id stringlengths 3 9 | source stringclasses 1 value | version stringclasses 1 value | text stringlengths 1.54k 298k | added stringdate 1993-11-25 05:05:38 2024-09-20 15:30:25 | created stringdate 1-01-01 00:00:00 2024-07-31 00:00:00 | metadata dict |
|---|---|---|---|---|---|---|
258478739 | pes2o/s2orc | v3-fos-license | MTHFR and MTRR Genetic Polymorphism of Methotrexate Therapy Outcomes in Early Rheumatoid Arthritis
Purpose Methotrexate (MTX) is used as an anchor drug for the treatment of rheumatoid arthritis (RA) and there may be differences in drug action between genotypes. The purpose of this study was to investigate the relationship between clinical efficacy response and disease activity of MTX monotherapy with methylenetetrahydrofolate reductase (MTHFR) and methionine synthase reductase (MTRR) polymorphisms. Patients and Methods In the study, a population of 32 patients in East China with early RA fulfilling the diagnostic standards of the American College of Rheumatology (ACR) were enrolled, all of them received MTX monotherapy. Genotyping of patients MTHFR C677T and A1298C, MTRR A66G using tetra-primer ARMS-PCR method and sanger sequencing to verify its accuracy. Results The distribution of three polymorphic genotypes that were studied is in accordance with the Hardy-Weinberg genetic equilibrium. The patient pathology variables smoke (OR = 0.088, P = 0.037), drink alcohol (OR = 0.039, P = 0.016) and males (OR = 0.088, P = 0.037) were significantly associated with non-response to MTX. Genotype, allele distribution and genetic statistical models were not found to be related to MTX treatment response and disease activity in both the response groups and non-response groups. Conclusion Our findings suggest that the MTHFR C677T, MTHFR A1298C and MTRR A66G polymorphisms may not predict MTX clinical treatment response and disease activity in patients with early RA. The study revealed that smoke, alcohol, and males were possible influential factors for MTX non-response.
Introduction
Rheumatoid arthritis (RA) is a chronic systemic autoimmune disease, mainly characterised by symmetrical pain and swelling of small joints in the hands and the feet. 1 When chronic inflammation is not properly controlled over a long period of time, it can eventually lead to irreversible damage to the joint structures. Methotrexate (MTX) is a folic acid inhibitor with structural and physicochemical properties similar to folic acid, which acts as an anti-inflammatory and antiproliferative agent in the body. 2 MTX is used extensively for the treatment of RA because of its efficacy, safety profile and affordability. The European League Against Rheumatism (EULAR) recommended MTX monotherapy for patients with early RA, and MTX in combination with other disease-modifying antirheumatic drugs (DMARDs) for patients with moderate or severe disease activity. 3 However, 30-50% of patients with RA still have poor response to MTX treatment or relapse with inadequate response to re-treatment, resulting in drug resistance, and have to stop treatment or change to other medicines. 4 The personalized response between patients may be due to differences in the gene expression or activity in the folic acid-MTX metabolic pathway, and such differences may arise to alter drug pharmacokinetics and affect MTX response. 5 The methylenetetrahydrofolate reductase (MTHFR) and methionine synthase reductase (MTRR) are closely related to the folic acid metabolic pathway. MTHFR catalyzes the transformation of 5,10-methylenetetrahydrofolate (5,10-CH2-THF) into 5-methyltetrahydrofolate (5-MTHF), which provides methionine-methylation for homocysteine (Hcy). 6 MTRR generates functionally active methionine synthase (MTR), and allows it to catalyse the conversion of Hcy to methionine, maintaining normal levels of Hcy. 7 These two enzymes are significant regulation factors, each regulated by its separate gene, and their associated regulatory genes are essential for folic acid metabolism and have therefore been extensively studied.
The MTHFR gene is located on chromosome 1p36.3 and the encoded production is a key enzyme in the metabolic process of the body. In existing studies, multiple polymorphic sites in the MTHFR gene have been reported, with the most widely studied SNP sites being mainly C677T (rs1801133) and A1298C (rs1801131). The early studies showed that the C677T mutation causes a decrease in enzyme activity, with carriers of the 677TT genotype and 677CT genotype having 30% and 65% of the enzyme activity of the 677CC genotype, respectively. 8 Ashfield-Watt et al reported that the C677T mutation resulted in reduced levels of ingested folic acid, and that 677TT had lower levels of folic acid than the 677CT or 677CC genotypes, requiring higher intake to achieve equilibrium Hcy concentrations. 9 Whether the enzyme activity was altered by the A1298C mutation remains controversial. 10,11 Neither homozygote nor heterozygote of A1298C resulted in increased or decreased in folic acid concentrations. 12 MTRR gene mutations are one of the main causes of Hcy and abnormal folic acid metabolism, with A66G (rs1801394) as the most prominent and most studied mutation site. MTRR gene is located on chromosome 5p15. 2-15.3, and A66G mutation results in methionine substitution at the 22nd isoleucine. The ability to metabolize folic acid and folic acid deficiency were associated with MTRR gene polymorphisms. 13 MTRR A66G mutation leads to abnormal plasma hcy levels, with significantly higher hcy concentrations in the 66AG and 66GG genotypes than in the 66AA genotype, which may have an impact on MTX treatment response. 14 In exploring the relationship between MTX therapeutic response with MTHFR C677T and MTHFR A1298C polymorphisms, it produced remarkably heterogeneous results, making this influential relationship full of uncertainty.
Therefore, in this study, we investigated a potential correlation between clinical efficacy response and disease activity of MTX monotherapy and MTHFR C677T, MTHFR A1298C and MTRR A66G polymorphisms in RA patients in East China for the first time, hoping to provide a possibility to achieve personalized treatment for RA patients.
Characteristics of the Population
During 2019-2021, we diagnosed 32 patients with RA in the Department of Rheumatology and Immunology of the Ningbo First Hospital according to the 1987 diagnostic standards of the ACR, 15 and all patients were diagnosed with RA for the first time. The early patients included in this study were treated with methotrexate monotherapy (no other DMARDs in combination) and initial methotrexate treatment dose was 7.5mg ± 2.5mg/week. To relieve the methotrexate negative effects, patients were supplemented with folic acid 10mg/week during treatment. The average treatment period was three months, according to the DAS28 score method and the standards for evaluating the degree of rheumatoid arthritis disease of ACR. 16 Quantitative indicators to assess patients at baseline and post-treatment, including tender joints counts (TJC), swollen joints counts (SJC), erythrocyte sedimentation rate (ESR) and pain visual analogue scales (VAS). Based on EULAR's response criteria according to DAS28, 17 MTX treatment response was determined by a combination of the DAS28 difference between the baseline and post-treatment periods, the ΔDAS28, and the DAS28 at post-treatment period, with ΔDAS28≤0.6 and ΔDAS28 > 0.6≤1.2 but DAS28 > 5.1 defined as treatment ineffective and the rest as moderate or good response. In this study, we divided patients into response and non-response groups according to treatment response, with moderate or good effect patients in the response group and ineffective patients in the non-response group. To explore the correlation between genotypes and response to MTX treatment by comparing the genotype distribution of patients in the response and non-response groups. The study was approved by the local ethics committee, complied with the standard procedures of the Declaration of Helsinki, and informed consent was obtained from all subjects.
DNA Extraction
Whole blood samples were taken intravenously from patients with EDTA anticoagulated blood collection tubes and marked with the date and patient information. Genomic DNA was extracted according to the kit manufacturer's recommendations (DNeasy Blood&Tissue Kit, QIAGEN, Germany). DNA was quantified using NanoDrop 2000 (Thermo Fisher Scientific, Massachusetts, US) and Qubit 2.0 (Thermo Fisher Scientific, Massachusetts, US). DNA integrity was tested by agarose gel electrophoresis. Purified gDNA samples were stored at −80°C.
Genotype Identification
Identification of allelic genotypes for the MTHFR C677T, MTHFR A1298C and MTRR A66G polymorphisms by primers designed based on the tetra-primer amplification refractory mutation system PCR (ARMS-PCR) proposed by Ye et al. 18 The 3' terminus of the inner primer was designed according to the mutation site, and allele-specific bases were introduced, as well as a mismatched base at the penultimate third of the 3' terminus to enhance the specificity of the PCR amplification. The primers sequences and working concentrations, and annealing temperatures are shown in Table S1. Both outer primer and inner primer concentrations were optimized for the system, and the amplification of the two shorter allele-specific products was enhanced at a final concentration ratio of 1:4, with clear genotyping results. The 10 µL PCR reaction system includes 10ng DNA, outer primer final concentration of 0.1-0.2µM, inter primer final concentration of 0.4-0.8µM, and 0.5U Taq polymerase (AmpliTaq Gold™ 360 DNA polymerase, Thermo Fisher Scientific, Massachusetts, US). The PCR amplification procedure was an initial denaturation at 95°C for 5 minutes, cycle conditions of denaturation at 95°C for 30 seconds, annealing temperature for 30 seconds depending on the polymorphic site, extension at 72°C for 30 seconds and final extension at 72°C for 7 minutes. Mixed 4µL of PCR products with 2µL of loading dye and analyzed genotypes in 8% polyacrylamide gel electrophoresis (PAGE).
Meta-Analysis
This section of the study was searched for literature using PubMed and Web of Science databases. Searches were conducted using the combination of keywords: "methotrexate" AND "rheumatoid arthritis" AND "polymorphism". The title and abstract were used to obtain information related to the study for literature screening. The criteria for inclusion in the literature were: (1) study of the association of MTHFR C677T, MTHFR A1298C and MTRR A66G with MTX treatment outcomes in RA patients; (2)
Statistical Analysis
Statistical analysis of data was performed with IBM SPSS Statistics for Windows, version 26 (IBM Corp., Armonk, NY, USA). The chi-square test assessed whether the genotype distribution was consistent with Hardy-Weinberg equilibrium (HWE), P > 0.05 indicated genetic equilibrium and the sample was statistically significant. Patients' sample measurement data were described with the means and standard deviations (±SD) or interquartile ranges (IQRs). Results were compared between two groups using the Student's t-test and between multiple groups (or three different SNPs analyzed in the same sample) with Kruskal-Wallis test. When the frequency was less than 5, we used Fisher's exact test instead of the chisquare test. P values were considered statistically significant at p < 0.05. Genotype and allele frequencies were compared based on chi-square tests, and allelic and genotypic risks were assessed with used odds ratios (ORs) and 95% confidence intervals (CIs).
Meta-analysis was conducted using STATA 17.0 (Stata Corporation, TX, USA) software. The Mantel-Haenszel test was used to statistically analyze the genetic models such as dominant model, recessive model and co-dominant model to determine the correlation between MTHFR C677T, MTHFR A1298C and MTRR A66G polymorphisms and MTX treatment efficacy. Risk estimates were expressed using OR and 95% CIs, and Z-test P values <0.05 were considered statistically significant.
Patient Genotype
To determine the patient genotype, we designed outer and inner primers for the C677T, A1298C and A66G sites, respectively. In the tetra-primer ARMS-PCR method, optimization of the inner and outer primer concentrations was essential for accurate genotyping. To further determine the optimal primer concentration, we used PCR amplification with different ratios of outer primers and inner primers of 1:1, 1:2, 1:4 and 1:10, respectively, and found that at the primer ratio of 1:4, the PCR products obtained by 8% PAGE resulted in clear separation of each genotype (Figure 2A-C) (size of PCR amplification products refer to Table S1). The genotype identification results of 32 patients with regard to C677T, A1298C and A66G are shown in Table S2. To verify the genotyping accuracy, PCR amplification was done using outer primers for each SNP site and sanger sequencing was performed on all samples. From further comparison, we found that the patient genotype obtained by the tetra-primer amplification method was 100% consistent with the sanger sequencing results ( Figure 2D-F). Therefore, we identified the genotype of the patient and provided a basis for subsequent correlation analysis.
Clinical Features of the Patients
A total of 32 patients with early RA were reported in this study, and the clinical characteristics of the patients at baseline and the variables assessed by DAS28 post-treatment period are listed in Table 1. All patients were treated with MTX monotherapy from the first date of diagnosis of RA, with a mean treatment period of 86 days. Based on ΔDAS28, 21 of the 32 patients were determined to be in the response group and 11 in the non-response group. Our results revealed no significant differences in age, BMI, Anti-CCP positivity, RF positivity, MTX treatment measures, or treatment duration between the response and non-response groups (p > 0.05). In contrast, smoke (current smokers at the time of data collection, OR = 0.088, P = 0.037), drink (alcohol consumption ≥1 unit, OR = 0.039, P = 0.016) and male (OR = 0.088, P = 0.037) were significantly associated with non-response to MTX. The average levels of TJC, SJC, ESR and DAS28 after the post-treatment in patients were 1 (0-2), 0 (0-1), 19.5 (10-33) mm/h and 3.12±1.55, respectively, which were clearly different in the response and non-response groups, but not statistically significant (P > 0.05). Table 2 shows the distribution of MTHFR C677T, MTHFR A1298C and MTRR A66G polymorphism genotypes between the response and non-response groups and comparison of statistical models. The results indicated that the
Association of Disease Activity with C677T, A1298C and A66G Polymorphisms
To determine the relationship between different genotypes and disease activity in patients, the distribution between MTHFR C677T, MTHFR A1298C and MTRR A66G polymorphisms genotypes and disease activity parameters were compared, as shown in Table 3. The results showed that ESR levels were not remarkably different across the C677T genotypes and were reduced in the 1298AC and 66GG genotypes (P > 0.05). DAS28 was decreased after the posttreatment in 677TT and 1298AC, but was not statistically significant. TJC and SJC were not found to be significantly different across genotypes in the three SNPs. To further compare the differences in each disease activity parameter between the response and non-response groups at baseline and after the post-treatment, Figure S1 shows that ESR, TJC, SJC and DAS28 were remarkably lower in the response group (p < 0.001), while the differences were not statistically significant in the non-response group.
Meta-Analysis
A total of 373 publications were identified after the initial search, and 37 publications were included after analysis of titles and abstracts. The full literature was interpreted in detail, and 13 of these publications met our inclusion criteria, with reasons for exclusion: (1) no detailed data for genotype; (2) letter or comment. MTHFR C677T: The meta-analysis of MTHFR C677T included 12 studies, [19][20][21][22][23][24][25][26][27][28][29][30] which included 958 responders and 840 non-responders, and the main characteristic information is presented in Table 4. Figure S2B) did not observe an association between the C677T polymorphism and MTX treatment response, nor was significant heterogeneity observed between studies. Study cohort stratified by ethnicity found that C677T polymorphism was significantly associated with MTX treatment response in the recessive model (OR = 0.497, 95% CI = 0.279-0.886, P = 0.018) and codominant model (OR = 1.492, 95% CI = 1.046-2.126, P = 0.027) in the European population, no association was found in other models and populations (Table 5).
MTHFR A1298C: The meta-analysis of MTHFR A1298C included 9 studies, 20-24,26-29 which included 688 responders and 615 non-responders, and the main characteristic information is presented in Table 6. (Figure 4) did not observe an association between the A1298C polymorphism and MTX treatment response. The study cohort was stratified by ethnicity, and there was a significant association between the A1298C polymorphism and MTX treatment response in the recessive model (OR = 0.432, 95% CI = 0.201-0.926, P = 0.031) and the codominant model (OR = 1.981, 95% CI=0.1.108-3.543, P = 0.021) in the South Asian population. In addition, there was significant between study heterogeneity in the recessive model (I 2 =55%, P = 0.023) ( Table 5).
Discussion
Since 1988, when the US Food and Drug Administration approved MTX as therapy for RA, 32 till the EULAR recognized it to be the anchor drug for the treatment of RA, 3 MTX has played an important role in the treatment of RA. Low-dose MTX has long been considered an effective and safe anti-rheumatic drug, as well as a base drug for other DMARDs combinations. 33 However, because of individual differences in clinical response to MTX, at least one-third of patients with RA have no response or significantly lower efficacy when they take it. 34 Similar differences in clinical response were observed in our study cohort of the Zhejiang, East China population, with 34.4% of patients with early RA poorly treated with MTX monotherapy. In this study, we explored the correlations between MTX therapy response and disease activity with MTHFR C677T, MTHFR A1298C and MTRR A66G polymorphisms.
Our patient clinical pathology characteristic statistics demonstrated that age and BMI were not relevant to clinical response to MTX, while smoke status, drink and male may be factors associated with non-response to MTX. In early studies, pathological variables were used as measures of MTX response prediction models, but with varying results. Among patient-related factors, males were connected with a good response to MTX treatment. 35 Smoking, alcohol, and high BMI may be risk factors for non-response to MTX in RA patients. 36 Whereas in our study, males had a poorer response to treatment. In addition, BMI was at normal levels in both the MTX response and non-response patient groups. Of the disease-related factors, the proportion of Anti-CCP positivity and RF positivity was similar in both groups initially.
Our study and meta-analysis displayed that genetic polymorphisms in MTHFR C677T and MTHFR A1298C were not associated with clinical efficacy response of MTX, and appeared consistent with our results in many studies. 20,37 In Western Algerian population studies, it was concluded that C677T and A1298C cannot predict clinical response and adverse drug reactions to MTX treatment outcomes. 22 Research on 120 patients with RA in the East Bohemian population has not found any correlation between the genotypes C677T and A1298C and ineffectiveness of MTX treatment in the respective genetic statistical models. 26 In an early study of the efficacy of MTX with folic acid supplementation, the C677T polymorphism was found to have failed to predicted toxicity or efficacy response to MTX treatment in patients with RA who received folic acid supplementation. At the same time, the
416
authors gave the explanation that it is possible that the anti-inflammatory effects are greater than the antiproliferative properties in the mechanism of action of low-dose MTX for the treatment of RA patients. 38 However, Lima et al in a Portuguese cohort of RA patients have found that 677TT was associated with an approximately more than 3-fold increased risk of non-response to MTX when compared to MTHFR 677CC and 677C carriers. 30 A recent meta-analysis describing MTX kinetics and efficacy characteristics, it was shown that genotypes 677CT and 677TT carriers had 30% and 65% lower enzyme activity, respectively, when the C677T allele was present, and both genotypes were associated with reduced MTX efficacy and increased toxicity. 39 Very few studies have shown that the A1298C SNP is associated with MTX efficacy. One of the studies was Lilya et al who observed that the A allele of the A1298C polymorphism was related to good and moderate response to MTX treatment. 23 Although marked differences in the frequency distribution of MTHFR A1298C genotypes were observed in our study, they were not correlated with MTX treatment response. Furthermore, the results of this study and the meta-analysis showed that the genotype frequency distribution of the MTRR A66G polymorphism was not significantly different between the response and non-response groups and could not predict response to MTX treatment. Consistent with our results, in the study of Chinese RA patients, Lv et al found no significant differences in the frequency distribution of MTRR A66G genotypes, alleles and haplotypes in the MTX response and non-response groups. Similarly, no differences were found in the five genetic statistical models. 21 Separate studies of RA patients and healthy people groups in Mexican and South Indian Tamil populations also did not find any association between the MTRR A66G polymorphism and response to MTX treatment. 31,40 In contrast to our findings, López-Rodríguez et al selected 25 relevant SNP sites and studied 956 RA patients from four regional groups, with the result that only MTRR A66G showed close correlation with response to MTX monotherapy. 41 Among Portuguese RA patients, MTRR 66A carriers are associated with an approximately 2-fold increased risk of MTX non-response. 24 Chaabane et al suggested that the MTRR A66G polymorphism may produce different MTX treatment responses and toxic effects in RA patients from different ethnic groups. 42 Our report of the correlation between the MTRR A66G SNP and MTX efficacy offers a possibility for studies in the Zhejiang RA population of China. On the study of the association of the three SNP sites with disease activity, we observed that ESR levels were not significantly different in the C677T polymorphism and were reduced in the 1298AC and 66GG genotypes. DAS28 was lowered in 677TT and 1298AC, TJC and SJC were not different among all genotypes, but neither was statistically different. Instead, each disease activity parameter was significantly lower in the response patient group compared to the baseline period (p < 0.001). Kurzawski et al found that ESR levels in the 677TT and 1298CC genotypes and TJC, SJC and DAS28 in patients with the 677TT and 1298AC genotypes were both significantly reduced. 43 Similar to our observed DAS28 with genotypes distribution changes. The current findings on the relationship between genetic polymorphisms and disease activity are varied, which may be related to factors such as patient cohorts, MTX dosage and different indicators of disease activity evaluation. Studies of Japanese RA patients reported that average DAS28 levels were markedly lower in patients with the MTHFR 1298AA genotype than in patients with the 1298AC/CC genotype. 44 González-Mercado et al evaluated the differences between polymorphic genotypes and DAS28, found a modest trend towards increased disease activity in patients heterozygous for A1298C and A66G (p > 0.05). There was no considerable difference between MTX monotherapy and combination therapy. 40 Gender, BMI, smoke status, RF-positive status, and age at onset all affect the response of RA patients to MTX treatment, leading to different levels of treatment response and disease activity. 45,46 This may also explain the heterogeneous results that occurred when explored SNP genotypes and MTX disease response. The small number of participants in our study makes the results have some limitations. To explore our findings further, we also look forward to confirming our findings in a larger sample size.
Conclusion
This study reported the association of MTHFR C677T, MTHFR A1298C and MTRR A66G polymorphisms with MTX treatment response and disease activity in early RA patients in the Chinese population of Zhejiang. We observed that smoke, alcohol consumption, and males were possible influential factors for MTX non-response. In contrast, the SNP sites we studied showed no correlation with MTX response and disease activity, and whether they can predict MTX treatment response needs to be repeated in a larger sample size. We also hope that this study may provide a possibility for personalized treatment of MTX in RA patients.
Ethics Statement
This study was approved by the Ethics Committee of Ningbo First Hospital and written informed consent was obtained from all participants. Subject identification information was not disclosed in the study, and patients were identified by numbers to indicate. | 2023-05-04T15:04:53.734Z | 2023-05-01T00:00:00.000 | {
"year": 2023,
"sha1": "7ad2a65bae3c2d45b2699bfa25d9f84ff61c128a",
"oa_license": "CCBYNC",
"oa_url": "https://doi.org/10.2147/pgpm.s404949",
"oa_status": "GOLD",
"pdf_src": "PubMedCentral",
"pdf_hash": "6a92caaf046eca2c4b591c1236fca6df0297d45b",
"s2fieldsofstudy": [
"Medicine"
],
"extfieldsofstudy": [
"Medicine"
]
} |
248595830 | pes2o/s2orc | v3-fos-license | Simultaneous Extraction of Planetary Boundary-Layer Height and Aerosol Optical Properties from Coherent Doppler Wind Lidar
Planetary boundary-layer height is an important physical quantity for weather forecasting models and atmosphere environment assessment. A method of simultaneously extracting the surface-layer height (SLH), mixed-layer height (MLH), and aerosol optical properties, which include aerosol extinction coefficient (AEC) and aerosol optical depth (AOD), based on the signal-to-noise ratio (SNR) of the same coherent Doppler wind lidar (CDWL) is proposed. The method employs wavelet covariance transform to locate the SLH and MLH using the local maximum positions and an automatic algorithm of dilation operation. AEC and AOD are determined by the fitting curve using the SNR equation. Furthermore, the method demonstrates the influential mechanism of optical properties on the SLH and MLH. MLH is linearly correlated with AEC and AOD because of solar heating increasing. The results were verified by the data of an ocean island site in China.
Introduction
The lowest atmospheric layer of the earth is marked by a planetary boundary layer (PBL). There is a variable daily convolution in the structure and composition of the PBL [1]. During the daytime, the PBL is mainly composed of the surface layer (SL), mixed layer (ML), and entrainment zone. During the nighttime, the ML collapses into the nocturnal boundary layer (NBL) and residual layer (RL) [2]. Furthermore, the mixing and residual layers coexist during the sunrise and sunset [1,3]. Atmospheric variables such as potential temperature, aerosol concentration, and specific humidity usually experience sharp gradients at the top of the PBL. Thus, some measurements of PBL height (PBLH) were proposed based on the characteristics of these variables [4]. Additionally, the optical properties including extinction coefficient and optical depth were employed to represent aerosols, including the total amount of pollutants [5,6], which were determined by the size distribution [7,8] of aerosol formation, which was affected by relative humidity (RH) and temperature (T) [9]. There is a complex interaction between PBL height and aerosols and statistical associations between PBL height and levels of pollutants [5,[10][11][12][13]. Thus, the PBLH is an important physical quantity for atmosphere environment assessment [14][15][16][17].
These measurement techniques were mainly implemented by microwave radiometer [14], ceilometers [18][19][20], and lidar, including Mie-scattering lidar [21,22] and coherent Doppler wind lidar (CDWL) [23]. The microwave radiometer is based on the thermodynamic properties of the atmosphere for potential temperature and specific humidity [24]. Ceilometers are single-wavelength micro-lidars intended for cloud-base height detection and are ubiquitous in airports and meteorological service centers worldwide [20]. The Mie-scattering lidar employs back-scattering signals to monitor aerosol concentrations [25]. CDWL data are related to the average wind speed [26]. These techniques have been proposed to combine with several algorithms to accurately detect the PBLH based on the sharp gradient. Some algorithms [4] include visual inspection, the threshold method, the gradient method, ideal profile fitting (FIT) [25], wavelet covariance transform (WCT), and variance (or standard deviation) analysis. Many studies have shown that the retrieved PBLH of lidar instruments is in good consistency with the radiometer [27]. However, the accuracy of the PBLH was influenced by multiple-layer aerosol layers and cloud layers [28]. Some methods were proposed to combine some different algorithms, such as combining WCT with the ideal curve-fitting (ICF) algorithm [25], combining WCT with the threshold for a range-corrected signal, and combining WCT with depolarization [3].
The PBL contains aerosols of the low troposphere. The optical properties of aerosols mainly include the aerosol extinction coefficient (AEC) and aerosol optical depth (AOD). It was pointed out that PBLH decreased sharply with the increase of aerosol load [29]. A two-component fitting method is employed to find an accurate AEC as the boundary value in Mie-scattering lidar [30]. However, the boundary value is determined by the empirical back-scattering ratio, which is measured by combining auxiliary sensors, such as a sun photometer [30]. Furthermore, the hundreds of meters of the blind zone and the transition zone in traditional Mie-scattering lidars [31,32] always lead to a difficulty in probing aerosols in the lower troposphere [33], since the biaxial lidars are in parallel to the laser and telescope axes. In addition, CDWL can also be used to estimate the MLH based on the signal-to-noise ratio (SNR) by combining with WCT [23]. However, there are multiple local maximum positions that are manually chosen to determine the PBL. Thus, an automatic PBL extracting algorithm is needed to speed up the determination process.
The existing studies on the interaction between aerosols and the PBL are mainly based on short-term numerical simulations [34] and long-term comprehensive observations [35]. The main influence of aerosols on the PBL is the cooling effect on the surface and the heating effect on the atmosphere. The aerosol extinction in the atmosphere (including the scattering and absorption of sunlight) will reduce the short-wave radiation of the sunlight reaching the surface, so the surface heat flux drives the development of the PBL. In these methods, a lidar and a sunphotometer were synthetically applied to monitor the PBLH, and AOD or AEC, respectively. The AEC and AOD depend on the wavelength of light, and the wavelengths of the sunphotometer and lidar are different. However, no attempt has been made to simultaneously determine the PBLH, AOD, and AEC based on the same lidar.
In this study, the atmospheric boundary layer and the optical properties of aerosols are implemented by employing CDWL and WCT based on two local maximum positions with an automatic algorithm. In this work, the surface-layer height (SLH) and mixed-layer height (MLH) were simultaneously extracted based on wavelet covariance transform with an automatic algorithm, due to the sharp gradient on the boundaries of SL and ML. Meanwhile, the optical properties including AEC were estimated by linear fitting in the range from SLH to MLH, and the AOD was calculated by AEC-times depth. Then, the relationship between optical properties, the SLH, and MLH were quantitatively characterized for an ocean island site in China.
Study Area
The measurements were carried out at the observation site in the ocean island site, which is located in the south of China with a tropical maritime monsoon climate. The weather around the site is summer-like the whole year, the highest temperature is 32 • C, and the lowest temperature is 20 • C due to the effect of the ocean. The prevailing period of the northeast monsoon is from October to March of the next year, and the prevailing period of the southwest monsoon is from May to September. The rainy period is from June to November and the dry period is from December to May of the next year. Rainless and sunny weather in December was selected as the observation object, and the observation site is far from the city and less affected by emissions from industries, vehicles, and other anthropogenic activities. The aerosols in ocean islands are mainly composed of sea salt aerosols.
Experimental Instruments
The measurements were performed by a CDWL (Windprint S4000, Qingdao Aerospace Seaglet Environmental Technology Ltd., Qingdao, Shandong, China), whose technical specification is shown in Table 1. The vertical resolution and temporal resolution of this CDWL are 30 m and 1 s, respectively. The telescope was designed with a diameter of 40 m and a focal length of 1000 m. The blind zone of CDWL is 60 m. The typical SNR image, which includes successive 180 measurements, is shown in Figure 1a. The SNR of one measurement and the average SNR of the successive 180 measurements are demonstrated in Figure 1b. The PBL is in the range of red rectangular area and the AEC of PBL is homogeneous. This work presents an automated algorithm to simultaneously extract the PBLH and AEC. The weather in December was chosen for typical case to verify the feasibility of the proposed method. The continuous sample data of 24 h by the CDWL was used to study the daily evolution of PBLH and the optical properties of the aerosol.
SNR of Coherent Doppler Wind Lidar (CDWL)
The SNR of the CDWL mainly depends on four factors: the average direct detection power, the heterodyne efficiency, the wavelength λ, and the receiver bandwidth B [36]. Under the conditions of negligible refractive-turbulence effects, the matched filter B = 1 τ , where τ is the pulse duration and far-field operation, the peak of SNR depends on the altitude z, and can be expressed as [37]: where η Q is the quantum efficiency of the detector, h is the Planck constant, U T is the transmitting pulse energy, β is the back-scattering coefficient, and D is the diameter of laser dr is the dimensionless one-way irradiance extinction at wavelength λ, and α(m −1 ) is the linear AEC along the propagation path. Figure 1b shows the typical SNR in terms of altitude z.
WCT
The Haar wavelet is discontinuous and usually applied to the location of the PBL due to its superior spatial location and computational efficiency. The Haar wavelet function can be expressed as: where z is the altitude, a is the dilation of the function, and b is the center of the Haar function. The Haar wavelet function is shown in Figure 2a and the WCT of the Haar function is defined using Equation [38]: where z t and z b are the spatial ranges in the profile, f (z) is the profile as a function of altitude and the normalization factor, and a −1 , is the inverse of the dilation. The first step in the algorithm to determine the PBLH is to define the dilation of the Haar function values. Figure 2b indicates the WCT of SNR with different dilation. The minimum of WCT was chosen as an objective parameter to find its optimal value of dilation. Figure 2c shows the minimum of WCT dependent on the dilation, and the position of the minimum value was chosen as the appropriate dilation for Haar function. The corresponding dilation is 60 m. The WCT was applied to the profile with the dilation of 60 m for the Haar function. Figure 3 demonstrates that the position P a is identified by the local minimum value in the resulting wavelet covariance profile and indicates the height of the strongest decrease of SNR. The altitude of 180 m can be considered as the SLH that is larger than the blind zone of 60 m. P b is determined by the local minimum value in absolute value of W f (a, b), which means the local minimum value of SNR, and the height could be seen as the MLH with an altitude of 840 m. The SLH and MLH are consistent with the results in reference [2]. The local maximum positions of absolute WCT can be automatically determined by dilation operation, which is defined as , where I represents the signal and E denotes the structuring element [39]. The dilation operation has a filtering effect that suppresses dark regions smaller than structuring elements and results in the enlargement of bright ones. The dilation operation can be recast into maximum operation on structuring elements.
AEC and AOD
The aerosols in the atmosphere in the range from P a to P b can be seen as roughly randomly distributed particles in PBL, and the corresponding linear extinction coefficients can be regarded as homogeneous [40]. Thus, the irradiance extinction T can be given by T = exp[−α(P b − P a )] at the PBL. The linear extinction coefficient α can be obtained by the fitting curve of Equation (1) when the boundaries of layers are obtained by local minimum values in the resulting wavelet covariance profile. Equation (1) made the logarithmic transform and can be expressed as: Furthermore, the corresponding AOD at the wavelength of 1550 nm is defined by [35]: To sum up, Figure 4 demonstrated the flowchart to determine the four parameters including SLH, MLH, AEC and AOD. Figure 5 demonstrates that the typical SLH and MLH, which are extracted from the mean of 180 measurements of SNR, depend on the local time during the whole day. During the daytime, the PBLH is identical to the MLH. During the nighttime, the MLH collapses into the nocturnal boundary layer (NBL) and residual layer (RL). The MLH is identical to the height of the NBL. In addition, Figure 5a,b indicate that the MLH is negatively correlated with AEC and positively correlated with AOD in terms of the local time. Figure 5c,d demonstrate that the linear fitting curves of the MLH depending on AEC and AOD can be expressed as: MLH = K 1 × AEC + C 1 , and MLH = K 2 × AOD + C 2 , where K 1 and K 2 are constants, and C 1 and C 2 denote constants which do not affect the result. Their correlation coefficients R are 0.67 and 0.65, respectively. The linear functions of the SLH dependent of AEC and AOD are given by: SLH = K 3 × AEC + C 3 , and SLH = K 4 × AOD + C 4 , where MLH is linearly correlated with AEC and AOD, and Figure 6a demonstrates that the slopes K 1 of the MLH dependent on AEC are negative, and the slopes of K 2 of the MLH linearly dependent on AOD are positive, which means that the MLH decreases while the AEC is increasing, and the MLH increases while the AOD is increasing. The reason is that solar heating increases in the ML while the strength of capping inversion decreases, leading to a rise in the MLH and decrements in AEC. There is a positive relationship between the MLH and AOD and a negative between MLH and AEC. The difference is that the effect of increment of MLH on AOD is greater than that of the decrement of AEC. Thus, the effect that solar heating increases in the MLH is greater than the effect of MLH on AEC.
Results
SLH is linearly correlated with AEC and AOD, and Figure 6b shows the distribution of the slopes K 3 and K 4 in eight successive days. The values are sometimes positive and sometimes negative, which means that the linear fitting curves of SLH dependent on AEC and AOD are complex. The reasons are the multiple factors such as the cooling effect of the surface enhanced with the increase of AOD and aerosols with human activity.
In order to study the factor of aerosols with different sizes on AEC, the data of PM 2.5 and PM 10 are obtained from the National Urban Air Quality data of the Ministry of Ecology and Environment, PRC [41]. Figure 7a indicates the positive correlation between AEC and aerosols (PM 2.5 and PM 10 ) during the local time. The Pearson correlation coefficient provides a measure of the strength of the linear association between two variables [42], and it is found that the correlation coefficient between the derived AEC and aerosols (PM 2.5 and PM 10 ) are 0.1026 and 0.5890, which suggested that aerosol of PM 2.5 plays an important role in the determination of AEC. Additionally, Figure 7b demonstrates that there are positive statistical associations between AEC and the mean of wind speed, which is estimated by the same CDWL. However, AOD is not positively related to the mean wind speed. Thus, the factors considered for AEC are much simpler than AOD. The comparison of the AEC with the optical absorption coefficient (OAC) is based on photoacoustic spectroscopy at the wavelength of 1064 nm [43], and it is found that the trend of the AEC is highly correlated with the OAC, shown in Figure 8a. Furthermore, the reference data of AOD and MLH were obtained from EAC4 (ECMWF Atmospheric Composition Reanalysis 4) [44], which is the fourth generation ECMWF global reanalysis of atmospheric composition, and reanalysis combines model data with observations from across the world into a globally complete and consistent dataset using a model of the atmosphere based on the laws of physics and chemistry. Figure 8b,c demonstrate that the trends of AOD and MLH are related to that in EAC4. Thus, it is feasible that the simultaneous extraction method of the planetary boundary-layer height and aerosol optical properties can be obtained from coherent Doppler wind lidar.
Discussion
AEC is the result of both absorption and scattering [40]: a ext = n(C abs + C sca ), where n is the number of particles per unit volume, and C abs and C sca are the absorption and scattering cross-sections, respectively. The light with a wavelength of 1550 nm passing through aerosols is attenuated almost entirely by scattering. The scattering cross-section depends on the size of the aerosols. It was found that the vertical meteorological parameters, such as relative humidity and temperature, and the aqueous and heterogeneous atmospheric chemical reactions altogether led to the aerosol formation [9] and resulted in different size distributions. Additionally, other parameters such as wind, rainfall, and even the emission rates will change the number of particles in unit volume n in the physical view. Thus, the AEC is affected by many parameters.
In this study, the signal-to-noise ratio of CDWL had been used to simultaneously extract four parameters, including SLH, MLH, AEC, and AOD, which simultaneously monitor the daily evolution of both the PBL height and the optical properties of aerosols and their relationships. Although the interaction between the aerosols and the PBL height is highly complicated, there is a positive relationship between MLH and AOD, and negative with AEC, which suggests that the effect of the increment of MLH on AOD is greater than that of the decrement of AEC. Thus, the effect that solar heating increases in the MLH is greater than the effect of MLH on AEC.
In this work, CDWL was used for measuring both PBLH and optical properties, since the system has a smaller blind zone than traditional Mie-scattering lidar due to the coaxial design of CDWL with the telescope axis. In addition, SLH can be extracted by the CDWL, which is difficult to estimate in Mie-scattering lidar.
Ruijun Dang, et al. [4] had made a review of techniques for measuring the atmospheric boundary-layer height (ABLH) or the MLH using aerosol lidar. In their review, many studies on measurements of ABLH were based on range-corrected SNR (RCSNR). The RCSNR can be obtained by Equation (1) multiplying z 2 , which can be expressed as [4]: Classical WCT methods were also applied for extracting the ABLH or MLH. When the Haar wavelet function h encounters a sharp drop in RCSNR, a local maximum in W f (a, b) occurs, indicating a step change in the RCSNR located at b with a coherent scale of a. Therefore, the ABLH is defined as the location of b, where the W f (a, b) reaches its maximum. Figure 9 shows the RCSNR and the corresponding WCT and demonstrates that the local maximum of WCT of RCSNR is at the altitude of 120 m. It is lower than 180 m, as shown in Figure 3. Thus, the local maximum of WCT of RCSNR is the location of SLH, which is consistent with the classical WCT method. However, the classical method cannot obtain the MLH, and the AEC between the SL and ML cannot be estimated. In order to overcome it, the local minimum values of WCT based on RCSNR are employed at the location of altitude of 900 m, which is smaller than the 840 m extracted by our algorithm. Therefore, the MLH can be defined as the local minimum values of the WCT of RCSNR.
In addition, the cloud has a strong effect on the accurate extraction of PBLH, and the opening filter, which is defined as the two sequential compositions of erosion and dilation, can be first applied to the SNR image to reduce the cloud before the mean of 180 measurements of SNR. Figure 10a shows that the bright spots are the clouds due to the strong scattering, and the clouds can be filtered with an opening operation, as shown in Figure 10b.
Conclusions
In this study, a method of simultaneously extracting the SLH, MLH, and optical properties based on the SNR of the same CDWL was presented. The method employed WCT to locate the SLH and MLH, and optical properties including AEC and AOD were determined by the fitting curve using the SNR equation. In addition, the effects of optical properties on the SLH and MLH were qualitatively studied for an ocean island site in China. The results preliminarily demonstrated that MLH is linearly correlated with AEC or AOD because of increasing solar heating. Furthermore, there is a positive relationship between MLH with AOD and negative with AEC, which suggests the effect that solar heating increases in the MLH are greater than the effect of MLH on AEC. However, the effect of optical properties on SLH is complex. Thus, this work provides an effective method for understanding the aerosol effect on PBL in the same location.
Institutional Review Board Statement: Not applicable.
Informed Consent Statement: Not applicable.
Data Availability Statement:
The data presented in this study are available on request from the corresponding author.
Conflicts of Interest:
The authors declare no conflict of interest. | 2022-05-10T16:29:30.823Z | 2022-04-29T00:00:00.000 | {
"year": 2022,
"sha1": "b2c26440223a0b500d9455fdc889f669fb852262",
"oa_license": "CCBY",
"oa_url": "https://www.mdpi.com/1424-8220/22/9/3412/pdf?version=1651224864",
"oa_status": "GOLD",
"pdf_src": "PubMedCentral",
"pdf_hash": "a83e4ec9331f3c0a0f4b5e74f46f1c5f211b3a73",
"s2fieldsofstudy": [
"Environmental Science",
"Physics"
],
"extfieldsofstudy": []
} |
264468680 | pes2o/s2orc | v3-fos-license | The Contemporary Japanese View of Life and Death as Seen through the Depictions of Reincarnation in Another World (Isekai Tensei) Anime
: The study discusses the Japanese view of life and death as seen in Isekai reincarnation anime. Isekai anime can be divided into two further categories, Isekai anime and Isekai reincarnation anime. Both genres are set in the world of swords and magic. However, the difference between them is how the protagonists are reincarnated into another world. The author conducted research how they were reincarnated into another world. It can be divided into five categories depending on the cir - cumstances and means of reincarnation. Of these, the author focused on the means of reincarnation after the deaths of the protagonists in this world. The concept of reincarnation in Isekai anime may appear similar to that of Buddhism (karma and saṃsāra). However, Buddhism has a negative view about reincarnation because Buddha tried to transcend reincarnation which was a traditional do - minant thought in Indian society at that time. In Isekai reincarnation anime, the protagonists are reincarnated in another world in which they gain magical powers and talents and can realize the unfulfilled dreams of their previous lives. We can see that this depictions of reincarnation in positive.
The Contemporary Japanese View of Life and Death as Seen through the Depictions...
Introduction
Japanese anime has long been gaining popularity around the world.In recent years, the genre of "Isekai (in English, "another world" or "a different world") anime" has emerged, and the term "Isekai" has already become commonplace among otaku, such as in the overseas otaku term "get isekaied." 1 Also, in the academic field, we will find many articles discussing Isekai anime (or manga and Japanese young adult novels, referred to as "light novels") written by foreign researchers, rather than Japanese-language articles. 2This is a good indication that people outside Japan are paying more attention to the "Isekai" genre than the Japanese themselves.
However, even for Japanese people, they don't know clearly what Isekai anime is.A closer look at "Isekai anime" reveals that by its content, it can be divided into two categories.One is stories set in the "world of swords and magic," and classified as a genre of fantasy.The other is stories in which people from this world reincarnate (transfer) into the "world of swords and magic" and face various ordeals or enjoy life in another world.In this study, the author will refer to the latter anime as "Isekai Reincarnation Anime." 3 Further research of this genre reveals that it can be divided into three main categories according to how the protagonist reincarnates into another world. 4 1. Summoned by someone in another world.2. Reincarnation in a different world after death in this world.3. Transference to the game world during or after playing a game.
According to Akiko Komura (2023), the most prominent manner of the protagonist's reincarnation is by being summoned by a person/persons in another world.Reincarnation in another world after the protagonist's death in this world is also often depicted. 5However, one question arises here.
For the Japanese, the concepts of "Isekai" and "Reincarnation" are not ideas that originated in Japanese subcultures such as manga or anime, but are Japanese religious traditions.First, in Japanese culture, "another world/a different world (他界 Takai, in Japanese)" is a traditional religious concept.For example, in Shintoism, the traditional Japanese religion, another world is "Yomi-no-Kuni (Underworld)."In folk beliefs, there is a world called "常世 (Tokoyo)" or "Takai," which appears in the folk legend, " (浦島太郎Urashima Tarō)," and means the eternal world beyond the sea and mountains.There is also a belief in another world (他界信仰Takai Shinkō) in which the spirits of ancestors stay and from where they return to their descendants during the Bon festival (around the middle of August).
In Buddhism, there is the concept of the endless cycle of transmigration in the six posthumous worlds (六道輪廻Rokudō Rinne).This is a very common religious concept that is familiar to the Japanese, who are traditionally Buddhist.
Is it possible to say that depictions in which the protagonists are reincarnated in another world after their deaths are based on this traditional Buddhist view of life and death?Or, does Isekai reincarnation anime create a unique and new concept of reincarnation as a belief of modern Japanese people?Needless to say, the reincarnation depicted in anime is completely different from the Buddhist concept of the endless cycle of transmigration in the six posthumous worlds.In many cases, a simplified concept of reincarnation is co-opted from traditional religions simply to enrich the storyline.However, as will be explained later, there are some anime that actually depict the concept of reincarnation expressed in the above-mentioned traditions.
In this study, the author will analyze the concept of reincarnation in anime about reincarnation, focusing on how people are reincarnated in another world and how they live in the reincarnated world.Then, by comparing the concept of Isekai reincarnation anime with the Buddhist concept of reincarnation, this study will investigate whether Isekai reincarnation anime represents a unique religious view of reincarnation by contemporary Japanese people.
About Prior Research
As mentioned above, there have been a variety of studies about Isekai anime published abroad.However, the author is not aware of any articles which discuss the relationship between anime and the Japanese view of religion, and this might be a topic of future research.If the research area is extended beyond the genre of Isekai anime to all genres in Japanese anime, several articles can be found.In particular, many of them are written about specific anime.For example, an academic article takes up religiosity in the film Spirited Away, directed by Hayao Miyazaki. 6However, it cannot be said that the articles that focus on a specific anime reveal the relationship between Japanese religious views or concepts, and anime.This is because, by focusing on a specific anime, they are merely describing the religious views of the anime's director or author.In recent years, there have been quite a few The Contemporary Japanese View of Life and Death as Seen through the Depictions... articles that discuss anime and religion in relative terms. 7However, the discussion of Isekai anime, which will be discussed in more detail below, will be studied and many articles will be published in the future due to the fact that Isekai anime has rapidly become popular since the 2010s, and various depictions of Isekai anime are still being produced today.Therefore, this paper can be said to be a pioneer in terms of discussing the Japanese view of religion as seen in Isekai anime.
