBER: NCT04973332.

Preliminary manifestation of the Yangtze River Protection Strategy in improving the carbon sink function of estuary wetlands.

In 2016, the Yangtze River Protection Strategy was proposed and a series of measures were applied to restore the health and function of the Yangtze River ecosystem. However, the impact of these measures on the carbon (C) sink capacity of the Yangtze River estuary wetlands has not been exhaustively studied. In this work, the effects of these measures on the C sink capacity of Yangtze River estuary wetlands were examined through the long-term monitoring of C fluxes, soil respiration, plant growth and water quality. The C flux of the Yangtze River estuary wetlands has become increasingly negative after the implementation of these measures, mainly owing to reduction in soil CO2 emission. The decrease in the chemical fertilizer release and returning farmland to wetland had led to the improvement of water quality in the estuary area, which further reduced soil heterotrophic microbial activity, and ultimately decreasing soil CO2 emissions of estuary wetlands.

Association between liver fibrosis and stroke recurrence in young patients with ischemic stroke.

BACKGROUND AND AIM: Stroke incidence rates are rising among young adults. Liver fibrosis has recently been recognized as a risk factor for cardiovascular events and stroke in the general population. It remains unclear whether liver fibrosis influences the prognosis of stroke. We aimed to evaluate the association between liver fibrosis and stroke recurrence in young stroke patients. METHODS AND RESULTS: Young adults with first-ever ischemic stroke were enrolled from a prospective stroke registry and were followed up for stroke recurrence. Liver fibrosis was evaluated by Fibrosis-4 (FIB-4) score and was stratified into three categories. Cox regression analysis was performed to assess the relationship between liver fibrosis and stroke recurrence. Over a median follow-up of 3.1 (1.7-4.6) years, 72 (11.6%) recurrent strokes occurred among 621 patients. According to the FIB-4 score, 73 (11.7%) patients had indeterminate fibrosis, while 11 (1.8%) had advanced fibrosis. Univariate Cox analysis revealed that patients with a high FIB-4 score were more likely to experience stroke recurrence than those with a low FIB-4 score (hazard ratio 3.748, 95% confidence interval 1.359-10.332, P = 0.011). After adjusting for potential confounders in the multivariate analysis, FIB-4 score remained an independent risk factor. CONCLUSIONS: Young stroke patients with advanced liver fibrosis were at a greater risk of stroke recurrence. Evaluating liver fibrosis may provide valuable information for stroke risk stratification, and the FIB-4 score could serve as a useful tool.

Quality consistency evaluation of chemical composition and pharmacology of Shaoyao-Gancao decoction dispensing granules and traditional decoction.

Dispensing granules of Chinese medicine (DGCM) have emerged as a more convenient alternative to traditional decoction (TD) of Chinese medicine, gaining popularity in recent years. However, the debate surrounding the consistency of DGCM compared to TD remains unresolved. In this study, three batches of Baishao and Gancao DGCM were obtained from manufacturers A, B, and C, and 15 batches of crude drugs were procured from hospital pharmacies for the preparation of dispensing granule decoction (DGD) and TD of Shaoyao-Gancao decoction (SGD). The HPLC-UV method was employed to determine the levels of gallic acid, paeoniflorin, albiflorin, liquiritin, liquiritin apioside, isoliquiritin apioside, isoliquiritin, glycyrrhizic acid, and isoliquiritigenin. The analgesic and antispasmodic effects were assessed using the hot plate and acetic acid writhing test in mice. To evaluate the consistency of chemical constituents and pharmacological effects between the two decoctions, the Criteria Importance Though Intercriteria Correlation (CRITIC) method combined with chemometrics was employed. Grey relation analysis (GRA) was used to assess the comprehensive quality consistency of the two decoctions. The CRITIC results revealed certain differences in chemical constituents and pharmacological effects between the selected DGCM and TD. Notably, DGD-A/C exhibited a significant difference from TD (p > 0.05), whereas DGD-B demonstrated no significant difference from TD (p > 0.05). The GRA analysis demonstrated that the overall quality consistency between DGD-B and TD was the highest among the three manufacturers. This study presents a method for evaluating the quality consistency of DGCM and TD of SGD, offering novel insights into the evaluation of consistency between DGCM and TD.