There are a few studies on the means of reincarnation in Isekai anime.For example, Paul S. Price (2020) also investigates the means of reincarnation in another world.Similarly, Akiko Komura (2023) also analyses Isekai reincarnation anime that aired on TV from the 1990s through the spring season (April to June) of 2022.
Furthermore, a few master's theses have mentioned escapism from the reality of Japanese society based on the idea of the connection between Japanese social issues and Isekai genre, in particular, Japan's suicide rate with Isekai reincarnation anime.For example, Curtis Lu (2020) mentions that Japan's current suicide rate has been decreasing but "the number of those under 20 who took their lives rose," also mentions that "the target demographic of most Japanese anime, manga, and light novels is aimed at young adults." 8It is not entirely accurate.Because we can say that middle-aged and older Japanese enjoy watching anime or reading manga and light novels rather than young adults. 9 The causal relationship between Japan's suicide rate and Isekai reincarnation anime has not been clearly proven.If we mention Isekai reincarnation anime connected with escapism from the real Japanese society, it is necessary to first understand the Japanese idea of life and death.
Purpose and Flow of This Study
As mentioned earlier, the purpose of this study is to examine whether or not the depiction of the protagonists' reincarnation into another world after their death in this world is based on the traditional Japanese view of life and death, such as the belief in another world and reincarnation.For this purpose, this paper will first examine what the characteristics of "Isekai reincarnation anime" are.Then the author will examine how the protagonists in Isekai anime are reincarnated into another world, have adventures in that world, and live their daily lives.
In writing this paper, based on Akiko Komura (2023), the author will also analyze and discuss Isekai reincarnation anime which aired on Japanese TV from the 1980s to the winter season of January to March 2023. 10The author will also discuss Isekai reincarnation anime from a new perspective, that of comparison with reincarnation in Buddhism.
The Characteristics of Isekai Reincarnation Anime
First of all, the author mentions the characteristics of Isekai reincarnation anime.We can say that Isekai anime are stories of a world of swords and magic.In fact, if we watch some of those anime works that are categorized as belonging to the Isekai anime genre, we will find that the majority of the stories are set in a world of swords and magic.Also, many stories can be seen in which the protagonists are depicted as brave warriors or wise persons in another world, who fight their enemies such as dragons, devilish beings, and so on by using swords and magic.
In recent years, however, a close examination of the stories of Isekai anime that have been aired on TV in Japan and other media such as the Internet reveals that the means of transferring or being reincarnated into another world and the way of being or lifestyle there are to some extent regulated. 11Therefore, we can further divide Isekai anime by the way they are depicted, and that is, the characteristic of Isekai reincarnation anime.
First, Isekai reincarnation anime are stories about how a person is transferred or reincarnated into another world.Because these stories are set in a different world, they have often been simply categorized as fantasy anime.However, in some Isekai anime, the story begins when the protagonists are transferred or reincarnated into another world, rather than being born and living there.Moreover, the means of transmigration (reincarnation) is not simply being summoned by someone in another world, but also being reincarnated into another world by a god or a god-like being upon the protagonist's death in this world.In addition, since some anime works are set up in such a way that a summoned individual cannot go back to this world, depending on one's point of view, a transfer to another world through a summons may also mean the death of the protagonists in this world.In other words, from the point of view of those who remain in this world, the protagonists will cease to exist, which indirectly means their death.In any case, works in which the protagonists are transferred or reincarnated into another world from which they can never return are defined as "Isekai reincarnation anime" for the sake of convenience in this paper.
In addition, the protagonists are reincarnated into another world with retaining their identities and memories of this world.Also, they can bring with them personal 10 There are two reasons why the author chose to study televised Japanese anime in this research.One is the unification of the subject matter of the study.Needless to say, the Internet did not exist in the 1980's.The second reason is the difference in medium to watch Japanese anime.Currently, Japanese young people are watching Japanese anime on the Internet, because they do not have a TV set.However, when they watch anime on the Internet, they tend to prefer watching free sites such as TVer provided by Japanese TV stations rather than the paid sites such as Amazon Prime or Netflix.And most of the free sites are aired on TV.The Contemporary Japanese View of Life and Death as Seen through the Depictions... belongings which they had in the previous world, such as a smartphone.In general idea of reincarnation, the dead person cannot retain their identities and memories of this world.Also, the dead person cannot bring his/her belongings which he/she used in our previous lives to another world.
The First Stage of the History of Isekai Reincarnation Anime before 2010
Before talking about the author's survey of Isekai reincarnation anime from 1980 to March 2023, the author would like to mention the first stage of the history of Isekai reincarnation anime.Because there hadn't been the word, "Isekai" before 2010.The term "Isekai" first appeared on the Internet website "Let's Become a Novelist (小説 家になろうShōsetsuka ni Narō)" around 2010.12However, if this term "Isekai" hadn't been appeared before 2010, we can see that there were some Isekai reincarnation anime aired on TV before this year.
According to Internet searches and social media postings, the first Isekai reincarnation anime appeared on TV is "Aura Battler Dunbine (聖戦士ダンバイン Seisenshi Danbain)" which aired in 1983.The protagonist of this anime work is transferred to another world together with his motorcycle on which he is riding just before transfer.Also, in "Shurato (天空戦記シュラト Tenkū Senki Shurato)" which was aired on TV from 1989 to 1990, the protagonist and his friend are transported by the god of harmony, Vishnu, into another world where other deities exist.This anime shows the world where Esoteric Buddhism and Hinduism are mixed.
Furthermore, the anime work "Escaflowne in the Sky (天空のエスカフローネ Tenkū no Esukafurōne)", which was produced after the Bubble economy collapsed (since around 1991) and aired in 1996, depicted the protagonist going to a different world and returning to this world.
Before 2010, in Isekai reincarnation anime works including the above-mentioned anime works, the protagonists are transferred into another world by summons by someone or a god/goddess in another world.And some protagonists can be back to this world.Therefore, we can say that the depictions of transition by summons and going back to this world are the characteristics of the first stage of Isekai reincarnation anime.
Case Studies: Isekai Reincarnation Anime Productions up to March 2023
As mentioned earlier, this paper expands the anime survey in Akiko Komura (2023) to include an analysis of anime works aired on Japanese TV between the 1980s and the winter season of March 2023.The author would like to begin this chapter by reviewing the yearly number of Isekai anime works aired during the above-mentioned period, and reconfirming how reincarnations in another world occur in the anime works.
Below, the table shows the number of Isekai reincarnation anime works aired on Japanese TV from the 1980s to the winter season of 2023.The total number of Isekai reincarnation anime aired on Japanese TV during this period is 76 anime works.The table also shows that since around 2011, the number of Isekai reincarnation anime aired on TV has increased dramatically.The number of anime aired in 2020 dropped for a period of time, but this was largely due to the COVID-19 pandemic, which hindered the production of anime in such areas as voice recording.The year 2023 counts anime aired from January to March during the winter season.By the author's own confirmation alone, seven anime works were aired during the same period.Therefore, the eventual number for 2023 broadcasts after the spring season (April to June) might be much higher than the previous year.
Figure 2 shows the results of classifying the means of transmigration and reincarnation into another world in Isekai anime aired during the same period as figure 1.
1: The protagonist is summoned by someone in another world.2: The protagonist dies in this world and is reincarnated in another world to start over.3: The protagonist is transferred into another world during or after playing a game.4: Great historical figures are summoned to another world or reincarnated for some reason.
5: Other
Based on the analysis of 76 anime works, about 40% of protagonists are summoned by someone in another world, whereas about 28% are reincarnated by a god or god-like being after death in this world.The most important means of reincarnation in another world for the aim of this paper is No. 2: "The protagonist dies in this world and is reincarnated in another world to start over."The first anime work that depicts reincarnation after the death of the protagonist is, as far as the author knows, "KonoSuba: God's Blessing on This Wonderful World! (この素晴らしい世界に祝 福を！Kono Subarashii Sekai ni Shukufuku wo!)" which aired from 2016.
Figure 3 shows how the protagonists in 21 works of Isekai reincarnation anime are depicted their life before and after reincarnation and their deaths.Based on this figure, the author would like to discuss how the 21 anime works depict the way of life and death in Isekai reincarnation anime.
Life before Reincarnation
First, we will discuss in detail how the 21 anime works depict the way of life before reincarnation.There are several patterns in the depiction of the protagonists before their reincarnation.For example, protagonists are depicted as NEET or hikikomori (a shut-in) e.g., "Mushoku Tensei: Jobless Reincarnation (無職転生-異世界いったら本 気出す-Mushoku Tensei: Isekai Ittara Honki Dasu)" and "KonoSuba: God's Blessing on This Wonderful World!", those depicted as ordinary people spending peaceful daily lives, e.g., "That Time I Got Reincarnated as a Slime (転生したらスライムだった件 Tensei Shitara Suraimu Datta Ken)", those depicted as hard working persons in this world, e.g., "By the Grace of the Gods (神達に拾われた男 Kami-tachi ni Hirowareta Otoko)" and "Wise Man's Grandchild (賢者の孫 Kenja no Mago)," and those depicted as superior-talented persons beings in present world, e.g., "The Saga of Tanya the Evil (幼女戦記 Yōjo Senki)", "Knight's & Magic (ナイツ＆マジックNaitsu ando Majikku)," and "The World's Finest Assassin Gets Reincarnated in Another World as an Aristocrat (世界最高の暗殺者、 異世界貴族に転生するSekai Saikō no Ansatsusha, Isekai Kizoku ni Tensei Suru)," etc.Of these 21 works, it can be said that most of them depict the actual social situations of modern Japanese people.
Causes of the Protagonists' Death
There are some regular patterns in the depiction of the death of the protagonist.Of the 21 anime works, 8 depict deaths due to car accidents.These are depictions in which the protagonist not only encounters a traffic accident himself/herself, but also sacrifices his/her own life in order to save others.Some of them depict helping elementary or high school students, e.g., "Mushoku Tensei: Jobless Reincarnation," "Didn't I Say to Make My Abilities Average in the Next Life?! (私、能力は平均値でって言 ったよね！Watashi, Nōryoku wa Heikinchi dette Ittayone!)," etc.There are also a few anime works depicting the protagonist's death from illness, e.g., "Farming Life in Another World (異世界のんびり農家Isekai Nonbiri Nōka)," and from political battles, e.g., "The Reincarnation of the Strongest Exorcist in Another World (最強陰陽師の異 世界転生記 Saikyō Onmyō-ji no Isekai Tensei-ki)."Furthermore, there is a depiction of protagonist's death by a god's mistake, e.g., "In Another World with My Smartphone (異世界はスマートフォンとともに。Isekai ha Sumātofon to Tomoni)."
Life after Reincarnation in Another World
Due to each storyline, it cannot be said that all anime works depict exactly the same way of life after reincarnation in another world.However, there is also a fixed flow or rule.First of all, reincarnated persons gain omnipotent magic power or special abilities in another world.In addition, they are able to make their best choices and good deeds in their lives easily so that they have vivid memories of their previous lives.In some Isekai reincarnation anime, male protagonists describe themselves by saying "My age is the same as the period in which I've never had a girlfriend."13It means that they were not able to realize their dreams in the previous life, especially dating a girlfriend, but they will come true in another world.Many female characters fall in love with male protagonists.
A few anime works depict that the protagonists enjoy living freely and with ease in another world, e.g., "I've Been Killing Slimes for 300 Years and Maxed out My Level (ス ライム倒して300年、 知らないうちにレベルMAXになってました Suraimu Taoshite 300nen, Shiranai Uchini Reberu Makkusu ni Nattemashita)."In this anime work, after reincarnation in another world, the protagonist practices a slow and leisurely life because she died from overwork in her previous life.In this depiction, we can say that the protagonist regrets the way she lived in the previous life and now lives a better life for herself in another world.
In general, Isekai (another world/a different world) is different from the real world, and the reincarnated person is depicted as a powerful or talented person whom everybody in another world admires.However, a closer look at those 21 anime works shows that no matter how talented protagonists are, they are repeatedly subjected to ordeals after their reincarnation in another world, then they finally achieve their dreams by overcoming ordeals.For example, "Ascendance of a Bookworm: I'll do anything to become a librarian (本好きの下剋上-司書になるためには手段を選んで いられません-Honzuki no Gekokujyō: Shisho ni Narutame niha Shudan wo Erande Iraremasen)" depicts the protagonist finding hope through maximum efforts to realize her dream.
Analysis: The Death of a Protagonist in This World
The death of a protagonist, as depicted in the above 21 works, is nothing more than the catalyst for the reincarnation into another world.Nevertheless, can it be regarded as a new modern Japanese idea of death?
First of all, not only human beings, but all living things must die.Therefore, every religion in the world teaches how to live one's life in this world.This is the so-called "view of life and death."If we analyze this view in Isekai reincarnation anime in detail, we might be able to see it as a brand-new view of life and death for contemporary Japanese people.In these 21 anime works, the depictions of life before reincarnation are very common depictions of the daily lives of people living in modern Japanese society.However, actual social problems, such as hikikomori (social withdrawal) due to bullying and death due to overworking or long working hours, are depicted in 21 anime works.We will take a closer look at the individual anime works.
First, "KonoSuba: God's Blessing on This Wonderful World!" features a hikikomori teenage boy who died of shock.He thought that he died because he tried to save a high school girl who was about to be run over by a truck, but he misunderstood about this truck accident.In fact, the truck was a farm tractor which slowly approached the high school girl.He saw the truck accident in a vision.When he was dead, he met a goddess and she told him that the real reason for his death was not the accident itself but the shock which the vision of the accident gave him.After that, she asked him to choose to be reincarnated in another world or to go to heaven.He made a choice to be reincarnated in another world and he became an adventurer with an unusually good fortune.
Second, "Mushoku Tensei: Jobless Reincarnation" features a 34-year-old Japanese man who becomes a shut-in after getting bullied at school and has never had a girlfriend.The death of his parents leads him to be kicked out of his home by his family.While wandering the streets, he saves some high school students who are about to be run over by a truck, but he dies.He is reborn as a new-born baby in a different world of swords and magic with vivid memories of his previous life, and he practices sword and magic himself as a daily routine, training to avoid repeating the mistakes of his previous life.
It is one of the characteristics of Isekai reincarnation anime that protagonists keep their previous identities with vivid memories of their previous lives even after they die and are reborn in the next life.By making use of the failures and experiences in the previous life, they can avoid failures in another world and turn into successful people.This is especially true in the case of a story in which a female is reincarnated as a villainess in a game, e.g., "I'm the Villainess, So I'm Taming the Final Boss (悪役令嬢なのでラスボスを飼ってみました Akuyaku Reijō Nanode Rasubosu wo Kattemimashita)," "My Next Life as a Villainess: All Routes Lead to Doom! (乙女ゲー ムの破滅フラグしかない悪役令嬢に転生してしまった… Otome Gēmu no Hametsufuragu Shikanai Akuyaku Reijō ni Tensei Shiteshimatta…)" and so on.
A further example related to the retention of memories of previous life is "That Time I Got Reincarnated as a Slime."The protagonist of this anime work is a company employee who is living a peaceful life, but has never had a girlfriend.One day, he is stabbed to death by a random attacker while defending his colleague.In the process of dying, all the feelings and thoughts that come into his head, and the deeds he had left unfinished in this world are transformed into various talents, and he is finally reborn as a slime in the world of swords and magic.
In "By the Grace of the Gods," we can see that the protagonist is working too hard and too much.In this world, the protagonist, a single company employee in his around 40, is always busy with his work and is constantly being scolded by his younger boss.One day, he falls out of bed and hits his head on the floor.As a result, he dies.After his death, he meets three gods.He learns of the cause of his death from the gods and complains to them because he is not convinced of the cause of his death.However, he is then reborn as an 8-year-old boy in another world by the gods, and unlike the way he lived in his previous life, he spends a leisurely and fulfilling life doing what he wants to do.
In "Didn't I Say to Make My Abilities Average in the Next Life?!" the protagonist was unable to make friends because she was much too excellent compared to her classmates.She wanted to make friends and have a normal life.One day, she was hit by a car when she tried to protect an elementary school child who was about to be run over by the car.When she was dead, she requested a god that her ability be average in the next world.However, what the god gave her was not an average ability as she had expected, but a destructive ability.
Such desires can be thought of as the desires of people in contemporary Japanese society.Wishes that protagonists are unable to fulfil during their earthly life are heard by gods or near-gods.It is a characteristic of principal characters of Isekai reincarnation anime that they retain their identities of their previous lives even after being reincarnated into another world.As they face death, they regret their failures and wish to live a better life in the next world by attaining talents or experiences that will make them perfect persons.
What is of interest here is whether these descriptions of reincarnation into another world reflect the Japanese view of life and death which is rooted in Buddhism.This is because Shintoism, the indigenous religion of Japan, regards death as impurity and tends to avoid dealing with death.Therefore, as a general religious tradition in Japan, the concept of death is addressed mainly in Buddhism.Buddhism also has a concept of reincarnation called "Rokudō Rinne" (the endless cycle of reincarnation in the six worlds, so-called "saṃsāra").Can it be said that reincarnation as depicted in Isekai reincarnation reflects such religious concepts as saṃsāra in Buddhism?In order to answer this question, in the following sections, we will discuss the Rokudō Rinne, which is rooted in Japanese religious culture.
Reincarnation in Buddhism
Various Buddhist scholars have discussed the endless cycle of reincarnation in Buddhism.There are also various discussions among scholars from different viewpoints of various sects of Japanese Buddhism.However, this paper is concerned with the concept of reincarnation in Isekai reincarnation anime works, not with reincarnation in Buddhism, which has been the subject of much debate.Therefore, the author would like to introduce a commonly accepted Buddhist idea and discuss reincarnation in Isekai reincarnation anime based on this idea.
First, we will discuss reincarnation in Buddhism.Gautama Siddhartha, who became enlightened and became Buddha, attempted to transcend reincarnation, which was a traditional dominant thought in Indian society at that time.While human beings are alive, they always face suffering such as illness, aging, and death.Everything in this world changes.Nothing can maintain the same form forever.This is what is called the impermanence of all things.Such suffering and transience are faced over and over again in each reincarnation.Therefore, in order to escape saṃsāra, one must be liberated.Buddha founded Buddhism in order to save people from this endless cycle of saṃsāra, and taught liberation from saṃsāra and its means (so-called the Four Noble Truths and the Noble Eightfold Path).Buddhism views reincarnation negatively and does not promote which world one will go to in the next life.However, human beings are always harassed by their desires and their spirits are fragile.Man's words and deeds in this life affect the next life (so-called karma, 業Gō in Japanese). 14This is further developed into the idea of "reincarnation of the six worlds," which is found in particular in Tibetan Buddhism, Japanese Esoteric Buddhism, and so on.
The six worlds of reincarnation are heaven, the world of war/anger (Asura), the human world, the animal world, the hungry ghost (preta) world, and hell.It is our deeds in this life that determine the world in which we will be reborn in the next life.Even if one is able to be reborn in heaven, he or she is not still liberated from saṃsāra.Therefore, Buddhists must make efforts to be liberated in heaven as well.In saṃsāra, if one does not reach enlightenment in any of the worlds, he or she will be reborn in some other world.This is the basic concept of saṃsāra.
The Concept of Karma and Saṃsāra Being Shown in Isekai Reincarnation Anime
In the 21 works of Isekai reincarnation anime, some seem to hold ideas similar to those of Buddhism.For example, some anime depict the sequence of what may 14 MINOWA, K. (ed).Encyclopedia of Buddhism in Japan (事典 日本の仏教).Tokyo : Yoshikawa-kōbunkan. 2014, p. 48, 507, 511.The influence which is brought about by karma is called "cause-and-effect (因果応報 Inga-ōhō in Japanese)."be called "karma."In the previous life, the protagonists died in a car accident in an attempt to save others.After reincarnation in another world, they spend a happy life or their dreams come true, because of their past self-sacrificing deeds.In some anime works, we can see that the protagonists met a god/goddess after their death and were told to be reincarnated in another world because of saving other lives.Also, if the protagonist had worked too hard for the company and died from overwork (karōshi), he/she was able to spend a leisurely life in another world.It shows that those who work hard in their lives receive a corresponding compensation after death.We can say that these stories are based on the idea of karma in Buddhism.As the author mentioned before, man's words and deeds in this life affect the next life.The protagonists' deeds such as saving other persons in the previous life bring them good fortunes in their next life.This is the basic idea of karma and we can see its depiction in some Isekai reincarnation anime works. 15owever, regarding saṃsāra, it may be difficult to depict the idea of the six worlds of reincarnation in anime works.As far as the author knows, only one Isekai reincarnation anime work, "The Saga of Tanya the Evil" attempts to depict the idea of saṃsāra.In this anime work the protagonist is a competent elite businessman.However, because he is a thorough rationalist, he ruthlessly punishes his colleagues who fail in their work.The protagonist's rationalistic attitude leads to his death.One day, he is pushed off a station platform by a man, whom the protagonist has fired, and is run over by a train entering the platform.Being confronted with death, he hears voices of the gods' pronouncing judgment on his past deeds and on his ungodly heart.Even though he argues with the gods, his outrageous attitude toward others results in his being reincarnated as a woman in another world where fierce battles and wars continue.In addition, if we consider the world in which the protagonist is reincarnated, it can be compared to the world of war (Asura) in the Buddhist view of reincarnation, where one is always in the midst of battle, whether one wants to be or not.
As the author mentioned before, the reception of the idea of reincarnation in Buddhism is fundamentally negative.However, many Isekai reincarnation anime show reincarnations as a positive thing.The portrayal of pre-reincarnation protagonists depicted in anime is often negative, such as NEETs or socially withdrawn individuals.For example, in the aforementioned "Mushoku Tensei: Jobless Reincarnation," the main character is a 34-year-old man who has been a long-term recluse since being bullied by his classmates at school.Although there are different perspectives on how to view such people, according to the Buddhist concept of reincarnation, one's words and actions in this life will have an effect on the next life.Even if a person in this world withdraws as a result of being bullied by others, this person is more likely to be regarded as lazy and be dropped into an even worse world.Even if we take into account some anime protagonists who die while saving someone in a car accident as if this atones for their sins in this life, their thoughts or desires in the next life are depicted as a priority rather than their words and actions in their previous life.In other words, when the characters are reincarnated in another world, they are given the maximum ability by gods and reborn as the strongest in another world.Instead of the actions in the previous life determining the world in which they will be reborn in the next life, they are simply portrayed as having a better life in the next.Such a depiction is also different from the reincarnation of the ancient Indian idea which Buddha wanted to overcome, because what is depicted here is not a simple reception of the idea of karma or saṃsāra but a more positive utilization of reincarnation.
There may be many more Isekai reincarnation anime produced in the future, having stories beginning with protagonists' deaths.And if the Japanese people continue liking such stories, we can say that reincarnation depicted in Isekai anime is regarded as the modern idea of reincarnation based on the ideas of Buddhism.
Conclusion
Isekai reincarnation anime are stories where the protagonists are reincarnated into another world of swords and magic.While they originally belonged to the fantasy genre, the term Isekai reincarnation anime began to be used as an original genre around 2010 and has been on the Internet since then.
Isekai reincarnation anime has some unique characteristics.First, the protagonists are reincarnated in another world either by summons by someone in another world or by the protagonists' deaths in this world.By being reincarnated in another world, the protagonists can become heroes/heroines in another world and fulfil wishes that cannot be fulfilled in their previous lives.And they can live the lives of their dreams.In addition, they can bring with them personal belongings which they had in the previous world, such as a smartphone.
A careful examination of these depictions of Isekai reincarnation anime reveals a characteristic depiction of reincarnation into another world through the death of the protagonist.The reason why the protagonists' deaths are depicted, not simply summons by someone in another world, is that reincarnation itself, even if it is seen in the anime, reflects the worldview of reincarnation in Buddhism.In some Isekai reincarnation anime, the protagonists sacrifice their own lives to save others from a car accident in this world, or died from overwork.The efforts of these protagonists in this life are rewarded in positive in the next life.In other words, if they make efforts and accumulate virtues in this life, they will receive good rewards in the next life.This is what Buddhism calls karma, the worldview of cause and effect.
As we discussed in the forgoing passages, reincarnation is viewed as a negative obstacle to transcendence in the original Buddhist teachings.By contrast, reincarnation in Isekai anime is viewed as a positive means to overcome the difficulties of life.This positive view of reincarnation is interpreted in a few academic papers as an escape from the reality of Japanese society.However, even before the appearance of the Isekai genre around 2010, Japanese society had always been inflicted with problems.In particular, after the collapse of the Bubble economy around 1991, Japanese
Figure 1 :Figure 2 :
Figure 1: Number of Isekai reincarnation anime aired on TV from 1980 to March 2023 (Source: Akiko Komura) The Darker Sides of the Isekai Genre: An Examination of the Power of Anime and Manga.USF Scholarship: a digital repository of The University of San Francisco.2020.[online].[cit.20.August 2023].Available at: https://repository.usfca.
Original Title English Title Year (Aired on TV) Life before Reincarnation Cause of the Protagonist's Death Life in Another World (after Reincarnation)
Figure 3: 21 works of Isekai reincarnation anime which the protagonist dies in this world and is reincarnated in another world to start over (Source: Akiko Komura) The | 2023-10-26T15:02:40.331Z | 2023-09-30T00:00:00.000 | {
"year": 2023,
"sha1": "04073b9f40abb9268cc77df39874b5b51d22bafd",
"oa_license": "CCBYNC",
"oa_url": "https://www.sav.sk/journals/uploads/10031232Akiko%20Komura.pdf",
"oa_status": "GOLD",
"pdf_src": "Anansi",
"pdf_hash": "dad5919e585ad234d42ced149d92da9e77f21a19",
"s2fieldsofstudy": [
"Art"
],
"extfieldsofstudy": []
} |
264462261 | pes2o/s2orc | v3-fos-license | Photovoltaic energy production systems and the carbon market in Maranhao: a case study
The dissertation deals with a research carried out with data from a 168.4kWcc/120kWca photovoltaic solar plant installed in Itapecuru Mirim, in the state of Maranhão, with the objective of modeling a system for parameterization and possible calculation of the effect of the solar photovoltaic system on the market of carbon credit. Throughout the
INTRODUCTION
According to Bondarik, Pilatti and Horst (2018) the rapid increase in global energy demand and consequent possible depletion of the main conventional energy resources, such as Oil, Coal and Natural Gas, combined with its negative environmental consequences, including the degradation of the environment through the emission of greenhouse gases (GHG), causing an increase in the need to use renewable energy sources that do not cause environmental damage or that cause less damage.
According to Barbosa (2016) Brazil contributed to the United Nations, committing itself to the reduction of greenhouse gas emissions, which reflects this demand.Solar energy emerges as an important ally from an environmental point of view, due to its low carbon footprint compared to fossil fuel technology and also because of the carbon credit as a way to reduce CO2 emissions in the environment.Poyer et al., (2020) describe in their study that carbon credits work as a currency and aim to reduce GHG emissions on the planet to combat climate change.This concept emerged in 1997, within the Kyoto Protocol.A carbon credit is generated for every ton of carbon that is not emitted.When the country manages to meet this reduction target and stop emitting 1 ton of CO2, it may receive Clean Development Mechanism (MDL) certification.
An example of this is presented in the research developed by Matsubara (2020), the shift to renewable energy options and low-carbon technologies, such as photovoltaics, is seen as a response to concerns about climate change.The primary source of energy for the photovoltaic system is solar, an option with good prospects for a country with high solar incidence, such as Brazil, which has an average annual irradiation that varies between 1200 and 2400 kWh/2 and mainly the northeast region, which It has greater energy availability due to its location closer to the equator.Chagas, Ferreira and Feitosa (2022) describe that the amount of carbon credit obtained is directly associated with the amount of solar electricity produced, which generally depends on the climatic conditions of the area and the efficiency of the cells.
Due to this fact, there are variations in the production of photovoltaic solar energy and, consequently, in the value of the carbon credit obtained.Despite this, Brazil has the advantage of having, in addition to clean fuel options, forests that remove carbon and, therefore, can generate more credits.
THE CLEAN DEVELOPMENT MECHANISM
According to Souza (2020) the concept of carbon credit emerged in 1997, under the Kyoto Protocol, in this same context other tools emerged in the fight against global warming by reducing greenhouse gas emissions.These include the Clean Development Mechanism (CDM) to help countries meet their emission reduction targets or offset them through carbon credits and certified emission reductions (CERs).
According to Souza et al., (2017) there are three points of the Kyoto Protocol that led to the emergence of the regulated carbon market: the joint implementation of countries, emissions trading -restricted to developed countries -and the CDM -which includes developing countries, enabling their inclusion in the global carbon market.Silva (2019) describes that the clean development mechanism allows interaction between developing countries by cooperating with developed countries.Annex 1 countries that emit the most greenhouse gases invest in technology, finance and support and non-Annex 1 countries execute emission reduction projects and provide these carbon credits and certified emission reductions (CERs).The primary source of energy for this system is solar, an option with good prospects for a country with a high incidence of electricity such as Brazil.Although the solar resource is considered unlimited, treating it is a challenge mainly due to the limited efficiency of photovoltaic cells.Furthermore, it is known that it is necessary to analyze the carbon footprint of this system when considering it as green energy.Machado and Miranda (2015) describe that the production of silicon cells from silica can be considered the non-green part of the technology.The use of solar energy is generally made using these photovoltaic cells and modules and it is considered that there In 2020 and mid-August 2021, renewable energies have fallen to second position in terms of volume, while forestry and land use projects are on the rise.While wind and solar projects, as shown below, have fallen comparatively between 2019 and 2021, they remain stable and ongoing as their crediting periods continue.While the prices of forestry credits are rising, those associated with renewable energy are going against the grain.
Countries in more developed regions have had difficulty certifying new wind and solar energy projects.Tomelin (2016) presents that renewable energies have become more competitive with other forms of energy, so that they no longer need carbon finance to survive.What makes them a problem regarding the criterion known as "additionality", since to issue a carbon credit, a project has to prove that it causes an impact that would not exist in the absence of the incentive given by the credit.One of the outputs/targets that are being considered and defined around the world so that renewable energy meets internal needs and external emission targets is grid connection.
POTENTIALITIES OF SOLAR ENERGY IN THE STATE OF MARANHÃO
Developing countries have large preserved areas of tropical forests and other ecosystems with a large capacity to store carbon.Latin America, in this context, is in a prominent position with great capacity to contribute to the reduction of GHG emissions on the planet due to its large area of tropical forest, with emphasis on Brazil with the Amazon.
According to data presented by Aneel (2023) that the northeast region of Brazil has potential for incidence, considering the Northeast area, 287,384km2 or 287,384,000,000m2, the incidence of 4000Wh/m2 per day and the use of 1/100 of territory and conversion efficiency of 20%, we would have 2.3 million MWh per day of production capacity or 96 thousand MW on average.This value would be comparable to the current Brazilian production capacity of 160,000 MW, according to ANEEL.
According to Lima (2016), the state of Maranhão is located two degrees from the equator, with an incidence of solar rays that penetrate the earth's surface almost horizontally.The state is considered one of the most promising in the generation of clean energy.
CLEAN DEVELOPMENT MECHANISM PROJECT CANDIDATE VALIDATION PROCESS
For the negotiation of carbon credits to be possible, it is necessary to design and implement clean development movement (CDM) projects, aimed at controlling and reducing greenhouse gas emissions in emerging countries.Brazilian institutions can benefit from obtaining carbon credits and selling them to developed countries, those that do not reach the agreed reduction targets can acquire these credits to partially prove their contribution to the environment.
Projects can be based on reforestation, energy conservation and alternatives, energy efficiency and renewable sources.There are clear and rigid stages and rules for the approval of these projects under the CDM.In Brazil, the designated national authority is the Interministerial Commission on Global Climate Change, which approves project implementation at the UNFCCC and the CDM Executive Board registers and issues the Certified Emissions Reductions (CERs) document.The agency's final decision is the Conference of the Parties to the UNFCCC.
For CDM project applicants, it is necessary to follow the steps of project design, preparation of the project design document (PDD), validation, obtaining host country approval, registration, project implementation, monitoring, verification and certification and, finally, the issuance of CERs (carbon credits).The figure below demonstrates the stages of the CDM project candidate validation process.CDM projects can be based on alternative and renewable sources of energy or reforestation.The country where the project will be implemented, through its Designated National Authority (DNA) and the government that will buy the generated carbon credits, must validate the project.The methodology adopted in each project must be approved and validated by Designated Operational Entities (DOEs).DNA in Brazil is the Climate Change and Green Growth policy, created by Decree No. 10,845, of October 25, 2021.
Plant Characterization
The study plant is a 168.
CALCULATION PROCEDURES
In the calculation procedure, it is important to consider the life cycleimplementation, operation and maintenanceof the photovoltaic solar system.The photovoltaic system applied in the context of distributed generation of electricity in Brazil allows a consumer unit to generate its own energy, switch between energy consumption and supply with the National Interconnected System (SIN), reducing the need to consume the energy generated by the SIN .
The generation of electricity in the SIN impacts the environment.This impact is measured using CO2 emission factors for electricity generation.The emission factor is calculated according to the methodology proposed by the CDM (Clean Development Mechanism), being in the public domain, and available monthly.
CARBON AVOIDED
The calculation of the amount of CO2 avoided by a photovoltaic installation is done through the electric energy generated in a period of time and the average CO2 emission factor of the SIN in this period.
LIFE CYCLE
Life cycle analysis (LCA) is an approach that allows assessing the environmental impact of a system throughout its entire life cycle, including production, use and disposal.This approach is used to assess the environmental impact of photovoltaic solar energy systems, with the objective of identifying the greenhouse gas emissions associated with each phase of the life cycle.Assessing the environmental impact of photovoltaic solar energy projects through life cycle analysis involves considering different phases of the system's life cycle, including implementation, operation and maintenance.
The implementation phase includes the production and installation of the system, which can generate greenhouse gas emissions such as CO2, mainly due to energy consumption during the production of components and installation of the system.Life cycle analysis makes it possible to assess the impact of these emissions on the reduction of CO2 emissions during system operation.
The operation phase includes the generation of electricity by the photovoltaic solar energy system, which does not emit greenhouse gases during operation, unlike fossil energy sources.It is during this phase that the main reduction in CO2 emissions occurs.
The maintenance phase includes activities necessary to keep the system running effectively over time, such as replacing worn components, cleaning solar panels, and repairs.This phase can also generate greenhouse gas emissions, mainly due to energy consumption during component production and maintenance activities.
Therefore, life cycle analysis allows a comprehensive assessment of the environmental impact of photovoltaic solar energy projects, considering all phases of the system's life cycle, from material production to final disposal.This approach can help identify opportunities to reduce greenhouse gas emissions over the life cycle of the system.
VERRA AND VCS ACM0002 METHODOLOGY
Verra (formerly known as Verified Carbon Standard) is a leading organization in the carbon credits industry that develops and administers standards and methodologies to validate and verify greenhouse gas emission reduction projects around the world.Verra works with a variety of sectors, including renewable energy, energy efficiency, waste management and reforestation, to validate projects and issue carbon credits.
The VCS (Verified Carbon Standard) methodology is a set of rules and requirements to validate projects to reduce greenhouse gas emissions.The ACM0002 methodology applies specifically to renewable energy projects, including solar energy projects, and aims to verify the reduction in greenhouse gas emissions generated by replacing non-renewable energy sources with renewable energy sources.Solar energy projects can qualify for carbon credits under the ACM0002 methodology, provided they meet the established criteria.To be eligible, projects must demonstrate that the energy generated by the solar energy system is additional to energy generated from non-renewable sources and that the reduction in greenhouse gas emissions is quantifiable, measurable and verifiable.
To validate the reduction in greenhouse gas emissions generated by the solar energy project, a series of steps must be followed, such as calculating the reduction in emissions, analyzing uncertainties and performing verification audits.Verra requires that these steps be performed by independent, Verra-accredited auditors to ensure the integrity of the validation and verification process.
In summary, Verra's ACM0002 methodology is an essential tool for validating the reduction in greenhouse gas emissions generated by solar energy projects and issuing carbon credits that can be traded and used by companies and organizations to offset their own carbon emissions.greenhouse gases.These carbon credits help drive the transition to a low-carbon economy and mitigate the effects of climate change.
DATA COLLECTION AND ANALYSIS
According to Lima (2016) in the state ranking of Distributed Generation, organized by ANEEL and ABSOLAR, the state of Maranhão ranks 15th, with an installed capacity of 454.5 MW, representing 2.1% of the country's total capacity.Currently, according to data from Aneel and Absolar (2023), the state of Maranhão has a wide presence of more than 100 solar plants in its territory.
Installed power: The photovoltaic solar project under study has a unit in the municipality of Itapecuru Mirim, in the state of Maranhao.The project has 169.4kWp of installed power.The system used as base has the following characteristics: plant for distributed generation, in remote self-consumption mode, use of Canadian modules, kUmax line, CS3U-380|385|390|395|400MS, 1000V/1500V, number of boards: 440 units , 22 units of 20-module strings and 2 units of Sungrow SG 60KTL inverters, total area: 0.31h, rated power: 1.36kWp, inverter power: 120Kw.
Renewable energy generation: The amount of electricity generated by the photovoltaic solar generation project with data from the end of 2020 to the beginning of 2022 can be seen in the tables below.The project's contribution to the production of renewable energy is not measured as a whole for the state of Maranhão, given that it is currently used for own consumption.The data shown in figure below represent the behavior of precipitation and temperature throughout the year in Itapecuru Mirim according to climatology.
Climatological averages are values calculated from a series of data observed over 30 years.It is possible to conclude a correlation between the months with the highest rainfall and the lowest performance of the solar plant.In the case of data from the plant used in the study, the emission of 61 tons of carbon was avoided.To better exemplify, the generation of photovoltaic solar energy at the plant under study can be compared with electrical and electronic equipment that The discussion on the role of photovoltaic solar generation systems in carbon credits reveals their significant potential for mitigating greenhouse gas emissions.These systems offer a sustainable and efficient alternative for the production of clean energy, contributing to the reduction of atmospheric emissions by avoiding the burning of fossil fuels.Furthermore, by being certified as emission reduction projects, they can generate carbon credits, encouraging their implementation and promoting investments in renewable energies.
The analysis of the photovoltaic solar generation system in the voluntary carbon market reveals its growing potential and global recognition.Models for reducing greenhouse gas (GHG) emissions were developed, an example is the study of the Estimate of carbon credits in the generation of energy with renewable sources in Santarem (SANTOS; BLANCO, 2016), where a reduction was estimated of 37.81 tCO2/year, totaling 378.10 tCO2 for a CDM project with a lifetime of 10 years.This resulted in US$ 4,964.45 (R$ 20,056.38)for the tons of CO2 that were no longer emitted.
CONCLUSION
Based on the desired objectives and results achieved and presented, the The discussion about the role of photovoltaic systems in carbon credit points to their potential as a sustainable and efficient alternative in the production of clean energy.
The certification of these projects as generators of carbon credits can boost their implementation and promote investments in renewable energies, aligning with the objectives of the Paris Agreement and boosting global sustainability.
However, it is important to emphasize that these conclusions are based on results during the interval of a little more than a year and a half and that a more lasting evaluation of the impact of photovoltaic solar generation systems on the carbon credit would require a more comprehensive research and the data collection over a longer period of time.The continuation of the study and the analysis of additional indicators will allow a deeper understanding of the potential of these systems in reducing carbon emissions and promoting sustainable development.
Figure 2 .
Figure 2. Market Sizes of Voluntary Carbon Traded by Major Project Types 2019 -August 2021
Figure 3 .
Figure 3. Stages of the CDM Project Candidate Validation Process 4kWcc/120kWac photovoltaic solar plant installed in the municipality of Itapecuru Mirim, in the midwest region of the state of Maranhão.The municipality's vegetation comprises the Cerrado and Mata dos Cocais ecosystems, which is a transitional vegetation between the Cerrado and the Amazon.The plant is of distributed generation, in remote self-consumption mode.It is composed of photovoltaic modules, with a power of 380Wp, mono technology.The modules used are from the Canadian line KuMax CS3U-380|385|390|395|400MS, 1000V and Sungrow SG 60KTL inverters.that the location of the photovoltaic solar plant, as it is located in the municipality of Itapecuru Mirim, which has a sub-humid tropical climate, with greater rainfall from January to June and a dry season from July to December, will have a large variation in production.The data shown in figure below represent the behavior of precipitation and temperature throughout the year in Itapecuru Mirim according to climatology.Climatological averages are values calculated from a series of data observed over 30 years.
Figure 5 .
Figure 5. Performance data in 2021 of mini photovoltaic solar plant.
consumption of shower baths: Number of showers possible with the total energy produced.1 bath of 10 min consumes approximately 1.105 kWh.-Consumption equivalence of the operation of a refrigerator: Number of months of operation of a refrigerator with the total energy produced.1 refrigerator consumes approximately 19.9 kWh per month.
photovoltaic solar plant project in Maranhão presented in the study demonstrated the ability to generate renewable energy and avoid the emission of 61 tons of carbon between the dates of July 2020 and March 2022.The equivalent of 121,000 units of cultivated trees and enough energy for more than 444 baths.This underscores the potential of these systems in reducing greenhouse gas emissions, contributing to climate change mitigation and the transition to a low-carbon economy.
Table 2 .
Produced Energy, Projected and Performance 2021 -First Semester.
Table 5 .
Carbon Avoided and and correspondence with electrical and electronic equipment. | 2023-10-26T15:09:49.579Z | 2023-10-23T00:00:00.000 | {
"year": 2023,
"sha1": "547d5f40c2c328248a3101ed01cead1674d6bf02",
"oa_license": "CCBYNC",
"oa_url": "https://ojs.observatoriolatinoamericano.com/ojs/index.php/olel/article/download/1719/1285",
"oa_status": "HYBRID",
"pdf_src": "Anansi",
"pdf_hash": "eca9cf2b748648f24f7eee2566da10ce9cb07910",
"s2fieldsofstudy": [
"Environmental Science",
"Engineering"
],
"extfieldsofstudy": []
} |
116666624 | pes2o/s2orc | v3-fos-license | ASTEC – MAAP Comparison of a 2 Inch Cold Leg LOCA until RPV Failure
A 2 inch, cold-leg loss-of-coolant accident (LOCA) in a 900 MWe generic Western PWR was simulated using ASTEC 2.1.1 and MAAP 5.02. The progression of the accident predicted by the two codes up to the time of vessel failure is compared. It includes the primary system depressurization, accumulator discharge, core heat-up, hydrogen generation, core relocation to lower plenum, and lower head breach.The purpose of the code comparison exercise is to identify modelling differences between the two codes and the user choices affecting the results.The two codes predict similar primary system depressurization behaviour until the accumulation injection, confirming similar break flow and primary system thermal-hydraulic response calculations between the two codes. The choice of the accumulator gas expansion model, either isentropic or isothermal, affects the rate and total amount of coolant injected and thereby determines whether the core is quenched or overheated and attains a noncoolable geometry during reflooding. A sensitivity case was additionally simulated by each code to allow comparisons to be made with either accumulator gas expansion models. The two codes predict similar amount of in-vessel hydrogen generated and core quench status for a given accumulator gas expansion model. ASTEC predicts much larger initial core relocation to lower plenum leading to an earlier vessel failure time. MAAP predicts more gradual core relocation to lower plenum, prolonging the lower plenum debris bed heat-up and time to vessel failure. Beside the effect of the code user in conducting severe accident simulations, some discrepancies are found in the modelling approaches in each code. The biggest differences are found in the in-vessel melt progression and relocation into the lower plenum, which deserve further research to reduce the uncertainties.
Introduction
After the nuclear events unfolded in Fukushima, the interest in severe accidents in nuclear power plants has increased greatly [1][2][3], both in severe accident management and in severe accident phenomena research.Because of high level of complexity and interplay between different phenomena, state-of-the-art integrated severe accident codes such as ASTEC [4], MAAP [5], and MELCOR [6] are often used to study and design accident management strategies [7].These simulation codes, which capture theoretical and experimental knowledge produced in the last four decades, predict progression of a postulated accident leading to fuel melting, core relocation to lower plenum, and vessel failure.
In addition to the input files describing the plant design, the code user has to choose among several models and correlations for individual phenomena in order to address uncertainties.Although there were several severe accident experiment campaigns [8][9][10][11][12] which produced valuable databases [13], there remain substantial uncertainties that need to be reduced further [14].In particular, there is scarcity of experiments with prototypic reactor components and materials.As a result, significant discrepancies are often observed in key quantities predicted by different codes such as hydrogen generation, coolability of the core during reflooding, vessel failure, and molten corium-concrete interaction.Attempts to identify research priorities needed to advance the severe accident knowledge and code validation are reported in [15,16].