Association of glycemic gap with stroke recurrence in patients with ischemic stroke.

BACKGROUND: Glycemic gap, as a novel index of acute glycemic excursion, is associated with poor prognosis of different diseases. This study aimed to explore the association of the glycemic gap with long-term stroke recurrence in patients with ischemic stroke. METHODS: This study included patients with ischemic stroke from the Nanjing Stroke Registry Program. The glycemic gap was calculated by subtracting the estimated average blood glucose from the blood glucose at admission. Multivariable Cox proportional hazards regression analysis was performed to explore the association between the glycemic gap and the risk of stroke recurrence. The Bayesian hierarchical logistic regression model was used to estimate the effects of the glycemic gap on stroke recurrence stratified by diabetes mellitus and atrial fibrillation. RESULTS: Among 2734 enrolled patients, 381 (13.9%) patients experienced stroke recurrence during a median follow-up of 3.02 years. In multivariate analysis, glycemic gap (high group vs. median group) was associated with significantly increased risk for stroke recurrence (adjusted hazard ratio, 1.488; 95% confidence interval, 1.140-1.942; p = .003) and had varying effects on stroke recurrence depending on atrial fibrillation. The restricted cubic spline curve showed a U-shaped relationship between the glycemic gap and stroke recurrence (p = .046 for nonlinearity). CONCLUSION: Our study found that the glycemic gap was significantly associated with stroke recurrence in patients with ischemic stroke. The glycemic gap was consistently associated with stroke recurrence across subgroups and had varying effects depending on atrial fibrillation.

Environmental risks of antibiotic resistance genes released from biological laboratories and its control measure.

Antibiotic resistance genes (ARGs) are a growing global threat to public health. Biological laboratory wastewater contains large amounts of free ARGs. It is important to assess the risk of free ARGs from biological laboratories and to find appropriate treatments to control their spread. The fate of plasmids in the environment and the effect of different thermal treatments on their persistence activity were tested. The results showed that untreated resistance plasmids could exist in water for more than 24 h (the special 245 bp fragment). Gel electrophoresis and transformation assays showed that the plasmids boiled for 20 min retained 3.65% ± 0.31% transformation activity of the intact plasmids, while autoclaving for 20 min at 121 °C could effectively degrade the plasmids and that NaCl, bovine serum albumin, and EDTA-2Na affected the degradation efficiency of the plasmids during boiling. In the simulated aquatic system, using 106 copy/µL of plasmids after autoclaving, only 102 copies/µL of the fragment after only 1-2 h could be detected. By contrast, boiled plasmids for 20 min were still detectable after plunging them into water for 24 h. These findings suggest that untreated and boiled plasmids can remain in the aquatic environment for a certain time resulting in the risk of disseminating ARGs. However, autoclaving is an effective way of degrading waste free resistance plasmids.

Systemic Immune-Inflammation Index and Long-Term Mortality in Patients with Stroke-Associated Pneumonia.

Background: Systemic immune inflammation has been investigated as a prognostic marker of different diseases. This study is designed to assess the association of systemic immune-inflammation index (SII) with long-term mortality of stroke-associated pneumonia (SAP) patients. Methods: Patients aged ≥18 years with SAP were selected from the Nanjing Stroke Registry Program in China. We retrospectively evaluated systemic immune-inflammation response with SII and pneumonia severity with the pneumonia severity index and the confusion, uremia, elevated respiratory rate, hypotension, and aged 65 years or older score. To explore the correlation between SII and mortality in SAP patients, multivariable Cox regressions and competing risk regressions were conducted. Mediation analysis was also performed to assess the role of pneumonia severity. Results: Among 611 patients in the SAP population, death occurred in 164 patients (26.8%) during the median follow-up of 3.0 (1.2-4.6) years. In multivariate analysis, higher SII scores could predict increased mortality in patients with SAP (adjusted hazard ratio 2.061; 95% confidence interval, 1.256-3.383; P = 0.004), and the association was mediated by pneumonia severity. Moreover, adding SII to traditional models improved their predictive ability for mortality. Conclusion: Our study displayed that SII was characterized in SAP patients with different prognoses. Elevated SII scores increased the risk of mortality. Further research is required for the clinical practice of the index among SAP patients.