Science and Technology of Nuclear Installations
To address the gap in the experimental database and in modelling and simulation, many multinational efforts were launched to guide research and development efforts.Several research activities in national and international framework were done and in progress in order to reduce the epistemic uncertainty in severe accident phenomena and to prevent or mitigate severe accidents.These efforts include the Code for European Severe Accident Management (CESAM) project developed under the 7 th Framework Programme of the European Commission [17], the In-Vessel Melt Retention Severe Accident Management Strategy for Existing and Future NPPs (IVMR) project, which is still in progress under the H2020 Framework Programme of the European Commission [18], and the European Corium Experimental Research Roadmap (SAFEST) [19], which is also funded under the auspices of the H2020 Framework Programme of the European Commission.The OECD/NEA/CSNI has also sponsored several projects such as the BSAF (Benchmark Study of the Accident at Fukushima) [20], Informing Severe Accident Management Guidance and Actions through Analytical Simulation [21], and the State-of-the-Art Report on Molten Corium Concrete Interaction and Ex-Vessel Molten Core Coolability [22].Under the IAEA framework, it is worth noting the technical meeting on the Status and Evaluation of Severe Accident Simulation Codes for Water Cooled Reactors held in Vienna on 9-12 Oct 2017 [23], whose main outcome will be a TEC-DOC on the status of severe accident codes and models.In addition, comprehensive, in-depth code-to-code comparison exercises, also called "crosswalk" activities, led by the code developer teams have been performed in order to identify key modelling differences between the codes.Good examples are the MAAP-MELCOR and the ASTEC-MELCOR crosswalks reported in [24,25], respectively.Another recent work addressing the uncertainties in integrated tools attempts to provide insights through Best-Estimate Plus Uncertainty approach [26].
The impact the code user has on the results of thermalhydraulic system codes is documented in [27][28][29].The main conclusion of these studies is that, in order to choose right models and model parameters appropriate for the specific accident scenario, the code user should have good understanding of the phenomena and processes involved.On the other hand, the recent OECD report "Benchmark Study of the Accident at the Fukushima Daiichi Nuclear Power Plant" highlights the difference in the code results after the core geometry is lost, attributing the cause to "each code's modelling approaches" [20].Therefore, different modelling approach after the loss of core geometry seems to be the leading cause of the persistent, nonreducible uncertainties in the code prediction.This conclusion is supported by the MAAP-MELCOR crosswalk study [24], where the major differences between the results are attributed to the different modelling approaches after core damage has occurred.The work presented here attempts to confirm this conclusion and to investigate to what extent the discrepancies can be attributed to the different modelling approaches versus user effects through user choices of appropriate models and model parameters.
A small break LOCA without active cooling before RPV failure is ranked among the highest risk-significant sequences in the severe accident domain of PWRs [30].The goal of the current work is to compare the results predicted by ASTEC and MAAP for a 2 inch, cold-leg LOCA without ECCS before RPV failure.Significant differences between the two codes are identified and the underlying causes for the differences are discussed.
The current work was performed as part of the European Commission Joint Research Centre activities within the Code for European Severe Accident Management (CESAM) project under the auspices of the 7 th Framework Programme of the European Commission.
Description of the Plant and Models
The reference plant used in the analysis is a generic, 900 MWe, 3-loop Western PWR with three passive accumulators connected to each of the three cold legs.
The ASTEC 2.1.1 model of the plant is based on the input deck provided by IRSN during the CESAM project [31,32].Figures 1, 2, and 3 show the containment, primary system, and secondary system model, respectively (regarding Figure 2, the primary system third loop (not shown), follows exactly the same nodalization that loop 2 (the loop without pressurizer)).The core is divided into 6 radial and 24 axial active nodes plus the upper and lower nonactive nodes for supporting structures.The core model includes the structural components such as the core barrel, baffle, support plates, and structural columns.
The MAAP 5.0.2 model of the plant is based on the information gathered in [31,32] and released to the CESAM partners.The MAAP core model was made consistent with the ASTEC model in decay power, fuel characteristics, mass distribution and relative elevations.Both models have similar initial water masses, dimensions, elevations, safety and relief valves, initial thermodynamic conditions and heat sinks in the primary and secondary systems.The safety systems were also implemented in a similar way.They include ECCS, accumulators, and containment spray system set points and flowrates.
The MAAP core model follows ASTEC's in terms of nodalization, elevations, and materials.It features 6 radial and 24 axial nodes for the active fuel region.In addition, 4 nonactive nodes below the active fuel region are to represent the nonfuel extension and lower support plates, and 2 nonactive nodes above the active core region are to represent the nonfuel extension and upper support plates.Figure 4 shows the elevations of the active fuel nodes (in red), upper nonactive fuel nodes (T-1 and T-2), and lower nonactive fuel nodes in the MAAP model (B-1 to B-4).The vessel internals such as the core barrel, baffle, plates, and columns are modelled separately with similar geometry, mass, and material in the two codes.
Both ASTEC and MAAP nodalize the lower head using 8 axial nodes and 5 radial nodes.
In MAAP the primary system nodalization is fixed whereas in ASTEC the user has control over nodalization.The primary system nodalization scheme is compared between ASTEC and MAAP in [31].It was found that the two models are similar in representing the individual components in In order to check the consistency between the two models, a steady-state case was simulated using the two codes, where the ASTEC steady-state data has been taken from [33].The results are in good agreement as shown in Table 1.
Preliminary Remarks on the Scenario
The scenario simulated is a 2 inch break in the cold leg.All active primary system coolant injection is assumed to be unavailable before RPV failure.Table 2 summarizes the key automatic and operator actions.
Since the standard reference SBLOCA experiments were conducted with break sizes ranging between 0.5% and 5% [34], the present 6%-equivalent break LOCA is expected to behave between an SBLOCA and MBLOCA in terms of primary system depressurization and accumulator discharge rate.The reactor thermal-hydraulic response to breaks of this size as initiating event has shown large sensitivity to the initial and plant-specific conditions.A small difference in the break size and location, thermal power, cross-over leg relative elevation, or manual actions taken on the secondary side can lead to a different plant evolution, affecting, among others, safety system actuations driving the plant to a safe state [35,36].Nevertheless, responses to relatively small break LOCAs share the following characteristics until the onset of core damage [34]: (1) Initially, since the break is not big enough to remove all the steam generated by decay heat, the primary system pressure stays slightly above the secondary side pressure so that the secondary side can act as a heat sink and remove the excess heat.
(2) If HPSI is not available and no manual actions are taken on the secondary side to depressurize the primary system, the core will eventually become uncovered and heat up before the steaming rate decreases and thereby depressurize the primary system enough to trigger LPSI.
(3) Once the RCPs are stopped and HPSI is not available, the primary system temperature will rise temporarily as a consequence of the loop seal in the cross-over leg; the primary pressure will also rise correspondingly.
(4) In the absence of a high pressure injection to the vessel, the steaming rate in the core will eventually start to fall and the pressure will start to decrease as the core becomes uncovered and the decay heat decreases.Eventually, the primary system pressure will decrease below the secondary side.Once the primary system pressure falls below the secondary side, the secondary side can no longer act as a heat sink, unless the secondary side is fully depressurized to near the atmospheric pressure.
(5) Provided no manual action is taken to depressurize the secondary side, the accumulators will start injecting water slightly before or after the fuel experiences temperature excursion, depending on the break size.If core damage has already occurred, it may have lost its original coolable geometry and the accumulator injection may not be able to cool the core.On the other hand, if the core retains its original geometry, the core can be cooled by the injection.
Introduction to the Analysis of the Scenario
To facilitate code comparison, the accident progression is divided into four phases as shown in Table 3 [13,37].The purpose of this study is to investigate code difference by focusing on the key driving phenomena and underlying models in each phase, not to perform an analytical decomposition of all phenomena characterising each phase.A detailed study about all the thermal-hydraulic phenomena in this kind of transient can be found in [38][39][40].As for the in-vessel core degradation phenomena, more detailed information about all the core degradation phenomena characterizing this kind of transient can be found in [10].Each accident progression phase will be analysed in detail, identifying the key driving phenomena.Then, conclusions will be drawn from the following three perspectives: (1) Code capabilities to simulate the key driving phenomena (2) Expected trends and values in the key driving phenomena (3) Significant differences between the two codes Table 4 summarizes the timing of key events predicted by each code whereas Table 5 shows the key results from the two reference and two sensitivity cases: In addition to Science and Technology of Nuclear Installations 7 6 shows the elapsed computing times and numerical time steps used in the four analysed cases by the two codes.
First Phase: Primary System Depressurization
5.1.Phenomenology Analysis.As shown in Figure 5, initially, the primary system depressurizes rapidly as it loses water mass through the break until flashing starts at around 90 seconds.
After the onset of flashing, the primary pressure stabilizes at a pressure slightly above the secondary side since the decay energy generated in the core cannot be removed fully by the steam flow through the break and the excess heat is removed by the secondary side.The excess heat is transferred to the secondary side by the two-phase natural circulation in the primary loop.
As voiding in the primary loop increases, the natural circulation stops due to the formation of loop seals.The primary pressure increases temporarily as the heat removal by the secondary side is lost and steaming in the core increases to compensate for losing the secondary side cooling.At the same time, the primary loop voiding and loss of natural circulation lead to phase separation, and single phase steam exits the break, resulting in a higher volumetric break flowrate.At around 55 seconds, the operator manually lowers the SG water level to 33% of the narrow range.Coincidently, the formation of loop seals causes temporary increase in the primary pressure.At around 2,800 seconds, one loop seal is cleared.From here on, the break flow can remove all the steam generated in the core.
As the primary coolant boil off continues, eventually the coolant level falls below the TAF and core becomes uncovered.The uncovered part of the core starts to heat up and eventually reaches the triggered temperature when rapid cladding oxidation occurs.At the same time, the steaming rate decreases as the covered fuel area decreases and the primary pressure starts to fall.Eventually the pressure falls below the accumulator set-point of 4.28e6 Pa at around 3800 seconds and the in-vessel passive injection starts.
Because the core uncovery and subsequent core heat up coincide with the primary system depressurization and accumulator injection, these two phenomena (i.e., damaged core and accumulator injection) are closely coupled and the interaction between the two phenomena plays an important role in determining the progression of the severe accident.
Code Capabilities.
Both codes incorporate suitable models and nodalization scheme to simulate and capture all relevant phenomena to predict consistent break flowrates, primary pressure response, natural circulation in the primary loop, formation of loop seal, phase separation in the primary loop, heat transfer to the secondary side, coolant boil off, and core uncover during the first phase of the accident.
Discussion on the Expected Trends and Values.
The two codes predict similar pressure transient and timing before and after the onset of flashing (see Figure 5), including the primary pressure stabilization at a pressure slightly above the secondary side.Both codes predict similar results for the key quantities such as pressures, temperatures, and coolant inventory.
Based on the fact that the primary pressure drops below the secondary pressure relatively quickly, a 2 inch break LOCA will probably be located at the high end of the SBLOCA break range; i.e., it behaves similar to a MBLOCA as discussed above.
Discussion on the Significant Discrepancies between the
Codes.As shown in Figure 5, no significant differences are found between the two codes, neither in magnitude (i.e., pressure) nor in width (i.e., timing of the key events).This phase ends at around 4000 seconds.
Second Phase: Accumulators Discharge
6.1.Phenomenology Analysis.Several studies stress the importance of passive safety injections on preventing core damage (see, e.g., [42,43]).The accumulators are designed as a Level 3 engineered safety feature, not as a Level 4 DiD barrier or mitigation system [44].Nevertheless, since they provide core cooling at a crucial point during an accident, albeit limited in duration and flowrate, they can play an important role in driving the severe accident evolution at least temporary, as demonstrated in the current study.
Consistent with the general understanding of the SBLOCA with no available HPSI and no manual depressurization through the secondary side, the accumulator discharge starts only after the core has uncovered and has been through substantial heat-up.When core reflooding starts at the beginning of core damage, before core relocation to the lower plenum has occurred, the accumulator injection flowrate and the primary pressure have positive feedback: the higher the accumulator injection flowrate is, the more effective the core is cooled, causing less cladding oxidation and core steaming rate.Consequently the primary pressure will decrease.The lower the primary pressure, the higher the accumulator injection flowrate in a positive feedback process.On the other hand, the lower the accumulator injection flowrate, the less effective the core is cooled, causing more cladding oxidation and core steaming rate.Consequently the primary pressure will increase.The higher the primary pressure, the lower the accumulator injection flowrate.
Since the accumulator water injected into the cold leg causes an increase in the core inlet flowrate, the boiling front in the core will rise and quench the uncovered core, increasing the steaming rate and supporting the primary pressure from falling.The behaviour of the primary pressure depends on the balance between break flowrate, steaming rate in the core, and condensation rate in SG.Therefore, aside from the core steaming rate, the break size has a strong influence on the primary pressure evolution for small to intermediate size break LOCAs as it primarily affects the break flowrate.Therefore, the break discharge coefficient [45] and the critical flow model greatly affect the response of the overheated core when the limited-degradedcore is reflooded by the accumulator injection.
In addition, the accumulator injection flowrate and duration depend on the thermodynamic expansion model of the gas in the accumulators, which can fall under the user effect.
Based on the results, the accumulator injection delays core damage but increases the cumulative hydrogen generated in the core, probably by providing additional source of steam to react with cladding.Depending on the accumulator injection flowrate and duration, coolability of damaged core, hydrogen generation, and core melt progression model, the core heat-up and degradation evolve differently.This phase ends up when the primary pressure falls below 15 bar and the accumulators are isolated.
Conclusions on the 2 𝑛𝑑 Phase 6.2.1. Code Capabilities.
Although the accumulator discharge rate and duration have been identified as the main driver for accident progression, the fact that core uncovery and overheating precede accumulator injection makes the coupling between the core damage and the accumulator injection become an early degraded core reflooding phenomenon.
Overall, for a given accumulator gas expansion model, both codes predict similar accumulator injection flowrate and duration, in-vessel FCI, hydrogen generation, core reflooding, core quenching, and delayed core degradation.
Discussion on the Expected Trends and Values.
For a given accumulator gas expansion model, both codes predict the expected trends in the primary pressure, temperature, and water inventory.The accumulator discharge rate and duration predicted by the two codes are discussed in detail in the next section.
Discussion on the Significant Discrepancies between the Codes.
The accumulator injection flow is driven by the pressure difference between the accumulator and the primary system.The accumulator injection flowrate and duration depend on the thermodynamic expansion of the gas stored in the accumulator [46].In ASTEC, the user can select the polytropic expansion coefficient which best describes the gas expansion process.In the current study, the polytropic coefficient of 1.4, corresponding to an isentropic expansion, was used for the reference case.In MAAP, the accumulator is modelled using a generic control volume.Hence, while ASTEC allows the user to select the polytropic constant, there is no such option in MAAP 5.02 (MAAP 5.04 is provided with this option).A MAAP sensitivity case was additionally run by imposing the same isentropic-type accumulator injection flowrate as calculated in the ASTEC reference case (called MAAP SEN).
For the given initial and final pressures, an isothermal expansion of the gas stored in the accumulators results in 35% higher final volume compared to an isentropic expansion as shown in According to the code results, ASTEC predicts a final gas volume of 27.935 m about 50% higher than ASTEC, resulting in a significantly prolonged injection.
Figure 6 shows the remaining mass of water in the accumulators.Figure 7 shows the RCS pressure evolution in the two reference cases and the two sensitivity cases.Figure 8 shows the discharged flowrate by a single accumulator.Figure 9 shows the cumulative mass of hydrogen generated inside the vessel.Figures 10 and 11 show the core exit thermocouple and peak cladding temperature in the fuel, respectively.
In MAAP, the accumulator gas expansion matches very well with an isothermal type even though not fully because the work exerted by the expanding gas to the liquid is not calculated by MAAP.Also, in MAAP the accumulator injection flowrate calculation does not consider the increase in the primary system pressure due to the increased steaming rate in the core.This causes injection flowrate overshooting as shown in Figures 6 and 8.It was found that limiting the time step size based on the maximum fractional change in the accumulator mass to 0.01% does eliminate the overshooting.Similar but less severe overshooting is observed in the ASTEC results as shown in Figures 6 and 8. Similar observation is reported in several published works including [47].This phenomenon occurs when partially uncovered and overheated core is quenched from below by core inlet flow.
The accumulator gas expansion process and the corresponding liquid discharge rate depend on the RCS depressurization rate and core overheating.If the core is partially uncovered and overheated, a rapid steaming occurs when the accumulators inject water into the RCS.This leads to a sudden increase in RCS pressure, which stops the accumulators discharge.As the rapid steaming stops in the core and steam is released through the break, the RCS pressure decreases gradually until it falls below the accumulator pressure.The accumulator injection starts again.For the gradual RCS depressurization and intermittent accumulator discharge in SBLOCAs, isothermal expansion of the accumulator gas would be more appropriate [48].On the other hand, for a rapid RCS depressurization such as in LBLOCAs, the accumulator gas expansion and liquid discharge will take place during a very short period of time.For this case, an isentropic expansion would be more appropriate.This discussion is not intended to provide guidance in choosing the right gas expansion model but to show how the code user choices can affect the results.
The following further observations can be made in the results: (1) Both codes predict the rapid depressurization subsiding when the accumulators start to inject water into the RCS.After the accumulators start to inject water, the RCS pressure decreases more gradually.The A REF run predicts a somewhat faster depressurization rate compared to the other cases.In this run, the core has been damaged and has formed a noncoolable geometry at the time the injection starts at 3824 seconds.Hence some of the overheated or molten core is not accessible to the water so that the steaming rate in the core is smaller than the other cases, resulting in (2) The two codes predict similar starting time for accumulator injection.This shows that the two codes predict similar break flowrate, natural circulation flowrate, heat transfer rate in SG, and heat transfer rate in the core until the accumulators are triggered.
(3) In all cases, the core is overheated and rapid oxidation of cladding has already started when the accumulators start injecting water as shown in Figure 9.Both ASTEC and MAAP predict that the overheated core will be quenched when the isothermal gas expansion model is used for the accumulator as shown in Figure 10 and clearer in Figure 11 (A SEN and M REF).Both ASTEC and MAAP predict that the overheated core will become damaged, and attain a noncoolable geometry when the isentropic gas expansion model is used for the accumulator as shown in Figure 11 (A REF and M SEN).Once the core attains a noncoolable geometry, the core interior is not accessible to the coolant and cannot be cooled by the large steam flow.Whether the overheated core is quenched or not depends on the initial flowrate of the accumulator.The initial accumulator flowrate is higher with the isothermal expansion compared to the isentropic expansion (not shown in Figure 6 but was confirmed in the expanded scale plot): between 4000 and 4400 seconds (the duration over which the core can either become quenched or form a molten pool), the total mass injected by the accumulators are 11000 kg in MAAP REF (isothermallike case).For MAAP SEN -just as for ASTEC REF-, the equivalent injected mass is instead equal to 7000 kg (hence 36% less).It appears that the accumulator injection rate, and the resulting steaming rate in the core, is barely sufficient to keep the quenched core cooled; the quenched core in M REF heats up again and temporarily reaches a noncoolable geometry during the accumulator injection period as shown in Figure 11.Once the accumulator injection is over, the quenched core heats up again.
(4) The rapid cladding oxidation and hydrogen generation occurs just before the accumulator injection starts as shown in Figure 9.The rapid oxidation and hydrogen generation stops when the core is quenched shortly after the accumulator injection starts as shown by the case A SEN and MAAP REF in Figure 9.The rapid cladding oxidation and hydrogen generation resume when the accumulator injection ends and the core heats up again after around 15000 seconds.As discussed before, the exception to this conclusion is M REF, which reheats and temporarily attains noncoolable geometry before the end of accumulator injection.
(5) If the core is not quenched at the start of the accumulator injection, the rapid cladding oxidation and hydrogen generation continues further until all the available fuel cladding surfaces are exhausted as shown by the cases A REF and M SEN in Figure 9.More gradual cladding oxidation and hydrogen generation resume when the accumulator injection ends at around 9000 seconds and continued coolant boil-off uncovers fresh fuel cladding.
Science and Technology of Nuclear Installations
As a conclusion, the two codes predict similar fuel cladding oxidation, core overheat, core damage, and thermalhydraulics response during the accumulator injection phase.In particular, quenching of the overheated core by bottom reflooding is realistically simulated by both codes.The melt progression model in MAAP was developed to be able to simulate formation of a molten pool in the core which is not coolable by late injection, as was observed during the TMI-2 accident.Although the current cases rely on the bottom reflooding and steam cooling, the code is able to predict both damaged core configurations; quenched core with the isothermal accumulator expansion model and damaged, uncoolable core with the isentropic accumulator expansion model.ASTEC also predicts the quenched core and damaged, uncoolable core depending on the accumulator gas expansion model.The results from this study also show that the accumulator injection rate is barely able to cool the core as shown in case M REF, and it can lead to either quenched or uncoolable core.The user should be aware of the uncertainty in the phenomenon and consider both accident progression paths.
Third Phase: Core Heat-Up and
Relocation to Lower Plenum 7.1.Phenomenology Analysis.When the accumulator injection begins, the core heat-up and hydrogen generation have already started.The fuel cladding oxidation and hydrogen generation depend on the fuel temperature, fuel cladding surface area available for reaction, and steam sources.Less damaged core produces more hydrogen.For the same reason, slower core heat-up and degradation processes produce more hydrogen than rapid core heat-up and degradation, typically found at low RCS pressures.In addition, since the cladding oxidation is an exothermal reaction, fuel heatup is accelerated once the cladding reaches a certain elevated temperature.Therefore, the in-vessel injection, if it is not introduced at sufficiently high and sustained flowrates, can accelerate core heat-up and lead to core degradation.
Depending on the accumulator injection rate, the core may either be quenched or attain an uncoolable geometry.Since the accumulator injection rate depends on the RCS pressure, and the RCS pressure in turn depends on the accumulator injection rate and steaming in the core, the accumulator injection rate and the rate at which the RCS pressure is decreasing depends ultimately on the break size.A large break will result in faster RCS pressure falling rate and higher accumulator injection rate, resulting in a quenched core.
The accumulation injection ends when the RCS pressure falls below 15 bar.The fact that the RCS pressure has been supported above the accumulator trip-off pressure means that the core is partially covered and the steaming is still continuing when the accumulator injection ends.Therefore, steaming continues at a lower rate due to the decay heat in the covered section of the core after the end of the injection.If the core is quenched, the uncovered section of the core will heat up again.When the cladding temperature reaches a certain point, oxidation of unreacted cladding will take off, accelerating the core heat-up.The oxidation rate in the uncovered core may be limited by the steaming rate in the covered part of the core.If the core is not quenched, most of the fuel cladding in the uncovered core is either already oxidized or is not accessible to steam because of the changed geometry.Therefore, additional cladding oxidation will be limited to the freshly uncovered core as the coolant boils off.Eventually the core will melt and start to relocate to the lower plenum.
The biggest uncertainty is expected in melt progression in the core and subsequent loss of geometry.The timing and the rate of core relocation to lower plenum will affect the behaviour of the debris bed in the lower plenum and failure of the lower head.
Conclusions on the 3 𝑟𝑑 Phase 7.2.1. Code Capabilities.
Both codes model oxidation of zircalloy cladding by steam at high temperatures.In MAAP the melt progression model combined with rules from subnodal physics creates a periphery of impenetrable, crust-like nodes surrounding the molten debris pool.Molten material cannot flow through a highly packed region, and upward gas flow is diverted around the crust.The outermost and top nodes can develop side and top crusts due to heat losses to the core former and baffle plates, as well as upward to the gas and upper plenum structures.A failure criterion is provided to determine the potential to reach the side crust.Separate relocation of control materials, metals, and oxides, including dissolution are considered.
In MAAP, there are two potential paths for corium to relocate from the core region into the lower plenum: sideward relocation and downward relocation.For sideward relocation, side crust around the corium pool in the core region fails.Only molten material is relocated.For downward relocation, the core support plate fails due to creep.Both molten and solid material is relocated.Hence, different relocation paths may lead to substantially different amounts of corium relocating into the lower plenum.
In ASTEC, in-vessel core degradation starts with highly exothermic zircalloy oxidation.As the core heats up, fuel rod ballooning and cladding rupture is modelled.Melting and relocation of control rod absorbers occur first.Formation of eutectic mixtures with lower melting point is considered.Fuel melting and loss of the original geometry occurs at around 2500 K after dissolution of UO2.In-core molten pool formation with crust is modelled.As the core heats up further due to decay heat, the crust is breached and corium relocation to the lower plenum occurs.Both sideward and downward relocations can be calculated by ASTEC code, depending on the corium melt progression.However, in both cases it is associated to the heat up and failure of the corium crust and only molten corium is relocated to the lower head.The mechanical failure of the core support plate being not simulated, the corium flows down through the holes in the plate.
Discussion on the Expected Trends and Values.
In this phase, the core is heating up by the decay heat.Cladding oxidation will add additional heat to the core.At some point, the fuel will start to melt and relocate within the core.When the fraction of core melted becomes sufficiently high, the core material will start to relocate to the lower plenum.Both codes are expected to track this integral behaviour with the same level of precision.
Because key phenomena such as cladding oxidation and melt progression have positive feedback, some uncertainty is expected in the accident progression in this phase.The total amount of zircalloy available for oxidation and the availability of steam provide some envelop on the total mass of hydrogen generated in the core.However, depending on the logic built in the models, the steam may flow through the damaged core and react with zircalloy or bypass the damaged core.
The melt progression in the core and relocation to the lower head also has uncertainties.First, the core geometry when it begins to melt and collapse (fuel rod collapse criteria may produce solid debris in addition to molten fuel) is subjected to uncertainties.Second, the way corium flows and solidifies in contact with colder structures is complex and is typically non-axisymmetric.Therefore, it is expected that there will be substantial uncertainty in the time and amount of initial corium relocation into the lower plenum.The total amount of hydrogen generated in the core may be somewhat more consistent but again will depend on the permeability of the damaged core to steam flow.
Discussion on the Significant Discrepancies between the Codes
(1) Core Heat-Up and Hydrogen Generation.A REF should be compared with M SEN for the accumulator isentropic gas expansion case and A SEN should be compared with M REF for the isothermal accumulator gas expansion case.
For the isentropic accumulator gas expansion case, the accumulator injection ends at around 9000 seconds and the core is not quenched in both codes.Substantial fraction of the cladding has been oxidized in both codes as well.
For the isothermal accumulator gas expansion case, the accumulator injection ends at around 14000 seconds and the core is quenched in both codes.Only a small fraction of the cladding has been oxidized in ASTEC.MAAP predicts a momentary temperature excursion during the accumulator injection period, which leads to a cladding oxidation spike.
The momentary temperature excursion predicted by MAAP is an exception and indicates the fine boundary between the coolable and uncoolable accumulator injection rate.A rapid cladding oxidation brings the core to melting and to form a molten pool.Subsequent melt relocation within the core brings it back to a coolable geometry.Hence, the initial condition of this phase is nearly identical between the two codes.
For the isentropic accumulator gas expansion case, the core is not quenched and hence there is no change in the peak cladding temperatures after the accumulator injection ends.However, cladding oxidation, which has been stopped due to the lack of available area of cladding to react with steam, resumes as the new cladding surface becomes uncovered once the water level in the core starts to decrease.At the end, both codes predict about 510 kg of H2 generated in the core.
For the isothermal accumulator gas expansion case, the quenched core starts to heat up after the accumulator injection ends.Shortly after it starts to heat up, rapid oxidation of cladding occurs and the fuel temperature increases rapidly.Note that the rapid oxidation of cladding is delayed for MAAP because some of the cladding has already been oxidized when the temporary heat up occurred before.At the end, both codes predict about 600 kg of H2 generated in the core.Compared to the isentropic accumulator gas expansion case, more hydrogen is generated at the end because the core is less severely damaged and more zircalloy is accessible to the steam flow.
Overall, both codes predict similar results in the core heat up and hydrogen generation after the accumulator injection ends.
(2) Core Material Relocation to Lower Plenum.Figure 12 depicts the debris bed layer temperatures predicted by the two codes.Figure 13 depicts the evolution of the relocated mass to the lower plenum.A REF should be compared with M SEN for the accumulator isentropic gas expansion case and A SEN should be compared with M REF for the isothermal accumulator gas expansion case.Both codes predict similar initial core slumping time.For the isentropic accumulator expansion case, the initial core slumping occurs at around 15500 seconds in both codes.For the isothermallike accumulator expansion case, the initial core slumping occurs at around 22500 seconds in MAAP and 21000 seconds in ASTEC.However, the amount of core initially slumped is significantly different between the two codes.For the isentropic accumulator expansion case, MAAP predicts around 70000 kg whereas ASTEC predicts 130000 kg.For the isothermal-like accumulator expansion case, the amount of core initially clumped is about 40000 kg for MAAP and 120000 kg for ASTEC.Hence, ASTEC predicts significantly higher initial mass of core material relocated into the lower plenum, which leads to an earlier vessel failure.
Note that significantly larger core material relocated into the lower plenum predicted by ASTEC does not lead to an equally larger RCS pressure spike as shown in Figure 14.ASTEC predicts a smaller heat transfer rate between the corium in lower plenum and the overlying water as it will be explained later.
Fourth Phase: Corium Interaction with
the RPV Lower Head and RPV Failure 8.1.Phenomenology Analysis.The initial molten corium relocated into the water-filled lower plenum will produce a huge steaming and pressure spike in the vessel.The molten corium jet will break up into particulates as it falls through the water in the lower plenum.The particulates will be quenched promptly, causing rapid steaming.Because of the magnitude of the steaming, the steam generated may not completely 2500 5000 7500 10000 12500 15000 17500 20000 22500 25000 27500 30000 32500 35000 0
Time [s]
Figure 12: ASTEC vs MAAP lower plenum corium layer temperature comparison (the steel layer temperature reading in ASTEC begins with the onset of core degradation even though only a few milligrams are being relocated before the significant slumping occurs at 15774 seconds).condense in the liquid pool before it is released to the gas space, causing a sudden pressure increase.Some unreacted zirconium in the particles may react with steam and produce hydrogen.Depending on the height of the water pool, the fall height, and the size of the jet, the molten jet may not break up completely.The intact jet arriving at the lower head will spread over the vessel bottom and form a contiguous layer.The quenched particles will fall on top of the contiguous layer and form a particle bed.The lighter metal and heavier oxides in the contiguous layer may separate and form a metal layer atop the oxide layer.If there is sufficient amount of unreacted zirconium present in the debris, some of the uranium oxide may be reduced into metallic uranium and, with other metals, form a heavy metal layer at the bottom.Thus, the debris bed will stratify according to the density of the material: heavy metal layer at the bottom, oxide layer in the middle, lighter metal layer at the top, and the particle bed submerged in the metal layer.Subsequent core relocations will disrupt the initial debris bed layers but they will gradually migrate towards the stratified configuration via continuous melting and freezing.
The molten corium that comes in contact with the vessel wall will freeze and form a crust.Similarly, an upper oxidic crust will form between the molten metal layer and the oxide layer because of the big difference in the melting temperatures of oxidic debris and metal.Thus the initial debris bed in the lower plenum will consist of the molten oxidic pool on the bottom surrounded by crust, a molten metal layer above the upper crust, and a particle bed partially submerged in the metal layer, with the possibility of a metallic-uranium-rich high density layer below the molten oxidic pool.There may not be a complete separation of metal at the time of corium delivery.Finally there will be an overlying water pool.
Before the lower plenum water is depleted, the debris bed will lose heat to the overlying water.The upward heat transfer rate will be limited by the counter-current flooding limitation in the particle bed.Once the overlying water has boiled off, the debris bed will start to heat up.The particle bed will melt due to its decay heat and gradually migrate to the oxidic pool.Since the decay heat in the debris bed is expected to be much larger than the heat loss to the vessel internals and to the below-vessel structures by thermal radiation, the debris bed will start to heat up.
As the debris bed heats up by the decay heat, the molten pool in the debris bed will get larger as the crust melts.A natural circulation in the pool will maintain the temperature distribution in the molten pool fairly uniform and close to the melting temperature of the oxidic pool.Hence, most of the decay heat in the early heat up phase goes into the latent heat of melting the debris or the crust and growing the molten pool.The boundary of the debris bed, the vessel wall and metal layer, are protected by the thick crust and is not affected by the debris bed heat up until the crust becomes thin.
As the molten pool grows and the crust becomes thin, the conduction heat transfer rate in the crust increases.The increasing heat flux to the vessel wall will eventually attack the vessel wall and penetrations and fail the vessel.The natural convection heat transfer coefficient in the molten pool is lowest at the bottom and highest near the top.Therefore, the bottom crust (i.e., the crust between the vessel wall and the oxidic layer) will be thickest at the bottom and thinnest at the top, and thermal attack and vessel failure is expected to occur near the top of the debris bed.
When the vessel wall is overheated, it usually fails by material creep.If penetrations are present in the lower head the weld holding the penetration tube to the lower head can fail and push out the penetration.Both vessel failure mechanisms are preceded by lower head overheating; presence of penetrations will accelerate vessel failure somewhat.
The vessel can also fail by metal layer attacking the vessel wall.Unlike the oxidic pool, the vessel wall adjacent to the metal layer is not protected by the crust.The upward heat transfer rate in the molten oxidic pool is fixed by the decay heat generated in the pool and the split between the upward and downward heat transfer rate in the pool.The upward thermal radiation heat transfer rate in the metal layer is also somewhat fixed because the metal layer temperature cannot be too far away from the steel melting temperature; otherwise the vessel wall adjacent to the metal layer will melt.The difference between the heat received from the oxidic pool and the heat rejected by thermal radiation to vessel internals is directed to the sideward heat transfer to the vessel wall.
Hence, the sideward heat flux depends on the thickness of the metal layer; the thinner the metal layer the higher the heat flux.This phenomenon is referred to as "focusing effect".When the top metal layer is sufficiently thin the adjacent vessel wall will melt and fail.
Beside the debris bed slowly heating up and eventually attacking the vessel wall or penetrations, the vessel can also fail by late arriving molten corium attacking the vessel wall or penetrations locally before exchanging heat with the preexisting debris bed.These late arrivals will not encounter water in the lower plenum and will remain molten.
With several chemical species competing for oxygen in the molten pool, some of the uranium oxide can lose its oxygen and become metallic uranium, especially when large amount of unoxidized zirconium is present.Depending on the amount of molten steel present in the pool compared to the metallic uranium, the metal phase can become heavier than the oxidic phase and it will separate out to form a heavy metal layer.The formation of a heavy metal layer will affect the heat transfer rate to the vessel wall and hence influence vessel failure.The greatest impact of the heavy metal layer is expected in transient situations when it is first formed and when it becomes lighter than the oxides, leading to a layer inversion.During a layer inversion, the metal can become significantly overheated when it rises to the top through the oxide layer and merges with the top metal layer.Also, the formation of heavy metal layer will take some material from the light metal layer, thereby reducing the light metal layer thickness and making the "focusing effect" worse.
Chemical equilibrium and phase diagram of the material present in the debris bed also affect physical properties such as specific heat, density, thermal conductivity, latent heat of melting, and melting temperature.Physical properties affect heat transfer and hence vessel failure.However, they are expected to have minor impact, within the uncertainty of the phenomena.The main factors affecting vessel failure are the time and amount of corium relocated to the lower plenum and the layer stratification affecting the top metal layer characteristics.The relocation time is important because of the decay heat; the early arriving corium will have significantly higher decay heat per unit mass.The amount of water in the lower plenum is also important.In MAAP, the corium jet breaks by entrainment in the liquid pool in the lower plenum.The particle size is determined by capillary effects.Steaming rate is determined by heat transfer in the interaction zone consisted of particles, steam, and water.Particle oxidation is considered.
In ASTEC, the fragmentation is calculated based on the same kind of model of jet break-up with debris formation.The heating and vaporization of the water is also determined.However, particles oxidation is not considered.This is done based on the fact that the uncertainties are high and this oxidation is expected to remain limited.In addition, it is conservative regarding the risk of heavy metal layer formation.
(1) The current plant uses two types of control rod material: AIC and stainless steel.MAAP cannot model stainless steel control rod.It also cannot model more than one cladding type; both Zr and stainless steel cladding cannot be used in a single model.Both features can be handled by ASTEC.
(2) Chemical equilibrium in the debris bed in lower plenum is not considered in MAAP; the initial material composition of the relocated corium is maintained in the lower plenum.MAAP has a simple heavy metal layer model to consider the effect of metallic uranium combining with other metals and forming a heavy metal layer below the oxide layer.Reduction of uranium oxide into metallic uranium is not explicitly modelled.Instead, when a certain temperature and mass composition criteria are met in the debris bed in lower plenum, some uranium oxide in the oxide layer is combined with some fraction of the metal layer to form a heavy metal layer.In ASTEC, chemical equilibrium is considered in the debris bed in lower plenum and the material composition in the debris bed is tracked.In addition fission products are distributed in both oxide and metallic phases based on their chemical affinities.This leads to a fraction of decay power in the metal layer.
(3) In version V2.1.1,ASTEC models the oxidation of metal components (U and Zr) in the lower plenum debris bed.However, the model is not activated by default and associated kinetics is found to be much longer than the time of vessel failure.
(4) Both codes separate the relocated corium into different layers, from top to bottom, particle bed, metal layer, and oxidic pool.However, as already anticipated above, ASTEC code evaluates phase separation based on thermochemical equilibrium in the quaternary system U-O-Zr-Fe, whereas MAAP code simply separates steel and Zr in the top metal layer plus a heavy metal layer (activated through a user option, as for the current simulations) at the bottom of the corium pool which is primarily consisted of metallic constituents of U, Zr, steel, and oxygen.Neither ASTEC nor MAAP tracks the corium at a node level but at a layer level.The properties of each layer, among which composition and chemical interactions, energy, and mass, are tracked individually throughout the accident though in a different way.Each code treats the heat transfer to the lower head vessel and heat transfer distribution, namely upwards and downwards, in a different way (even if both modelling approaches rest on the use of heat transfer correlations): MAAP starts by initially imposing a 10 centimetre thickness node of solid crust attached to the lower head vessel while in ASTEC the crust is evaluated based on heat fluxes equality from the corium to the vessel, considering the crust as an additional thermal resistance.Either way, the corium composition computation across the different layers is fundamental as it greatly impacts the initial melting temperature (solidus temperature) calculation and the relative thickness of each layer.
MAAP considers RPV failure in two steps.Several vessel failure mechanisms are considered for the initial failure: creep rupture, ejection of penetration tubes when the closure weld fails, melt ingress into penetration tubes outside of the vessel, melt jet impingement of the lower head, and overlying molten metal layer attacking the vessel wall.After the initial failure, debris above the failure location is discharged, leaving behind rest of the debris inside the vessel.The location, initial radius, and growth of the failure opening are tracked.The remaining debris in the vessel continues to heat up and eventually the entire lower head fails by creep.The subsequent failure is considered catastrophic and the remaining debris in the lower plenum is dumped into the reactor cavity.
The ASTEC V2.1.1 model does not make an explicit distinction between a first localized failure and a second extended one but these are taken into account using the rupture criterion, which may be local, and the collapse criterion, which is associated to a generalized failure of the vessel.The RPV failure criteria consist of a set of userinput threshold values of temperature, molten fraction, and mechanical stress, plus more mechanistic models of plastic strain and creep.In particular, for hemispherical lower head, a specific rupture model is available based on a Norton-Bailey type creep law and the coupled damage-viscoplasticity model developed by Lemaître and Caboche.A refined submeshing of the vessel wall is considered in this mechanical model.
In MAAP, creep rupture of the lower head is modelled using the Larson-Miller parameter method [49], which gives the failure time of a metal subjected to a constant tensile load at an elevated temperature.The temperature gradient in the lower head is considered by distributing the tensile load among individual laminae (i.e., radial nodes) in the wall such that each laminae experiences the equal strain.That is, the coldest laminae will bear the most stress.The timevarying temperature and stress are considered by considering a fractional contribution to rupture at each time step.The ultimate tensile strength is considered.The partially molten wall thickness is considered.
It is also worth noting a modelling gap in the ASTEC 2.1.1 version of the code related to the failure of mechanical penetrations, whether because of losing the welding material attached to the vessel or because of the corium penetrating inside the penetration, located at the lower head of PWR reactors.
Lastly, the lack of a thermochemical model in MAAP code may lead to incorrect compositions in stratified layers, neglecting decay power in the top metal layer due to the metallic uranium in Zr-Fe-U.Also, MAAP models the molten steel relocation up to the top metal layer at its melting temperature independently from its initial localization, which may lead to an underestimation of the top metal layer temperature.
Discussion on the Expected Trends and Values.
As discussed in the previous section, there is a large uncertainty in melt progression in the core which leads to a large uncertainty in corium relocation to the lower plenum, both in timing and in quantity.Hence, the discrepancies in the debris bed behaviour in the lower plenum do not reflect exclusively the difference in modelling but are mainly the results of different initial condition.Therefore, the lower plenum debris bed response predicted by the two code, including vessel failure, should be compared in qualitative and relative terms considering the timing and quantity of corium relocated from the core predicted by each code.15 and 16 show the corium mass in the lower plenum predicted by ASTEC and MAAP, respectively, for the isentropic accumulator gas expansion case.ASTEC predicts about 130,000 kg of core slumping initially at around 16,000 seconds followed by a gradual relocation whereas MAAP predicts about 70,000 kg of core slumping initially at around 16,000 seconds followed by a gradual relocation.Both codes predict little or no particle bed formation.Both codes separate metal from the relocating corium at the time of corium relocation and put it in a separate metal layer.Both codes predict that there is no heavy metal layer formed in the lower plenum.MAAP predicts lower and upper crust surrounding the oxidic pool whereas in ASTEC the mass of crust is not tracked explicitly but is included in the mass of oxidic pool.As shown in Figure 12, MAAP predicts the metal layer quenching by the overlying water in the lower plenum.Once the water in the lower plenum has boiled off (see Figure 14) the metal layer starts to heat up.The oxidic pool is not quenched but is cooled somewhat initially and then heats up steadily as subsequent relocation is added to the oxidic pool.ASTEC predicts gradual cooling of the metal layer by the overlying water in the lower plenum.As shown in Figure 14, ASTEC predicts that most of the water remains in the lower plenum when the vessel fails.Hence, ASTEC predicts much smaller heat transfer rate between the metal layer and the overlying water in the lower plenum compared to MAAP.The heat transfer model between the metal layer and the overlying water is improved in ASTEC V2.1.1.4.
Discussion on the Significant Discrepancies between the Codes. Figures
MAAP predicts vessel failure due to creep rupture 0.55 m below the cylindrical portion of the vessel 7324 seconds after the initial core slumping.ASTEC predicts vessel failure due to a melt-through 0.4 m below the cylindrical portion of the vessel 1090 seconds after the initial core slumping.The significantly earlier vessel failure predicted by ASTEC can be explained as following: (1) ASTEC predicts a significantly larger initial core slumping compared to MAAP.Hence the decay heat in the debris bed in lower plenum is much higher in ASTEC.Consequently the thermal load to the lower head is greater and the lower head is expected to fail earlier in ASTEC.This dominant factor affecting debris behaviour in the lower plenum is dependent on the melt progression model in the core.
(2) ASTEC predicts much smaller heat transfer rate from the top metal layer to the overlying water in the lower plenum.Consequently the corium retains most of its sensible heat such that the thermal attack to the vessel wall is greater.
(3) The debris bed model in the lower plenum, including the vessel failure mechanisms considered, affects the vessel failure time.In particular, the treatment of lower crust can be important because heat conduction through the lower crust exerts thermal load to the lower head so that the thickness of the crust can modify the temperature evolution of the lower head vessel.In MAAP, vessel failure due to creep is always preceded by the thinning of the lower crust as shown in Figure 16.
The above explanation for the discrepancy in the vessel failure time is based on the depicted results, i.e. tables and figures.Nevertheless, it is worth noting that ASTEC's prediction of a very high top metal layer temperature can be tied to the earlier vessel failure.The reasons for ASTEC to predict such a high temperature can be related to the following modelling details (these issues are discussed in more detail in [50]): (1) ASTEC assumes that the molten steel relocated to the top of the corium pool has same temperature as the oxidic pool because it originates from melting of the internal structures surrounding by the oxide pool or from the ablation of the vessel wall in contact with the oxide.This leads to an overheated metal layer compared to MAAP, which accelerates ablation of the vessel wall adjacent to the metal layer.The difference in the metal layer temperature is clearly shown in Figure 12.
(2) ASTEC considers thermochemical equilibrium in the lower plenum debris bed, which leads to the presence of metallic uranium in the metal layer along with Fe and Zr.This adds decay heat in the metal layer and increases heat flux to the vessel wall.
As a conclusion, the response of the debris bed in lower plenum is primarily determined by the initial delivery of corium from the core: ASTEC predicts a significantly larger initial core slumping, which leads to earlier vessel failure.
In addition, ASTEC's modelling capabilities predict an overheated metal layer, which causes earlier vessel failure.Both codes predict vessel failure at the highest elevation in the molten pool.Both codes predict vessel failure shortly after the average temperature of the oxidic layer exceeds the liquidus temperature.
Summary and Conclusions
A code comparison has been performed between ASTEC and MAAP.The selected scenario is a 2 inch cold leg SBLOCA in a 900 MWe PWR.All safety injections except the accumulators were assumed to be unavailable.Two sensitivity cases were added so that comparisons were able to be made with the same boundary conditions (i.e., the accumulator gas expansion model).The accident evolution predicted by the two codes up to the time of RPV failure was compared.The goal of this study is to identify key differences in the results and to explain the differences in terms of model differences and code user choices.The following conclusions can be derived from this study: (1) When the same accumulator gas expansion model is used, the two codes predict similar thermal-hydraulic response until core melting and relocation.They include break flow, steaming in the core, RCS depressurization, core uncovery, natural circulation and heat transfer between the vessel and SG, core heat up, cladding oxidation, and hydrogen generation.Even the intricate feedback between the RCS pressure and the accumulator injection predicted by the two codes is consistent.
(2) The code comparison exercise highlights the importance of the user choice on model parameters.Once the same accumulator gas expansion model was selected, the overall results look similar and minor differences are explainable.
(3) Both codes predict that the core has already uncovered and is heating up with cladding oxidation when the accumulator injection begins.Moreover, both codes predict that the injection may or may not quench the core.With isothermal gas expansion, the accumulator injection rate is a little higher at the beginning and both codes predict core quenching.With isentropic gas expansion, the accumulator injection rate is a little lower at the beginning and both codes predict the core is not quenched.