Cadmium stimulated cooperation between bacterial endophytes and plant intrinsic detoxification mechanism in Lonicera japonica thunb.

Due to the intimate association between plant physiology and metabolism, the internal colonizing microbe (endophytes) community must be adjusted to support plant productivity in response to cell damage in plants under stress. However, how endophytes coordinate their activities with plant intrinsic mechanisms such as antioxidative systems and detoxification pathways during Cd accumulation remains unknown. In this hydroponic pot study, we investigated how exposure of Lonicera japonica. thunb. to different levels of Cd (0.5, 2.5, 5, 10, and 20 mg kg-1) affected plant growth, metabolic pathways, and endophyte community structure and function. Although Cd accumulation increased at 5 mg kg-1 Cd, the biomass and height of L. japonica increased in association with elevated endophyte-involved plant detoxification activities. Endophytes, such as Sphingomonas, Klenkia, and Modestobacter, expressed major antioxidative regulators (superoxide dismutase and ascorbate acid) to detoxify Cd in L. japonica. Furthermore, L. japonica and its endophytes synergistically regulated the toxic effects of Cd accumulation via multiple plant metabolic defensive pathways to increase resistance to metal-induced stress.

The high organic carbon accumulation in estuarine wetlands necessarily does not represent a high CO2 sequestration capacity.

Estuarine wetlands with high organic carbon (OC) accumulation rates due to their high plant biomass and interception of tide-derived OC are generally considered as large CO2 sinks. However, our previous study found that tidal OC input seems to stimulate soil CO2 emissions, potentially weakening CO2 sequestration in estuarine wetlands. To further verify this phenomenon, we first established a structural equation model, which confirmed a positive correlation between tidal OC input and soil organic carbon (SOC) and soil respiration. We then performed trace analysis to determine the stability of SOC derived from different sources and its effect on soil CO2 emissions by analyzing the input and retention of OC derived from tides and plants in the Yangtze Estuary wetlands. From upstream to downstream, as tidal OC input decreased, the relative retention ratio of the tidal OC in wetland soil increased from 1.259 to 2.148, whereas the relative retention ratio of plant OC in the soil decreased from 61.5% to 14.8%. Our findings indicated that the degradability of tidal OC was higher upstream than that downstream, but both inhibited plant OC degradation, thus providing an important reason for the higher CO2 emissions upstream of wetlands (with higher tidal OC input). In addition, the primarily contributor to CO2 (δ13) emissions' transforming from plant SOC (81.35%) to tidal SOC (91.18%) was an increase in organic matter input from the tide in a microcosm system. Consequently, a higher CO2 output than CO2 input (plant OC) due to the ready degradation of tidal OC consequently weakens the CO2 sequestration capacity of the estuarine wetlands. This phenomenon is cause for concern regarding the CO2 sink function of estuarine wetlands intercepting large amounts of organic matter.

[Analysis on "the essence of tuina, arrival of qi ensuring curative effect"].

In reference with the systematic review of the thought of deqi (arrival of qi) put forward in Huangdi Neijing (Internal Classic of Yellow Emperor) and other classic books of traditional Chinese medicine, in view of detecting qi and identifying qi before treatment, as well as the prerequisites of deqi in tuina, meaning the accurate syndrome differentiation and manipulations, the importance of deqi in treatment with tuina is expounded. In association with clinical experience, the specific manifestations of deqi in patients during tuina are summarized, e.g. soreness, distention, pain, numbness, warm feeling and slight sweating, local changes in intestinal sound and skin color, as well as mind regulation. It is anticipated that deqi of tuina may be drawn the attention in clinical practice, and the relevant study be expanded.

[Chinese medicine dispensing granules: history, status quo, and development prospect in "post-pilot era"].