(4) Both codes are capable of predicting the formation of a molten pool in the core which cannot be cooled by a late injection of coolant.
(5) The major difference between the two predictions is found in the melt progression and core relocation to the lower plenum.ASTEC predicts a significantly larger core relocation mass to the lower plenum compared to MAAP during the first instants.On the other hand, the timing of core relocation is similar, about 7,000 seconds after the accumulator trip off.
(6) The two codes predict similar but somewhat different debris bed behaviour in the lower plenum.ASTEC predicts much lower heat transfer rate between the top metal layer and the remaining water in the lower plenum.Consequently the upper metal layer temperature decreases at a slower rate and the oxidic pool remains hot until vessel failure.There is still water in the lower plenum when the vessel fails.On the other hand, MAAP predicts the top metal layer and the oxidic pool to cool down faster.Once the water in the lower plenum has boiled off, the metal layer and oxidic pool start to heat up until the vessel fails.ASTEC assumes that the molten steel relocated to the metal layer is in thermal equilibrium with the oxidic layer.It also considers chemical equilibrium in the debris bed, predicting presence of metallic uranium in the metal layer and associated decay heat.Hence it predicts higher metal layer temperature compared to MAAP.Nevertheless, keeping in mind such differences in the lower plenum debris bed modelling, their impact on the vessel failure time is not discernible in this study.Therefore, the earlier vessel failure time predicted by ASTEC should be mostly assigned to the larger core relocation.
Nomenclature
Accum: Accumulator AFW: Auxiliary feedwater system AIC: Silver Indium Cadmium CET: Core exit thermocouples (temperature) d: Derivative DiD: Defence in depth ECCS: Emergency core cooling system EFW: Emergency Feedwater FCI: Fuel coolant interaction HPSI: High pressure safety injection IVMR: In-vessel melt retention LPSI: Low pressure safety injection m: Meter ṁ: M a s sfl o w r a t e MBLOCA: Medium break loss of coolant accident MCCI: Molten corium concrete interaction MFW: Main feedwater system MWe: Electric megawatt P: Pressure PCT: Peak clad temperature PWR: Pressurized water reactor RCP: Reactor coolant pump RCS: Reactor coolant system SBLOCA: Small break loss of coolant accident SG: Steam generator SI: Safety injection TAF: Top of active fuel V: Volume
Figure 5 :
Figure 5: Primary and Secondary (SG1) Pressure Evolutions, 1 st Phase (all the figures will follow the same pattern codification: continuous line for the reference case (REF) and dotted line for the sensitivity case (SEN); blue colour for ASTEC (A) and red for MAAP (M)).
Figure 13 :
Figure13: Evolution of the relocated mass to the lower plenum (at around 20760 seconds, there seems to be a spurious value of the relocated mass into the lower plenum in the ASTEC sensitivity case).
Figure 14 :
Figure 14: Primary pressure vs primary water mass and corium relocated mass to the lower plenum.
8. 2 .
Conclusions on the 4 ℎ Phase 8.2.1.Code Capabilities.Both codes have enough capabilities to consider all the important phenomena in the lower plenum.However, the way they treat individual phenomena is somewhat different between the two codes.
Figure 15 :
Figure 15: ASTEC corium stratification in the lower plenum (in this figure, the mass of the crusts is included in the mass of the oxide layer).
Table 1 :
Comparison of the steady state operational conditions predicted by ASTEC and MAAP.
Table 2 :
Key automatic and operator actions.
the primary system: hot leg, SG primary and secondary components (water box, U tubes, etc.), cold leg, intermediate leg (loop seal leg), pressurizer, etc.The containment compartments, junctions, heat sinks, and reactor cavity configuration in the ASTEC model were implemented in the MAAP model.
Table 3 :
Phenomenological windows identified and dominant driving phenomena investigated.
Table 4 :
Timing of key events in the SBLOCA predicted by ASTEC and MAAP.
Table 5 :
Key results in the SBLOCA predicted by ASTEC and MAAP.
Table 6 :
Computational times and time steps in the analysed cases. | 2019-04-16T13:29:12.256Z | 2018-12-02T00:00:00.000 | {
"year": 2018,
"sha1": "a643d5179b3fbc37a820648a723934be8f38da41",
"oa_license": "CCBY",
"oa_url": "https://downloads.hindawi.com/journals/stni/2018/9189010.pdf",
"oa_status": "GOLD",
"pdf_src": "Anansi",
"pdf_hash": "a643d5179b3fbc37a820648a723934be8f38da41",
"s2fieldsofstudy": [
"Engineering",
"Environmental Science"
],
"extfieldsofstudy": [
"Engineering"
]
} |
244438753 | pes2o/s2orc | v3-fos-license | Outcomes of Impella compared with intra-aortic balloon pump in ST-elevation myocardial infarction complicated by cardiogenic shock
Background Despite limited randomized trial data demonstrating clinical efficacy, the utilization of Impella in ST-elevation myocardial infarction (STEMI) patients complicated with cardiogenic shock (CS) has increased over time. Methods We identified 75,769 hospitalizations with STEMI complicated by CS between October 2015 and December 2018 using International Classification of Diseases, Tenth Revision, Clinical Modification (ICD-10-CM) codes. From this cohort, hospitalizations were stratified according to IABP or Impella placement. The primary outcome was all-cause in-hospital mortality. Secondary outcomes were divided into efficacy, safety, and device-related complications. Propensity-score matching was used to account for differences in the baseline characteristics between the groups. Logistic regression was performed to get the odds ratio and confidence intervals. Results Among 75,769 admissions with STEMI and CS, hospitalizations with <18 years old, both IABP and Impella placement, and who underwent ECMO and/or LVAD implantation were excluded. After the exclusion, out of 72,791 admissions, 25,260 (34.70%) hospitalizations received IABP, and 7825 (10.75%) received Impella support. After propensity score-matched analysis, 7345 hospitalizations were included in each group. All-cause in-hospital mortality was higher in the hospitalizations requiring Impella support as compared to IABP (42.10% vs. 31.54%, adjusted OR 1.71; 95% confidence interval (CI) 1.60–1.84, P < 0.0001). Impella was associated with a higher risk of in-hospital complications and hospitalization cost compared with IABP. Conclusion Impella compared with IABP in STEMI patients with CS was associated with higher in-hospital mortality and other adverse clinical and procedural outcomes.
Impella IABP STEMI Cardiogenic shock Mortality A B S T R A C T
Background: Despite limited randomized trial data demonstrating clinical efficacy, the utilization of Impella in ST-elevation myocardial infarction (STEMI) patients complicated with cardiogenic shock (CS) has increased over time.Methods: We identified 75,769 hospitalizations with STEMI complicated by CS between October 2015 and December 2018 using International Classification of Diseases, Tenth Revision, Clinical Modification (ICD-10-CM) codes.From this cohort, hospitalizations were stratified according to IABP or Impella placement.The primary outcome was all-cause in-hospital mortality.Secondary outcomes were divided into efficacy, safety, and devicerelated complications.Propensity-score matching was used to account for differences in the baseline characteristics between the groups.Logistic regression was performed to get the odds ratio and confidence intervals.Results: Among 75,769 admissions with STEMI and CS, hospitalizations with <18 years old, both IABP and Impella placement, and who underwent ECMO and/or LVAD implantation were excluded.After the exclusion, out of 72,791 admissions, 25,260 (34.70%) hospitalizations received IABP, and 7825 (10.75%) received Impella support.After propensity score-matched analysis, 7345 hospitalizations were included in each group.All-cause in-hospital mortality was higher in the hospitalizations requiring Impella support as compared to IABP (42.10% vs. 31.54%,adjusted OR 1.71; 95% confidence interval (CI) 1.60-1.84,P < 0.0001).Impella was associated with a higher risk of in-hospital complications and hospitalization cost compared with IABP.Conclusion: Impella compared with IABP in STEMI patients with CS was associated with higher in-hospital mortality and other adverse clinical and procedural outcomes.
Introduction
Despite advances in treatment, cardiogenic shock (CS) remains the foremost cause of in-hospital mortality among patients presenting with acute myocardial infarction (AMI), with a mortality rate of approximately 50% [1].Studies have reported the incidence of CS complicating AMI, ranging from 4% to 14% [1][2][3].Furthermore, patients with STelevation myocardial infarction (STEMI) are more likely to develop CS than non-ST-elevation myocardial infarction (NSTEMI) [1].
While immediate revascularization in AMI patients with CS is lifesaving, controversy remains regarding the role of mechanical circulatory support (MCS) devices in such patients.Data from randomized controlled trials have not found a consistent benefit of routine use of temporary MCS devices in AMI patients.The routine use of intra-aortic balloon pump (IABP) did not reduce mortality in patients with AMI and CS treated with immediate revascularization [4].Although Impella has been shown to improve hemodynamic parameters such as blood pressure and cardiac output, the IMPRESS trial and other retrospective analyses comparing IABP vs. Impella in STEMI patients complicated by severe CS did not show a significant difference in 30-day mortality [5,6].Recent reports from National Cardiovascular Data Registry and Premier Healthcare Database have suggested higher mortality and risk of complications with Impella compared with IABP [7,8].In contrast, recent reports from National Cardiogenic Shock Initiative (NCSI) have suggested improvement in survival rates in acute MI and CS patients with Impella [9].Despite limited data exhibiting improvements in clinical outcomes relative to IABP, the trend in the utilization of Impella devices has increased over time in CS [10][11][12].Hence; we sought to demonstrate the comprehensive nationwide data on the overall outcomes associated with the use of Impella versus IABP specifically in STEMI complicated by CS hospitalizations, using the most recent National Inpatient Sample (NIS) database.
Data sources
We used the National Inpatient Sample (NIS) database between October 2015 and December 2018 for the present analysis.The NIS is the largest publicly available all-payer inpatient care database in the United States.Unweighted, it contains data from more than 7 million hospital stays each year, and weighted, it estimates more than 35 million hospitalizations nationally.The NIS is maintained by the Healthcare Cost and Utilization Project (HCUP) through a Federal-State-Industry partnership sponsored by the Agency for Healthcare Research and Quality (AHRQ) [13].Its large sample size is ideal for developing national and regional estimates and enables analyses of rare conditions, uncommon treatments, and special populations.Since the NIS uses deidentified hospital discharges as samples with prior ethical committee approval, no additional ethical committee approval was required for the present analysis.The NIS contains information regarding patient demographics, primary and secondary diagnosis at discharge, hospital characteristics, payment source, total charge, discharge status, length of stay and severity, and comorbidity measures.The Reporting of studies Conducted using Observational Routinely-collected health Data (RE-CORD) Statementa checklist of items is provided as Supplementary Table 1.
Study cohort
We identified hospitalizations with STEMI complicated by CS, with either IABP or Impella implanted, between October 2015 and December 2018 using appropriate International Classification of Diseases, Tenth Revision, Clinical Modification (ICD-10-CM) codes (Supplementary Tables 2 and 3).We excluded the hospitalizations with <18 years old, who received both Impella and IABP, as well as underwent extracorporeal membrane oxygenation (ECMO) and/or left ventricular assist device (LVAD) placement (Fig. 1).The primary outcome was all-cause in-hospital mortality.The secondary outcomes were classified as efficacy, safety, and device-related complications endpoint as well as hospitalization cost.To obtain the cost of hospitalization, hospital charges were multiplied with the cost-to-charge ratios for each hospital for a given year and indexing to the year 2018 to adjust for inflation [14].We also performed the subgroup analysis looking at the outcomes in hospitalizations who underwent PCI only as well as who did not have any intervention in terms of CABG or PCI (medically managed only).
Statistical analysis
We compared hospitalizations for age, gender, comorbidities, use of vasopressor support, and outcomes between patients who received IABP vs. Impella during the hospitalization.The Elixhauser Comorbidity Index was used to identify comorbid disorders.
Categorical variables are presented as numbers and percentages and compared using the Chi-square test.Numerical variables are presented as the median and interquartile range (IQR) and were compared using the Wilcoxon test.The P value of <0.05 was set as a level of significance.Discharge weights provided by the HCUP were applied to generate the national estimates as recommended [15].Next, we examined whether the use of IABP, compared with Impella, was associated with a lower risk of our study endpoints.Since patients who receive IABP may differ from patients who receive Impella, we used a matched propensity score analysis to account for confounding.The propensity score was estimated using a non-parsimonious logistic regression model where the outcome was a receipt of IABP or Impella.The model included comorbidities and the use of vasopressors as variables and estimated the probability of receiving Impella for a given patient.We created matched cohorts using propensity score matching to balance the differences in comorbidities between the two groups.After logistic regression, propensity matching was performed using a one-to-one scheme without replacement using the nearest neighbor matching and a caliper width of 0.05 on the probability scale.We followed the methodology for analysis recommended by the NIS-HCUP as described in previous studies and included in Supplementary Table 4 [10,16].All statistical analysis was performed with SAS 9.4 (SAS Institute, Inc., Cary, North Carolina).
Discussion
The present study compared in-hospital outcomes of MCS using IABP versus Impella in STEMI complicated by CS using the most recent national database.The main findings of our study are: (a) Impella was used in younger hospitalizations that were more likely to be male in Fig. 1.Flow chart for participant inclusion.
S. Thakkar et al. comparison to IABP (b) The use of IABP was 3.2 times more common than Impella in STEMI hospitalizations complicated by CS.(c) Impella was associated with higher in-hospital mortality, AKI and AKI requiring dialysis (d) Impella was associated with a higher incidence of ischemic and hemorrhagic stroke, gastrointestinal bleeding, blood transfusion, and acquired hemolytic anemia compared with IABP.(e) The devicerelated complications were also higher with Impella as compared with IABP.(f) Hospitalizations in the Impella cohort were associated with significantly higher total hospital charges compared with IABP.
Previous clinical trials and observational studies have failed to show a significant improvement in mortality in patients with acute myocardial infarction complicated by CS who received IABP versus Impella [5,6,[17][18][19].The major limitation was each of these randomized controlled trials (RCTs) enrolled a limited number of patients.Additionally, as noted many times in the past, it is exceptionally difficult to start an RCT in an emergency setting like STEMI CS.Basir et al. and colleagues from the Detroit Cardiogenic shock initiative revealed mortality rates as low as 28% in patients who received Impella [20], compared to 42% in the present analysis, which can be explained by the Impella use by the organized and standardized shock team approach which incorporates hemodynamics and real time multidisciplinary discussions as part of an algorithm, strict exclusion criteria including several conditions that are independently associated with increased inhospital mortality (i.e.unwitnessed arrest) designed for early response to MI and shock.However, it is not always the case in the real world, especially in small centers with limited pLVAD experience.Another plausible explanation for higher mortality in our study is that we only included patients with STEMI, compared to the study by Basir et al., which had both STEMI (71%) and NSTEMI (29%).Moreover, the 30-day mortality analysis of 237 patients from the IABP-SHOCK II trial revealed all-cause mortality in the Impella arm to be 48.5% which is even higher than our study [6].A 2018 meta-analysis of five randomized controlled trials and one observational study comparing IABP and percutaneous ventricular assist devices (Impella & TandemHeart) included a total of 674 patients and found no significant differences with regards to 30-day or long-term mortality [21].In a propensity-matched analysis utilizing the Premier Healthcare Database (2004-2016), an all-payer database representing approximately 20% of all acute care hospitalizations in the U.S., Amin et al. reported that Impella use was associated with a higher risk of in-hospital mortality compared with IABP in patients who underwent percutaneous coronary intervention requiring MCS [7].Similar to the findings by Amin et al., the use of Impella was associated with worse all-cause in-hospital mortality compared with IABP in STEMI patients complicated by CS in our analysis as well.
The use of IABP was associated with a lower incidence of AKI compared with Impella, likely due to the diastolic augmentation of renal perfusion by IABP, thereby reducing rates of pre-renal AKI.This could also explain the higher mortality with Impella than IABP, similar to that seen with radial vs. femoral access in STEMI [22].Another factor could be a higher volume of contrast exposure with Impella placement increasing the risk of contrast-induced nephropathy and AKI [23].A higher incidence of hemolytic anemia due to mechanical hemolysis seen in the Impella cohort could also potentially account for AKI rates which were also observed in the previous studies [19,24,25].Cerebrovascular accidents, including ischemic stroke, are rare with IABP since the balloon is positioned distal to the left subclavian artery, except if it is accidentally placed or migrates proximally [26].Impella placement, on the other hand, inevitably requires crossing the aortic arch and the aortic valve, which could potentially account for the higher rates of embolic stroke seen with Impella in our study.Adequate anticoagulation is a must with Impella to maintain the patency of the purge pathway in the event blood enters the motor, which is not the case with IABP where anticoagulation can be omitted as suggested in previous studies [27,28].This, along with a higher rate of vascular complications, could partly explain the higher rate of bleeding complications along with the higher incidence of blood transfusion with Impella in our study, similar to the retrospective analysis by Amin et al. [7].Access site-related hematoma/hemorrhage was more common with Impella use.This finding is intuitive as Impella requires a bigger access catheter size compared with IABP.The total hospital charges that were higher with the Impella group can be related to the higher cost of the Impella device and higher complication rates than IABP, as seen in this study [29].Amin et al. also found higher incremental hospitalization costs associated with Impella use in their analysis [7].
The present analysis has several limitations.The NIS database is an administrative database and hence subject to under-coding, over-coding, or erroneous coding.However, since coding is the means of obtaining reimbursement, the error in coding for procedures like IABP and Impella implantation is less likely.While the ICD-10 PCS code only provides codes for percutaneous left ventricular assist device (pLVAD) placement, of which Impella forms the majority, the effect of other pLVAD cannot be excluded from the present analysis.However, these coding strategies have been utilized in prior studies and hence are validated.Second, we couldn't assess the severity of CS and thus the acuteness of the clinical picture due to the inherent limitation of the database, which can have a significant effect on the selection of the device and in-hospital outcomes.Along with CS severity, the NIS cannot assess the timing of Impella placement since the earlier placement of Impella has been linked with better outcomes [30].Third, long-term outcomes in the two cohorts were not evaluated, which can substantially influence the choice of the MCS.Recent reports from registries like Abbreviations: AKI = acute kidney injury.Abbreviations: AKI = acute kidney injury.
the NCSI have reported significant improvements in survival rates in AMI CS patients when best practices like early initiation of MCS, following CS protocols, and large bore access care are followed.Whether the lack of such best practices and operator experience across multiple centers in the nation resulted in worse outcomes in Impella cohort cannot be addressed in a retrospective study.Fourth, there could be selection bias as this is not a randomized controlled trial, and the selection of the MCS devices may have been done at the operator's discretion.However, the use of Impella is expanding nationally, and our results do not support that this has been accompanied by improvement in outcomes.Standardization of CS protocols besides demonstration of benefit in randomized control trials is vital to improve outcomes in CS patients.The strengths of our analysis include using previously validated ICD-10 codes to identify conditions and propensity-score-matched analysis to reduce the effect of confounders.
In conclusion, the present analysis reported that in hospitalizations with STEMI complicated by CS, Impella compared with IABP was associated with higher in-hospital mortality, morbidity, and proceduralrelated outcomes.To further evaluate this, large-scale randomized studies are warranted to determine the effect of the Impella in this acutely sick population.Until then, the best approach would be to use team-based approaches to evaluate as much information as possible to help guide device selection on an individual patient basis, taking into account center and operator experience.
Table 1
Baseline characteristics of hospitalizations with IABP compared with IMPELLA placements.
Table 2
Baseline characteristics of hospitalizations with IABP compared with IMPELLA placements (Propensity Score Matched).
Table 3
Outcomes of hospitalizations with IABP compared with IMPELLA placements (Propensity Score Matched).
Table 4
Odds ratio for outcomes of hospitalizations with IMPELLA compared with IABP placements (Propensity Score Matched). | 2021-11-21T16:15:34.711Z | 2021-11-01T00:00:00.000 | {
"year": 2021,
"sha1": "72795052d0b74291a5f3206bacada1a6cc879775",
"oa_license": "CCBYNCND",
"oa_url": "https://doi.org/10.1016/j.ahjo.2021.100067",
"oa_status": "GOLD",
"pdf_src": "PubMedCentral",
"pdf_hash": "0fe403c410c2bbb1dd18b7eff323be2bbc188b54",
"s2fieldsofstudy": [
"Medicine"
],
"extfieldsofstudy": []
} |
232483110 | pes2o/s2orc | v3-fos-license | Long-range optofluidic control with plasmon heating
Using light to manipulate fluids has been a long-sought-after goal for lab-on-a-chip applications to address the size mismatch between bulky external fluid controllers and microfluidic devices. Yet, this goal has remained elusive due to the complexity of thermally driven fluid dynamic phenomena, and the lack of approaches that allow comprehensive multiscale and multiparameter studies. Here, we report an innovative optofluidic platform that fulfills this need by combining digital holographic microscopy with state-of-the-art thermoplasmonics, allowing us to identify the different contributions from thermophoresis, thermo-osmosis, convection, and radiation pressure. In our experiments, we demonstrate that a local thermal perturbation at the microscale can lead to mm-scale changes in both the particle and fluid dynamics, thus achieving long-range transport. Furthermore, thanks to a comprehensive parameter study involving sample geometry, temperature increase, light fluence, and size of the heat source, we showcase an integrated and reconfigurable all-optical control strategy for microfluidic devices, thereby opening new frontiers in fluid actuation technology.
4.
Only water was used for the flow studies in the manuscript. Cell culture media consists of many different types of proteins that must be flowed to cells. The authors should comment on how they believe their system would be applied to other biologically relevant media. This would strengthen the applicability of their work.
REVIEWER COMMENTS: NCOMMS-20-18044
Reviewer #1 (Remarks to the Author): The manuscript by Ciraulo et al. presents a method by which they induce a liquid flow due to the local heating of a thermoplasmonic substrate with gold nanorods, which is in contact with a liquid in a chamber of a few 10 µm in height. The authors measure the temperature distribution inside the fluid chamber as well as the local flow velocity by a 3D holographic tracking of particles. Fluid manipulation techniques that allow a freely configurable manipulation of liquids by means other than a mechanical actuation are powerful and very interesting, and the presented method of convection based fluidics belongs to that category. The new contribution is mainly the simultaneous measurement of 3D fluid flow and temperature distribution. Overall this is a nice experimental demonstration, which might be suitable for Nat. Comm. Before publication, however, I suggest addressing the issues listed below. As convection is a well-understood effect, the authors especially could be more quantitative in comparing their observations to expectations and discussing possible deviations.
We thank the reviewer for taking the time to evaluate our manuscript and provide insight to improve our work. The issues pointed out are addressed below: 1) I think the authors should work out in the introductory text the distinction between different thermally driven effects. As mentioned in the manuscript, different groups have shown in the past various types of motions driven by local heat sources. A part of these publications describes experiments that move objects in liquids, but not the liquid; others do move the liquid. Moving the liquid will always yield a long-range flow in the given geometry and is therefore not surprising.
We thank the reviewer for this suggestion and we have now clarified this point in the introductory text.
Action taken: We have added the following text to the introduction section: Thermal gradients, broadly speaking, alter the dynamics of the particles in solution at two distinct levels: at the particle and at the fluid level, respectively. At the particle level, the motion of objects in solution along or away the thermal gradient, thermophoresis, is determined by their interactions with the solvent and leads mostly to short-range motion 1 . At the fluid level, thermal gradients can induce long-range motion of particles by either convection or thermoviscous flow.
2) The group of Dieter Braun has been working with convective effects as well, including modeling of the thermogravimetric flows. Even though these experiments do not rely on localized heat sources, I think this work needs to be considered when addressing the characterization of convective flows. We refer the referee to response 2.2 to reviewer 2, where we detail the major differences between locally and uniformly heated systems. Unfortunately given the word-limit constraints of the article, we believe that this discussion is best addressed in the supplementary information.
Action taken: The following paragraph has been changed to make reference to the supplementary note and the following citations have been added: This phenomena, in the case of uniformly heated interfaces, has been previously observed in the seminal works from Braun and Mast et al [33][34][35] ; nonetheless, there are key differences in the system as a whole when localized heat sources are involved (Supplementary Note), which to the best of our knowledge have not been reported before.
Corresponding Supplementary Note added to the SI document:
Differences between locally and uniformly heated systems:
Focussing on the model flow cell system presented in our work, the first key difference between locally and uniformly heated systems is the resulting temperature field in the water layer, which determines the resulting fluid and particle dynamics. Here, a critical observation is that the heat diffuses through the surrounding water and glass sidewalls and dissipates to the environment (air) by natural convection from the glass surfaces.
In the case of a locally heated system, despite the low heat exchange rate of the external air convection, it is possible to reach a steady state temperature distribution thanks to the relatively large size of the thermal bath (surrounding glass and water) compared to the heat source. Thus, at steady-state, the temperature at any point X in the sample at a distance r much greater than the size of the heat source is well approximated as T0/r, where To, is the temperature at the heat source. In the case of a uniformly heated system 2,3 , there is no such additional thermal bath within the flow cell, so heat can only diffuse perpendicularly to the heated surface. Therefore, to obtain a steady state temperature field in the water layer, the system requires a heat sink on opposing sidewall, which can be achieved by either using a material with a very high thermal conductivity 4 (silicon, sapphire), and/or keeping the surface at a fixed temperature via active cooling. The resulting temperature field will be linear, with the distance from the heated surface all across the sample, i.e. proportional to r.
A second key difference is the nature of flow expected in the case of uniformly heated surfaces. In the parallel orientation, the fluid flows upwards close to the hot surface, and downwards close to the cold surface. In the perpendicular orientation, specifically when the system is heated from the bottom, convection arises from an instability when the Rayleigh number exceeds a critical value 5 . This type of convection goes by the name of Reynard-Bénard convection, and leads to the appearance of local convection cells located throughout the sample. Both these scenarios are very different from locally heated systems, such as those presented in the work.
A third key difference involves the thermal inertia of the system, which determines the cooling and heating dynamics, which in turn affects the particle and fluid dynamics. Namely, for locally heated systems, the thermal inertia is small, leading to faster heating and cooling dynamics, which result in a faster system response. This translates into greater reconfigurability and finer control compared to uniformly heated ones. As shown in Fig. S7, the onset of convection-based flow takes place at most within the first 10 s after heating A fourth key difference is the contribution from particle specific dynamics, which determine whether particles move along or against the thermal gradient 1 . These particle transport mechanisms depend on the temperature gradients, which in the case of locally heated systems are localized around the heat source and tend to be greater compared to the uniformly heated system. These particle specific dynamics are evident close to the heat source as shown in the spatial maps in Figure 3c.
To conclude, although locally and uniformly heated systems can achieve similar long-range fluid actuation, the underlying temperature field distribution, fluid transport mechanisms microscale dynamics, and conditions that lead to such behaviour are very different.
3) On page 9, the authors state that for a parallel orientation of the sample, the flow along the y-direction is caused by radiation pressure and thermo-viscous flow. According to Weinert et al., the thermo-viscous effects require a moving heat source, which is not valid in this case. How could the thermo-viscous effects then contribute?
We had initially considered thermoviscous flow as a possible contributor to the dynamics, if we looked at our system from the frame of reference of the steady state fluid moving upwards due to convection; i.e. the fluid is static and it is the heat source that moves downwards. However, the reviewer is indeed correct in pointing out our misunderstanding of the nature of thermo-viscous flow, which should not be valid along the y-axis. As a result, our phenomenological explanation is missing the dominant effect that contributes to the observed flow along the y-direction. This represents a deviation from the expected convection driven dynamics, which the simulations also fail to account for.
Action taken:
We have amended the referring text in page 9 to make the reader aware of this deviation, as follows: This behavior follows the same trend as Uz in Fig. 3b. yet we cannot account for the major phenomena responsible for it. On the one hand, no buoyancy forces act along this direction and the heat source is stationary, thereby ruling out convection and thermos-viscous flow. On the other hand, although radiation pressure acts along this direction, it is not the dominant effect for the observed dynamics as shown in Supplementary Fig. 5b. 4) A few sentences below, the authors state about the thermophilic nature of the tracer particles. As the particles should follow the flow (as ideal tracer particles), I would not call them thermophilic, since moving towards the heat source is not a property of the tracers but the flow.
Although the tracer particles do indeed follow the flow, this does not imply that they do not exhibit dynamics caused by thermal non-equilibrium conditions. At no moment do we claim that the tracers are ideal. In fact polystyrene particles such as the ones used in this work, have been reported to exhibit either thermophobic or thermophilic behaviour depending on the surface coating-solvent interactions and the temperature of the solvent itself 6 .
To ascertain experimentally whether the particles exhibit any significant contributions from particledependent dynamics (not flow) we minimize the convective-driven contribution by making the channel height from the flow cell as small as possible (20 μm) under the parallel orientation. Under these conditions ( Figure R1), we observe that the tracer particles migrate towards the heat source at low channel depths (0-5 μm) and especially near the heat source. However, at channel depths beyond 5 microns the directionality switches with the particles migrating away from the heat source. This behaviour is also visible in Figure 3c in the main text, for components Ux and Uz. This contrasts with our fluid dynamics simulations, where the dominant contribution is an overall upwards fluid motion regardless of the sample depth position or distance away from the heat source. A more detailed discussion is reported in response 1.7.
To summarize, upon more careful consideration of our data, we agree with the referee that we cannot claim that the observed particle behaviour is either thermophilic or thermophobic. Nonetheless, what we can indeed claim is that these observations arise from a significant contribution from dynamics at the particle level rather than exclusively at the fluid level.
Action taken:
The excerpt indicated by the referee has now been modified as follows: At sample depths below 5 µm, the flow is strongly focused towards the heat source analogous to the Ux component, indicative of significant contributions from particle driven dynamics. To remark, this non-trivial particle behaviour is also present in the perpendicular orientation.
Also we've adjusted the abstract to reflect the above point: Here we report an innovative optofluidic platform that fulfills this need by combining digital holographic microscopy with state-of-the-art thermoplasmonics allowing us to identify the different contributions from particle-specific dynamics, convection, and radiation pressure. 5) As the temperature field is known from the experiments, I would ask the authors to at least give an estimate of the thermophoretic velocity of the tracers. Similarly, there should be a way to estimate the velocity due to radiation pressure, which is mentioned to contribute to the dynamics. The thermophoretic velocity, ut, for colloidal particles under a temperature gradient, ∇ , is given as: where Dt, is the thermophoretic mobility which is related to the Soret coefficient, St, as follows = /D. From Fig. S2, we extract the D = 0.52 μm 2 s -1 . On the one hand, in the absence of any fluid motion contributions, one could estimate the thermophoretic velocity from the instantaneous displacements from particle trajectories upon suppressing the Brownian motion contribution as shown in Figure 3a. On the other hand the Soret coefficient can be approximated by determining Dt from the slope of a plot of ut vs ∇ . In the context of experiments, we decoupled the contributions attributed to the motion of the fluid by minimizing the convective-driven dynamics, i.e. by making the channel height as small as possible (20 μm) under the parallel orientation. Under these conditions, the velocity maps presented in Fig R1, would thus approximate the normalized thermophoretic velocity. Here the normalization is performed with respect to the maximum temperature change induced in the flow cell system. If we consider the data, where induce a maximum temperature difference of 43K, a plot of ut vs ∇ within the first 0-5 μm away from substrate with the heat source yields Figure R2 with Dt = -7.5 and -3.4 μm 2 s -1 K -1 ; and corresponding to = -14.4 and -6.5 K -1 , respectively.
One can immediately notice that such plot is dependent on the channel depth, and if we were to plot additional sample depth positions we would arrive to the same conclusion stated in response 1.4, namely that there is an inversion in the directionality of the particle flow. We believe that our data, taken under the given experimental conditions, does not provide sufficient insight at this stage to extract meaningful Soret coefficients or to assign whether there is a thermophobic or thermophilic behaviour, since the dynamics are the result of multiple contributing phenomena occurring in the presence of thermal gradients. Regarding the second point of the reviewer's comment, i.e. estimation of the velocity attributed to radiation pressure, we refer the referee to Figure S5 where we experimentally determined such contribution. Here the experiment is performed in the absence of any gold nanorods on the substrate and in a perpendicular geometry, therefore no thermally driven contributions are present. As a result, the only forces acting on the tracer particles are attributed to the pump beam illuminating the sample in a Koehler configuration, namely radiation pressure (Figure 5a, velocity component Uz) and optical gradient ( Figure S5a, velocity components Ux/Uy) . The average velocity due to radiation pressure is shown in Figure S5B alongside other contributions for comparison. In this experiment, we found the radiation pressure contribution to be approximately 0.7 um/s at an illumination power at the sample of 15 mW. 6) I find the notion parallel and perpendicular a bit confusing as the convective effects rely on the direction of the gravitational acceleration, and I would refer to this axis.
We thank the referee for this comment; however, we would like to point out that in the manuscript we explicitly defined the orientation of the system according to the reviewer's suggestion, specifically: "For this we consider two orientations: perpendicular (Fig. 3b) and parallel (Fig. 3c) with respect to the direction of gravity. By orientation, we use as a reference the plane at which the heat source is located."
Action taken:
In Figure 3b and 3c, we have moved the location of the arrow indicating the direction of gravity closer to the cartoon representation to clarify the convention taken in this work.
7) The authors mention theoretical predictions and also present numerical calculations of the flow in the supplementary information. I would have liked to see at least some of the data in SFig. 7 in comparison to the experimental data in the main text. Similar quantitative comparisons should be possible for the velocity along the substrate to show the scaling of the velocity with the distance from the heat source. I feel Fig. 4 can be condensed to contain this information.
Simulations of our system are consistent with the experimental data in the regime where convective flow is the main phenomena driving the underlying dynamics. This is best observed when we compare our empirical figure of merit, (<Uz>/<|U|>), between experiments and simulations. Here we assume that buoyancy driven dynamics is the major component in <Uz> . Looking closely at the simulations, we notice that <Uz> accounts for more than 90% of the observed velocity, regardless of sample depth position and chamber height. This discrepancy between simulations and experiments is not surprising, as it can be explained by the fact that the simulations do not capture the motion of the particles in response to thermal non-equilibrium conditions and repulsive interactions with the substrate; whereas the experiments do. Similarly, if we now plot the figure of merit as a function of the other two axes (xand z-axis), we again note a discrepancy between simulations and experiments close to the heat source. Nevertheless the mm-scale, long-range fluid actuation, remains comparable amongst simulation and experiments.
In short, the presence of a thermal gradient, gives rise to particle-specific dynamics that make the motion of particles in the fluid much more complex around the heat source as discussed in response 1.2. In our opinion the best way to capture such rich behaviour is through the spatial maps shown in Figure 3c. Furthermore, we would like to highlight that because the flow profile, Uz, is fairly homogenous along the x-z plane; it is thus possible to collapse the dominant flow profile behaviour to the plots shown in figure 4 and 5; which is not the case for the Ux and Uy components. We have focussed our attention to the convective-term because this is the driving mechanism behind long-range transport --the thesis of our manuscript.
Action taken:
Modified figure 4 and accompanying text in the main document Since buoyancy-driven convection is not the only active process affecting the non-equilibrium dynamics, we use an empirical figure of merit to quantify its overall contribution as a function of each axis position and channel height (Fig. 4c). 8) It would be nice if the authors could comment on the dynamics, e.g., the speed at which the convective flows built up.
Upon inspection of the videos provided as supplementary information, one can notice that the onset of the convective flow occurs within the first seconds upon heating. Although in principle this information can be obtained experimentally, in practice this requires either the collection of a large amount of data to segment the particle displacements as a function of time bins to obtain an equivalent signal to noise ratio. This arises from the fact that we rely on temporal and spatial averaging of particle displacements to suppress the Brownian contribution from the observed dynamics. As such, to address this comment we rely on simulations, which show excellent agreement with experiments in the regime where convection-driven dynamics dominate; and more importantly enable us to access early time dynamics, which are inaccessible in our system experimentally, given our limited to time resolution of 10 ms, provided by detector's frame rate.
We compare the temporal evolution of the dominant effect in these flow channels, which is the vertical flow, given by convective flow. From the simulations, we determine that the build up of the convective flow occurs within the first seconds after heating, and reaches a steady value within the first 10 s irrespective of the channel height in this work, in agreement with experimental observations.
Action taken:
We replaced Figure S7 with Fig R3 shown above, and make reference to it in the results section as follows: We also corroborate via simulations that the onset of convection occurs within seconds upon heating ( Supplementary Fig. 7) 9) It would also be nice to have the scale information for the velocity maps in Figure 3 b and c. Given that the experiments that led to Figure 3b and 3c had slightly different absolute temperature profiles, we opted for ease of comparison between different geometries, as stated in the caption, to normalize the corresponding spatial maps by the maximum temperature increase in the system.
Action taken:
We have added a label on the magnitude scale to clarify that the temperature and velocity components are normalized to the maximum temperature change of the system. Furthermore, we have added the missing scale bars for the spatial maps of 3b and 3c.
Reviewer #2 (Remarks to the Author): The authors reported an elegant approach to initiate large scale fluidic motion with a localized heat source by simply changing the orientation of the channel relative to the direction of gravity. The reported approach could be utilized to achieve fluidic pumping across a microfluidic network with controllable flow direction that is achieved by moving the position of the laser heating beam. The technical content of the work is well presented and includes simulation results that support the experimental findings. The work is an important contribution to the field of optofluidics and I believe that it is a good fit for Nature Communications. Below are some comments for the authors to address: We thank the reviewer for supporting the publication of our manuscript. The remaining concerns are addressed below:
1.) The authors should comment on what flow behavior would be expected if the channel was tilted say by 45 degrees instead of being vertical.
We have investigated via simulations given their excellent agreement with experimental results, what would occur if the microfluidic circuit was tilted by a certain angle. We focussed on two rotation axes, specifically along the x-and y-axis, which are illustrated in Figure R4.In both cases, the overall direction of flow is preserved for 45-degree rotations, although the magnitude is reduced by about 30%. The latter is to be expected when one decomposes the resulting flow velocities into their respective axis components <U x,Uy,Uz> and note that the predominant flow contribution, buoyancy-driven, is along the z-direction, so cos(π/4) ≅ 0.7 . A further 45-degree rotation in either cases, a total of a 90-degree rotation, leads to the behaviour reported for the perpendicular geometry, i.e. little to no overall fluid circulation. 2.) How is the flow field behavior described in this work using a focused heating source different from the case of a uniformly heated surface under the parallel configuration?
To address this question, we would first like to elaborate on the key differences that exist between the two systems. Focussing on the model flow cell system presented in our work, the first key difference between locally and uniformly heated systems is the resulting temperature field in the water layer, which determines the resulting fluid and particle dynamics. Here, a critical observation is that the heat diffuses through the surrounding water and glass sidewalls and dissipates to the environment (air) by natural convection from the glass surfaces.
In the case of a locally heated system, despite the low heat exchange rate of the external air convection, it is possible to reach a steady state temperature distribution thanks to the relatively large size of the thermal bath (surrounding glass and water) compared to the heat source. Thus, at steady-state, the temperature at any point X in the sample at a distance r much greater than the size of the heat source is well approximated as T0/r, where To, is the temperature at the heat source. In the case of a uniformly heated system 2,3 , there is no such additional thermal bath within the flow cell, and the heat can only diffuse perpendicularly to the heated surface. Therefore, to obtain a steady state temperature field in the water layer, the system requires a heat sink on opposing sidewall, which can be achieved by either using a material with a very high thermal conductivity 4 (silicon, sapphire), and/or keeping the surface at a fixed temperature via active cooling. The resulting temperature field will be linear, with the distance from the heated surface all across the sample, i.e. proportional to r.
A second key difference is the nature of flow expected in the case of uniformly heated surfaces. In the parallel orientation, the fluid flows upwards close to the hot surface, and downwards close to the cold surface. In the perpendicular orientation, specifically when the system is heated from the bottom, convection arises from an instability when the Rayleigh number exceeds a critical value 5 . This type of convection goes by the name of Reynard-Bénard convection, and leads to the appearance of local convection cells located throughout the sample. Both these scenarios are very different from locally heated systems such as those presented in the work.
A third key difference involves thermal inertia of the system, which determines the cooling and heating dynamics, which in turn affects the particle and fluid dynamics. Namely, for locally heated systems, the thermal inertia is small, leading to faster heating and cooling dynamics, which result in a faster system response. This translates into greater reconfigurability and finer control compared to uniformly heated ones. As shown in Fig. S7, the onset of convection-based flow, takes place at most within the first 10 s after heating A fourth key difference is the contribution from particle specific dynamics, which determine whether particles move along or against the thermal gradient 1 . These particle transport mechanisms depend on the temperature gradients, which in the case of locally heated systems are localized around the heat source and tend to be greater compared to the uniformly heated system. These particle specific dynamics are evident close to the heat source as shown in the spatial maps in Figure 3c.
To conclude, although locally and uniformly heated systems can achieve similar long-range fluid actuation in the parallel configuration, the underlying temperature field distribution, fluid transport mechanisms microscale dynamics, and conditions that lead to such behaviour are very different.
Action taken: A Supplementary Note detailed the above discussion has been added in the supporting information document.
The manuscript presents a carefully constructed experimental and numerical study of multiparameter, multiscale (micro-to mm) thermally driven fluid and particle transport. The authors argue that, unlike previous studies that focus on local fluid/particle manipulation over short ranges, their approach permits control over short and long range. The novelty in their work is two-fold. First, it is the unique combination of a thermoplasmonic platform comprising Au nanorods with a digital holographic microscopy platform, as well as 3D particle tracking, to systematically study the effects of thermal perturbation on fluid dynamics from micron to mm-scales. Secondly, the authors show the effects of orientation of a thermoplasmonic platform, either perpendicular or parallel to gravity, on fluid dynamics. The manuscript is well written and interesting. For consideration of publication the authors should address the comments below.
We thank the reviewer for supporting the publication of our manuscript. We address the remaining concerns point-by-point below: 1. Digital holography, and the use of an off-resonance beam, is used to map the temperature-dependent refractive-index profile of the fluid. However, there is often a modulo-2*pi phase ambiguity using such interferometric methods. The authors should explain how they calibrated for this effect, at least in the Methods section.
The reviewer is indeed correct in pointing out the intrinsic issue of phase unwrapping associated with interferometric based methods. Nonetheless, these issues are only present when the measured phase difference is greater than 2π. In all the experiments reported in this manuscript, given the weak thermooptic coefficient of water and the relatively small height of the chamber, we determine the optical phase difference attributed to a thermal perturbation to be below 2π even for temperature changes approaching 100°C, which is consistent with previous works 7,8 . Therefore, there is no need to calibrate for this effect. In addition, we provide the reviewer with the following plot that compares the maximum phase difference detected as a function of light fluence on the sample, alongside the retrieved temperature increase that was presented in Figure 5a in the main text. Action taken: We added the following segment into the corresponding methods section.
"In all the experiments reported here, given the weak thermo-optic coefficient of water and the relatively small height of the chamber, the measured phase difference was smaller than 2π, even for temperature changes approaching 100°C. As a result there are no issues regarding phase unwrapping that are intrinsic to off-axis digital holography" 2. The authors would benefit from better articulating the potential applications of their work. If the work is truly for lab-on-a-chip applications, which is often thought of in a biological context, then it should explain, at least briefly, how it translates to biologically relevant media.
We appreciate the referee for pointing this out and allowing us to improve the visibility and applicability of our work.
In general, by using more complex media, such as biologically relevant ones, one is bound to introduce additional contributions affecting mass and fluid transport in thermal non-equilibrium systems. As a result, the particle and fluid dynamics become more complex, and there are additional parameters available to tune the system 1 . For instance, the presence of ions with different thermal diffusivities could lead to the optothermoelectric effect recently reported 9 , whereby a local electric field is induced by charge separation of these ions in solution. This in turn leads to enhanced particle motion for electrically charged species in solution.
Irrespective of how complex the system may be, the platform and analytical tools presented here, allow a detailed characterization of the thermal non-equilibrium dynamics of the system based on relatively straightforward and short experiments. This on the one hand enables to study systems that may be too complex or too computationally demanding to simulate. On the other hand, it allows to fully account for experimental parameters that are often ignored in simulations such as radiation pressure, sedimentation, and in situ temperature profile.
In this work, we mainly focussed our attention to a simple model system to appeal to the broad readership of the journal. Yet, in response to the referee's suggestion, we performed additional experiments using either a phosphate buffer saline (PBS) solution or cell culture media as the aqueous media using polystyrene tracer particles; and cells rather than polystyrene tracer particles. In these experiments, we observed no significant differences in the dynamics of the tracers particles to those obtained with a just water. Likewise, the cells behaved similarly to the tracer particles with the exception that there is a considerable contribution due to sedimentation. Together these observations pave the way towards labon-a-chip applications, as the conclusions derived from the model water-based system can be equally extended to biologically relevant media.
Furthermore we refer the reviewer to a list of possible applications stated at the end of the discussion: "As such, we envision that plasmonic-based heating together with the platform presented in this work will serve as a foundational basis for the development of new technologies that either require fast response times, high and rapid reconfigurability, or involve the delivery, transfer or manipulation of temperature sensitive samples such as proteins, DNA or cells from micro-to mm-length scales." Action taken: We added the following segments to the main text: Results section: Fluid dynamics characterization and influence of sample orientation with respect to gravity To verify that this observation is also applicable to more biologically-relevant media, we performed additional experiments using either phosphate buffer saline solution or cell culture media as the aqueous media, and observed no significant differences in the dynamics of the tracer particles (Supplementary Movie S3). We also observe a similar behavior if we swap the polystyrene tracer particles for cells, with the caveat that the cell dynamics exhibit a significant sedimentation contribution (Supplementary Movie S4).
Discussion section: Furthermore, we highlight that the general dynamics studied in detail for our model system also apply to biological compatible aqueous media and systems, thereby making our platform suitable for lab-on-a-chip applications.
Materials and methods section:
For experiments shown in Movie S3, the tracer particles were diluted in either phosphate buffer saline solution or Eagle's minimum essential medium; both at pH=7.4. For the experiments shown in Movie S4, we used human HEK293 cells obtained from American ATCC Cell Line Center (CRL-1573), which where diluted in Eagle's minimum essential medium.
Added Supplementary Movie S3 and S4, and adjusted the ordering of the remaining movies accordingly.
Movie S3.
Effect of different aqueous media on the fluid dynamics for a sample orientated parallel to gravity and with a depth of 100 μm. Scale bar: 10 μm. Movie S4. Evidence for hydrodynamic-based cell manipulation with a localized thermal gradient when the is sample orientated parallel to gravity and with a depth of 100 μm. Scale bar: 10 μm.
3. Related to comment #2 above, such platforms like the one the authors describe could potentially be really useful for cell manipulation. Thus, the authors should comment on how their platform might perform when applied to cells, which are much larger (> 20 microns) and deformable than the tracer particles used in their study. Can they manipulate cell trajectories?
We thank the reviewer for asking this question. As detailed in the response above (response 3.2), we performed experiments in the parallel configuration using cell culture media and cells. In short, we show that the platform can indeed manipulate cells (See Supporting Movie XX). Interestingly, cells given their greater mass, experience a non-negligible sedimentation contribution that acts in the direction of gravity and is thus opposite to the convective-driven flow. As such, depending on the location of the cell with respect to the depth of the flow chamber, its mass and the applied temperature difference, one can either slow down, cancel, or even overcome the sedimentation contribution; thus leading to interesting applications regarding cell manipulation using hydrodynamic forces. However, we would like to stress that a detailed account of the cell manipulation mechanism is beyond the scope of this work, and we consider it better suited if its covered in a separate manuscript, which is in preparation.
I thank the authors for their revision and response to my comments and questions. I am actually satisfied with most oft the responses and modifications the authors have made.
Yet, I still have a question/comment concerning the response to comment 1.4 and 1.5.
The authors describe in their response the inversion of the direction of motion of the tracer particles with respect to the heat source, when changing the cell height to exclude convective effects. They report the variation of an apparently effective Soret coefficient. For small cell height (<5 um), the tracers seem to be attracted to the heat source, while they are repelled if the sample height get larger than 5 um. If I understand the observations and reference 18 correctly, then this seems to comply with an interfacial thermo-osmotic flow setting up a flow field in the cell together with the thermophoretic effects. It would be good if the authors consider such contributions in their discussion, especially as they are still missing a contribution and the corresponding paper is actually cited already.
Reviewer #2 (Remarks to the Author): I thank the authors for addressing my comments. I am satisfied with the revised version of the manuscript.
Reviewer #3 (Remarks to the Author): The authors have sufficiently responded to my concerns. I recommend the manuscript for publication in Nature Communications.
REVIEWER COMMENTS: NCOMMS-20-18044
Reviewer #1 (Remarks to the Author): I thank the authors for their revision and response to my comments and questions. I am actually satisfied with most of the responses and modifications the authors have made.
Yet, I still have a question/comment concerning the response to comment 1.4 and 1.5.
The authors describe in their response the inversion of the direction of motion of the tracer particles with respect to the heat source, when changing the cell height to exclude convective effects. They report the variation of an apparently effective Soret coefficient. For small cell height (<5 um), the tracers seem to be attracted to the heat source, while they are repelled if the sample height get larger than 5 um. If I understand the observations and reference 18 correctly, then this seems to comply with an interfacial thermo-osmotic flow setting up a flow field in the cell together with the thermophoretic effects. It would be good if the authors consider such contributions in their discussion, especially as they are still missing a contribution and the corresponding paper is actually cited already.
We would really like to thank the reviewer for this comment as it has helped us unravel the different contributing phenomena to the observed dynamics. The reviewer is indeed correct in pointing out the presence of thermo-osmotic flow contributions at the glass water-interface, which we had erroneously overlooked in our initial analysis. This phenomena together with a description of the thermophoretic behaviour of the tracer particles used in our assays now allows us to fully explain the observed fluid and particle transport.
To confirm the presence of thermo-osmotic flow, we first determined the thermophoretic nature of the tracer particles. To do so we performed long term assays with thin flow cell channels (50 µm) in the perpendicular configuration to highlight whether the concentration of particles around the localized temperature profile increased or decreased, corresponding to either thermophilic or thermophobic behaviour 1 . These experiments confirmed that our tracer particles are indeed thermophobic and are presented in the form of a supplementary video (Movie S2). Therefore the in-plane motion of particles towards the heat source in the proximity of the interface can't be assigned to particle driven dynamics. Instead this motion fits the description of thermo-osmotic flow, which stems from presence of a solid boundary (for instance glass water interface) and a thermal gradient; both of which are present in our model system. Specifically our system counts with a glass substrate that has a uniform coating of PSS (see Material and Methods section), a polar, ionic molecule that give rise to an electric double layer in the water, hence creating very similar experimental conditions to that of a bare glass conditions shown in the original reference 2 .
As a consequence of thermo-osmosis, the inwards flow of the liquid towards the heat source must, by mass conservation, lead to outwards flow of the fluid at the centre of the heat source in the perpendicular. This latter contribution accounts for the missing contribution in the U y component in Figure 3c. Also, both thermo-osmotic and thermophoretic transport mechanisms are expected to be more relevant at lower sample depths because of the closer proximity to the heat source, which results in greater thermal gradients and a reduction in the convection contributions. Our experiments agree with this as evidenced in Fig. 3b and 3c for sample depths below 5 µm.
To demonstrate that thermo-osmotic flow, convective flow and thermophoresis are competing transport mechanisms at low sample depth, we present a supplementary movie (Movie S4) where the balance the balance of the three contributions leads to an accumulation of particles below the heat source.
Action taken:
We have modified the section of Fluid dynamics characterization influence of sample orientation with respect to gravity of the main text as follows: The intensity maps for the U z component as a function of sample depth describe a strong upwards flow in the volume immediately above the heat source that decreases and spreads out with increasing sample depth. Surrounding this volume, there is also a significantly weaker flow in the opposite direction. Ultimately these intensity maps capture the dynamics observed in Movie S1, which are primarily dominated by convection and thermo-osmotic flow. Furthermore, experiments probing longer time-scales, show a depletion in particle concentration around the induced temperature field, indicative of thermophobic behaviour 3 (Movie S2).
Changing the sample orientation to a parallel configuration leads to a significant change in the dynamics ( Supplementary Fig. 6, Movie S3), namely, an overall upwards motion of the particles. In this configuration, the intensity maps represent slices along the XZ plane, while the sample depth is directed along the Y-axis (Fig. 3c). For the U x component, the velocity distribution follows the same trend as in the perpendicular arrangement, with flow towards the heat source at short depth-wise distances away from it, followed by a reversal in direction at depths beyond 10 µm, as expected from thermo-osmotic flow 18 . The U y component exhibits a strong flow perpendicularly away from the heat source that decays and spreads with increasing depth. This behavior is similar to U z in Fig. 3b., yet no buoyancy forces act along this direction and the heat source is stationary, thereby ruling out convection and thermo-viscous flow.
Although radiation pressure also acts along this direction, it is not the dominant effect ( Supplementary Fig. 5B); thus leaving thermophoresis and thermo-osmosis as the main contributors. The U z component distribution captures the greatest difference between the two orientations. Namely, at sample depths below 5 µm, the flow is strongly focused towards the heat source analogous to the U x component; whereas, above 5 µm, an asymmetry develops leading to a strong upwards flow. This upwards flow dominant across the field of view for depths above 10 µm and reaches a maximum at 20 µm. Contrary to the perpendicular orientation, this flow extends over an area much larger than the heat source; thereby making it a more suitable orientation for fluid actuation.
In summary, the dynamics in the parallel configuration result from the superposition various competing phenomena ( Supplementary Fig. 7), with the three main contributors being: convection, responsible for the overall upwards motion (U z ); thermo-osmosis, responsible for the short range in-plane movement of fluid towards the heat source at low sample depths (U x and U z ), which by mass conservation leads to fluid flowing along the optical axis away from the heat source (U y ); and thermophoresis, responsible for the short range movement of particles away from the heat source. Under particular experimental conditions these three phenomena can lead to regions with no net motion of the tracer particles as shown in Movie S4, where particles accumulate below the heat source as a result of thermophoresis counteracting out the convection and thermo-osmosis contributions. | 2021-04-02T06:17:55.014Z | 2021-03-31T00:00:00.000 | {
"year": 2021,
"sha1": "cd38b10599798d0661753d35aa542344793a7438",
"oa_license": "CCBY",
"oa_url": "https://www.nature.com/articles/s41467-021-22280-3.pdf",
"oa_status": "GOLD",
"pdf_src": "PubMedCentral",
"pdf_hash": "d5d79c29dac2da1d39c0152c211e31bea0fc4fca",
"s2fieldsofstudy": [
"Physics"
],
"extfieldsofstudy": [
"Medicine"
]
} |
2004781 | pes2o/s2orc | v3-fos-license | Determination of the Processes Driving the Acquisition of Immunity to Malaria Using a Mathematical Transmission Model
Acquisition of partially protective immunity is a dominant feature of the epidemiology of malaria among exposed individuals. The processes that determine the acquisition of immunity to clinical disease and to asymptomatic carriage of malaria parasites are poorly understood, in part because of a lack of validated immunological markers of protection. Using mathematical models, we seek to better understand the processes that determine observed epidemiological patterns. We have developed an age-structured mathematical model of malaria transmission in which acquired immunity can act in three ways (“immunity functions”): reducing the probability of clinical disease, speeding the clearance of parasites, and increasing tolerance to subpatent infections. Each immunity function was allowed to vary in efficacy depending on both age and malaria transmission intensity. The results were compared to age patterns of parasite prevalence and clinical disease in endemic settings in northeastern Tanzania and The Gambia. Two types of immune function were required to reproduce the epidemiological age-prevalence curves seen in the empirical data; a form of clinical immunity that reduces susceptibility to clinical disease and develops with age and exposure (with half-life of the order of five years or more) and a form of anti-parasite immunity which results in more rapid clearance of parasitaemia, is acquired later in life and is longer lasting (half-life of >20 y). The development of anti-parasite immunity better reproduced observed epidemiological patterns if it was dominated by age-dependent physiological processes rather than by the magnitude of exposure (provided some exposure occurs). Tolerance to subpatent infections was not required to explain the empirical data. The model comprising immunity to clinical disease which develops early in life and is exposure-dependent, and anti-parasite immunity which develops later in life and is not dependent on the magnitude of exposure, appears to best reproduce the pattern of parasite prevalence and clinical disease by age in different malaria transmission settings. Understanding the effector mechanisms underlying these two immune functions will assist in the design of transmission-reducing interventions against malaria.
Introduction
Plasmodium falciparum malaria continues to be a major cause of human morbidity and mortality, especially in Africa, but varies greatly in endemicity across the continent and elsewhere [1]. The consequent variation in levels of acquired immunity and age-specific disease patterns complicates malaria epidemiology and means that control policies that are optimal for one setting are not easily translated to other settings. In highly endemic areas where clinical immunity develops rapidly [2]; there is concern that interventions which reduce transmission could also affect the development of immunity [3][4][5][6]. A delay in the acquisition of immunity beyond early life has the potential to change the spectrum of serious clinical symptoms [7,8] and the lifetime risk of disease [4].
While the processes that determine the acquisition of immunity to P. falciparum clearly impact on the epidemiology of the disease, they are complex and poorly understood due to the unclear relationship between immunological markers and functional immunity [9][10][11]. However, there is evidence to suggest that both clinical (anti-disease) immunity and antiparasite immunity develop at different rates. For example, in people who emigrate from malaria endemic settings, clinical disease appears to emerge only in those who remain away for at least 3-5 y [7,12]. Furthermore, these emigrants also present clinically with lower parasite densities than those who travel from non-endemic areas, suggesting that an additional component of immunity that regulates parasite densities may be longer-lived. This hypothesis is also supported by analysis of age-stratified anti-malarial antibody seropositivity rates which gives estimates of half-lives that span decades [13]. There is also evidence to suggest that acquired immunity does not only depend on exposure but is also influenced independently by age. For example, there is evidence for an age-dependent exposure-independent maturation of the antibody response to malaria [14], and this may in part explain the observation that the proportion of severe malaria cases presenting with severe malarial anaemia is more closely associated with age than with transmission intensity [15,16]. Immune responses which affect subpatent parasitaemia may influence malaria transmission, but high rates of subpatent infection in high transmission areas suggest that acquired immune mechanisms capable of complete parasite clearance rarely develop in naturally exposed populations, so we allow for the possibility of subpatent tolerance.
Here we develop a mathematical model to better understand the impact of the development of immunity on observed epidemiological patterns, and also aspects of the immunology which might be inferred from the epidemiology such as time scales of acquisition and loss. Whilst a number of malaria transmission models have been developed in the past which incorporate immunity [17][18][19][20][21][22][23][24][25], each do so in different ways and hence make comparison between model structures difficult. In contrast, we systematically explore the impact of immune responses at different points of the host's natural history of infection which are then tested by comparing model output with epidemiological observations. Our results demonstrate that more than one type of age-and transmission intensity-specific response are necessary to predict malaria epidemiological patterns, in line with current immunological understanding [7,9,10,26]. (B) Clinical episodes by age and altitude for region 2 (Usambara mountains) from severe malaria admissions to district, regional, and referral hospitals. (C,D) Prevalence of parasitaemia by age, year, and season (wet/dry) from North Bank (C) and South Bank (D) of River Gambia. doi: 10
Author Summary
Whilst it is clear that natural immunity to malaria infection develops in those living in malaria-endemic regions of the world, the precise way in which it is acquired and the duration of immune memory are less-well-understood. We used a mathematical model that mimics malaria transmission between humans and mosquitoes in endemic settings to explore what epidemiological data, and in particular the prevalence of malaria in different aged individuals, can tell us about how immunity might develop. We explored three different parts of the transmission cycle at which immunity could act: 1) reducing the likelihood that an infected person develops symptomatic disease; 2) increasing the rate at which infection is cleared, and 3) increasing the duration of low-level (subpatent) infections that would continue to boost the immune system and hence protect against further disease. Our results show that the first two mechanisms together give rise to patterns of malaria by age group that are consistent with those observed in different malaria endemic settings in Africa. Our model also suggests that immunity to symptomatic disease lasts for at least five years, develops faster if there are higher levels of infection in the population, and increases with age. On the other hand, our model suggests that immunity that helps to clear infection lasts longer (20 years or more), develops later in life, and does not depend on the amount of transmission in the population.
Immunity Functions Required To Reproduce Observed Age-Prevalence Patterns
We first developed an age-structured transmission model for malaria in which acquired immunity acts at three different stages of a host's history of infection: 1) susceptibility to symptomatic disease (severe and clinical cases) upon infection or re-infection, assuming susceptibility decreases with cumulative exposure to infectious bites (e.g., as a result of antibodymediated strain-specific immunity); 2) natural recovery from ; (E,F) immunity acting on clearance of detectable parasites (immunity function 2); (G,H) immunity acting on susceptibility to clinical disease (immunity function 1); (I,J) immunity acting on clearance of detectable parasites and susceptibility to clinical disease (immunity functions 1 and 2). Parameters are as shown in Table 1. doi:10.1371/journal.pcbi.0030255.g002 asymptomatic to undetectable infection (i.e., effective clearance of parasites), which increases with cumulative exposure to infectious bites after a delay during childhood representing maturation of the immune system, 3) natural clearance of undetectable subpatent infection, assuming increased tolerance and slower clearance of such infection.
Each response, which we call an immunity function, is allowed to change with age and malaria transmission intensity (commonly expressed as the entomological inoculation (EIR)) and hence represents the acquisition and loss of immunity dependent upon exposure. The first two immunity functions incorporate a memory component (i.e., allow for gradual loss in the absence of reinfection) [27], whereas the final immunity function (associated with regulation of parasite density) is assumed independent of acquired immunity, as subpatent parasites (if any) are kept subpatent by an effective immune response. Figure 1A shows the patterns of parasitaemia and clinical disease by age observed in northern Tanzania. These data were collected from 24 villages at three different altitude levels (,600 m, 600-1200 m, and .1200 m) and in two different regions [28]. In one of the regions (region 2), estimates of malaria transmission intensity as measured by the EIR were also collected. These varied by altitude with the highest transmission intensity occurring at low altitude (56 infectious bites per person years (ibbpy), range 28-108 at ,600 m, 3 ibppy, range 0.4-7.6 at 600-1200 m, and 0.12 ibppy, range 0.01-0.032 at .1200 m). Although these data were not available in the other region, the patterns of parasite prevalence by age and altitude are similar. Clinical data from severe malaria admissions to district, regional, and referral hospitals serving the Usambara mountain region (region 2) are shown in Figure 1B [15]. Figure 1C-1D shows the prevalence of parasiteamia by age in locations on the north bank and south bank of River Gambia, The Gambia [29]. Transmission in The Gambia is highly seasonal, and transmission intensity differs between the settings with higher intensity on the south bank. The estimates are presented separately for the dry and wet seasons, with higher prevalence observed during peak transmission in the wet season. The corresponding patterns predicted by different versions of the model are shown in Figure 2. If the model does not incorporate immunity at any point, we observe a rise in the prevalence of parasitaemia or clinical disease which saturates at older ages (Figure 2A and 2B). This clearly does not match the decline in both parasitaemia and clinical disease at older ages observed in data ( Figure 1). Allowing the model to incorporate immunity that results in increased persistence of subpatent infections (immunity function 3) gives rise to profiles that either peak too early in life and decay too rapidly at high EIRs or which saturate for low EIRs ( Figure 2C and 2D). Allowing the model to incorporate immunity resulting in more rapid recovery from asymptomatic infections or symptomatic disease (immunity function 2) gives rise to patterns of parasitaemia that match those observed reasonably well. However, the patterns of symptomatic disease decay too slowly with age ( Figure 2E-2F). Finally, allowing the model to incorporate immunity that reduces the proportion of infections that result in clinical disease (immunity function 1) results in patterns of clinical disease that closely match those observed in the data but fails to reproduce the decline in parasitaemia with age ( Figure 2G-2H). Other discrepancies between the model predictions and observed patterns of parasitaemia and disease by EIR and inconsistencies in lifetime episodes were also observed for each immunity function (see Protocol S1). We next considered combining the different functions to identify which combination best reproduces the observed age-prevalence patterns in Figure 1. Combining immunity functions 1 and 2 (i.e., allowing a reduction in the proportion of infections that give rise to clinical disease and an increase in the rate of recovery from asymptomatic infection to subpatent infection) reproduces well the age-prevalence of parasitaemia and severe disease observed in the study data ( Figure 2I and 2J). It also reproduces the observed decrease in clinical cases in older ages as the EIR is increased (see Protocol S1). Adding the third immunity function (increasing persistence of subpatent infection) results in patterns that more closely resemble those observed if this function alone drives immunity ( Figure 2C and 2D) and therefore lessens the agreement between model predictions and observed data.
Improved Model for the Impact of Immunity on Recovery from Asymptomatic Infection The age-prevalence patterns in Figure 2I and 2J resemble but do not exactly match those observed in data ( Figure 1). There are many reasons for not expecting an exact match: estimates of EIR are imprecise, and quoted values are averages over surveys and locations within altitude ranges; there may be random variation and unaccounted factors, such as bias in data sampling among age groups; and parasite density and detection at a given age may differ among sites. However, we note that the model predicts age-parasitaemia curves which saturate with age for medium-to-low EIR, which is not observed in data. Adjusting parameters does not seem to alter this feature. However, if natural recovery from infection (e.g., from asymptomatic to subpatent) is solely determined by age (via physiological processes, provided there is exposure on which infection is conditional), we obtain patterns closer to those observed ( Figure 3). This suggests that parasite immunity in non-naïve individuals may be controlled by physiological development rather than by the amount of natural exposure (provided there is exposure) [7][8][9]14,15,30].
Patterns of Infectivity by Age
An alternative way of testing the immunity functions (conditional on the remaining model structure and assumptions being valid) is to compare the predicted mean (A) Predicted infectivity by age from the model with different immunity functions. If1¼ immunity function 1 (susceptibility to clinical disease); If2 ¼ immunity function 2 (clearance of detectable parasites); If3 ¼ immunity function 3 (clearance of subpatent infection), If2* denotes EIR-independent version of If2. Parasitaemia is calculated in the model as symptomatic cases plus asymptomatic infections (D H þA H ). All runs assume an annual EIR ¼ 40 ibppy and that parameters are as before (Table 1), except c D is adjusted (for If2 and If3) to make comparable the curves corresponding to different immunity function models. Table 1 infectivity by age, which may be regarded as the probability of carrying gametocytes (although not all gametocyte carriers will be infectious), with the observed age-prevalence of gametocytes. The patterns predicted by our best model (incorporating immunity functions 1 and 2) closely match the patterns observed in northern Tanzania and The Gambia ( Figure 4). Since the model parameters were fixed or fitted to asexual parasite data, these results are an independent test of the model's ability to reproduce observed epidemiological patterns.
Duration of Clinical and Parasite Immunity
Our determined half-lives of clinical and parasite immunity were 5 y and 20 y, respectively. By varying these parameters, we explored whether patterns of age-prevalence can inform possible bounds for these parameters.
Reducing the half-life for the duration of clinical immunity below 5 y results in a sharp increase in the proportion of all infections that are symptomatic cases and, in addition, results in less-pronounced age-prevalence peaks which begin to deviate from those observed in data. Increasing the duration of clinical immunity does not substantially change age-prevalence patterns but does have an impact on the proportion of infections that are symptomatic cases ( Figure 5A and 5B).
Reducing the half-life for the duration of parasite immunity below 20 y similarly has an impact on the ageprevalence curves and at very low values (,10 y) gives rise to curves that saturate rather than decline at older ages. The proportion of infections that are asymptomatic and parasitaemic is also increased. However, increasing the duration of parasite immunity has little impact on either outcome ( Figure 5C and 5D).
Discussion
Our results demonstrate that, while distinct models can explain patterns of parasitaemia observed in individuals aged 0-5 y, in order to reproduce full age-prevalence patterns of parasitaemia and clinical disease observed in endemic malaria settings at least two distinct acquired immunity processes are required: 1) an early age (or early exposure) reduction in clinical susceptibility, and 2) a process of parasite immunity that increases the rate of natural recovery from infection and which develops substantially later in life (late childhood to adolescence). Adopting one of these processes in isolation does not reproduce observed patterns of age-prevalence of asexual parasitaemia, disease, and infectivity (gametocytaemia) across different endemicities (as measured by EIR). Moreover, while both clinical and parasite immunity were allowed to vary with age and EIR, the model in which natural recovery from infection (e.g., asymptomatic to subpatent) is determined solely by age better matches observed patterns than a model in which this is also determined by the intensity of exposure (EIR). This suggests parasite immunity in non-naïve individuals may be controlled by physiological processes rather than by amount of exposure (provided there is exposure). These findings agree with the current view that parasite immunity may require ageing to develop, but subsequently can persist without high antibody titres and therefore be maintained by occasional infrequent boosting [7][8][9]. Peaks in parasitaemia above 30 y of age present across endemic levels in eastern Tanzania might reflect malaria-HIV co-infection [31] and are not expected to be captured by the model. Incorporating a prolonged duration of (subpatent) infections, i.e., continual reinfection that prolongs infection and boosts an immune response that allows parasitaemia to persist at subpatent levels, worsened the model predictions. However, we cannot exclude that an overall immunemodulated increase in duration of infections takes place, as suggested by recent hypotheses from within-host models [32] and in transmission models with fewer immunity components [17,18]. This is because the increase in duration of subpatent infection with increasing EIR could be weaker than considered by us. Furthermore, interpretation of this immunity function may depend on our model structure: we assume (via immunity functions 1 and 2) that a host returning to the noninfected state (S H ) is likely to rapidly become asymptomatic with subpatent parasitaemia upon reexposure (i.e., is immune to symptomatic and to patent asymptomatic infection), tantamount to frequent subpatent infection but with recovery and reinfection modelled explicitly. Other models [17,18] assume persistent asymptomatic infection (though patency status may not be specified) which may be regarded as an implicit way of modelling this reinfection cycle.
Our model additionally allowed us to explore what ageprevalence patterns can tell us about the duration of clinical and parasite immunity. Our results suggest that clinical immunity has shorter memory (with a half-life of the order 5 y or more), while parasite immunity is effectively everlasting (with a half-life of 20 y or more after onset in adolescence). These durations are in line with evidence that migrating adults returning to endemic areas tend to become more sensitive to clinical attack but have lower parasite levels than children [8]; they are also in line with immunological studies in which one postulated mechanism of clinical immunity (antibodies to parasite phospholipids) has been shown to have a rather short half-life [33,34].
There are limitations in the epidemiological data that are available to inform model parameters. In particular, there are few and uncertain estimates of EIR by altitude range [28,35], as mentioned earlier. Furthermore, EIR estimates were not obtained from the same villages that were parasitologically surveyed, and the local history of interventions (which might affect the EIR) is not known. Therefore, discrepancies between observed and estimated EIR values are to be expected, especially in low-transmission areas where mosquito sampling is more difficult.
The model presented here clearly makes a number of simplifying assumptions. One of the main limitations is that the immunity functions, whilst generated based on current immunological understanding, could not be constrained by data. Further data on the way in which immunity develops and on the factors driving its development could help to refine these functions. The model also does not allow for partial immunity to reinfection, which would be relevant from the point of view of treating or vaccinating against preerythrocyte stages. While sterilising or partial pre-erythrocyte immunity are likely to be rare [7], it could be useful to extend the model to explore this possibility. Thirdly, we have not explicitly modelled the effects of parasite genetic diversity and have thus, strictly speaking, treated infections as monoclonal. However, the widely accepted hypothesis that immune development is regulated by antigenic variation and cumulative exposure to inoculations of differing parasite strains [20,22,26,32,36] is analogous to our definition of immunity levels in terms of cumulative exposure with finite memory. Our model is therefore consistent with theories in which immunity is strain-specific whilst integrating other aspects of acquired immunity development supported by cross-sectional data and current immunological understanding.
This age-structured malaria transmission model shares many features with existing models [17][18][19][20][21][22][23][24][25] but is novel in the K H /K M is the force of infection on the human and mosquito populations, respectively, 1/h is the mean latent period in humans, 1/g the mean latent period in mosquitoes, / is the proportion of human infections that develop disease, f the proportion of symptomatic cases that receive effective drug treatment, r T the rate of recovery on treatment, r D the rate of recovery without treatment, r A the rate at which asymptomatic infections become subpatent, and r U the rate at which subpatent infections are cleared. The coloured circles denote the stages at which acquired immunity can have an effect (modifying /, r A , and r U ). The parameters and their values are described in Table 1 way it combines epidemiological and immunological processes. Previous models have considered immune responses of types similar to those studied here (especially immunity that acts on the duration of asymptomatic and subpatent infections) [17,18,21,22,24], whilst others have represented acquired immunity through increased ability to reduce blood-stage parasite density [18,22,23,25]. Clearly, it is never possible to determine whether the structural assumptions behind any model represent the true processes generating the observed data, and it is likely that more complex model structures could also generate similar patterns. One alternative method that could be employed is to track parasite density rather than infection alone. Such an approach explicitly acknowledges variation in parasite load between individuals, and this variation may influence the development of immunity. However, such an approach also has its limitations. In particular, the distinction between disease and asymptomatic and subpatent infection requires definition of arbitrary parasite density thresholds for becoming diseased once infected and for detection by microscopy. Our assumption that susceptibility and recovery vary continuously via dependence on cumulative exposure is, however, analogous to the effect of immunity in bringing parasite density below such thresholds.
A second alternative method for incorporating immunity into mathematical models is to explicitly model strains and hence incorporate long-lasting strain-specific immunity. As noted above, our assumption that immunity develops with exposure and has finite memory essentially reproduces the patterns that would be obtained from such a model. The model does not imply that parasite density or strain-specific immunity are unimportant; as indeed there is strong evidence to support both playing a role in the development of immunity. Rather, our simpler model structure which [21] implicitly incorporates these processes through immunity functions allows us to explore the timescales over which clinical and parasite immunity develop and are lost as well as the role of ageing and exposure on these functions. Few previous models have been consistent in checking that they can reproduce the patterns of infection observed across a range of endemicities. By validating output against such patterns, we have sought to develop a model that is both informative about the impact of immunity on falciparum malaria epidemiology and also forms a solid basis with which to explore the impact of interventions. Having a robust framework which adequately captures the development of immunity with exposure and age is particularly important in exploring the impact of interventions such as insecticide treated nets (ITNs) and intermittent preventive therapy (IPT) in infants and children for which there is the potential to delay immunological development.
Materials and Methods
Mathematical transmission model. We model a human population with continuous age structure in which individuals of a given age can be in one of the following states: susceptible or not infected (S H ), latent infection (E H ), infected with symptomatic disease (including severe and clinical cases) (D H ), asymptomatic with detectable parasites (A H ), and asymptomatic infection with undetectable (subpatent) parasite density (U H ). The main distinction between states D H and A H is that individuals in state A H do not prompt treatment that leads to a change in infection state. The state U H is included to account for the fact that measured parasitemia often decays with age, while highly sensitive parasite detection techniques suggest parasitemia continues increasing with age nearing 100% in highly endemic areas [37]. In tandem, we consider a mosquito population whose individuals can be susceptible (S M ), exposed (latent) (E M ), or infectious (I M ). Figure 6 shows the transitions between states in each population (without displaying ageing). Susceptible humans move to latent infection at rate K, the force of infection on the human population. Individuals remain in this state for a mean duration 1/h (the mean latent period). A proportion / develop disease whilst the remainder (1À/) move to the asymptomatic infection category. A proportion f of symptomatic schematically how each model assumes that immunity is developed (through exposure and/or age) and lost. (B,D,F) Show the resulting effect of these immunity levels on (B) susceptibility to clinical disease, (D) the rate of clearance of detectable parasites, and (F) the clearance of subpatent infection as people age and for five different transmission settings (identified by the EIR in ibppy). Further mathematical details are given in Protocol S1. doi:10.1371/journal.pcbi.0030255.g007 cases (D H ) receive effective drug treatment and recover at rate r T , while the remaining cases recover naturally without treatment at rate r D . If clinical treatment or natural recovery is fully successful at removing parasites (with probability /), the host returns to the susceptible state and otherwise moves to the asymptomatic state. Asymptomatic infections become subpatent at rate r A , and these subpatent infections are cleared at rate r U with individuals returning to the susceptible state. Those in the asymptomatic state may additionally develop disease through superinfection at rate /K. Each human infection state, namely D H , A H , and U H , has a specific level of infectivity (transmission of mature gametocytes) to biting mosquitoes. The full equations for this model and further parameter definitions are given in Protocol S1. Table 1 summarises variables, parameters, and the values used to generate the model outcomes presented in Results. Sensitivity analyses of model output to these parameters are presented in Protocol S1. In our analysis, we focus on results obtained once endemic levels are reached. Model outputs are generated by fixing the EIR or by fixing mosquito density (m) and calculating the EIR via the equations describing the mosquito section of the parasite's transmission cycle (see Protocol S1). We ignore any possible dependence of infectivity in the different infection states (D H ,A H ,U H ) on age and EIR because this is currently less-well-understood [38]. For simplicity, we assume that the rate of natural recovery from clinical disease (r D ) in the absence of treatment is identical to that from asymptomatic infection (r A ), and that the rate of recovery of treated cases (r T ) is determined by treatment only.
Parameter estimation. Unknown parameters (Table 1) were estimated by running the model over a wide range of plausible values and excluding values which lead to epidemiological patterns that clearly failed to visually match observed patterns. Our aim was to identify model structures and parameters values based on their ability to reproduce patterns and relationships. Given the many uncertainties in model structure, large number of parameters, and limited data available, it would have been very difficult to implement a more formal and rigorous statistical approach. Rather, we have focused on qualitative comparison and understanding. The sensitivity analyses to key parameters (in Protocol S1) give an idea of uncertainty and ranges of parameter values that might be expected on the basis of this model and datasets.
Incorporation of acquired immunity. To explore the impact that acquired immunity can have on patterns of age prevalence in endemic settings, we extend the basic transmission model above to incorporate immunity acting at three different stages of a host's history of infection. Mathematical details of the functions, described in brief below, are given in Protocol S1.
1. Susceptibility to symptomatic disease, / (immunity function 1). We assume that individuals are born with maternally acquired immunity which is determined by the endemic level of disease and decays with a half-life d M . Following birth, clinical immunity accumulates due to exposure at a rate dependent on the force of infection in the population, K. This acquired immunity decays with a half-life d S . The schematic for this model is shown in Figure 7A. Susceptibility to symptomatic disease is then assumed to decrease in a nonlinear way as levels of clinical immunity increase. The overall dependence of susceptibility / on age and EIR resulting from this model is shown in Figure 7B.
2. Rate of natural recovery from asymptomatic to undetectable infection, r A (immunity function 2). The parasite immunity level associated with this response is similarly assumed to accumulate at a rate dependent on the force of infection in the population, K. The onset of parasite immunity is further assumed to have an age-related delay with mean d l , and any maternal immunity is lost during this period. Parasite immunity then decays with half-life d A . The schematic for this model is shown in Figure 7C. The recovery rate r A is assumed to increase with levels of parasite immunity through a nonlinear function which saturates at higher levels of immunity. The overall dependence of recovery on age and EIR resulting from this model is shown in Figure 7D, where change with age follows from age-dependent exposure (see Protocol S1).
As an alternative, we also consider a model in which parasite immunity is determined only by age (given some exposure to infection) and not by EIR.
3. Rate of natural clearance of undetectable infection, r U (immunity function 3). We assume that the duration of undetectable infection is boosted by continual reexposure and therefore not directly dependent on age. The onset of immunity is therefore dependent on the force of infection, K, and decays with half-life d U as in previous models of superinfection [17,18]. A schematic for this is shown in Figure 7E and the resulting recovery rate as a function of the force of infection in Figure 7F.
Supporting Information
Protocol S1. Mathematical Details and Sensitivity Analyses for Key Model Parameters Found at doi:10.1371/journal.pcbi.0030255.sd001 | 2014-10-01T00:00:00.000Z | 2007-11-14T00:00:00.000 | {
"year": 2007,
"sha1": "1a81439a290a9762d71e2473c59428a6b27dc745",
"oa_license": "CCBY",
"oa_url": "https://journals.plos.org/ploscompbiol/article/file?id=10.1371/journal.pcbi.0030255&type=printable",
"oa_status": "GOLD",
"pdf_src": "PubMedCentral",
"pdf_hash": "4dacb6b776b1dd0456640426f0666159b6dc5b68",
"s2fieldsofstudy": [
"Medicine",
"Biology"
],
"extfieldsofstudy": [
"Medicine",
"Computer Science",
"Biology"
]
} |
212557767 | pes2o/s2orc | v3-fos-license | Stroke, Carotid Thrombosis and Other Thrombotic Events in Essential Thrombocythemia Patient with a High Quantity of Micromegakaryocytes
Essential thrombocythemia is a myeloproliferative neoplasm characterized by thrombocytosis and possible complications such as thrombosis, hemorrhage, splenomegaly, bone marrow failure and acute leukemia. In the myeloproliferatives neoplasms, there is an increase number of dysplastic megakaryocytes, known as micromegakaryocytes and there is an association between the high expression of the JAK2V617F mutation and the presence of micromegakaryocytes in bone marrow. To date, reports and researches involving essential thrombocythemia and micromegakaryocytes are exceedingly scarce. We report a peculiar and unique case of a death of a 55-year-old man, due to stroke and carotid thrombosis, preceded by successive thrombotic events in a patient with essential thrombocythemia, JAK2 positive, with no additional risk factor, who did not respond well to standard treatment and had a high amount of circulating micromegakaryocytes in peripheral blood. This case goes beyond the known prognosis of ET and raises the discussion of new prognostic markers, such as the quantification of micromegakaryocytes and reaffirms the importance of the JAK2 mutation in the evolution of the disease. We also discuss the involvement of micromegakaryocytes in the probable mechanisms leading to our patient’s thrombotic state.
Introduction
Essential Thrombocythemia (ET) is a Myeloproliferative Neoplasm (MPN) characterized by thrombocytosis and, like others MPNs, has complications such as thrombosis, hemorrhage, splenomegaly, bone marrow failure and a possible course to acute leukemia [1]. In addition, there are reports of patients with ET that have developed from thrombosis of celiac and superior mesenteric arteries [2], thrombosis of cerebral venous sinuses [3] to pericarditis [4].
In MPNs, abnormal transient myelopoiesis and, more characteristically, myelodysplastic syndrome, there is an increase in the number of dysplastic megakaryocytes, known as micromegakaryocytes (mMK). The International Working Group on Morphology of MDS (IWGM-MDS) defined them as generally diploid, mononuclear cells with a nucleus similar in size to that of a myeloblast or promyelocyte and less than 30 µm in diameter [5].
In a study conducted by Pich, it was found that the high expression of the JAK2 V617F mutation in patients with ET is associated with a higher presence of mMK in bone marrow biopsies [6]. However, little is known about the relationship between the presence of mMK in peripheral blood and the occurrence of such thrombotic events.
Here we present the case of a patient diagnosed with ET, who died after successive thrombotic events, both arterial and venous, with no additional predisposing factor, in a short period of time. The large amount of mMK and the presence of the JAK2 mutation are the main peculiarities of the case.
Case Presentation
A 55-year-old white male was admitted in November 2012 to investigate a thrombocytosis detected on a routine exam. The patient had no symptoms or any other complaint. The possible cause of thrombocytosis was identified in 2013 when the diagnosis of ET was given. The patient's diagnosis complies with the WHO criteria, which are based on clinical and laboratory characteristics [7]. Their initial platelet count was greater than 450 × 10 9 /L, a bone marrow biopsy (BMB) confirmed the ET. The presence of the JAK2 V617F mutation was also evaluated (positive).
Figure 1
Bone marrow biopsy of the patient (HE staining, original magnification 40x). The major arrow points to a micromegacarocyte, while the smaller one, a binucleate megakaryocyte.
Therefor cytoreductive therapy with anagrelide (0.5 mg/2 × day), hydroxyurea (1.5 mg/day) and anti-aggregative with acetylsalicylic acid (100 mg/day) was started, which maintained his platelet count controlled. The patient did not report a history of alcohol or tobacco use and did not present any other comorbidities. However, within a few months, despite the standard therapy, the patient presented ulcers and thrombosis of both lower limbs, as well as ischemia of the fifth toe. In July 2016, another BMB confirmed the myeloproliferative disease, type ET, with mild fibrosis (grade 1). It was also excluded other myeloid malignancies and other causes of reactive thrombocytosis. Small, monolobular megakaryocytes with high nucleus/cytoplasm ratio were found on the slides, compatible with micromegacarocytes ( Figure 1).
In February 2017, the patient was recruited for a quantification study of mMK in peripheral blood using flow cytometry ( Figure 2) and almost 7 times the amount of these cells was observed compared to a healthy person [8]. At the same time, their blood counts revealed LDH and platelet counts within the reference values, but red blood cell counts, lymphocytes, and leukocytes were reduced, as well as hemoglobin levels ( Table 1). The patient died in June 2017 after an ischemic stroke associated with carotid thrombosis.
Discussion
Hitherto it is unusual for a case to report so many thrombotic events in the same patient with ET and /or to relate them to the high amount of mMK in peripheral blood. However, the medical literature is rich in the collection of thrombo-hemorrhagic events. In a systematic review of Panayiotis D. Ziakas, thrombotic events were observed in 31.8% of cases positive for the JAK2 mutation [9]. While in the analysis conducted by Lussana, such thrombotic events, whether arterial or venous, occurred in 32% of patients with the JAK2 mutation [1]. In addition, the literature presents a greater number of cases of thrombosis in patients with ET and mutation carriers, splanchnic veins (including the hepatic portal vein) and deep venous system, with an incidence of 11% and 23%, respectively [10].
It is also worth mentioning that the current risk stratification for patients with ET is based on predictors of arterial thrombosis, such as age less than 60 years, history of thrombosis, cardiovascular risk factors (CV). In addition to the use of tobacco, hypertension or diabetes mellitus, leukocytosis (>11 × 10 9 /L) and the presence of the JAK2 V617F mutation [11].
The patient not only had deep vein involvement but also developed the curious event of carotid thrombosis associated with a stroke. Although there is no CV risk factor, no tobacco or alcohol use, no history of previous surgeries, splenomegaly, hypertension, overweight or diabetes mellitus, and did not have a leukometry lower than 15,000/mm 3 , indicated as an isolated risk factor for thrombosis [12]. A recent study revealed the association between monocytosis and deep venous thrombosis, but the patient did not present monocytosis at any time (Table 1) [13][14][15][16][17].
The patient survived a little more than 3 years after his diagnosis, not corresponding to the average survival of 33 years for patients with ET less than 60 years of age [13]. In addition, several thrombotic events occurred in this interval, even with the standard treatment for patients with high-risk ET, which consists of systemic anticoagulation conjugated to a cytoreductive therapy, as already mentioned. The uniqueness of the case is attributed to the JAK2 V617F mutation and a high number of mMK observed in the peripheral blood of the patient. The pathogenesis of vascular thrombosis associated with JAK2 V617F mutations is still not well understood. However, the cause appears to be associated with increased heparanase activity under the JAK2 mutation, which would directly increase Tissue Factor concentration (TF) and decrease Tissue Factor Pathway Inhibitor (TFPI) activity leading to increased factor Xa and subsequent activation of the coagulation cascade. This situation implies an increased thrombosis [14,15].
In addition to this evidence, there are studies in animal models with the JAK2 mutation, showing that megakaryocytes become hypersensitive to fibrinogen, thrombopoietin and other endogenous stimulant compounds. Thus, this alteration in the physiology of megakaryocytes could be related to thrombocytosis and to thrombotic events due to a higher reactivity also of platelets to thrombin [16,17].
The patient on this case had mMK-compatible cells in the bone marrow while demonstrating an increase in the number of these cells in the circulation. In this way, what would be the influence of the large amount of mMK? Can mMK be associated with a greater predisposition to thrombotic events? Since they could respond in the same or similar way to normal megakaryocytes in the condition of the JAK2 mutation ( Figure 3), we need to analyze what is the impact on patient survival?
Conclusion
The presentation and outcome of the case did not correspond to many of the proposed paradigms for essential thrombocythemia and raised questions about the role of mMK in the pathogenesis of the disease. Therefore, there is a need for more related studies in order to prove the importance of the quantification of mMK in peripheral blood of patients with ET. Perhaps in the future, we will use quantification of mMK in the clinic, with new perspectives for the prognosis of these patients.
Author's Contributions
MAP wrote the case report and was the principal investigator; SN was responsible for the acquisition of data and drafting the manuscript; BP recruited the patient and elaborated the consent form; HK evaluated critically and designed the manuscript. All authors read and approved the manuscript.
Funding
Not applicable. | 2020-03-07T13:51:18.322Z | 2019-01-01T00:00:00.000 | {
"year": 2019,
"sha1": "14dd23edde415655ad1f137a3039903a48687a86",
"oa_license": "CCBY",
"oa_url": "https://www.archivesofmedicine.com/medicine/pstroke-carotid-thrombosis-and-other-thrombotic-events-in-essential-thrombocythemia-patient-with-a-high-quantity-of-micromegakaryo.pdf",
"oa_status": "HYBRID",
"pdf_src": "Anansi",
"pdf_hash": "634159a6b414314aa3730963416c233f8620c9ad",
"s2fieldsofstudy": [
"Medicine",
"Biology"
],
"extfieldsofstudy": [
"Medicine"
]
} |
221091954 | pes2o/s2orc | v3-fos-license | Age Dynamics of Wind Risk and Tree Sway Characteristics in a Softwood Plantation
Quantifying wind risk in planted forests is critical to ensure the provision of wood-based biomaterials and forest ecosystem services in the future. Tree sway dynamics play a major role in determining their vulnerability to wind storms. While structural engineering theory can greatly assist in predicting tree and branch motion accurately, the effects of forest aging and silvicultural treatments on motion and the likelihood of mechanical failure are still poorly understood. In this study, we simulate the growth of an individual model tree and its architecture for a managed, even-aged pure Cryptomeria japonica D. Don. stand. The dynamic behavior of the tree is predicted for a rotation period at a 5-year interval using finite element analysis. Results show that tree natural sway frequency is subject to a 5-fold decrease over the tree's lifetime. The tree is thus more susceptible to absorb turbulent kinetic energy from the wind as it grows older. The damping ratio, which measures how rapidly the tree can return to its rest position after being deflected, is predicted to increase by a factor of four. By simulating the mechanical response to a wind gust, the model predicts a quick transition from a low risk of failure in the juvenile phase to a high-risk situation in the mature phase. The transition occurs between ages 15 and 30 when canopy closure takes place, when juvenile wood formation stops in the lower stem region and when the first thinning is carried out. Wind risk plateaus after the transition period. The age-risk relationship shown by the model is consistent with post-typhoon damage patterns documented by inventories in Japanese planted forests, albeit with a sharper transition.
INTRODUCTION
Trees are the longest-living organisms on Earth. To ensure that longevity, they must withstand the forces exerted on them by the physical environment throughout their lifetime. Although tree mechanical failure is a natural disturbance process important to forest dynamics and biodiversity (Thompson et al., 2009;Mitchell, 2013), large-scale damage is mostly detrimental in forestry because of the associated economic losses (Gardiner et al., 2013) and forest health issues (Stadelmann et al., 2014). As trees grow in size and exposure, they are subject to continuously increasing mechanical forces that should eventually compromise their likelihood of survival. Studies have shown that tree allometric patterns ensures that mechanical stability is less limiting for tree development than other constraints such as hydraulic transport (Koch et al., 2004;Niklas and Spatz, 2004). Often, those studies only consider elastic instability (buckling) under self-weight and neglect other agents and processes that can cause failure.