Chinese medicine dispensing granules, the result of the efforts to transform Chinese medicinal decoction pieces in China, features portability and ease of storage. Thus, it is destined to be an indispensible dosage form in the modernization drive of Chinese medicine. The Announcement on Ending the Pilot Project of Chinese Medicine Dispensing Granules was released in February 2021 and relevant regulations went into force in November 2021, which marks the a new journey for the development of Chinese medicine dispensing granules and the beginning of the "post-pilot era". However, it faces the challenges in quality and standard. This study reviewed the history of Chinese medicine dispensing granules, analyzed the technical progress, market, and main problems in development, and proposed suggestions and prospects for its development in the "post-pilot era", which is expected to serve as a reference for its industry development and rational use.

Differential responses of polysaccharides and antioxidant enzymes in alleviating cadmium toxicity of tuber traditional Chinese medicinal materials.

Polygonatum cyrtonema Hua (PC) and Bletilla striata (BS) are widely used and planted as tuber traditional Chinese medicinal materials (TCMMs). Cadmium (Cd) is one of the major causes of soil pollution and challenge to the quality and safety of TCMMs. Understanding the absorption and distribution of Cd is important for addressing the risks posed by its residues. As a result, the higher Cd translocation factor (TF) results in the lower Cd bioconcentration factor (BCF) in the PC tuber than that of BS attributed to a lower Cd concentration in the PC tuber, which guaranteed its safe utilization and edible safety under 1 mg·kg-1 Cd soil. Cd stress overall activated peroxidase (POD), catalase (CAT), and water-extractable polysaccharides in PC (PCP1) to exhibit better antioxidation, while the superoxide dismutase (SOD) in BS increased by approximately 206-277% to alleviate more severe oxidative damage. Particularly, Cd induced an increase in PCP1 higher than that of water-extractable polysaccharides of BS (BSP1) by approximately 335% to 1351%. PC exhibited effective strategies for alleviating Cd toxicity, including transferring Cd to nonmedicinal parts, increasing polysaccharides, and synergistically activating the enzymatic antioxidant system. This study expands the application for the safe utilization of low-Cd contaminated soil and provides novel insights for tuber TCMMs to alleviate Cd toxicity.

Antioxidant system response, mineral element uptake and safe utilization of Polygonatum sibiricum in cadmium-contaminated soil.

Chinese herbal medicine is widely cultivated in Southwest China, where the soil cadmium (Cd) contamination of farmland is more serious than that in China as a whole. In this study, Polygonatum sibiricum was exposed to Cd at concentrations of e-1, e0, e2, and e4 mg/kg for 30, 60, and 90 days, and the physiological stress responses, Cd and mineral element uptake, antioxidant enzyme activities, and content changes of pharmaceutical ingredients (polysaccharides) were analyzed to decipher the feasibility of safe utilization in Cd-contaminated soil. The results show that the activity of antioxidant enzymes (SOD and CAT) in the aboveground part was always higher than that in the underground part. The underground part of Polygonatum sibiricum mobilizes nonenzymatic systems to facilitate the synthesis of polysaccharides (PCP1, PCP2) with antioxidant properties to cope with Cd stress. Mineral elements (P, K, Ca, Mg, Fe, Cu, and Zn) significantly (p < 0.05) changed after 90 d of cultivation. In particular, the changes in the iron and zinc content were significantly correlated (p < 0.05) with the activities of SOD and POD. Soil Cd at e0 mg/kg can guarantee the safe production and utilization of Polygonatum sibiricum, and the stimulation of Cd promotes polysaccharide synthesis and biomass growth.

[Change laws of water absorption in Chinese herbal pieces and prediction model of relative density for Chinese medicine decoction].