As top-loaded, tall and slender mechanical structures, forest trees are inherently unstable. To counteract this, they have evolved adaptive growth mechanisms limiting the effects of chronic and acute mechanical loading. A large focus in the study of tree biomechanics has been posture control and gravitropism, an active reorientation process to keep the tree upright thus minimizing the effects of weight (Fourcaud et al., 2003;Coutand et al., 2007;Alméras and Fournier, 2009). Other key adaptive mechanisms are thigmomorphogenesis (Jaffe, 1973) and thigmotropism (Telewski, 2012). They involve regulatory cambial growth to increase resistance and decrease exposure to mechanical loads. Trees also display passive mechanisms such as streamlining (Harder et al., 2004;Butler et al., 2012) and branch damping (Théckès et al., 2015). They allow reducing excess drag and improving kinetic energy dissipation in the swaying tree, respectively. They also make aerial architecture, (i.e., the geometrical and topological configuration of the branch system) a significant actor in tree biomechanical function and safety. The contribution of all those mechanisms is essential to ensure long-term tree stability.
Wind is a major abiotic agent of damage in forest plantations. While mechanical stability to wind loading has been studied in relation to growth (Ancelin et al., 2004;Gardiner et al., 2008), the effects of aging on tree vibrational properties and aerial architecture are rarely included. Instead, the dynamic behavior, which is essential to quantify the risk of windthrow and windbreak, is often investigated at a fixed point of a tree's lifetime (Moore and Maguire, 2008;Sellier and Fourcaud, 2009). As a result, the effects of canopy closure, silviculture, and juvenile wood formation on tree structural dynamics are poorly understood. That knowledge is critical to evaluate the vulnerability of forest plantations to extreme storm events. Extreme storm systems are expected to become regionally more intense and more frequent in future climate projections (Ulbrich et al., 2008;Gastineau and Soden, 2009). Because those events are rare, it is difficult to expand our knowledge about the damage they cause to forests.
In this study, we investigate how tree sway properties and the dynamic response to wind loading vary with tree age on a theoretical basis. The case study is an individual Cryptomeria japonica D. Don. tree representing managed, even-aged pure stand conditions. As Japan is annually subject to several tropical cyclonic storms (typhoons) and has ∼10 million hectares (30% of land area) of planted forests (MAFF, Japan, 2017), extensive information on tree growth and wind damage was available to support model development and evaluate the predicted age patterns of wind risk.
MATERIALS AND METHODS
The aerial system of a C. japonica tree was reconstructed from age 5 (sapling) to age 55. The common age of harvest in Japanese forestry is 60. The model tree was assembled using distinct experimental datasets and relationships for tree stem, architecture, foliage, and wood properties. A complete dataset was unavailable, even for a single age. The dynamic behavior of the tree was evaluated under impulse loading and wind loading using finite element analysis.
Tree Stem
The shape of the stem corresponds to a measured C. japonica tree named Sugi 2 (Minato et al., 1989). The tree was planted in the Wakayama experimental forest (135.40E 33.4N, Honshu island, Japan). It was 53 years old, 26.8 m tall and had a DBH of 28.5 cm when it was felled. The stem taper (diameter variation over height) was reconstructed from the internal ring structure at a 5-years interval. Stem taper at age 55 was obtained in this study by extrapolating ring width using the last known growth rates.
Crown Structure
The branching structure was modeled using the crown patterns measured in 9, 16, and 22-year-old stands (Hashimoto, 1991). Those ages represent the main phases of crown development from a young crown covering the full length of the stem to a mature crown after canopy closure. The number of branches borne per unit length of stem was given by a random Gaussian distribution (22.5 ± 3.3 branch m −1 ) (Hashimoto, 1991). Branches were uniformly positioned over the growth unit as C. japonica does not form whorls. Each branch was assigned a random growth potential at formation which acts as a scaling factor for initial length and growth rate. The upper crown length was defined as the topmost 4 m of the crown (Kajihara, 1976). In the lower crown region, branches were assigned a probability p = 0.1 to die every year because of limited light availability. Overall crown length increased with age up to a maximum of 9 m (Kajihara, 1976). The azimuthal orientation of branches was predicted using a phyllotactic pattern with a divergence angle equal to 139.5 • . The branch angle with the vertical is higher in older trees (Hashimoto, 1991), which was represented using θ = 71 − 0.5n where n is tree age at time of branch formation.
Branch primary growth was modeled using an age-dependent yearly length increment: L = a n b e c n + d (Hashimoto, 1991), with a = 0.06; b = 2; c = −0.5; d = 0.2. Branch basal diameter, D, was increased with branch length as D = 0.28L. The number of trees per hectare (stocking), s, ultimately limits the lateral extent of branches. For a circular crown, maximum length is equal to L max = 1 cos θ 10 4 π s . A thinning event causes s to decrease, allowing branch elongation to resume to take advantage of newly available space. At the Wakayama site, thinning events took place at age 20, 27, and 47 (Minato et al., 1989). In this approach, crown size expansion can resume or accelerate after thinning occurs. Modeling thinning was essential to synchronize crown growth with stem growth. The architecture of the model tree during growth is shown in Figure 1.
Foliage Properties
Tree's foliage mass, m F (n), is required to simulate tree mechanical behavior at any age n. It allows the inertia, the weight and the sail area of the crown to be calculated. In absence of repeated measurements during the growth of the tree Sugi 2, foliage mass was estimated using allometric models. Two models were considered. Both predicted foliage mass in relation to stem size or mass, which were measured (Minato et al., 1989). Only these two models provided a realistic trend of foliage biomass among considered models.
The first model relates directly foliage mass to stem diameter at breast height, D (cm). The model (Hashimoto and Gyokusen, 1995) predicts foliage mass as: It was retained as the best model describing the functional relationship between foliage mass and shoot cross-sectional area derived from plant hydraulic principles. The second model, purely empirical, was developed to obtain the most realistic allometric for the studied species. It predicts the foliage-tostem mass ratio as a function of age. It was obtained by fitting a power law on the biomass ratio using measurements across nine C. japonica forest sites at multiple times during the rotation. Those sites were Gunma/Ibaraki, Saitama, and Nara (Ando et al., 1968), Chiba (Negishi et al., 1988), Gifu (Watanabe and Moteki, 2007), Miyazaki, Nagasaki, Oita (Tadaki et al., 1965), and Toyama (Kato et al., 2005). They cover a broad range of silvicultural and environmental conditions, including different stocking densities and thinning schedules. The power law was fitted using Standardized Major Axis (SMA) regression as provided by the smatr R package (Warton et al., 2012). With both foliar models, the effects of thinning on total foliage mass result from the effects of thinning on stem growth (diameter or mass).
The amount of foliage borne by each branch was a fraction of the total crown foliage weighted by the basal diameter of the branch. Foliage was then distributed over the five most recent growth units of each branch to match the average lifespan of needles in C. japonica (5 years). The vertical distributions of foliage mass were in good agreement with profiles measured at ages 9, 16, and 22 (Hashimoto, 1991).
The foliage area, A F , is used for calculating the drag force applied to the crown of the tree. For an individual tree, it is approximated by A = m F (n) a F , where a F = 1.667 [m 2 kg −1 (green weight)] is the specific leaf area (SLA) (Tadaki, 1966).
Wood Properties
Three material properties were used to define wood mechanical behavior in the model. Those are the longitudinal Young's modulus, E (GPa), the density ρ (kg m −3 ), and the damping ratio, ξ 0 . E and ξ 0 are often known for dry wood. However, their values differ for green material in trees. Green wood is typically less stiff (lower E) and more viscous (higher ξ 0 ) than dry wood.
Most softwood species exhibit a typical radial pattern (TRP) of wood properties. The TRP consists of an increase, or a decrease depending on the property considered, followed by a plateau as the age of wood formation increases. In C. japonica, E follows the TRP (Nakatani et al., 1988;Zhu et al., 2005) and can be expressed as a function of the tree-ring number k (Zhu et al., 2005) as E k = c G (2.369 + 2.263 ln k ) where c G = 0.7 is the green-to-dry ratio E (arbitrary value for a 30% reduction in stiffness). Wood forming higher in the tree is stiffer than in the lower stem region (Yamashita et al., 2009). The effect of the height at which the wood is formed z was modeled by E k, z = E k (0.722 + 0.169 ln (5.204z) ). The cross-section of any stem or branch element is composed of several rings. The effective modulus of elasticity (MOE) of a cross-section was calculated as the sum of each ring's E value weighted by ring's moment of inertia: where R k is the outer radius of the kth ring. Unlike E, basic density remains relatively constant with cambial age in C. japonica (Minato et al., 1989;Yamashita et al., 2009). It was thus modeled as a homogeneous quantity. The green density value, 1,213 kg m −3 , was obtained using the average basic density (ρ= 438 kg m −3 ) and the average moisture content (w = 1.77) of Sugi 2 (Minato et al., 1989). The density value of woody elements bearing foliage was increased by foliage mass divided by the element's volume. The damping ratio of wood depends on the ratio of Young's modulus to basic density (Ono and Norimoto, 1983;Brémaud et al., 2013). For the kth ring, it was predicted using: where the specific density γ is equal to the ratio of basic density to water density. The factor d G = 1.27 corrects the material viscosity for the fact it is greater for saturated cell walls. The value of d G was calculated from damping experiments in relation to moisture content (Obataya et al., 1998). The effective damping ratio of a cross-section ξ 0 was calculated as the sum of each ring's ξ 0 value weighted by ring area. Material damping was then modeled using Rayleigh's model, which linearly relates damping to mass using the coefficient α R = 4πf 0 ξ 0 where f 0 is the natural sway frequency of the tree.
Numerical Model
The mechanical behavior of the tree was simulated using a finite element (FE) model developed and tested to study tree dynamics (Sellier et al., 2006(Sellier et al., , 2008. The model uses ABAQUS software with the AQUA module (SIMULIA, Dassault systèmes, France). Tree stem and primary branches are meshed using beam elements. Foliage was modeled by modifying the drag area and the density of elements bearing foliage. Dynamic analyses were carried out by direct integration of the equations of movement using an implicit solver. Unlike with a modal procedure, the geometry of the structure is updated at every time step of a simulation using non-linear analysis. This is essential to simulate large structural displacements, which may occur in response to gale force winds. Furthermore, geometrical non-linearity accounts for the streamlining phenomenon, (i.e., a reduced drag area when the crown is deformed by wind action). There is no need for ad hoc modifications of the drag equation.
Stability Criteria
Two types of simulations were carried out with the FE model. For both types, the mechanical behavior is simulated every 5 years. The first experiment was a virtual pull-and-release experiment. The tree was initially displaced by applying static wind loading on the tree's crown, then released suddenly to induce movement. A 100 tree architectures were randomly generated and tested. The crown features affected by random state were: number of branches borne by a growth unit, height of insertion, growth potential, and branch survival in the lower crown. The foliar model fitted on the experimental datasets was used to predict foliage biomass in this experiment because of its stronger empirical basis. Two important sway characteristics were calculated from the free oscillation response: • f 0 , the natural sway frequency of the tree. It is the inverse of the period required by a tree to sway back-and-forth once. f 0 is also referred to as the frequency of the fundamental (i.e., of lowest frequency) mode of vibration. Although the presence of modes of higher frequency has sometimes been observed in tree motion, the fundamental mode typically dominates the response. • ξ 0 , the damping ratio of the fundamental mode. ξ 0 measures the decrease of sway amplitude every cycle. For example, a structure would perpetually oscillate for ξ 0 = 0; the amplitude of lateral movement is divided by two every sway when ξ 0 ∼ 0.11; sway amplitude is divided by four every cycle when ξ 0 ∼ 0.22; and there is no oscillation (single cycle) when damping is critical (ξ 0 = 1). ξ 0 was calculated by fitting the theoretical motion of a simple damped oscillator to simulated tree response using least-squares optimization.
In the second numerical experiment, the behavior under wind loading was simulated. Because of the computational cost of those simulations, only one tree architecture was tested under wind loading: that of the tree with the sway frequency and the damping ratio closest to the mean values obtained in the first experiment. Simulations were done with both foliar models (section foliage properties). Two criteria were used to characterize the tree response.
• M 0 , the bending moment at the base of the tree. If M 0 exceeds the anchorage strength of the soil/root plate, structural failure by uprooting occurs. Anchorage strength is well-predicted by stem mass m S or, equivalently, volume (Moore and Quine, 2000;Cucchi et al., 2005;Kitagawa et al., 2010). The ratio M 0 m S was used to indicate the relative risk of uprooting. Other aspects not considered here, such as soil water logging or limiting rooting depth, may further reduce anchorage strength and thus increase the relative risk. • σ (z) the longitudinal stress along the tree stem. Breakage can occur at any point on the stem where σ exceeds the Modulus of Rupture (MOR). Thus, it is max (σ ) and not the mean stress that is relevant to analyze the risk of structural failure by windbreak. A single point of failure on the stem is sufficient to cause failure. MOR = 30 MPa is used as reference for green C. japonica wood. Natural variation is ∼± 50% of that value (Nakatani et al., 1988). It is consistent with MOR values from bending tests on green logs from a 33-year-old plantation (mean 34.9 MPa, min. 25.8 MPa max. 49 MPa, n = 36) (Suzuki and Ito, 1994).
Wind Loading
Wind exerts a force called aerodynamic drag (Cullen, 2005) on any tree element that is function of the speed of the wind relative Frontiers in Forests and Global Change | www.frontiersin.org to the structure: where ρ is the air density (1.25 kg m −3 ), c D is the drag coefficient (0.8), A is the projected area exposed to the moving fluid (m 2 ), u (z, t) the horizontal wind speed (m s −1 ) andq the speed of displacement (m s −1 ) of the tree element considered. A was calculated as the product of foliage area per unit length and element's length projected on the vertical plane normal to wind direction. The value of A was updated during simulations using the current element orientation. u (z, t) was decomposed into a spatial component u(z) and a temporal component u(t). To derive the spatial component of wind speed inside a forest stand, one must first define the wind speed when unperturbed by the forest (at the edge). Horizontal wind speed varies with height (Peltola et al., 1999;Gardiner et al., 2008). At the edge of a forest stand, the profile of u(z) is logarithmic (Ancelin et al., 2004): where h is the mean height of the canopy and z 0 is the roughness length (z 0 = 0.06h). u E h was calculated for each tree age using the above equation with u E (30) = 20 m s −1 for reference. At a distance more than 8-10 h away from the edge downwind, forest stand conditions apply (Dupont et al., 2011). Due to the presence of vegetation, the wind profile in the stand u S (z) is rapidly attenuated below the top of the canopy. Here the wind profile is represented by a power law: where u S h was obtained with a logarithmic profile above the canopy and u E = u S at 1 km altitude (Ancelin et al., 2004). The attenuation coefficient α was chosen as: where z C is the height at the base of the crown and the ratio u (z C )/u(h) is equal to 0.2. The resulting wind speed profiles are shown in Figure 1.
The time-dependent component of wind speed u (t) corresponds to a single gust event. In forest canopies, gusts are characterized by periodic ramp patterns of turbulent quantities with large deviation from the mean (Raupach et al., 1996). In the case of horizontal velocity, a gust is associated with air movement slowing down followed by a strong acceleration (Collineau and Brunet, 1993). In this study, corresponds to a 30-s event measured below canopy top (Collineau and Brunet, 1993). A maximum velocity equal to twice the mean is typical of wind storm conditions (Oliver and Mayhead, 1974). Figure 2 shows the evolution of stem biomass for 60 years in nine different C. japonica plantations. At a young age (9-yearold), allocation was prioritized toward foliage production with nearly 60% of biomass allocated to it. This was measured at two forest sites: Saitama (Nishikawa management, 4,000-4,500 trees ha −1 ) and Gunma/Ibaraki (National Forest management, 3,000 trees ha −1 ) (Ando et al., 1968). The fitted model predicted up to 75% of biomass allocated to foliage for a 5-year-old tree. The situation changed rapidly: stem biomass was three times higher than foliage biomass at age of 20. The relative stem biomass kept increasing as the forest grew older, but more slowly. At harvest age, stem biomass was ∼6 times the foliage biomass.
Above-Ground Growth Allocation
The trend of allocation between foliage and stem was consistent for all sites despite varied initial stocking (up to 10,000 trees ha −1 at the Nara site under Yoshino management), cultivars and geographical locations (Kyushu and Honshu islands). Unlike the foliage:stem biomass ratio, the absolute foliage biomass productivity is highly variable across sites (not shown). The allometric relationship (Hashimoto and Gyokusen, 1995) predicted a evolution of allocation similar to the national trend obtained with the fitted model although it overestimated the amount of foliage at early age and underestimated it later on. It performed remarkably well for a simple relationship based on functional hydraulics without any age information. It also predicted more short-term variation in foliage biomass.
Natural Sway Frequency
The modeled tree displayed a general decrease in natural sway frequency with age ( Figure 3A). The decrease was observed whether the tree was debranched, defoliated, or complete. There was an exception for the complete tree which, at age 5, was found to sway more slowly (f 0 = 0.85 Hz) than at age 10 (f 0 = 0.93 Hz). Afterwards, f 0 decreased until it reached 0.19 Hz at age 55. Defoliation had for effect to increase f 0 with respect to the intact tree. The relative increase was greatest at age 5 (52%) and less but notable (19-30%) at older ages. Branch removal caused an additional increase in f 0 (i.e., the tree stem swayed faster without any crown). The increase due to debranching in addition to defoliating was smallest (12%) at age 10-15 and largest (27%) at age 35.
Damping Ratio
The damping ratio ξ 0 increased with tree age (Figure 3B). Damping was relatively low (ξ 0 = 0.05) when the tree was young (15-yo or less). It increased until the tree was 40-yo and stabilized at ξ 0 ∼ 0.2. The amount of variation in the damping ratio due to architectural differences increased as the tree got older. The quality of the fit used for determining ξ 0 was found unsatisfying at age 5 and at ages above 35 for different reasons. At age 5, a shift in natural sway frequency occurred while the tree was oscillating. A single degree-of-freedom (1-DOF) model cannot describe that phenomenon (Figure 4). At age 50, oscillation damping exhibited a distinctive non-linear behavior. Motion was strongly damped during the first cycle, but residual oscillations persisted for a long time afterwards. As a result, the 1-DOF model with linear damping under-then over-estimated the amplitude of tree sways during free vibrations. The r 2 value was high because the model performed well-quantitatively on average but less so qualitatively. This occurred from age 40 onwards. At age 20, a transient component was found in tree sway response (Figure 4). It probably resulted from the initial activation of a secondary vibration mode of higher frequency. The 1-DOF model could not represent that additional contribution and overestimated the amplitude of displacement during the first cycle. However, the quality of the fit is considered acceptable because the r 2 value was high and the signal perturbation was short-lived.
Sway Frequency and Crown Structure
Variability in branch positions in the crown caused variation in sway frequency. At age 5, f 0 ranged from 0.73 to 0.93 Hz FIGURE 4 | Relative lateral displacement of a fixed point on the tree stem during free oscillations at a young age (5), a mature age (20) and near harvest (50). Free oscillations were simulated by finite element analysis (blue line) for trees bearing individual first-order branches. A one-degree-of-freedom oscillator model (black line) was fitted to the simulated displacement time series to calculate the damping ratio. During the test, the initial deflection before release was applied to the tree crown using static wind loading.
depending on the crown arrangement, which corresponds to 22.6% of the mean. The range of variation was lower from age 10 onwards, between 2.5 and 8% of the mean. Figure 5 shows the relationship between f 0 (normalized by the sway frequency of the stem alone, f stem 0 ) and simple crown descriptors as the crown transitioned to a mature stage (age 25). At age 5, f 0 and crown mass m C were independent as f 0 varied by more than 20% for the same m C value (Figure 5A). At ages 10 and 15, no clear relationship was observed. At ages 20 and 25, a linear relationship was found. At those ages, it must be noted that f 0 was 30% less than for the stem alone whereas the crown represented 15% of tree mass. A better predictor of the influence of the crown on f 0 was found to be the change in height of center mass, z M , associated to the presence of the crown. The relationship between f 0 and z M were distinctively linear at all ages but 5 (Figure 5B). At 5, a crown of similar mass could either lower or heighten the center of mass depending on how the branches were positioned. As a result, f 0 values were quite dispersed. In older trees, a heavier crown always translated to a center of mass located higher than for the stem alone.
Response to Wind Loading
The bending moment and bending stress applied to the tree during a wind gust varied with tree age and size. The basal bending moment increased throughout the rotation period (Figure 6). The overall increase was three orders of magnitude for both the mean and the maximum values. Although the increase slowed down over time, the moment reached the highest value at the oldest age.
The magnitude of bending stress increased by a factor of 3-4 during the entire rotation (Figure 7). Unlike the bending moment, the mean bending stress peaked 5 years before the oldest age. The maximum bending stress caused by the gust event peaked even earlier, at age 40. Both mean and maximum stress values transitioned from low stress levels in the early phase to high stress levels that plateau later. In the case of the maximum stress, the transition was rapid, between age 10 and 25, whereas it took longer for the mean stress.
Both predicted basal bending moment and bending stress were higher when using the model describing the national trend of fractional foliage biomass. This stemed from the additional foliar biomass and crown area predicted by that model. Despite differences in load magnitude, the age dynamics of wind loading were similar for both foliar models. FIGURE 6 | Bending moment at the base of the tree induced by a wind gust as a function of tree age. The mean and maximum moment values recorded during the gust are given. Two model trees with different models of foliar allometry are considered: a fit of experimental allometric ratios and an empirical power law (Hashimoto and Gyokusen, 1995). Figure 8 shows peak tree deflection during a wind gust at three different tree ages. When the tree was 15-or 25-yo, the lateral displacement was moderate. However, the stress profiles were completely different. Bending stress was uniformly distributed along the length of the stem at age 15 whereas it is concentrated in the upper crown region at age 25. At age 45, the windinduced lateral displacement became large and the curvature more pronounced. Bending stress was high over most of stem length excluding the lower region extending from tree base and the very top of the tree. Figure 9 shows how wind risk evolved with the age of the plantation forest using the maximum loads obtained under wind loading (Figures 6, 7). Risk of structural failure is given as a ratio of load to notional resistances to windthrow and windbreak. The risk of windthrow ( Figure 9A) increased up to age 30 with both foliar models considered. The likelihood of overturning remained high in older trees without increasing further. The fitted foliar model yielded a risk of windthrow that was 10.9% higher on average, all other factors being equal, which is consistent with higher foliage area.
Age-Risk Relationship
The stress maximum in the stem did not reach the lower end of the MOR range in young trees ( Figure 9B). Because compression strength is between 50 and 60% of the MOR value (Chiba, 2000;Zhu et al., 2005), local compression failures could happen. After the age of 20, the maximum bending stress reached a level which implies windbreak would occur in trees with weak wood. In the later growth phases (age ≥ 30), the maximum stress reached a FIGURE 7 | Bending stress induced by a wind gust in the tree stem as a function of tree age. The mean and maximum stress values recorded during the gust are given. The mean value is averaged in time and along stem length. The maximum value is the overall maximum recorded at any stem height at any time during the gust. Two model trees with different models of foliar allometry are considered: a fit of experimental allometric ratios and an empirical power law (Hashimoto and Gyokusen, 1995).
value sufficient to induce stem failure in most trees. However, tree stems with the strongest wood and no point of weakness could theoretically resist those stress levels. Like the risk of windthrow, the risk of windbreak was found high and stable after a 10-year period of transition (age 15-25) from a low risk level. The same evolution of windbreak risk was obtained independently of the foliar model, except for an average increase in maximum stress of 10.6% associated to using the fitted model for foliage allocation.
Evolution of Natural Sway Frequency With Age
The model predicted a 6-fold decrease of natural sway frequency over the lifetime of the studied C. japonica tree. The overall range 0.18-0.94 Hz, lies within the global 0.16-1.39 Hz range known for trees (Moore and Maguire, 2004). Albeit shifted toward higher values, the predicted natural frequency evolved with age similarly to measurements: 0.62-0.78 Hz in 4-yo Pinus pinaster Ait. (Sellier and Fourcaud, 2005), 0.65 Hz in a 6-yo C. japonica (Suzuki, 2012), 0.18-19 Hz for 36-yo P. pinaster (Sellier et al., 2008) and 0.11 Hz in a 63-yo C. japonica (Suzuki, 2020). Age is useful to define a main trend in sway frequency evolution during growth. However, frequency depends principally on size. Silviculture through thinning schedules and stocking densities FIGURE 8 | Tree maximum deflection during a gust event at ages 15, 25, and 45. Grayed silhouettes represent trees at rest. Color scale, from blue (low) to red (high) corresponds to the relative magnitude of longitudinal stress under wind loading.
will modify the age-size relationship and, therefore, the agefrequency relationship.
The decrease of f 0 with age is consistent with mechanical theory which predicts that f 0 varies as the square root of the stiffness to mass ratio [i.e., a specific stiffness (∼ E 1/2 D L 2 for a cantilever beam)]. As the structure grows heavier and taller, mass scales faster than bending stiffness and the frequency decreases. Those effects are mostly size effects dictated by growth rates. Trees of different ages with similar dimensions will oscillate at similar natural frequencies. Age might have a direct effect on f 0 through the modulus of elasticity, E. The progressive stiffening of wood with age of formation would increase f 0 . That effect must be minor because it is insufficient to counteract the general decrease of f 0 observed in the numerical simulations.
The sway frequency helps in estimating the stability of an individual tree. Movement is amplified when the frequency of a dynamic load is similar the frequency of one mode of vibration of the structure (Wood, 1995). Trees are more sensitive to the turbulent kinetic energy of wind in the frequency range close to their natural frequency. In non-storm conditions, the peak frequency of forest canopy turbulence is less (0.02-0.05 Hz) than that of tree sways (Amiro, 1990). Higher peak frequency values equal to 0.1 Hz have been recorded during a typhoon (Yamanoi et al., 2005). Trees with the lowest f 0 values, (i.e., the older and larger ones), are more at risk of experiencing large sway displacements due to dynamic amplification and resonance. The decrease of f 0 with age is asymptotic (0.25 Hz at age 35 vs. 0.18 Hz at age 55). The transition to a less stable state, with respect to sway frequency, occurs rather early and rapidly. The windthrow risk indicator is given as the ratio of the basal bending moment to stem mass, which predicts well tree overturning strength. (B) The relative windbreak risk is given as the ratio of maximum mechanical stress in tree stem to the wood's Modulus of Rupture (MoR). The grayed area in (B) indicates ±50% of reference MoR; it covers most of the natural range of variation for green C. japonica (Nakatani et al., 1988). Results shown for both foliar models.
Evolution of Damping Ratio With Age
Between ages 5 and 40, the tree structure transitioned from being lightly damped (ξ 0 ≈ 0.05) to being heavily dampened (ξ 0 ≥ 0.2). That transition corresponds to a positive effect of aging on tree stability to wind forces. A higher damping limits the dynamic amplification of tree sways under dynamic loading more effectively. This positive effect contrasts with the negative effect of aging on frequency and wind load magnitudes. Values predicted for ξ 0 lie within the 0.03-0.25 range documented by (Moore and Maguire, 2004). However, measured ξ 0 values are more commonly found between 0.05 and 0.1. Furthermore, no relationship has been documented between ξ 0 and tree size and, a fortiori, age (Moore and Maguire, 2004). ξ 0 can also vary substantially among trees of similar age and dimensions [e.g., 0.05-0.12 in pine saplings (Sellier and Fourcaud, 2005)]. Because of lack of data, it is not possible to assert that the simulated increase of ξ 0 occurs in nature.
The predicted increase of damping with age is consistent under the hypothesis that aerodynamic drag is the main source of damping in trees. The exponential increase of foliage biomass in the early stage of tree growth would translate to a rapidly increasing aerodynamic damping. That increase would cease after canopy closure unless crown growth is released by thinning events. Once crown size and foliage mass have stabilized after release, the aerodynamic damping would plateau as it is observed in the numerical simulations. The higher ξ 0 value predicted at age 50 is likely a response to thinning at age 47. Trees also dampen motion thanks to branch vibrations (Sellier et al., 2006;Spatz et al., 2007). The efficiency of branch damping does not depend on crown size but on a fine-tuning of branch natural frequencies (Scannell, 1984;Théckès et al., 2015). Viscous damping, which dissipates motion though internal friction in wood, is known to have a minor contribution to overall damping (Milne, 1991;Sellier and Fourcaud, 2005). Viscosity should decrease for wood formed later in tree lifetime declines because of the inverse relationship between viscous damping and specific modulus (Ono and Norimoto, 1983).
Two important aspects have emerged about damping in this study. Firstly, the absolute damping rate varied little with tree age. It means that the tree deflected by a wind gust remains swaying for a similar time window (20-30 s) whether it was 5, 20, or 50 years old. During that window before the tree returns to rest, it is temporarily more exposed to the arrival of another gust. Secondly, the nature of tree sways changed as the tree grew. At a young age, tree's response exhibited a frequency shift. At an intermediate age, a secondary mode of vibration was excited. That mode limited the lateral displacement during the first cycles. At an old age, damping appeared quadratic (i.e., drag-dominated) with a relatively higher dissipation of initial cycles followed by persistent small-scale vibrations. In all those cases, the notional simple oscillator model used for calculating ξ 0 was insufficient to represent the aspects of damping at each age. However, ξ 0 remains useful to provide a gross estimate of the structure's ability to dissipate motion.
The Role of Crown Architecture in Tree Dynamics
From the numerical experiments manipulating the crown status, we find that the presence of branches and foliage slows down tree sways. In absence of any crown, at age 5, the tree swayed twice as fast as the complete tree. A similar ratio was measured for a 4-years-old P. pinaster sapling (Sellier and Fourcaud, 2005). The frequency ratio between the debranched tree and the complete tree continuously decreased during growth until it was 1.25 at harvest age. Thus, the crown's influence on tree sway frequency is reduced with tree age. The crown slows down tree motion because it adds mass onto the stem without contributing any stiffness. Despite that, the relationship between crown mass and frequency is weak. Most of the crown must be removed to significantly increase f 0 (Moore and Maguire, 2005). It is also found that the relationship between crown and sway frequency was better represented when the spatial distribution of crown mass was taken into account, for instance by using the height of the center of mass as a predictor of f 0 changes. The effects of branch distribution along the stem on natural sway frequency was found to be age-related too, with the youngest trees affected most. Integrated silviculture could become a tool for wind damage risk management by influencing the sway frequency of planted trees because it can change crown structure and allocation between compartments (Ando et al., 1968).
Even the center of mass was a poor predictor of the influence of crown on f 0 at age 5. At that age, the spatial distribution and the amount of crown biomass were loosely coupled. All other things being equal, a sapling with a heavy crown could have a center of mass at the same height as a sapling with a light crown. Inversely, two saplings with crowns of equal mass could have centers of mass at a different height. It is possible to accurately predict f 0 in the case of saplings, but those predictions require to describe tree's aerial architecture extensively (Sellier et al., 2006). This is in extenso also the case of mature opengrown angiosperm trees, where large branches have a significant impact on the dynamics of the whole tree (Rudnicki et al., 2012;Ciftci et al., 2013). The emergence of laser scanning technology to measure tree architecture will play a major role to systematically perform complex analysis like finite element modeling (Jackson et al., 2019) and to characterize the dynamical properties of trees during their development.
Aerodynamic Behavior: Juvenile and Mature
Wind forces continuously increase as the tree grows larger and taller. The height growth translates to exposure to higher wind speed and an increased lever arm for the bending moment. Foliage growth and branch growth cause greater sail area and drag. Despite tree dimensions and exposure continually increasing because of growth, the increase in wind forces becomes gradually smaller in the late stages of the rotation. This behavior may result from the shift in carbon allocation from foliage to woody biomass due to aging. The shifted allocation translates to relatively less crown sail area and a stronger stem. If that is the case, then there is a clear need to capture precisely allocation trends and to investigate the role of forest management on those trends in future studies of wind risk. We have presented two allometric models that predict reasonably well foliage mass and crown area across a wide range of sites, even for a single individual tree. They are a practical alternative to repetitive foliage measurements for a plantation lifetime. Hashimoto and Gyokusen (1995)'s model is interesting in that it describes the functional relationship between foliage area and sapwood area promoted by the pipe model theory (Lehnebach et al., 2018). In the future, conceptual models of allometry based on the constant-stress theory (Morgan and Cannell, 1994) or source-sink relationships (White et al., 2016) could be used or tested.
In the juvenile tree, mechanical stresses are approximately uniform along the length of the stem. This agrees with the uniform stress theory that is a well-known paradigm in plant biomechanics. Stresses induced by wind loading are expected to be uniform below the crown and above the butt swell (Morgan and Cannell, 1994). The mature tree also presents a long stem section where bending stresses are uniform. This corresponds to the stem region where breakage is expected to occur (Chiba, 2000). That section extends well into the lower crown region. The modulus of rupture of wood increases with height in trees (Nakatani et al., 1988), which might be a beneficial trait to withstand higher stress levels in the stem region where branches are borne. Breakage of the top part of old trees during typhoons sometime occur in old C. japonica specimens growing in natural forests (Takashima et al., 2009). That type of windbreak could be associated to a decurrent tree form, however, and is atypical in plantations. The tree at age 25 presented a significant deviation from uniformity with very high bending stresses near tree top. The exception can be explained as a response to thinning at age 20 with crown growth in width temporarily exceeding stem radial growth.
Age-Risk Pattern and Consequences for Future Forestry
The risk of wind damage was found to strongly depend on which age class the tree belongs to. In the simulations, trees <20-years-old are submitted to low-magnitude wind loads. All the criteria considered, except for damping, indicate that juvenile trees can be considered as stable under wind loading. During an intermediate phase (age 20-30), the tree transitioned from a situation of low risk to one of high risk. Several events occurred during that period: LAI peaked, the canopy closed, wood properties plateaued, and two thinning events were applied. Any of those events would be detrimental to stability on their own. Because all those changes were concurrent, it could explain why the transition to a situation of high risk was particularly rapid and pronounced. In future studies, it would be interesting to quantify how successive thinning events interact with canopy growth and, by releasing growth, if they contribute to an artificially heightened or precocious risk of wind damage. This would allow the onset and magnitude of high wind risk to be tied with silvicultural planning and productivity curves. This and the role of planting density could be investigated using a combination of growth and yield models and theoretical allometric models.
In the second half of the rotation period (after age 30), the predicted level of wind risk remained approximately constant despite the continuously increasing wind loading on the structure. It appears that the strength of the stem and the strength of the root system increased in proportion to the applied loads. Dedicated models of stem and root plate mechanics should be employed to investigate the scaling of strength with age in more detail.
Wind damage recorded in planted forests after typhoon events has a distinctive pattern. Minimal damage is reported for trees 20years-old or younger. Later, the amount and likelihood of damage increase to a peak value followed by a decrease. Numerous damage inventories reported this "bell-shaped" pattern (Nakao et al., 1993;Fujimori, 1995;Abe, 2005;Sato and Abe, 2006). It is observed across different regions, from Kyushu to Hokkaido, and across different planted species [C. japonica, C. obtusa, Larix kaempferi (Lamb.) Carr.]. The age of peak damage can vary significantly across sites from 35 (Nakao et al., 1993) to 80 (Sato and Abe, 2006), commonly between 50 and 60 (Fujimori, 1995;Abe, 2005). Deviation from the general pattern may happen. For example, important damage has been recorded in the sub-20 age classes at one site (Nakao et al., 1993). Damage inventories for natural forests have shown an altogether different age-risk relationship with a steadily increasing likelihood of damage over a 100 of year (Fujimori, 1995). Similarly, in the natural forests of the Pacific northwest (North America), wind becomes a significant cause of tree mortality only after age 20 and becomes the primary cause in age classes older than 100 (Franklin et al., 1987). The differences between natural forests and plantation forests indicate that the age-risk relationship shown in this study is not inherent to tree's increase in size during growth in general. Whether the influence of forest management on risk derives from the impact on tree growth (slenderness, taper, allocation) or the impact on forest structure heterogeneity remains to be investigated.
In Japan, the dominant age class in plantation forests was 50-55 in 2017. It is of paramount importance to determine whether the age of peak damage likelihood has been reached to decide if the rotation period can be extended without increasing the vulnerability of forests to wind storms nationwide. Age 50 is approximately the mean reported age for peak damage but, because that age is variable, it is difficult to estimate if the risk associated to windstorms has already peaked. Although the present model can, to a large extent, reproduce the documented age-risk pattern, the lack of available growth data unfortunately did not allow predicting tree mechanical behavior beyond age 55. Assessing wind risk beyond the current traditional age of harvest remains difficult because experimental data on biomass spatial patterns (including architecture) is scarce past that age and because of the strong coupling between those patterns and tree aerodynamic behavior. Linking tree mechanical behavior to the age dynamics of forest aboveground biomass allocation as a function of climate and forest management is an important challenge for future research on wind-tree interactions.
DATA AVAILABILITY STATEMENT
The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.
AUTHOR CONTRIBUTIONS
DS developed and implemented the growth model, performed the finite element analyses, analyzed the results, and wrote the manuscript. SS sourced published and unpublished experimental data sets, translated scientific literature, analyzed the results, and edited the manuscript. All authors contributed to the article and approved the submitted version.
FUNDING
The Japanese Society for the Promotion of Science (Grant no. S12707) provided a short-term fellowship funding to support DS 5-weeks stay at FFPRI, Tsukuba, Japan in 2012. | 2020-08-11T13:09:15.965Z | 2020-08-11T00:00:00.000 | {
"year": 2020,
"sha1": "cf8af536e87f4dc8b6c2667aae5a6e541638ec15",
"oa_license": "CCBY",
"oa_url": "https://www.frontiersin.org/articles/10.3389/ffgc.2020.00089/pdf",
"oa_status": "GOLD",
"pdf_src": "Frontier",
"pdf_hash": "cf8af536e87f4dc8b6c2667aae5a6e541638ec15",
"s2fieldsofstudy": [
"Environmental Science"
],
"extfieldsofstudy": [
"Environmental Science"
]
} |
227247792 | pes2o/s2orc | v3-fos-license | Labor Reforms in Rajasthan: A boon or a bane?
We examine the impact of labour law deregulations in the Indian state of Rajasthan on plant employment and productivity. In 2014, after a long time, Rajasthan was the first Indian state that introduced labour reforms in the Industrial Disputes Act (1947), the Factories Act (1948), the Contract Labour (Regulation and Abolition) Act (1970), and the Apprentices Act (1961). Exploiting this unique quasi-natural experiment, we apply a difference-in-difference framework using the Annual Survey of Industries panel data of manufacturing plants. Our results show that reforms had an unintended consequence of the decline in labour use. Also, worryingly, the flexibility resulted in the disproportionate decline in the directly employed worker. Evidence suggests that the reforms positively impacted the plants' value-added and productivity. The strength of these effects varies depending on the underlying industry and reform structure. These findings prove robust to a set of specifications.
Introduction
Internationally, Indian labor laws are considered rigid and complex. In this vein, recently, the Indian government passed three major labor code bills by the Parliament: The Industrial Relations Code Bill, 2020; the Code on Social Security Bill, 2020; and the Occupational Safety, Health, and Working Conditions Code Bill, 2020 along with the Code on Wages Bill enacted in 2019. After a long time, the government introduced these new laws to reduce complexities, bring more transparency and accountability, and help both employers and workers. These reforms in the labor laws with a high degree of both political and public interest started back in 2014, with Rajasthan being the first Indian state to deregulate the labor laws in four major Acts (Government of India, 2018).
The reforms in India's labor laws resulted from a rigorous debate. One strand of literature argued the restrictive labor laws hurt the firms by forcing them to remain small and use contractual workers or capital-intensive technologies (Amirapu and Gechter 2020;Hasan, Mehta, and Sundaram 2020;Hasan, Kapoor, Mehta, and Sundaram 2017;Ahluwalia, Hasan, Kapoor, and Panagariya 2018). In contrast, the other strand of literature opined the labor laws could not be held responsible for the Indian economy's sluggish growth (D'Souza 2010; Chatterjee and Kanbur 2015;Roychowdhury 2014;Roy, Dubey, and Ramaiah 2020;Deakin and Haldar 2015). These two strands of opinion on the Indian labor market flexibility differ in various theoretical understanding, methodological details, and empirical ground.
Believing that the strict labor laws are detrimental to the Indian economy, the Indian government started relaxing some of the major Indian labor laws both at the national and sub-national levels (Government of India, 2018). These important reforms in the Indian labor market require careful independent evaluation. The labor reforms in 2014 in the Indian state of Rajasthan provides a natural experiment to understand the impact of such reforms. In particular, in this paper, we find the causal impact of the labor law reforms in Rajasthan on the plant's employment and performances. Rajasthan deregulated the labor laws in the Industrial Disputes Act (1947), the Factories Act (1948), the Contract Labor (Regulation and Abolition) Act (1970), and the Apprentices Act (1961). These reforms in the labor laws in Rajasthan allowed us to utilize a quasi-natural experimental research design. We use a difference-indifference specification to the establishment-level Annual Survey of Industries (ASI) longitudinal data from 2011-12 to 2016-17 to examine the effects of Rajasthan's pro-employer reforms on employees, direct and contractual workers, capital, inputs, gross value added (GVA), total factor productivity (TFP), profits, and workers' emoluments. As the existing literature does not provide any clear-cut opinion of the benefits of the labor market flexibility in the Indian economy, it is not surprising that the empirical outcome of the newly introduced labor reforms on plant employment and performances are ambiguous. Our work contributes to the existing literature by providing evidence on whether the newly introduced flexibility in the labor laws is gainful or not. The novelty of our work is that understanding the impact of deregulations in the Indian state of Rajasthan will help predict the implications of the recent national amendments in labor laws that affect nearly 425 million Indian working-age population. Thus our study on the causal effect of deregulations in the labor laws generates important policyrelevant insights.
Our article is not the first to examine the effect of the amendments in labor laws in Rajasthan. Most notably, the Economic Survey 2018-19 of India (Government of India, 2018) exploited the ASI data from 2011 to 2017 to analyze the reforms in Rajasthan. Our work is distinguished from the Economic Survey (Government of India, 2018) by the aggregation level and the outcome variables. In particular, the Economic Survey finds a macro impact (extensive margin) of the reforms on the total manufacturing employment and other variables like the number of factories with greater than 100 workers, total output, total wages, output per factory, and wages per factory using 146 observations at the state level. In contrast, our article finds the micro impact (intensive margin) of the reform on the plant's employment and performances. Provided the heterogeneity among the manufacturing plants at the aggregate level, our study provides a deeper understanding of the reforms (Mathew 2017;Padmaja and Sasidharan 2016).
Our empirical analysis provides evidence that the reforms had an unintended consequence of the decline in labor use. The implications regarding employment change are similar to Roy, Dubey, and Ramaiah (2020), Deakin and Haldar (2015), Roychowdhury (2019a); Chatterjee and Kanbur (2015), D'Souza (2010), Kapoor (2014), Chandru (2014) in the sense that higher flexibility causes weaker employment growth. Also, worryingly, the increased flexibility resulted in the decline in directly employed workers. We find the plants that directly fall under the Industrial Disputes Act (1947) reforms experience an expansion in labor compared to the plants that are not under the direct ambit of the Industrial Disputes Act (1947) reforms. Moreover, our data show that the plants that fall directly under the Contract Labor (Regulation and Abolition) Act (1970) reforms experience greater use of contractual workers than the plants that are likely to be unaffected. We also find reforms to cause the plants in the labor-intensive industries to restructure their production mix by reducing their labor use. In contrast, the newly introduced labor laws' flexibility caused the plants in export-oriented industries to use more contractual workers. Regarding the impact of the reforms on plant inputs and performances, we find the reforms to positively impact the plants' value added and productivity. The parallel test result indicates no change in the plant outcomes before the reforms in the treatment and control states. Furthermore, the authors find that the reforms did not cause new manufacturing plants in Rajasthan.
Literature Review
Indian labor laws have been the focus of many debates. One strand of literature argues against labor protection on the grounds of strict labor laws; i) directly ii) indirectly reducing the economy's efficiency, and iii) increasing labor substitution with capital or contractual workers. In contrast, the other literature opines that the labor laws could not be held responsible for the Indian economy's sluggish growth.
i) Direct adverse impact of the strict labor regulation on plant performances and economic outcomes
One of the influential studies on the detrimental effects of labor protection on the Indian economy is Besley and Burgess (2004). They find the Indian states' pro-worker regulations to cause lower output, employment, productivity, and investment in the formal manufacturing sector. Another similar study by Ahsan and Pagés (2009), find an adverse impact of employment protection and cost of dispute resolution on employment and output. Moreover, this adverse impact is more for the states and timeframe, where the cost to resolve a dispute is high. Workers do not benefit from these protections as the authors do not find an increase in labor share or wage bill. Bhattacharya, Narayan, Popp, and Rath (2011) find the rigid labor market in India to hinder the multinationals from operating in the laborintensive production process compared to countries like China and the Philippines. Also, Lee (2019) finds a lack of labor demand in rigid labor markets in India. This strand of literature opines labor market reforms to arrest the detrimental effects of high labor cost and rigidity. The reforms will improve wage share, control the increase of informal employment, and increase aggregate productivity (Dougherty 2009). Amin (2009) analyze the impact of labor laws on the employment of 1948 retail stores in India. He reports that 27% of the stores find labor regulation as a hinder to their business activities. He finds that the labor reforms will increase employment by 22% for an average store. Further he finds the strict labor laws to increase labor costs, resulting in firms substituting labor with the computer (Amin 2007). Pro-worker legislation or labor unrest also adversely impact the location choice and investments (Menon and Sanyal 2007;Sanyal and Menon 2005). Dougherty, Robles, and Krishna (2011) find that strict labor regulations are likely to harm industries with high labor intensity or high sales volatility.
They estimate that firms experience a 14% higher TFP in labor-intensive industries and the states with the flexible labor market than the firms in the labor-intensive but rigid labor market. Similarly, they experience 11% higher TFP in a pro-employer state than in the pro-worker state for the volatile industries. One recent study by Hasan, Mehta, and Sundaram (2020) finds that the rigid labor regulations to adversely impact the exporters by reducing the output. The rigid firing restriction also reduces the firm's employment responses to temporary shocks (Adhvaryu, Chari, and Sharma 2013). Another impact of rigid labor laws is an increase in corruption. A recent study by Amirapu and Gechter (2020) estimates that regulations increase firms' labor costs by around 35%, which increases the possibility of corruption 3 . Aghion, Burgess, Redding, and Zilibotti (2008) find the dismantling of License Raj helps the industries in pro-employer states to grow more quickly than in pro-worker states. Mitra and Ural (2008) find the positive impact of trade liberalization on productivity more pronounced for states with a flexible labor market. They also find that trade liberalization helps the export-oriented industries in states that have flexible labor laws. Labor demand elasticity is also higher with trade liberalization for the states with flexible labor markets (Hasan, Mitra, and Ramaswamy 2007). Labor regulations are not only limited to generate gains from trade liberalization and are also crucial in the firm size distribution. Larger sized firms are prevalent in the states with flexible labor regulation. The prevalence of large-sized firms in flexible states is more prominent among the firms that started production after 1982, when labor laws were tightened (Hasan and Jandoc 2012). Thus, Hasan and Jandoc (2012) claim labor regulations to affect the firm's size adversely. Hasan, Kapoor, Mehta, and Sundaram (2017) emphasize that even if India is one of the largest producers and exporter of apparel, the sector is still to operate at its potential. They point this incapability to the labor regulations that cause the firms to operate at scales which are insufficient to use the advanced techniques. Another recent study by Ahluwalia, Hasan, Kapoor, and Panagariya (2018) analyzes the impact of labor regulations on employment and wages. They use the 2005 abolition of the quota restrictions on the export of apparel and textile products from developing to developed countries and the variation in the labor regulations across the Indian state as a natural experiment to find the effect of labor regulation. They find significant benefits in employment and wages post 2005 in the apparel and textile industries in states with flexible labor laws. Hasan, Mitra, and Sundaram (2013b) find the capital intensity higher for India's firms than other countries with the same level of economic development or factor endowments. They find strict labor regulations as one of the primary reasons for the high capital intensity. The rigid labor laws do not help trade gains based on factor abundance comparative advantage (Hasan, Mitra, and Sundaram 2013a). Hasan, Mehta, and Sundaram (2020) find that producers in pro-worker states replace labor with capital. Firms use contractual or fixed-term workers for many reasons (Singh, Das, Abhishek, and Kukreja 2019). Some of the reasons are; to reduce the high labor cost (Sapkal 2016;Basu, Chau, and Soundararajan 2018), reduce the bargaining power of the permanent workers (Saha, Sen, and Maiti 2013), stay away from the legal establishment size threshold of 100 workers (Ramaswamy 2013a;Ramaswamy 2013b), increase flexibility as the employers are free to hire and fire the contract workers (Srivastava 2016), deal with temporary shocks (Chaurey 2015), and many more. Ramaswamy (2013a) finds the strict labor laws to cause higher intensity of contract workers for the size group of 55-99 workers and in labor-intensive inflexible states. A regional case study by Barnes, Das, and Pratap (2015) in North India's automotive components production shows how a regional contract labor system has helped the employers to keep the wages low, enjoy more flexibility, skip the burden of monitoring and controlling the workers, and weaken the labor rights. However, the use of a contract worker has its demerits. As it is an 'incomplete contract', the workers' underinvest in specific skills (Singh, Das, Abhishek, and Kukreja 2019;Singh, Das, Kukreja, and Abhishek 2017).
Criticism of the view that Indian labor regulations harm the Indian economy
The regulations on job security do not negatively affect as (D'Souza 2010) as firms transform the work practices and make it flexible through non-compliance or weak enforcement of laws (Chatterjee and Kanbur 2015). Badigannavar and Kelly (2012) finds that even a pro-worker state like Maharashtra provides weak protection to the formal sector workers and the labor unions. A recent study by Roy, Dubey, and Ramaiah (2020) finds no evidence of the spatial variation in labor regulations flexibility in explaining employment growth variation. They find that higher flexibility associates with weaker employment growth. In a similar vein, Roychowdhury (2019a) and Roychowdhury (2019b) explain that the labor laws cannot be held responsible for the employment stagnation in India's organized manufacturing as it applies to less than 35% of aggregate employment. He further finds that the worker's bargaining power is declining in Indian manufacturing. A study on Gujarat's deregulatory reforms by Deakin and Haldar (2015) proposes very little evidence linking law deregulation to growth. In response to the belief that employment protection legislation restricts employment adjustment from demand shock, Sofi and Kunroo (2018) find no evidence from 2000-01 to 2011-12. Moreover, Rodgers and Menon (2013) find that employment adjustment and dispute settlement restrictions cause higher job quality for women.
One of the most influential studies by Besley and Burgess (2004) has been criticized on various conceptual and measurement issues, coding errors, methodological problems, failure to replicate the findings (Bhattacharjea 2006;Bhattacharjea 2009;Jha and Golder 2008;Storm 2019;D'Souza 2010) and, difficulties in the enforcement of the labor laws in India (Fagernäs 2010). Some scholars find the labor laws changes are endogenous to several other economic factors and do not explicitly determine economic indicators (Dutta Roy 2004;Deakin, and Sarkar 2011). Another study that has been severely criticized is Basu, Fields, and Debgupta (2009), which find flexible labor laws beneficial to workers' wages and employment. Roychowdhury (2014) examine their theoretical argument and find their policy conclusion to be unsustainable.
Thus, the impact of strict labor laws on the Indian economy is inconclusive. One group of scholars advocate relaxation in labor laws, while the other group claims does not. The differences in opinion stem from both analytical as well as methodological understanding. In this context, the Indian government deregulated the labor laws in recent years, believing that the pre-existing labor laws are detrimental (Government of India, 2018). We analyze the impact of these recent relaxations in labor laws on plant employment and performances. Thus, this paper adds to the existing literature by finding whether the recent relaxations in the labor laws that one group of scholars have been advocating over the years have been gainful or not. This paper contributes to the literature on the Indian institutional reform effects, plant employment and performances in Indian manufacturing, quasi-natural experiment, and on the recent developments in the Indian labor market.
Background
Under the Constitution of India, labor is a subject in the concurrent list where both the Central and the state governments are capable of enacting legislation. None of the Indian states adopted labor reforms previous to 2014 (Government of India, 2018). After a long time, Rajasthan was the first state that introduced labor reforms in four majors Acts: The Industrial Disputes Act (1947), The Factories Act Table 1 describes the amendments in each of these Acts. (1947) i.
Government approval is not required for companies with 300 or fewer workers to shut down or retrench workers. The earlier limit was 100 workers. ii.
The membership requirement to form a union has increased from 15% to 30% of the total workmen. iii.
The time limit for any worker to object has been reduced to three years from an indefinite period.
The Factories Act (1948) i. The threshold limit increased from 10 or more workers with the power to 20 or more workers with power, and 20 or more workers without power to 40 or more workers without power. ii.
Any complaint against the employer about the violation of this Act will not receive cognizance by a court without prior permission from the state government. The Contract Labor (Regulation and Abolition) Act (1970) i.
Applicable to establishments that employ 50 or more workers on contract against the former 20 or more workers.
The Apprentices
Act (1961) i. Apprentice's stipend will be no less than the minimum wage. ii.
Government to bear part of the costs of apprentice training in order to encourage skilling.
All these reforms were pro-employer. These deregulations in the labor laws provide an interesting setup to examine the impact of the reforms on plant's employment and performances.
Diluting of the labor laws can have an ambiguous impact on the plant's employment, performances, and inputs. The flexibility in labor laws can increase plant employment as employers prefer labor over other production factors. As the reform reduces the hiring and firing cost, employers can adjust the laborers according to their requirements and prefer cheap labor. The labor's bargaining power also reduces, and this might act as a further boost to increase employment. These reforms can also increase contractual workers' use because of their added advantage (Drager and Marx 2017;Kuroki 2012). However, these pro-employer reforms can also cause employment to decline as employers get the authority to shed workers. The lack of powerful labor unions further makes the dismissal process easy (Watanabe 2018; Roychowdhury 2019a; Roychowdhury 2019b). As the labor cost declines with the reforms, plants might increase capital investments to complement the labor. Also, flexible labor laws result in less costly bank loans as the borrower's default risk declines due to the increased flexibility to adjust labor (Alimov 2015). On the contrary, the low cost of labor can cause employers to substitute labor for capital (Hasan, Mitra, and Sundaram 2013a;Hasan, Mitra, and Sundaram 2013b;Hasan, Mehta, and Sundaram 2020). Plant productivity may increase because employers can adjust laborers and lay off unproductive workers resulting in the most productive skill matches (Caballero, Cowan, Engel, Micco 2013;Maida and Tealdi 2020). The worker's effort can also increase because of the fear of dismissal (Bradley, Green, and Leeves 2014). On the contrary, productivity can decrease as low job security might cause the workers to invest less in plant-specific human capital value addition (Acharya, Baghai, and Subramanian 2013), discourage the workers from providing effort, and high wage inequality among the workers (Shimizutani, and Yokoyama 2009;Silva, Martins and Lopes 2018). Thus, the effect of the labor laws reforms on plant employment, inputs, and performances warrants an empirical examination.
Empirical Methodology
We use difference-in-difference (DID) framework to compare the plant outcomes before and after the reforms in the treatment and the control state. To the best of our knowledge, we find no other policy or reforms implemented in Rajasthan in 2014 that impacted plant-level outcomes differentially more or less than in the control state. This will help us identify the treatment effect of labor reforms in Rajasthan.
Ideally, we would like to compare the plants in Rajasthan with an observationally similar control group. We choose the establishments in Punjab as a control group because of the following reasons: i) Punjab is a neighboring state of Rajasthan and have similar characteristics in various aspects ii) Among the other neighboring states of Rajasthan, i.e., Gujarat, Madhya Pradesh, Uttar Pradesh, and Haryana had also started to think of introducing flexibility in labor laws around 2014 mostly because of the similar political affiliation in those states (ruling party). Thus, we would not get correct estimates if we use these states as a control group. iii) Punjab and Rajasthan experience a similar degree of flexibility in labor restrictions before the reform (Government of India, 2018). Furthermore, as a robustness check, we choose establishments from other states as a control group.
The identifying assumption of the DID estimator is that the treatment and the control group should have similar trends before the reform. In the subsequent section, we show that Rajasthan and Punjab have similar trends in pre-2014. We estimate the following plant level regression specification in Equation (l) to find the impact of the amendments in labor laws on various plant outcomes. represents plant level outcomes like employers, direct workers, contractual workers, capital, inputs, GVA, TFP, profit, and emoluments.
is an indicator variable that takes the value of 1 for the years after the amendments (2014-15 to 2016-17) and 0 otherwise.
is an indicator variable that takes the value of 1 if the plant is in the treated state Rajasthan and 0 if the plant belongs to Punjab. is the plant fixed effect that controls for any timeinvariant unobserved heterogeneity. is the year fixed effect that controls the year specific unobserved changes. We should keep in mind that will be completely absorbed by the year fixed effects whereas, will be completely absorbed by the plant fixed effects. are the controls, namely, age of the plant, percentage of the output that the plant export, import dummy trend, GVA, capital, inputs, profit, emoluments, and workers 4 .
represents industry trends and is the stochastic error term. We cluster standard errors at the state levels. The coefficient of the interaction of and , 2 is the coefficient of our interest that captures the causal impact of the labor laws reform on the plant outcome variables. We consider the entrants, incumbents, and exiters during the sample period.
The reforms in the labor laws may be more pronounced for the "affected plants". Affected plants are those that are most likely to be affected by the labor laws reforms. We identify affected plants in two ways; i) Plants that fall directly under the Industrial Disputes Act (1947) reforms, and ii) Plants that fall directly under the Contract Labor (Regulation and Abolition) Act (1970) reform, in the pre-treatment period. To find the impact of the reforms on the affected plants, we use triple difference and estimate the following regression specification: The coefficient, 6 captures whether the affected and the non-affected plants responded differently after the reform to before in Rajasthan compared to Punjab. A significant 6 indicates that the law changes were effective in impacting those plants that were intended to.
We further analyze the impact of the reform on heterogeneous industry categories. Plants in laborintensive industries or export-oriented industries are more likely to be impacted by increased flexibility in labor laws. Many studies have found the strict labor regulations to affect the exporters and the laborintensive industries adversely (Hasan, Mehta, and Sundaram 2020;Mitra and Ural 2008;Saha, Sen, and Maiti 2013;Dougherty, Robles, and Krishna 2011;Hasan, Mitra, and Sundaram 2013a;Ramaswamy 2013a). Therefore, in a similar vein, the increase in flexibility in labor laws should be differentially larger in these types of industries. To test this, we estimate the following regression specification: = 0 + 1 + 2 + 3 * + 4 * + 5 * + 6 * * + + + + (2) 6 finds the heterogeneous impact of the changes in labor laws on plant outcomes. is an indicator variable that takes a value of 1 if a plant is in labor-intensive/export-oriented industries and otherwise zero.
Data
We use the plant longitudinal data of the Indian manufacturing from the Annual Survey of Industries (ASI) provided by the Ministry of Statistics and Programme Implementation, Government of India. The ASI is a nationally representative survey of plants/establishments that are registered under The Factories Act, 1949. The Factories Act, 1949, is important legislation that regulates India's manufacturing activities for and all establishments that employ 10 or more workers (with electricity) or 20 or more workers (without electricity). Since the labor reforms pertain to the formal sector, our data set covers the formal manufacturing in India. The establishments in ASI data are divided into a census sector and a survey sector. The plants with more than 100 workers or that file joint returns in the ASI survey or are situated in some industrially backward states like Manipur, Meghalaya, Nagaland, Tripura, Andaman, and Nicobar Island are surveyed every year and hence are called census sector. Plants that do not fall in the census sector are randomly sampled using a systematic circular sampling technique within each state*Industry*Sector*4 digit stratum and form the survey sector. We use the information from both the census and the sample sector for manufacturing plants. Furthermore, as a robustness check, we restrict our sample with the census sector's establishments and use a balanced panel. Table 2 presents the number of observations by the census and the sample sector in the treatment and control group. In this study, we utilize the ASI dataset from 2011-12 to 2016-17. The reference period of the ASI data is a fiscal year between April to March. We use plant-level information on employees, direct and contractual labor, capital, inputs, profits, emoluments, GVA, and TFP. Capital is measured as the average of fixed capital's net book value at the beginning and the end of the fiscal year. The labor input is measured as either the average number of person worked. The average number of person worked is the ratio of total person-days to the number of working days. We estimate TFP using the methodologies proposed by Woolridge (2009) and Levinsohn and Petrin (2003) as TFP (method 1) and TFP (method 2), respectively. The procedure for estimating the TFP is presented in the Appendix. GVA is deflated by the suitable wholesale price index (WPI) by groups using 2005 as the base year. Matching the detailed categories of WPI with the 2-digit industry classification was impossible due to data limitations. However, a close and mindful comparison of the groups was undertaken to choose appropriate price deflators. Fixed capital is deflated using WPI for machinery and equipment. The consumer price index (CPI) of rural laborers and industrial workers is used as a deflator for workers' emoluments. We classify an industry as labor intensive if the capital intensity 5 is below the median of the total manufacturing (Kapoor 2015). To find the export-oriented industries, we use the 2 digit industry trade information from U.N. Comtrade and calculate the value of T, where T = M−X Q−X+M , M is import, X is export, and Q is production. If T's values is negative, then that particular industry is exportoriented (Erlat 2000;Krueger, Lary, Monson, and Akrasanee 1981). We use the output data (Q) from the United Nations Industrial Development Organization. We use the information from the pretreatment years (2011-12 to 2013-14) to categorize the industries. Table 3 summarises the data used in our analysis for the treatment and the control groups pre and post-reform.
Results
This section presents the empirical evidence regarding the causal impact of the reforms in labor laws on various plant's employment and performances. We compare the difference in the outcome variables for the plants in Rajasthan relative to Punjab before and after the amendments. We then confirm that the reform had a differentially larger effect on the plants that are most likely to be "affected" than the "unaffected" using a triple difference framework. We estimate the triple difference specifications across the capital and labor-intensive industries and exporting and non-exporting industries to find the heterogeneous impact of the reform.
Parallel Trends
We establish that the parallel trends assumption between the treatment and the control group holds using two approaches. First, we visually inspect the parallel trend assumption. Figure 1 provides the detrended values of employees, both directly employed as well as contractual workers for treated and the control states. Visually, the trends are parallel until 2014-15 and diverge thereafter. Second, we use a formal placebo regression, to check the potential treatment effects before the labor laws amendments (2011-12 to 2013-14).
where is a continuous variable (0,1,2) for the year 2011-12, 2012-13 and 2013-14 respectively. If the treatment and the control state holds parallel trends, then 1 should be zero. In Table 4 and Table 5, we find the coefficient of the interaction term * to be statistically insignificant. Thus the parallel trend assumption holds and confirms that the results are not driven by spurious effects. Table 6 presents the impact of the reforms in labor laws on employment outcomes. The outcome variables are log employees, log contractual workers, log direct workers, contractual to total workers, and direct to total workers. We find that the plants in Rajasthan differentially reduced total employees by around 3% compared to plants in Punjab, after relative to before the reforms. This suggests that flexibility in the labor laws resulted in the decline in plant employment. Furthermore, the plants responded to the reforms by decreasing the number of direct workers by around 2% whereas the decline in contract workers is insignificant. We notice that the ratio of both contractual to total workers and direct to total workers declined significantly by around 1% in Rajasthan after the reforms. The impact of the reforms is different for the plants that are most likely to be "affected" from the "unaffected". According to the Industrial Disputes Act (1947) reforms, we notice that the plants with workers greater than 100 and less than 300, and plants with less than or equal to 100 workers in 2014 are most likely to be affected. The triple difference estimates from Equation 2 in panel A of Table 7 indicate that the plants that had workers between 100 and 300 in 2014 experienced a significant increase in total employment and contractual workers, compared to the plants that had greater than 300 workers in Rajasthan compared to Punjab, in post compared to pre-treatment period. However, worryingly, these types of plants experienced a significant decline in direct to total workers. The plants with less than 100 workers in 2014 experienced a significant increase in total employment and direct employment compared to the plants with greater than 300 workers in Rajasthan compared to Punjab, in post compared to the pre-treatment period. Thus, the Industrial Disputes Act (1947) reforms successfully impacted those targeted plants.
Effect of Labor Law Amendments on Employment
According to the Contract Labor (Regulation and Abolition) Act (1970) reforms, we find that the plants with contractual workers greater than 20 and less than 50 in 2014 are most likely to be "affected". Consistent with the hypothesis, the affected plants experienced a significant increase in the ratio of contractual to total workers compared to the "unaffected" in Rajasthan compared to Punjab in post compared to the pre-treatment period (panel B of Table 7). Thus the reforms caused these "affected" plants to have a higher proportion of contractual workers. Also, these affected plants experienced a significant decline in both direct and contractual workers. Thus the effect on the "affected" plants is twofold i) experienced a decline in both contractual and direct workers ii) The proportion of contractual workers to total workers increased. The results indicate that total employees and contractual to total employees declined whereas direct to total workers increased for the plants in labor-intensive industries compared to capital intensive industries in Rajasthan versus Punjab, and after compared to before the reform. These industries are preferring direct workers. This might be because of the plant-specific skill that the direct workers entail. Thus, the reforms helped the plants in the labor-intensive industries restructure the production factors according to their requirements.
As expected, plants in export-oriented industries experienced an increase in contractual to total workers than non-exporting industries in Rajasthan versus Punjab, and after compared to before the reform. The flexibility in the labor laws caused the plants in export-oriented industries to use contractual workers. The export market volatility causes this type of plant to choose contractual workers and thus reduce fixed cost of labor.
Effect of Labor Law Amendments on Plant Performances and Inputs
We estimate Equation 1 to find the effect of deregulating the labor laws on various plant input and performances like capital, inputs, GVA, TFP, profits, and emoluments. The estimates are presented in Table 9. We do not find any significant impact of the labor laws on capital, inputs, profit, and emoluments. However, we find that the plants' productivity significantly increased by around 3% due to the increased flexibility in labor laws in Rajasthan compared to Punjab. We present the estimates of the impact of the the reform on plant inputs and performances by "affected" plants and industry heterogeneities in the Appendix.
Plant Entry
Is there more new plants' entry due to the increased flexibility in labor laws post 2014 in Rajasthan? To examine this change at the extensive margin, we estimate a separate regression at the 2-digit industry*state*year level. We identify a plant's birth from the "year of initial production" in the ASI data.
= 0 + 1 + 2 * + + + + + + + where is the total entry of new plants in a year t, state s, and industry j. is an indicator variable that takes on the value 1 if the plant is in the treated state Rajasthan and 0 if the plant belongs to Punjab.
is a indicator variable that takes on the value 1 for the years after the amendments (2014-15 to 2016-17) and 0 otherwise. is the industry fixed effect that control for any time invariant unobserved heterogeneity at the industry level. is the year fixed effect and is the state fixed effects.
is the industry trend and is the state trend. are the control variables, namely, age of the plant, percentage of the output that the plant export, import dummy trend, gva,capital, inputs, profit, emoluments and workers. 2 is the coefficient of interest that finds the impact of the reforms on the entry of new plants. We find from Table 10 that 2 is statistically insignificant, and thus the impact of the reforms on plant employment and performances is from the incumbent plants in Rajasthan. We do not find the reforms to cause entry of new plants.
Robustness Checks
In this section, we perform several robustness checks of the main results presented above. For robustness, we use the establishments in the Indian states of Gujarat, Madhya Pradesh, Haryana, Uttar Pradesh as a control group (Panel A, B, C, D, and E of Table 11). We also estimate the DID in Equation 1 by making the control group as all the formal manufacturing establishments in India except those in Rajasthan (Panel F of Table 11). The results indicate that Rajasthan's labor reforms negatively impacted the total number of employees in an establishment. Moreover, this decline in employment is primarily through the decline in employment for direct workers. These results are qualitatively and quantitatively similar to the main results in Table 6, which indicate the labor laws' deregulations to cause a decline in employment.
We also limit the sample of establishments to the census sector and a balanced panel (Panel G and Panel H of Table 11). These establishments are larger, with greater than 100 employees. We donot notice a significant fall in the number of employees, but we notice a shift in the usage of directly employed workers to contractual workers. Worryingly, we find the labor deregulations in Rajasthan cause a decline in the direct workers and this has been offset by an increase in the contractual workers.
Conclusion
In this paper, we empirically examined the impact of the 2014 labor laws deregulations in the Indian state of Rajasthan on plant employment and performances. The reform in the labor laws allowed us to utilize a quasi-natural experimental research design. We use a difference-in-difference specification to the establishment-level ASI panel data to examine the effects of Rajasthan's pro-employer reforms.
Our empirical analysis showed that the reforms had an unintended consequence of the decline in labor use. The implications regarding employment are similar to those presented by Roy, Dubey, and Ramaiah (2020), Deakin and Haldar (2015), Roychowdhury (2019a), Chatterjee and Kanbur (2015), D'Souza (2010), Kapoor (2014), Chandru(2014) in the sense that higher flexibility is associated with weaker employment growth. Also, worryingly, the increased flexibility resulted in the reduction in the directly employed workers. Heyes and Lewis (2015) and Avdagic (2015) find similar results for the European Union. If we consider plants as those that directly fall under the Industrial Disputes Act (1947) reforms, then we find these "affected" plants expand in labor use due to the reforms. If we consider the plants that fall directly under the Contract Labor (Regulation and Abolition) Act (1970) reforms, then we find that these "affected" plants experience greater use of contractual workers. We also find the reforms to cause the plants in the labor-intensive industries to restructure its production mix by reducing the labor use and preferring more direct workers. On the other hand, the labor laws' flexibility caused the plants in export-oriented industries to use more contractual workers. We also evaluate the reforms in labor laws on the plants' outcomes beyond the employment effects and find the reforms to positively impact GVA and productivity. | 2020-12-03T02:47:47.183Z | 2020-12-01T00:00:00.000 | {
"year": 2020,
"sha1": "f1def8312f86977633cbca9ff8d10b14d269bc41",
"oa_license": null,
"oa_url": "http://arxiv.org/pdf/2012.01016",
"oa_status": "GREEN",
"pdf_src": "Arxiv",
"pdf_hash": "f1def8312f86977633cbca9ff8d10b14d269bc41",
"s2fieldsofstudy": [
"Economics",
"Law"
],
"extfieldsofstudy": [
"Economics"
]
} |
53696028 | pes2o/s2orc | v3-fos-license | Frequency mixing at an electromagnetically induced transparency like metasurface loaded with gas as a nonlinear element
Local electromagnetic field enhancement in resonant metamaterials is useful for efficient generation of nonlinear phenomena; however, the field enhancement is suppressed by losses of nonlinear elements in metamaterials. For overcoming this issue, we investigate the nonlinear response of an electromagnetically induced transparency like metasurface loaded with gas as the nonlinear element. To induce nonlinearity in the gas associated with discharges, an electromagnetic wave with a modulated amplitude is incident on the metasurface. The measured waveform and spectrum of the transmitted electromagnetic wave, along with light emission from the discharge microplasma, reveal that frequency mixing can occur on the metasurface. The parameter dependence of the conversion efficiency of the frequency mixing phenomenon shows that the efficiency is determined almost entirely by the ratio of the duration of microplasma generation to the modulation period of the incident wave amplitude. This result implies that the frequency mixing is derived from a binary change in the transmittance of the metasurface caused by the generation and quenching of the microplasma.
Local electromagnetic field enhancement in resonant metamaterials is useful for efficient generation of nonlinear phenomena; however, the field enhancement is suppressed by losses of nonlinear elements in metamaterials. For overcoming this issue, we investigate the nonlinear response of an electromagnetically induced transparency like metasurface loaded with gas as the nonlinear element. To induce nonlinearity in the gas associated with discharges, an electromagnetic wave with a modulated amplitude is incident on the metasurface. The measured waveform and spectrum of the transmitted electromagnetic wave, along with light emission from the discharge microplasma, reveal that frequency mixing can occur on the metasurface. The parameter dependence of the conversion efficiency of the frequency mixing phenomenon shows that the efficiency is determined almost entirely by the ratio of the duration of microplasma generation to the modulation period of the incident wave amplitude. This result implies that the frequency mixing is derived from a binary change in the transmittance of the metasurface caused by the generation and quenching of the microplasma.
The enhancement factor of the local electromagnetic field in a resonant metamaterial is inversely related with the losses in its unit structure. In the optical region, nonlinear dielectric resonators 7-9 would be suitable for the unit structure, since nonlinear dielectrics possess small losses. In contrast, lower frequency regions, including microwave and terahertz, require electronic devices, such as varactor diodes, to be introduced as nonlinear elements. 1 These electronic devices have relatively high losses, which suppress the local electromagnetic field enhancement.
In this study, we investigate a metasurface loaded with gas as a nonlinear element to develop a method for preventing the suppression of the resonant enhancement of local electromagnetic fields. Gases have low linear losses, and exhibit discharges under the application of a strong electric field. Thus, we consider gases to be suitable for the efficient generation of nonlinear phenomena in resonant metamaterials, especially at lower frequencies. In addition, discharges cause abrupt changes in the electromagnetic characteristics of gases. Therefore, it may be possible to generate highly nonlinear phenomena in gas-loaded resonant metamaterials. In this paper, we show that frequency mixing can be generated using the a) Electronic mail: tamayama@vos.nagaokaut.ac.jp dynamic changes in the electromagnetic response of a metasurface associated with a gas discharge in its unit structure. Figure 1(a) shows the metasurface structure used in this study. The details of the electromagnetic response of the metasurface are described in our previous papers, 32,33 and we briefly review them here. This metasurface is composed of two radiatively coupled cut-wire resonators with slightly different resonance frequencies, and exhibits an electromagnetically induced transparency (EIT) 34,35 -like electromagnetic response. 36 The EIT-like transmission peak frequency is f 0 = 3.031 GHz, which is approximately the average of the resonance frequencies of the two cut-wire resonators, 3.016 GHz and 3.059 GHz. When an electromagnetic wave with a frequency around f 0 is incident on the metasurface, a strongly enhanced local electric field is induced at the gaps of the cut-wire resonators. The amplitude of the local electric field at the gaps is about 300 times larger than that of the incident field. Because of the strong enhancement of the local electric field, an electromagnetic wave with relatively low power can generate a discharge microplasma at the gap of the cutwire resonator with higher (lower) resonance frequency when the incident frequency is larger (smaller) than f 0 . After the generation of a microplasma at either of the gaps, the electric field enhancement factor at the other gap drops to one eleventh of its original value, because the microplasma itself behaves as a lossy dielectric and changes the characteristics of the metasurface. Although microplasmas could be generated at both gaps simultaneously by using higher incident power, high-powered electromagnetic waves were not used in study and a microplasma was generated only at one gap.
We consider a condition in which the EIT-like metasurface behaves as a nonlinear medium. Harmonic generation was not previously observed on the EIT-like metasurface with a microplasma, as described in our previous paper. 32 This implies that the metasurface with a microplasma behaves as a linear medium for continuous waves. The electromagnetic response of the metasurface must be dependent on the incident field to induce a nonlinear phenomenon. That is, the generation and quenching of the microplasma needs to be dependent on the incident field. Therefore, we hypothesize that the metasurface would behave as a nonlinear medium when exposed to electromagnetic waves with temporally varying amplitudes.
We measured the transmission characteristics of the metasurface for electromagnetic waves with sinusoidally modulated amplitudes to investigate the nonlinear response of the metasurface. Figure 1 waves with the same power were generated by two signal generators, and these waves were combined to generate a sinusoidally modulated incident wave. The frequencies of these two continuous waves were f 1 = f 0 = 3.031 GHz and f 2 = f 1 + 10 kHz. For this frequency condition, a microplasma was generated at the gap of the cut-wire resonator with the higher resonance frequency, which is at the left side of Fig. 1(a). The sinusoidally modulated wave was amplified using an amplifier. The power of each continuous wave after amplification was defined as P 0 . The amplified wave was introduced into the waveguide through a coaxial-to-waveguide transformer, and was incident on the metasurface. The transmitted wave was received by an ultra-wideband dipole antenna. 37 The received wave was split into two halves. One half was detected by a microwave diode detector to observe the envelope of the transmitted wave using an oscilloscope. The other half was fed into a spectrum analyzer. The microplasma emission at the gap of the cut-wire resonator with the higher resonance frequency was observed through an aperture fabricated in the receiving antenna substrate, as shown in Fig. 1(c), and detected by an avalanche photodiode. The output signal of the photodiode was fed into the oscilloscope. We measured the envelope of the transmitted wave and the light intensity of the microplasma emission, as well as the spectrum of the transmitted wave as P 0 and p Ar were varied. Figure 2 shows examples of the measured characteristics of the transmitted wave and microplasma emission. The upper and middle panels show the envelope of the transmitted wave and the intensity of the microplasma emission, respectively. As the incident amplitude increases from zero, the transmission amplitude first increases and then drops sharply. Further increases and subsequent decreases in the incident amplitude cause little change in the transmission amplitude. After the incident amplitude becomes smaller than a certain value, the transmission amplitude varies with the incident amplitude again. The light intensity of the microplasma emission varies periodically, with a period equal to the modulation period of the incident amplitude. The duration of the low-transmittance state agrees with the time for microplasma emission. Thus, the temporal variation of the transmittance is confirmed to be caused by the generation and quenching of the microplasma. The result that the transmittance becomes low during microplasma generation agrees with our previous results. 32,33 As P 0 increases, the ratio of the duration of microplasma generation to the modulation period increases. This is because the duration that the incident amplitude exceeds the threshold value for plasma ignition increases with P 0 . The lower panel of Fig. 2 shows the spectrum of the transmitted wave. Many sidebands are observed in the spectrum and the power of each sideband varies with P 0 . The frequency separation between the sidebands is 10 kHz, which equals f 2 − f 1 . These results in both the time and frequency domains clarify that frequency mixing can be realized using the generation and quenching of a microplasma on the EIT-like metasurface. Next, we investigate the frequency mixing phenomenon in detail. Figure 3 shows the incident power dependence of the frequency conversion efficiency. Here, we define the frequency conversion efficiency as η {L,H}n = P {L,H}n /P 0 (n = 1, 2, 3, · · · ), where P {L,H}n is the power of a sideband with frequency f {L,H}n , as indicated in Fig. 2. The conversion efficiency is increased or decreased by increasing the incident power, depending on P 0 and n. This observation is quite different compared with typical nonlinear phenomena, in which frequency conversion efficiency always increases with incident power.
Let us consider a simple model of the EIT-like metasurface to understand the dependence of the frequency conversion efficiency on P 0 . The transmittance of the metasurface with a microplasma is relatively low, and nearly independent of the incident amplitude. Therefore, we assume that the transmittance of the metasurface only depends on whether the microplasma is generated or not. That is, the amplitude transmittances with and without the microplasma are assumed to be a 1 and a 2 (a 2 > a 1 ), respectively. The time dependence of the amplitude transmittance a(t) is a periodic function with a period of T = 100 µs = (f 2 − f 1 ) −1 in this experiment. Thus, a(t) can be expanded into Fourier series as follows: [c n exp (−i2nπt/T ) + c * n exp (i2nπt/T )], where D is the ratio of the duration of microplasma generation to T , |c n | = [(a 2 − a 1 )/(nπ)]| sin [nπ(1 − D)]|, and * indicates the complex conjugate. The time dependence of the transmitted amplitude is described by the product of the incident amplitude and a(t). Therefore, the spectrum of the transmitted wave can be expressed as the convolution of the Fourier transforms of the incident wave and a(t). Assuming that the lower (upper) sidebands can be expressed as the convolution of the Fourier transforms of a continuous wave with frequency of f 1 (f 2 ) and a(t) for simplicity, the powers of the sidebands are described by |c n |, where the only parameter that depends on the experimental condition is D.
To compare this model with the experiment, we show in Fig. 4 the dependence of η {L,H}n (n = 1, 2, 3) on D that was measured for various values of P 0 (≤ 32.1 dBm) at p Ar = 0.4 kPa, 0.8 kPa, and 1.2 kPa. Here, D was determined from the light intensity of the microplasma emission. The conversion efficiencies are found to be determined almost entirely by D, even though P 0 and p Ar are tested at various values. In addition, the values of D for which η {L,H}n becomes local maxima or minima in the experiment roughly agree with those calculated from the model. This implies that the frequency mixing is derived from the binary change in the transmittance, which is caused by the generation and quenching of the microplasma. The discrepancy between the experiment and model in D for which η {L,H}n becomes a local maximum or minimum is relatively large in the region of larger D values, which may be caused by oversimplifications in the model.
The nonlinear response in the present experiment cannot be expressed as a n-th order nonlinearity. In typical nonlinear media, the ratio of the polarization density to the electric field depends on the electric field. However, the ratio in the metasurface depends instead on the amplitude of the electric field. In this way, the nonlinear response at this metasurface is similar to that of a rf-SQUID metamaterial. 15 The abrupt change in the electromagnetic response observed in these metamaterials may be useful for the efficient generation of various nonlinear phenomena.
In conclusion, we investigated the nonlinear response of an EIT-like metasurface loaded with gas as a nonlinear element to develop a method for preventing the suppression of the enhancement of the local electromagnetic field in a resonant metamaterial. We measured the transmission characteristics of the metasurface and the light intensity of the microplasma emission for an incident wave with a sinusoidally modulated amplitude to verify the generation of nonlinear phenomenon. The measured envelope and spectrum of the transmitted wave, as well as the temporal dependence of the microplasma emission, revealed that frequency mixing occurred at the metasurface because of the generation and quenching of the microplasma. The parameter dependence of the frequency conversion efficiency showed that the efficiency was determined almost entirely by the ratio of duration of the microplasma generation to the modulation period. The metasurface may be classified into a kind of waveform selective metasurface 38,39 because the time variation of the incident amplitude is essential for the generation of nonlinear phenomenon. Note that the nonlinear phenomenon in this study is not caused by a nonlinearity in the plasma, in contrast with some previous works. [40][41][42] Although harmonic generation has not been observed in this metasurface with microplasma, it can be generated in principle and should be investigated in a future study. It should be added that the interactions between free electrons in conductors and the enhanced local magnetic field can be used as another way to prevent the suppression of the enhancement of the local electromagnetic field for efficient generation of nonlinear phenomena. 43 This research was supported in part by JSPS KAKENHI Grant Number JP16K14249, and by a re-search grant from The Murata Science Foundation. | 2018-11-14T08:42:05.000Z | 2018-08-06T00:00:00.000 | {
"year": 2018,
"sha1": "26eb8e7a59f76c66127aa7fcd40455b23cd9de0f",
"oa_license": null,
"oa_url": "http://arxiv.org/pdf/1811.05687",
"oa_status": "GREEN",
"pdf_src": "Arxiv",
"pdf_hash": "26eb8e7a59f76c66127aa7fcd40455b23cd9de0f",
"s2fieldsofstudy": [
"Physics"
],
"extfieldsofstudy": [
"Physics",
"Materials Science"
]
} |
234046799 | pes2o/s2orc | v3-fos-license | SYNTHESIS, STRUCTURE, AND BIOLOGICAL ACTIVITY OF NOVEL BISPIDINE DERIVATIVES
Objective: Derivatives of 3,7-diazabicyclo[3.3.1]nonan-9-one attract considerable attention from pharmacists for the treatment of a wide rangeof diseases. According to this interest, the novel derivatives of 3-cyclopropanmethyl-7-alkoxyalkyl-3,7-diazabicyclo[3.3.1]nonan-9-one withisopropoxypropyl and ethoxypropyl substituents in the position 7 had been synthesized to study their biological activity and toxicity. The practicalsignificance of the work is in the accumulation and development of scientific representations about diazabicyclic compounds, methods for theirsynthesis, structure, and properties, which can subsequently be used in a targeted design and identification of even more complex systems, as wellas in the development of further research in the field of 3,7-diazabicyclo[3.3.1]nonanes. For this purpose, complexes of the synthesized compoundswith β-cyclodextrin are obtained and their biological activity is investigated at the Department of Pharmacology of S.D. Asfendiyarov Kazakh NationalMedical University with the aid of the pharmacological tests.Methods: An experimental study of local anesthetic activity on the models of infiltration, conduction anesthesia, and acute toxicity of synthesizedmolecules was carried out using primary screening methods.Results: As a result of pharmacological screening, it has been found that the compounds exhibit local anesthetic activity and low toxicity and wasrecommended for in-depth study of their pharmacological properties.Conclusion: It turned out that a nature of the N-alkoxyalkyl radical does not affect the toxicity of cyclopropanmethyl- substituted bispidines. In theseries of O-benzoyloximes of bispidinones, the isopropoxypropyl- substituted analog is 1.3 times less toxic than ethoxypropyl- one.
INTRODUCTION
An analysis of current trends in the medical use of drugs indicates the ongoing gradual replacement of obsolete drugs with more effective and safe drugs of novel generations. Therefore, research on the search for novel potentially biologically active substances is relevant.
The aim of research is the synthesis of novel potentially pharmacologically active derivatives of 3-cyclopropanmethyl-7alkoxyalkyl-3,7-diazabicyclo[3.3.1]nonan-9-one, as well as the establishment of the structure and evaluation of their biological activity.
The reaction products were obtained with high yields as viscous oils. Monitoring of the progress of the reaction was carried out by TLC on alumina. The structure of bispidinone derivatives (3)(4)(5)(6)(7)(8)(9)(10) was determined by nuclear magnetic resonance (NMR) and infrared (IR) spectroscopies.
Experimental chemical part
NMR spectra of the studied compounds were recorded on a JEOL JNM-ECA400 spectrometer with an operating frequency on carbon nuclei of 100.53 MHz in CDCl 3 with hexamethyldisiloxane as internal standard. Elemental analysis data were consistent with calculated values. IR spectra were recorded on a Nicolet 5700 instrument between KBr plates. Column chromatography and thin-layer chromatography were carried out on alumina (Al 2 O 3 ) of the third degree of activity, R f of the compounds is provided for this type of plate. The spots were developed in iodine vapors.
Fig. 1: Synthetic routes to the target bispidines
hydrate (99% solution) in 52.5 ml of triethylene glycol at 60°C add 12.5 g (0.22 mol) of KOH. The reaction mixture was heated to 150°C and stirred at this temperature for 4 h. At a temperature of 190-200°C, water and excess hydrazine were removed by evaporation. After cooling, 64 ml of distilled water was added, extracted with diethyl ether, and dried over anhydrous MgSO 4 . The solvent was evaporated, the obtained product was purified by column chromatography on Al 2 O 3 , benzene:dioxane 5:1. (6) Oxime (3) in 60 ml of ethyl alcohol and 1.23 g (0.0153 mol) of pyridine were placed in a three-necked flask equipped with a mechanical stirrer, reflux condenser with a calcium chloride tube, and dropping funnel. While stirring, 1.84 g (0.0265 mol) of hydroxylamine hydrochloride was added. The reaction mixture was heated at 110-120°C for 20 h, the solvent was evaporated, and the residue was dissolved in 15 ml of water, alkalized with NaOH to pH 12, extracted with chloroform, and dried over anhydrous MgSO 4 . The solvent was evaporated, the residue was purified by column chromatography on Al 2 O 3 , benzene:dioxane =5:1. (9) 1.5 g (0.0048 mol) of oxime of 3-cyclopropanmethyl-7-(3isopropoxypropyl)-3,7-diazabicyclo[3.3.1]nonan-9-one (7) was mixed with 15 ml of absolute benzene and a mixture of 2 ml of absolute benzene and 0.6 ml (0.0048 mol) of benzoyl chloride was added dropwise. Reaction took place at room temperature. At the end, 10 ml of distilled water was added to the reaction mixture and neutralized with potash to pH 10-11, the product was extracted with chloroform, the combined extracts were dried over anhydrous MgSO 4 . The solvent was evaporated and residue was distilled in vacuo.
The evaluation of infiltration anesthesia was studied on guinea pigs according to the Bulbring-Wade method. The following values were determined: The depth of anesthesia (anesthesia index), the duration of deep anesthesia, and the total duration of the anesthetic effect.
The conduction anesthesia was examined through the modified tail flick method on rats. It allows to measure the duration of deep anesthesia and the total duration of the anesthetic effect of the compound.
The study of acute toxicity was carried out on mice of the same species, sex, age, and weighing 18-22 g. Acute toxicity (LD 50 ) was determined by a single subcutaneous injection of aqueous solutions of the compounds.
The local anesthetic effect and acute toxicity of novel derivatives of 3,7-diazabicyclo[3.3.1] nonan-9-one in the form of complexes with β-cyclodextrin were studied under laboratory codes LA 1-4:
RESULTS AND DISCUSSION
The infiltration anesthesia It turned out that on the model of infiltration anesthesia in a bispidines family, the most active is LA-2, the anesthesia index of which is 35.60±0.19 (Table 1). In addition, it caused a longer complete anesthesia of 34.00±1.26 min, while the total duration of action was 43.00±1.84 min. Replacing the ethoxypropyl radical in the LA-2 compound with an isopropoxypropyl-one led to a three-fold decrease in activity.
Among the two of O-benzoyloximes of 3,7-diazabicyclo[3.3.1]nonan-9ones, LA-3 with cyclopropanmethyl and isopropoxypropyl radicals at nitrogen atoms was active, its anesthesia index was 35.60±1.18, and the duration of deep anesthesia was equal to 34.80±0.98 min. The total duration of action was 43.10±1.17 min. Replacing the isopropoxypropyl radical with a ethoxypropyl-one led to a decrease in activity (LA-4) (anesthesia index was 29.16±1.45; the duration of deep anesthesia was 16.00±2.90 min; and the total analgesic effect lasted (29.16±2.06 min). However, it should be noted that it was weaker than comparison preparations (не знаю, говорят ли так, я предлагаю язамену) for the reference compounds.
Conduction anesthesia
It should be noted that bispidines and O-benzoyloximes on the model of conduction anesthesia did not show any significant effect. However, LA-1 can be distinguished, which in terms of total duration of action is comparable to novocaine and inferior to trimecaine and lidocaine.
In the group of O-benzoyloximes, according to the duration of total anesthesia, LA-4 exceeded trimecaine and novocaine (Table 2).
CONCLUSION
In this research, derivatives of 3,7-diazabicyclo[3.3.1]nonan-9-ones were synthesized and the biological properties were studied for their complexes with β-cyclodextrin.
Thus, as a result of structural modifications of bicyclic piperidines, novel low toxic substances possessing a local anesthetic effect had been obtained, the activity of which depends on the nature of the N-alkoxyalkyl-and substituents at the position 9 of the 3,7-diazabicyclo[3.3.1]nonane ring. The most harmless is the complex of O-benzoyloxy-3-cycloproylmethyl-7-(3-isopropoxypropyl)-3,7diazabicyclo[3.3.1]nonan-9-ketoxime with β-CD, where the LD 50 is 825 mg/kg. The compound was recommended for in-depth study of its pharmacological properties.