This study aims to explore the change laws of water absorption in Chinese herbal pieces and establish the prediction model of relative density for Chinese medicine compound decoction. Firstly, fitted equations of water absorption and decocting time was established by observing the change laws of water absorption in 36 kinds of Chinese herbal pieces in 12 groups(according to the drug-parts) with decocting time. The r value of the mineral group and other type group was 0.691 2 and 0.663 3, respectively. The r value of the remaining 10 groups was 0.802 2-0.925 4. All P values were less than 0.05(n=21). The formula of the amount of water added was optimized by combining the fitted equations with determined water absorption, and the liquid yield could be controlled in a range of 100%±10%. Secondly, it was determined that the liquid density tester could be used for the rapid determination of relative density of Chinese medicine decoction after methodological study and comparison with the pycnometer method. The linear regression equation between the corrected relative density(y) and extraction ratio(%, x) was built by measuring and analyzing the related parameters such as liquid yield, relative density and extraction ratio in 46 kinds of Chinese herbal pieces. The established equation was y=0.041 3x+1.003 7, r=0.930 9(P <0.01, n=46), with linear range of 1.94%-65.75%. Based on this, the prototype model for predicting relative density of Chinese medicine decoction was established, and the relative densities of 8 Chinese medicine decoctions were within the prediction interval of this model in verification. This study lays a foundation for database construction of Chinese medicine decoction, implementation of personalized decocting mode and rapid quality control of Chinese medicine decoction.

Metabolic responses and their correlations with phytochelatins in Amaranthus hypochondriacus under cadmium stress.

Phytochelatins (PCs) play a vital role in the tolerance and enrichment of cadmium (Cd) in higher plants by chelating with Cd2+. The aim of this study was to perform a full-scale metabolomics analysis of metabolic responses highly correlated with PCs generation. These metabolites and metabolic pathways were expected to promote PCs generation and further optimize Cd absorption in plants. In the current study, Amaranthus hypochondriacus, a potential species for phytoremediation, was first adopted to investigate physiological responses to Cd stress via LCMS/MS-based metabolomics and the HPLC based determination of thiol compounds. The results showed that the leaves of A. hypochondriacus under high Cd stress accumulated 40 times the amount of Cd compared to the leaves of the plants not under Cd stress and had an increased content of three types of PCs. Metabolomics qualitatively identified 12084 substances in total, among which 41 were significantly different metabolites (SDMs) between the two groups and involved in 7 metabolic pathways. Among the SDMs, 12 metabolites were highly linearly correlated with PCs involved in three pathways (Val, Leu and Ile biosynthesis; Ala, Asp and Glu metabolism; and Arg and Pro metabolism). These results provide an innovative method to promote PCs synthesis for the restoration of Cd-contaminated-soil.

Predicting the toxic effects of Cu and Cd on Chlamydomonas reinhardtii with a DEBtox model.

Predicting the sublethal effects of pollutants to aquatic organism is essential in realistic chemical risk assessment. However, only a few dynamics models for sublethal endpoints are available. Here, we investigated the toxic effects of the essential metal Cu and the nonessential metal Cd on Chlamydomonas reinhardtiiunder both single and combined exposure, compared the effectiveness of different effect endpoints as toxic effect factors, and developed a Dynamic Energy Budget toxicology (DEBtox) model to predict the sublethal effects of Cu and Cd on C. reinhardtii. The results showed that the chlorophyll fluorescence parameter is a better toxic effect indicator than others for short-term exposure (<24 h), while algal cell growth is preferred for long-term exposure (2-6 days). The developed DEBtox model could successfully predict single metal toxicity to C. reinhardtii, while the combined metal DEBtox model slightly overestimates the joint toxicity of Cu-Cd due to the antagonistic effect of Cu-Cd on C. reinhardtii. This study is helpful to understanding and better predictions of metal sublethal toxic effects on aquatic organisms.

A Comprehensive Motion Estimation Technique for the Improvement of EIS Methods Based on the SURF Algorithm and Kalman Filter.

Video stabilization is an important technology for removing undesired motion in videos. This paper presents a comprehensive motion estimation method for electronic image stabilization techniques, integrating the speeded up robust features (SURF) algorithm, modified random sample consensus (RANSAC), and the Kalman filter, and also taking camera scaling and conventional camera translation and rotation into full consideration. Using SURF in sub-pixel space, feature points were located and then matched. The false matched points were removed by modified RANSAC. Global motion was estimated by using the feature points and modified cascading parameters, which reduced the accumulated errors in a series of frames and improved the peak signal to noise ratio (PSNR) by 8.2 dB. A specific Kalman filter model was established by considering the movement and scaling of scenes. Finally, video stabilization was achieved with filtered motion parameters using the modified adjacent frame compensation. The experimental results proved that the target images were stabilized even when the vibrating amplitudes of the video become increasingly large.