Moreover, the results of research will be used in a targeted design and identification of even more complex systems on the base of 3,7-diazabicyclo[3.3.1]nonan-9-ones with different biological activity. | 2021-05-10T00:04:09.016Z | 2021-01-31T00:00:00.000 | {
"year": 2021,
"sha1": "3b5a7259d1cebc39b42c5a682c46f7dd5cff275d",
"oa_license": "CCBYNC",
"oa_url": "https://innovareacademics.in/journals/index.php/ijap/article/download/40954/24291",
"oa_status": "GOLD",
"pdf_src": "Adhoc",
"pdf_hash": "a299613466290e7f3948c57e13fb7a8c42a0ffb3",
"s2fieldsofstudy": [
"Chemistry",
"Medicine"
],
"extfieldsofstudy": [
"Chemistry"
]
} |
47792755 | pes2o/s2orc | v3-fos-license | Few smooth d-polytopes with n lattice points
We prove that, for fixed n there exist only finitely many embeddings of Q-factorial toric varieties X into P^n that are induced by a complete linear system. The proof is based on a combinatorial result that for fixed nonnegative integers d and n, there are only finitely many smooth d-polytopes with n lattice points. We also enumerate all smooth 3-polytopes with at most 12 lattice points. In fact, it is sufficient to bound the singularities and the number of lattice points on edges to prove finiteness.
Introduction
The present note has two target audiences: combinatorialists and algebraic geometers. We give combinatorial proofs of results motivated by the algebraic geometry of toric varieties. We provide two introductions with statements of the main results in the language of divisors on toric manifolds on the one hand, and in the language of lattice polytopes on the other. In Section 2, we collect the relevant entries from the dictionary translating between the two worlds. In Section 3 we prove our theorems, and in Section 4 we report on first classification results.
1.1. Introduction (for algebraic geometers). The purpose of this note is to show that there are very few embeddings of Q-factorial toric varieties 1 into projective space that are induced by a complete linear series.
Theorem 1. Let N be a nonnegative integer. Then there exist only finitely many embeddings of Q-factorial toric varieties X into P N that are induced by a complete linear series.
Note however, that every Hirzebruch surface F a admits an embedding into P 5 (see Example 17), so we cannot omit the hypothesis that the embedding is induced by a complete linear series. Moreover, for non-Qfactorial toric varieties the embedding dimension does not even bound the degree, see Example 15.
Date: October 19, 2010. 1 All toric varieties appearing in this paper are normal by construction.
Using Oda's classification of smooth 3-dimensional toric varieties that are minimal with respect to equivariant blow ups, we classify all 3-dimensional embeddings of smooth toric varieties into P ≤11 using a complete linear series. In the appendix we present the complete list of the corresponding 3-polytopes with ≤ 12 lattice points up to equivalence (see Definition 3).
One possible application of this classification is a long standing open question by Oda [Oda08] whether every embedding of a smooth toric variety that is induced by a complete linear series is projectively normal. If we require our embedding to be projectively normal, then standard arguments from algebraic enumerative combinatorics imply finiteness (see Remark 14).
There is an entire hierarchy of successively stronger conjectures concerning embeddings of smooth projective toric varieties which are open even in dimension 3, (compare [MFO07, p.2313]). The principal obstacle to theoretical progress on Oda's question and the related conjectures is a serious lack of well understood examples. Recently Gubeladze [Gub09] has shown that any lattice polytope with sufficiently long edges (depending on the dimension) gives rise to a projectively normal embedding. In view of this result, if there exists a counterexample, it is more likely to be a small polytope. Yet, all polytopes in our classification up to 12 lattice points satisfy the even strongest of these conjectures (see Corollary 24).
1.2. Introduction (for polytope people). The purpose of this note is to show that there are very few lattice polytopes with a unimodular normal fan. More generally, we prove the following.
Theorem 2. Let N be a nonnegative integer, and let F be a finite family of rational cones. Then there are only finitely many lattice polytopes with N + 1 lattice points all whose normal cones are integrally equivalent to cones belonging to F .
In this theorem it would not be enough to fix the combinatorial type of the polytope (see Examples 15 & 16). The relevance of this result in algebraic geometry stems from the fact that a lattice polytope corresponds to an ample line bundle on a projective toric variety (see Section 2).
Oda classified 3-dimensional unimodular fans with ≤ 8 rays that are minimal with respect to stellar subdivisions. Based on this classification, we classify all 3-dimensional polytopes with unimodular normal fan and ≤ 12 lattice points up to equivalence (see Definition 3). They are listed in the appendix.
One possible application of this classification is a long standing open question by Oda [Oda08] whether every polytope P with a unimodular normal fan is integrally closed: for k ∈ Z ≥2 every lattice point in the dilation kP should be the sum of k lattice points in P . If we require P to be integrally closed, then standard arguments from algebraic enumerative combinatorics imply finiteness (see Remark 14).
There is an entire hierarchy of successively stronger conjectures concerning polytopes with unimodular normal fan which are open, even in dimension 3. (Compare [MFO07, p.2313].) The principal obstacle to theoretical progress on Oda's question and the related conjectures is a serious lack of well understood examples. Recently Gubeladze [Gub09] has shown that any lattice polytope with sufficiently long edges (depending on the dimension) is integrally closed. In view of this result, if there exists a counterexample, it is more likely to be a small polytope. Yet, all polytopes in our classification up to 12 lattice points satisfy the even strongest of these conjectures (see Theorem 23).
Related Results. Let us briefly give an overview over related classification results. Most of them concern toric Fano varieties. In this case, the primitive ray generators of the corresponding fans form the vertices of a convex polytope. In dimension two, Q-Gorenstein toric Fano surfaces are known up to Gorenstein index 17 [KKN10]. In dimension three, the finite list of canonical toric Fano varieties was obtained by A. Kasprzyk [Kas06]. We refer the interested reader to the Graded Ring Database grdb.lboro.ac.uk for these and other classification results. Gorenstein toric Fano varieties, corresponding to so-called reflexive polytopes [Bat94], are completely classified up to dimension four [KS98,KS00]. Toric Fano manifolds are classified up to dimension 8 [Bat99, Sat00, KN09, Øbr07]; recently, B. Lorenz computed dimension 9. Smooth reflexive polytopes up to dimension 9 can be found in the data base at www.polymake.de. Higher-dimensional classification results of toric varieties are only known in two cases: in the Gorenstein Fano case under strong symmetry assumptions [VK85,Ewa96,Nil06] or if the Picard number of a toric manifold is at most 3, i.e., the ddimensional fan has at most d + 3 rays, in which case the variety is automatically projective [KS91,Bat91].
Polarized toric varieties and lattice polytopes.
In this section we introduce notation and recall some basic facts about toric varieties. For more details we refer to [CLS10,§2.3] or [Ful93, Section 3.4].
2.1. Line bundles and polytopes. Let k be an arbitrary field. Let N ∼ = Z d be a lattice, and let Σ be a fan of dimension d in N R := N ⊗ Z R. Let X = X(Σ) be the associated toric variety, a normal equivariant compactification of the algebraic torus T ∼ = (k * ) d . The lattice M = Hom(N, Z) is naturally isomorphic to the character lattice of T . Assume that X is projective, and let L be an ample line bundle on X. The polarized toric variety (X, L) corresponds to a lattice polytope P ⊆ M ⊗ Z R of dimension d such that the normal fan to P is equal to Σ. Moreover, we have an isomorphism For a subset S of R d , let aff(S) denote the affine span of S.
Definition 3. We say that two lattice polytopes P ⊂ R d and P ′ ⊂ R d ′ are integrally equivalent if there is a lattice preserving affine map aff P → aff P ′ that maps Z d ∩ aff P bijectively to Z d ′ ∩ aff P ′ and P to P ′ .
Up to this lattice equivalence, we can always assume that our polytope P is full dimensional. Moreover, if P and P ′ are equivalent, and if (X, L) and (X ′ , L ′ ) are the corresponding polarized toric varieties, then there exists a torus equivariant isomorphism φ : X → X ′ such that φ * L ′ ∼ = L.
2.2. Singularities and cones. Let L be an ample line bundle on the toric variety X(Σ) with corresponding lattice polytope P . Then X(Σ) is covered by torus invariant affine pieces U u which correspond to the vertices u of P . For a vertex u of P , let T u P = cone(u ′ − u | u ′ ∈ P ) be the tangent cone to P at u. It is dual to the normal cone σ(P, u) of P at u. The semigroup T u P ∩ M is finitely generated. The unique minimal set of generators Hilb(T u P ) for this semigroup is called the Hilbert basis of the cone [CLS10, Proposition 1.2.22]. The coordinate ring of the affine variety U u is the semigroup ring k[ The line bundle L is called very ample if its global sections induce an embedding into projective space. The combinatorial condition for L to be very ample is that for every vertex u of P , the shifted polytope P − u contains the Hilbert basis, i.e., Hilb( In this case, there is a well defined linear functional called height ht σ ∈ M R on σ which takes the value 1 on all v i . The index of σ is the smallest k ∈ Z >0 so that k · ht σ ∈ M. We call σ Gorenstein if this index is equal to 1. These notions agree with the notions (Q-)Gorenstein and index for the toric singularity associated with σ. We define the multiplicity mult(σ) as the normalized volume of the nib of σ which equals the product of the index with the normalized volume of conv(v 1 , . . . , v r ).
Let P be a lattice polytope with Q-Gorenstein normal fan. We define the multiplicity of P to be mult(P ) = max u mult(σ(P, u)), the maximal multiplicity of a normal cone to P .
Note that for a toric variety X, the multiplicity does not depend on the polarization, so we can define the multiplicity mult(X) = mult(P ), where P is a lattice polytope corresponding to an ample line bundle on X.
2.2.2. Simplicial cones. The toric singularity U u is Q-factorial if the tangent cone T u P of P at u is simplicial, that is, it is generated by a linearly independent set {v 1 , . . . , v d } of primitive vectors. In this case, the singularity U u is a quotient k d /G of affine space by a finite abelian group, and the multiplicity is the cardinality of that group.
Such cones are automatically Q-Gorenstein. The box of T u P is the half open parallelepiped and a box point is one of the mult(T u P ) many lattice points in (T u P ). Every Hilbert basis element that is not one of the generators of T u P is a box point, and has smaller height than d. In particular, we have A cone is called unimodular if its primitive minimal generators form a lattice basis. Unimodularity is equivalent to having multiplicity 1.
Definition 4. We call a lattice polytope P smooth if every cone in its normal fan is unimodular.
A lattice polytope is smooth if and only if the if the associated projective toric variety X is smooth (see for example [Ful93, Section 2.1]). Moreover, every ample line bundle on a smooth toric variety is very ample.
Finiteness Theorems
Before we embark on a proof of our main theorem, let us recall some known finiteness theorems from the literature. The first such theorem goes back to Hensley [Hen83]. The currently best bound is due to Pikhurko [Pik01, (9)].
Theorem 5. For a positive integer d, there is a bound V (d) so that the volume of every lattice d-polytope with k ≥ 1 interior lattice points is bounded by k · V (d).
The second result, due to Lagarias and Ziegler [LZ91, Theorem 2], shows that it is enough to bound the volume if we want to show that only finitely many lattice polytopes satisfy our conditions. Theorem 6. A family of lattice d-polytopes with bounded volume contains only a finite number of equivalence classes. Theorem 8. For a nonnegative integer d and a finite family F of rational d-cones, there are only finitely many lattice d-polytopes with N + 1 lattice points all whose normal cones are equivalent to cones belonging to F .
We first prove that this is true in dimension two.
Lemma 9. For a finite family F of rational 2-dimensional cones, there are only finitely many lattice polygons with N +1 lattice points all whose normal cones are equivalent to cones belonging to F . Proof. Let P be a lattice polygon, and let u be a vertex of P with normal cone σ. Up to equivalence, we can assume u = 0 and σ = cone[(0, 1), (p, q)] with 0 ≤ q < p. Because σ is equivalent to a cone in the finite family F , the multiplicity p is bounded, and there are only finitely many choices for p and q.
With this notation, P contains x ′ + 1 lattice points on the edge uu ′ , and the segment in P parallel to uu ′ at distance one has length ℓ := x ′ + q ′ /p ′ + q/p, and thus contains more than ℓ − 1 lattice points. Now the inequalities x ′ ≥ 1, ℓ ≥ 0 and x ′ + ℓ ≤ N + 1 together leave only finitely many choices for x ′ and q ′ . u u ′ Figure 3: Lots of lattice points at distance one.
The normal fan together with all edge lengths determines P up to equivalence.
Proof of Theorem 8. The number of combinatorial types of fans with ≤ N + 1 maximal cones is finite. Here, the combinatorial type is given by the set of faces, partially ordered by inclusion. For each combinatorial type there are only finitely many choices of cones from F for its maximal faces. So we can restrict to a class of normal fans with a fixed combinatorial type and with given equivalence class for each maximal face.
For instance, in dimension two, the fans of Hirzebruch surfaces (compare Example 17) all have the same combinatorial type and all maximal faces of the fan are unimodular.
By Lemma 9, we can also fix the integral type for every 2-face of P . We claim that these choices determine P up to equivalence.
To this end, fix a vertex u of P with normal cone σ ∈ F . This determines all 2-dimensional faces of P incident to u. In particular, if u ′ is another vertex of P adjacent to u, then u ′ together with all edges which are incident to u ′ and contained in a common 2-face with u are determined. The directions of these edges together with the edge uu ′ span R d as a vector space. They thus pin down the normal cone at u ′ .
In summary, fixing a vertex and its tangent cone also fixes all adjacent vertices and their tangent cones. As the vertex-edge graph of P is connected, this determines P .
Corollary 10. For nonnegative integers d and N + 1, there are only finitely many equivalence classes of smooth d-polytopes with N +1 lattice points.
Proof. In the case of smooth polytopes, F contains only one cone: the standard orthant.
Corollary 11. For nonnegative integers d, m and N, there are only finitely many d-polytopes with Q-Gorenstein normal cones of multiplicity bounded by m and with N + 1 lattice points.
Proof. Applying Theorem 6 to the convex hull of 0 and the primitive generators of a Q-Gorenstein cone, we see that the family of Q-Gorenstein cones with multiplicity ≤ m contains only finitely many equivalence classes.
Using the dictionary between toric morphisms and lattice polytopes, Corollary 11 implies the following corollary. We consider two morphisms to P N the same if they differ by an automorphism of P N .
Corollary 12. Let N and m be nonnegative integers. There are finitely many morphisms from some Q-Gorenstein toric variety X of dimension less than N with mult(X) ≤ m to P N that are induced by a complete linear series.
In order to deduce Theorem 1 from Corollary 12, we need another lemma.
Lemma 13. Let N and d be nonnegative integers. Then there are up to equivalence only finitely many Q-Gorenstein cones σ ⊂ R d so that Proof. We will show by induction on d that ( * ) implies that mult(σ) is bounded. Then Theorem 6 implies that there are only finitely many choices for σ. For d = 1 there is only one cone. For d = 2, Pick's formula [Pic99] tells us that mult(σ) ≤ 2#(nib(σ) ∩ Z 2 ) − 5. So let us assume that the lemma is true for d − 1. Because of Corollary 7, we can assume that nib(σ) has no interior lattice points. This implies that all interior Hilbert basis elements of σ have height ≥ 1. By induction, there is a minimal height ǫ(d − 1, N) > 0, depending only on d − 1 and N, of a Hilbert basis element in the boundary of σ. Let ǫ = min{ǫ(d−1, N), 1}.
Triangulate σ = ∪ r i=1 σ i into simplicial cones using only rays of σ. Every Hilbert basis element of σ is a box point of one of the σ i . As σ has at most N rays, every box point belongs to less than N d of the σ i . Now, every box point of every σ i has a representation v∈Hilb(σ) a v v with a v ∈ Z ≥0 . On the other hand, any box point has height < d, so that in the above representation we must have ǫ · v∈Hilb(σ) a v < d which leaves at most N +⌊d/ǫ⌋ N possibilities. In other words, Proof of Theorem 1. Since X is Q-factorial, P is simple. This means that every tangent cone to P is simplicial, in particular, Q-Gorenstein. Moreover, since P corresponds to a very ample line bundle, a translate of the Hilbert basis for each tangent cone is a subset of the lattice points of P . Since P has N + 1 lattice points, it follows from Lemma 13 that there are only finitely many equivalence classes of tangent cones. So there are only finitely many equivalence classes of normal cones. Now the claim follows from Theorem 8.
Remark 14. If we require the embedding of X given by P to be projectively normal, then the homogeneous coordinate ring is a Cohen-Macaulay standard graded algebra [Hoc72] with ≤ N + 1 generators. Thus, its Hilbert function (the Ehrhart series of P ) is bounded (compare, e.g. [Hib92, Lemma 18.1]). This bounds the degree (the normalized volume of P ). By Theorem 6, there are only finitely many such P .
The following examples show that we need to assume that X is Qfactorial in Theorem 1 (Example 15) and that the multiplicities are bounded in Corollary 11/12 (Example 16).
Example 16. In [Ree57] John Reeve constructs the simplices P k = conv 0 1 0 1 0 0 1 1 0 0 0 k with 4 lattice points but unbounded volume. In particular, this family of polytopes shows that the number of lattice points of a lattice polytope does not give a bound on its volume. On the other hand, note that the line bundle corresponding to P k is not very ample. The line bundle corresponding to 2P k is normally generated, so in particular it is very ample (see [BGT97,Theorem 1.3.3] or [ON02]). It induces an embedding into P k+8 .
The finiteness theorem does not mean that there are only finitely many projective torus equivariant embeddings into a fixed projective space.
Example 17. Figure 4 shows how to embed an arbitrary Hirzebruch surface torically into P 5 . Corollary 10 can also be strengthened.
Theorem 18. For nonnegative integers d and N, there are only finitely many equivalence classes of smooth d-polytopes with N +1 lattice points on edges.
Again, the proof relies on the study of the two-dimensional situation.
Lemma 19. There are only finitely many smooth polygons with N + 1 lattice points on the boundary. Moreover, where a denotes the number of vertices and b the length of a maximal edge.
Example 20. Here is an example, due to Paco Santos, with all edges of length one whose volume grows exponentially with the number of vertices. This shows that the upper bound 4 a b 2 above is actually not that bad. The construction gives a lower bound of 1.272 a b 2 .
Every consecutive pair of segments is unimodular, because of the above relations. The signs in the right-hand sides say that the chain is convex. Since the chain starts with (1, 0) and ends with (0, 1), one can build a convex smooth polygon by reflecting it horizontally and vertically. This polygon has a = 8k + 4 vertices and volume Θ(F 2k+2 F 2k ) = Θ(τ 4k ) where τ = (1 + √ 5)/2 is the golden ratio. The latter comes from the fact that the sum of the first k−2 Fibonacci numbers equals (almost) the k-th Fibonacci number. Hence, the polygon is inscribed in a rectangle of sides about 2F 2k+2 and 2F 2k , and it contains the mid-points of the edges of this rectangle, so it contains at least half of the rectangle. Since the Fibonacci sequence grows exponentially, we are done.
Proof of Lemma 19. By Theorem 6 we only have to show the inequalities. For this, we induct on the number of vertices a.
From the classification of two-dimensional complete unimodular fans (compare [Ewa96, Theorem 6.6]) we can easily check the statement for a ≤ 4 (here the fan corresponds either to the projective plane or a Hirzebruch surface).
For a ≥ 5, there exist three consecutive rays in the fan such that the corresponding primitive lattice points [Ewa96,Theorem 6.6]). As Figure 6 shows this implies that there is a smooth polygon P ′ containing P and having a − 1 vertices whose maximal edge length equals at most 2b. This yields the statement by induction. More precisely, the length of the edge blown down equals the difference in boundary lattice points.
The upper bound can be improved to 2 a b 2 . The idea is that there has to be more than one edge that can be "blown down". In fact, there must always be at least three and (as soon as a > 6) one can always find two of them "at distance at least three", meaning that the two are incident to four other edges in total. Those two edges can be blown down simultaneously to get a polygon with a−2 vertices and maximum length at most 2b.
Proof of Theorem 18. Since P contains only a bounded number of vertices, there are only finitely many possible combinatorial types for P . Let us fix a vertex u with unimodular normal cone σ = cone(v 1 , . . . , v d ). By Lemma 19, there are only finitely many choices for the d 2 many 2-faces incident to u. Any such choice determines the positions of all vertices of P adjacent to u as well as their normal cones. Since the vertex-edge-graph of P is connected, this finishes the proof.
Classification in Dimension 3
This section summarizes the strategy to classify smooth 3-polytopes with at most 12 lattice points, implementing the proof of Corollary 10. For full details, including source code, see [Lor09,HLP10]. 4.1. Generating Normal Fans. Katsuya Miyake and Tadao Oda have classified smooth 3-dimensional fans which are minimal with respect to equivariant blow-ups [Oda88, Theorem 1.34]. This classification goes up to at most eight rays or equivalently, 12 full-dimensional cones. Starting from this list, all possible sequences of blow-ups had to be enumerated until no fan of a polytope with ≤ 12 lattice points could occur further down the search tree. In order to prune the search tree, we used bounds based on the two-dimensional classification.
4.2. Generating Polytopes. The next step is to find the polytopes, given the normal fan Σ. We write the primitive generators of the rays of Σ as rows of the matrix A, and describe the polytopes with inequalities The edge lengths of P (A, b) depend linearly on the right-hand side vector b -provided we stay inside the chamber of all b yielding a polytope with normal fan Σ (the nef-cone of the toric variety X(Σ)). If we require these edge length functionals to be positive, we obtain an inequality description of the chamber. Bounding the sum of the edge lengths, the search space of possible b-vectors which yield at most N +1 lattice points becomes itself the set of lattice points in a polytope.
The last step is to remove all polytopes that are lattice-isomorphic to another one in the list.
All these computations can be done with the polymake lattice polytope package by Benjamin Lorenz, Andreas Paffenholz and Michael Joswig [GJ,JMP09] Lists of all smooth polygons and smooth 3-polytopes with at most 12 lattice points can be found in the appendix. 4.4. Comments. We now have a list of smooth lattice polytopes in dimensions two and three with at most 12 lattice points. The bound 12 may seem rather low -the smallest 3-polytope with one interior lattice point has 21 lattice points total [Kas08]. The classification carried out here serves as a proof of concept -it can be done. There are several points in the algorithm where it could be improved (compare [Lun10]). One easily proves by inspection of the list in the appendix that all these polytopes satisfy all the ideal theoretic conjectures mentioned in the introduction. In particular, the homogeneous coordinate ring is a Koszul algebra.
Theorem 23. If P is a 3-dimensional smooth polytope with at most 12 lattice points, then P has a regular unimodular triangulation with minimal non-faces of size two.
Corollary 24. Let X be a toric threefold embedded in P ≤11 using a complete linear series. Then the defining ideal of X has an initial ideal generated by square-free quadratic monomials.
In the current implementation, the generation of the normal fans is the bottleneck. By implementing a different way to directly generate all smooth normal fans one could skip the big recursion calculating all blowing-ups, as well as overcome the limits of at most 12 vertices imposed by the Miyake/Oda classification. The second point to work on is the calculation of lattice points of the polytope containing all right-hand sides b. The dimension of this polytope is equal to the Picard number of the toric variety: the number of rays of the fan minus the ambient dimension. Of course, better theoretical bounds for all steps of the algorithm will directly impact the performance. | 2013-07-17T13:41:11.000Z | 2010-10-19T00:00:00.000 | {
"year": 2010,
"sha1": "adeb6229bf50ccf2eedf32db79c646592f32130a",
"oa_license": null,
"oa_url": "http://arxiv.org/pdf/1010.3887",
"oa_status": "GREEN",
"pdf_src": "Arxiv",
"pdf_hash": "21a5bc4794ae859d16298e40473df7f53f2890f4",
"s2fieldsofstudy": [
"Mathematics"
],
"extfieldsofstudy": [
"Mathematics"
]
} |
259194271 | pes2o/s2orc | v3-fos-license | Dynamic interplay between IL-1 and WNT pathways in regulating dermal adipocyte lineage cells during skin development and wound regeneration
SUMMARY Dermal adipocyte lineage cells are highly plastic and can undergo reversible differentiation and dedifferentiation in response to various stimuli. Using single-cell RNA sequencing of developing or wounded mouse skin, we classify dermal fibroblasts (dFBs) into distinct non-adipogenic and adipogenic cell states. Cell differentiation trajectory analyses identify IL-1-NF-κB and WNT-β-catenin as top signaling pathways that positively and negatively associate with adipogenesis, respectively. Upon wounding, activation of adipocyte progenitors and wound-induced adipogenesis are mediated in part by neutrophils through the IL-1R-NF-κB-CREB signaling axis. In contrast, WNT activation, by WNT ligand and/or ablation of Gsk3, inhibits the adipogenic potential of dFBs but promotes lipolysis and dedifferentiation of mature adipocytes, contributing to myofibroblast formation. Finally, sustained WNT activation and inhibition of adipogenesis is seen in human keloids. These data reveal molecular mechanisms underlying the plasticity of dermal adipocyte lineage cells, defining potential therapeutic targets for defective wound healing and scar formation.
INTRODUCTION
The skin, comprised of layers that include the epidermis, dermis, and dermal white adipose tissue (dWAT), is the largest organ of the human body and a critical barrier against external insults.2][3] However, the cellular and molecular mechanisms underlying dWAT remodeling are poorly understood.
Dermal fibroblasts (dFBs), the most prevalent cell type in the dermis, are highly heterogeneous.5][6] Adipocyte progenitors (APs) and/or preadipocytes (pAds) are present in both reticular and hypodermal layers, and these adipocyte-lineage cells are poised to differentiate into adipocytes, forming the dWAT layer. 4However, the origin, cellular complexity/heterogeneity, and molecular mechanism of dermal adipocyte differentiation from their heterogeneous progenitors are only partially understood.
Skin wound healing is a highly orchestrated process; it includes an early hemostatic and inflammatory phase, then a proliferative phase, and finally a remodeling phase that can last weeks to years. 7During the inflammatory phase, myeloid immune cells are recruited, and fibroblasts migrate from the wound periphery, forming granulation tissue (GT), a temporary scaffold holding the wound. 8During the wound proliferative phase, myofibroblasts are transdifferentiated from fibroblasts to synthesize extracellular matrix (ECM) molecules; however, sustained activation of myofibroblasts leads to the development of fibrotic diseases like keloids. 9erging roles of adipocyte-lineage cells in skin wound healing have been reported.Following inflammation, APs proliferate, and new lipid-laden adipocytes are generated in wound GT, and inhibition of adipogenesis impairs dermal repair. 10Recently, a subset of hypodermal DPP4 + LY6A + fascia fibroblasts with remarkable reparative potential was identified, and genetic ablation or blocking of their migration into wound sites led to chronic wounds.In adipose tissue, DPP4 + LY6A + fascia fibroblasts have been identified as APs that give rise to ICAM1 + DLK1 + pAds. 11,12However, whether skin DPP4 + LY6A + dFBs are adipogenic and their cell differentiation trajectory during wound repair remain largely unknown.Furthermore, the molecular cues that trigger wound-related adipogenesis responses remain undefined.
Here, we obtained skin samples from developing, homeostatic, and wounded mice and applied single-cell RNA sequencing (scRNA-seq) followed by immunostaining (immunohistochemistry [IHC]) and cell sorting to define dFB populations.scRNA-seq predicted the cellular trajectories through which dermal adipocytes differentiated from their heterogeneous APs.The behavior of these cells was examined during wound repair.Finally human keloid samples were analyzed to determine the human relevance of our findings.
Our study provides insight into the molecular and cellular mechanisms underlying dermal adipogenesis and/or lipolysis during skin development and wound regeneration.
Characterization of heterogeneous dermal adipocyte-lineage cells in developing neonatal skin
We performed scRNA-seq of cells isolated from neonatal mouse skin, in which dFBs are actively developing from their precursors, 4 allowing us to capture a continuum of cell states spanning differentiation and specification.Pdgfra + (a pan-fibroblast marker 4,13 ) dFBs, Krt10/14 + keratinocytes, Ptprc/Cd45 + immune cells, and Rgs5 + pericytes were identified as the major cell types present in neonatal mouse skin (Figures 1A and S1A).
Selected Pdgfra + dFBs were reclustered into 12 clusters (C0-C11) (Figures 1A, S1B, and S1C), which were grouped into three distinct developmental cell states (Figures 1B, 1C, and S1D) and defined based on marker gene expression as shown by bubble and/or violin plots (Figures 1C and S1E).Specifically, state 1 cells (C9, C2, C5) expressed high levels of Cd24 and Trps1 but lacked Ly6a (Figures 1C and S1E).IHC analysis of neonatal skin with distinct anatomical layers (Figures S1F-S1G) showed that CD24 and TRPS1 were co-expressed at high levels in PDGFRA + dFBs located around hair follicles at the upper dermis or in the hair bulb DP structure (Figure 1D), where DPP4, LY6A (Figure 1E), and THY1 (Figure S1H) were not expressed or expressed at low levels.ALPL, another marker enriched in state 1 cells (Figures 1C and S1E), co-expressed specifically with TRPS1 in the DP (Figure S1H).We therefore termed the state 1 cluster cells as perifollicular dFBs.
In line with these functional results, trajectory analysis predicted that C11 (HI-AP) was the origin of the differentiation trajectory of adipocyte lineage cells (Figures 1B and S1S).In addition, RNA velocity analysis (Figure 1M) predicted that both HI-pAd/Areg and RET-AP dFBs actively differentiated into pAd/Ad, and C0, C1, C4, and C9 further converge into C2 − perifollicular dFBs.These trajectories need to be validated by lineage tracing studies in the future.
Together, using scRNA-seq, we classified the neonatal developing dFBs into three distinct states (Table S1) and defined the heterogeneous adipocyte-lineage cells located in the RET dermis, dWAT, and HI (Figure 1N).
The antagonistic interplay between WNT-β-catenin and IL-1-immune signaling pathways in regulating dermal adipogenesis
We next sought to identify the key molecular pathways that regulate adipogenesis in the developing skin.Gene ontology (GO) analyses identified the IL-1 and WNT pathways as among the top activated or silenced pathways, respectively, during the conversion of various dFB subsets to pAd/Ad (Figures 2A and S2A).Gene expression plots (Figures 2B and S2B) showed that WNT-related genes were specifically enriched in the perifollicular and/or PAP dFB clusters, whereas a panel of immune-and inflammation-related genes was selectively enriched in the pAd/Ad-related clusters.In addition, transcription factor (TF) analysis identified Cebpb, Pparg, and several inflammatory TFs (Nfkb1 and Junb) as top TFs activated in pAd/Ad-related clusters (Figure S2C).
To determine the direct effect of WNT and IL-1 on adipocyte differentiation, neonatal dFBs were treated with WNT3A, lithium, and/or IL-1β.Lithium, a GSK3β inhibitor, is commonly used to achieve broad-spectrum activation of the WNT-β-catenin pathway. 24RNA-seq results revealed that WNT3A and lithium treatment robustly induced the expression of genes associated with perifollicular dFBs (Cd24, Alpl), whereas IL-1β treatment induced the expression of a panel of pro-inflammatory genes (Cxcl12, Il1r1) and several pAd/Ad marker genes (Pparg2, Fabp4) (Figure S2D).Venn diagram analysis showed that genes upregulated by IL-1β shared a high degree of similarity with genes downregulated by WNT3A + lithium (Figure S2E).Functionally, WNT3A potently suppressed the adipogenic potential of neonatal dFBs (Figures 2C and 2D) and secretion of CAMP and FABP4, which can be partially restored by adding IL-1β (Figure 2E).Furthermore, WNT3A-mediated activation of β-catenin (Figures 2F and 2G), suppression of adipocyte-or immune-related genes, and induction of Axin2, Acta2, and Col7a1 were partially reversed by IL-1β (Figures 2H and
We found that AKT inhibition by wortmannin promoted loss of β-catenin expression, whereas p38 inhibition by SB202190 or NF-κB inhibition by Bay117028 led to a decrease in the phosphorylation levels of both CREB and CEBPβ (Figures 2K and S2K-S2M).In line with these results, the induction of adipocyte-or immune-related genes by IL-1β was largely blocked by p38 or NF-κB inhibitors (Figure 2L).These results indicate that IL-1β promotes β-catenin degradation by inhibiting AKT and promotes activation of proadipogenic molecules by stimulating p38 and NF-κB (Figure 2M).
We next analyzed skin biopsies from neonatal Il1r1 −/− and Il1r1 +/+ littermates to determine the role of IL-1 signaling in adipogenesis.We found that Il1r1 deficiency led to a blockage of CREB phosphorylation in the neonatal mouse skin (Figure 2N).As the result, formation of BODIPY + adipocytes (Figure 2O) and the expression of adipocyte-related genes and the inflammatory cytokine Cxcl5 were suppressed, whereas expression of WNT-related Axin2 was enhanced upon Il1r1 deletion (Figure 2P).Taken together, our results indicate that activation of the IL-1R-CREB signaling axis may play a role in promoting dermal adipogenesis during skin development.
Tracking dermal adipogenesis and fibrogenesis during skin maturation
To track dermal adipogenesis during skin maturation and aging, dorsal skin samples were collected from male mouse skin throughout the postnatal lifespan for analysis.Lipid staining and bulk RNA-seq of skin samples (Figures 3A and S3A) showed that dermal adipocytes progressed from an immature state at birth (characterized by small lipid droplets and high expression levels of Camp and Mest), to a mature state at 3 weeks (large lipid droplets), following which dermal cells and adipocytes were gradually lost during skin maturation and aging from 2 months to 1-2 years.In contrast, dermal collagen content and the expression levels of several ECM molecules increased up to 2 months of age and then progressively decreased during aging (Figures 3A and S3A).
To determine the cellular and molecular changes in dermal adipocyte-lineage cells during skin maturation, scRNA-seq was performed on skin cells isolated from mice at neonatal stage (P1) and 3 weeks and 2 months of age (Figure 3B).Clustering analysis identified 30 clusters, which were classified into nine cell types (Figures 3C-3F and S3B-S3D).Interestingly, we found that among all cell types, Pdgfra + dFBs contained several age-unique clusters (Figure 3F).
To further inspect dFB heterogeneity, Pdgfra + dFBs were reclustered (Figure 3G), and these dFB clusters from the three age groups were clearly separated on the tSNE (t-distributed stochastic neighbor embedding) plots in an age-dependent manner (Figure 3H).Based on the established neonatal dFB markers (Figure 1), we identified the corresponding adult dFB clusters (Figures 3H, 3I, and S3E).In contrast to neonatal DP cells that expressed Alpl, Sox2, and Lef1, adult DP cells lost activated WNT signals (Lef1, Nkd2) and expressed Crapb1 and the WNT antagonist Dkk2, in line with previous studies 27,28 (Figures 3I and S3E).Similar to neonatal dFBs, adult PAP dFBs were enriched in Grem1 and Entpd1 (Figures 3I and S3E), and ENTPD1 (also known as CD39) has been shown to mark human adult skin PAP dFBs. 29In adult skin, Trps1 was also an exclusive marker for all Ly6a + adipocyte-lineage cells (Figures 3I and S3E).Interestingly, we found that HI-pAd/Areg progressively lost Dlk1 but gained Areg signature gene signatures (F3, Fmo2, Mgp, and Gdf10) during maturation (Figures 3I and S3E).
Next, we examined how genes related to the IL-1, WNT, and TGF-β pathways were altered in key dFB clusters during skin maturation.First, analysis of bulk skin RNA-seq showed that the expression of WNT-and IL-1-related genes was progressively lost, whereas TGF-βrelated genes were induced postnatally during skin maturation (Figure 3J).In line with these results, analysis of key dFB clusters showed that WNT-or IL-1-related genes were inhibited in pAd/Ad and HI-AP, whereas TGF-β-related genes were notably induced in PAP/RET and pAd/Ad but not HI-AP clusters age dependently (Figure 3K).As a result, ECM genes were induced and adipogenesis-related genes were inhibited in PAP/RET or pAd/Ad clusters age dependently, whereas HI-APs started to lose ECM gene expression at 2 months of age (Figure 3K).These age-dependent changes in WNT, IL-1, and TGF-β signaling activities in adipocyte-lineage cells may contribute to the adipogenic-to-fibrogenic switch of these cells during skin maturation.
IHC confirmed that, similar to neonatal skin, the fascia layer of adult skin was enriched with LY6A hi DPP4 hi HI-APs, and LY6A + DPP4 − pAds were detected exclusively in the dWAT layer of adult skin (Figure 3L).In addition, DLK1 + cells were detected only within the interstitial space around the p.c. muscle layer in the 3-week skin and were largely lost by 2 months (Figure 3M), which is consistent with the Dlk1 expression pattern shown by scRNAseq (Figures 3I and S3E).scRNA-seq showed that HI-AP% increased during skin maturation (Figure S3F).FACS analysis of PDGFRA + dFBs validated that, while pAd% increased postnatally for up to 3 weeks, HI-AP% continued to increase for up to 2 months and then declined in 1-year-old skin (Figures S3G, 3N, and 3O).In contrast, CD24 + LY6A − dFBs, identified as the neonatal perifollicular dFBs, were rapidly lost postnatally (Figures S3H and S3I), which is consistent with the Cd24 scRNA-seq plots (Figures 3I and S3E).Together, these results showed that skin maturation is associated with age-dependent inactivation of WNT and IL-1 signaling, depletion of adipogenic pAd, and accumulation of inactive HI-APs.
Tracking dermal adipogenesis and fibrogenesis during skin wound healing
Next, we studied how dermal adipogenesis may be reinitiated upon wounding in adult homeostatic skin.BODIPY staining showed that numerous adipocytes were transiently detected in the wound center GT, peaking at wound day (w.d.) 3, and lipolysis was apparent at w.d.5-7, indicated by the presence of dispersed small lipid droplets, and by w.d.14, adipocytes were cleared from the wound bed (Figure 4A).To determine whether woundassociated adipocytes were generated de novo from adipogenesis, we induced CRE activity in Adipoq-CreERT2;mTmG mice by tamoxifen (TAM) application prior to wounding to label preexisting adipocytes with GFP (Figure S4A).Results showed that more than onethird of wound-associated COLIV + FABP4 + adipocytes were GFP − (Figures S4B and S4C), demonstrating that newly differentiated adipocytes partially contribute to wound-induced adipogenesis.
During the wound-proliferative phase, fibroblasts are activated and differentiate into ACTA2 + myofibroblasts. 9Here, IHC showed that at w.d. 3, DPP4 + LY6A + HI-APs were detected at the wound periphery and bottom, whereas DPP4 − LY6A + pAds/Ads were mainly detected in the wound mid-region (Figures 4B and 4C).By w.d. 7, pAds/Ads were largely lost, and ACTA2 + DPP4 − LY6A − myofibroblasts were abundantly detected in the upper wound bed (Figures 4B and 4C).By day 14, myofibroblasts were largely lost, and DPP4 − LY6A + pAds were regenerated above the HI-AP fascia layer (Figure 4B).scRNA-seq analysis of wound skin cells was next performed to determine the cell differentiation trajectory (Figures 4D and 4E).A total of 15 different cell types, including dFBs, neutrophils, macrophages, T cells, and keratinocytes (KCs), were identified (Figures 4D, 4E, S4D, and S4E).We found that neutrophils (~30% of all cells) and dFB clusters (~50% of all cells) were the major cell types that infiltrated wound GT on w.d. 2, and a subsequent decrease in neutrophils at w.d.7 was accompanied by an increase in dFB heterogeneity and regeneration of other cell types, such as KCs and vessel cells (Figure 4F).
To define heterogeneous dFBs, Pdgfra + dFBs were reclustered into 13 subclusters (r0-r12; Figures 4G-4H and S4F-S4G), which were defined based on the expression of dFB marker genes identified in Figure 1 and myofibroblast markers (Acta2, Tagln).We found that adipocyte-lineage cells, including HI-AP (r0, r2, r12) and pAd/Ad (r3) cell clusters, represented >85% of dFBs in w.d. 2 GT, whereas by w.d. 7, pAd/Ad cells were largely lost, and Acta2 + myofibroblasts and Cd24 hi Alpl + dFBs represented ~1/3 of the w.d.7 dFBs (Figure S4H).Gene expression plots (Figures 4H and S4F-S4J) showed that the r2 HI-AP cluster, which was transiently generated in w.d. 2 GTs, expressed high levels of proliferation marker genes (Ki67 and Pcna) and IL-1-inducible chemokine genes (Cxcl2/5/12), suggesting that these cells were highly proliferative and inflammatory.RNA velocity analysis (Figure 4I) and cell-rank analysis (for calculation of cell-cell transition probabilities 30 ) (Figure 4J) predicted that at w.d. 2, the activated r2 HI-APs were poised to differentiate toward the r3 pAds/Ads, which were in turn poised to differentiate into other dFB clusters, including the r7 myofibroblasts.Together, these results suggest that activated HI-APs may contribute to pAd/Ad generation during the wound-inflammatory phase, and pAd/Ad-to-myofibroblast transdifferentiation may occur during the subsequent wound-proliferative phase.Future studies using lineage tracing mouse models specific for HI-APs and pAd/Ad are needed to confirm these cell differentiation trajectories.
Wound-induced adipogenesis is regulated by neutrophils and the IL-1 signaling axis
FACS analysis confirmed that the percentage of LY6A + DPP4 + HI-AP peaked at approximately w.d. 2, and loss of HI-APs was accompanied by gain of ACTA2 + LY6A + myofibroblasts at w.d.7 in wound GTs (Figures 5A-5C and S5A).The transition from HI-AP (Dpp4 + ) to pAd/Ad (Cebpb hi Fabp4 + ) and myofibroblasts (Acta2 + ) during wounding can also be visualized at the single-cell level (Figure S5B).SCENIC TF analysis identified several key adipogenesis TFs (Cebpb, Creb, Srebf1) as the top enriched TFs driving dFB activation at w.d. 2 (Figure 5D).In addition, GO pathway analysis of the r0 HI-AP cluster identified cellular responses to stress/stimuli and IL-1 signaling as the top upregulated pathways after wounding (Figure 5E).Furthermore, violin plots showed that wounding led to a decrease in the expression of HI-AP marker genes (Smpd3, Dpp4) and an increase in the expression of IL-1-and adipogenesis-related genes in HI-APs.(Figure 5F).In addition, CellChat analysis identified IL-1 as one of the top incoming signals for HI-AP dFBs (Figure S5C), and IL-1-IL-1R signaling network analysis identified neutrophils as the most prominent source of IL-1 ligand acting on dFB clusters at w.d. 2 (Figures 5G and S5D).Expression plots confirmed that Il1b was predominantly produced by neutrophils, whereas the IL-1 receptor (Il1r1) and IL-1-inducible chemokines (Cxcl1/12/5) were expressed primarily by dFBs (Figures 5H, S5E, and S5F).Together, these results suggest that IL-1 released from neutrophils may drive the wound-induced adipogenesis response during the early wound-inflammatory phase.S5G, S5H, and 5I) confirmed that Ly6G + neutrophils were the dominant infiltrating immune cell type (>60% of all CD11B + myeloid cells) during w.d.2-3, and neutrophils were cleared from the wound by w.d. 7. Depletion of neutrophils (Figures S5I and S5J) led to a significant reduction in the DPP4 + wound GT area (Figure 5J) and impaired wound-induced adipogenesis (Figure 5K), decreased CAMP protein expression (Figure S5K), and decreased pCREB signal (Figures S5L and S5M) in the migrating DPP4 + HI-APs.In addition, depletion of neutrophils led to decreased wound-induced Il1b expression (Figure 5K), demonstrating that neutrophils are the major cellular source of IL-1β during the early stages of wound healing.
FACS and immunostaining analyses (Figures
Wild-type (WT) and Il1r1 −/− mice were subjected to skin wounding to validate the role of IL-1 in wound-induced adipogenesis.We found that Il1r1 deficiency led to profound inhibition of pCREB activation in DPP4 + HI-APs (Figures 5L and 5M), but not in the migrating epidermal KCs (white arrow) or other DPP4 − dermal cells, indicating that this IL-1-pCREB signaling axis is specific for the activation of hypodermal cells.In line with pCREB inhibition, the wound-mediated induction of Cxcl5, Cxcl12, and adipogenesisrelated genes was significantly reduced in Il1r1 −/− mice compared with WT controls (Figure 5N).Furthermore, administration of an NF-κB inhibitor to skin wounds also led to decreased pCREB expression in HI-APs and pAds (Figure S5N).These results indicate that neutrophils contribute to HI-AP activation and wound-induced adipogenesis by activating the IL-1R-NF-κB-CREB pathway.
Activation of WNT and TGF-β signaling pathways in adipocyte-lineage cells during the wound-proliferative phase
We found that a peak in the expression of lipogenesis genes at w.d. 3 was followed by the induction of lipolysis genes (Atgl and Hsl) at approximately w.d. 5, prior to the induction of Acta2 and ECM genes around w.d.5-7 (Figures 6A and S6A).In addition, loss of FABP4 + adipocytes was accompanied by transient formation of ACTA2 + myofibroblasts on w.d.7 (Figure S6B), suggesting that adipocyte lipolysis and dedifferentiation may contribute to myofibroblast formation during the wound-proliferative phase.
To determine whether adipocytes (preexisting or newly generated) contribute to the formation of myofibroblasts, we induced CRE activity in Adipoq-CreERT2;mTmG mice by TAM application during w.d.0-6, thereby investigating both the inflammatory and the proliferative phases of wounding (Figure 6B).Mature adipocytes labeled with GFP were detected in the upper wound bed region, where these GFP + cells co-expressed ACTA2 (Figures 6B and S6C), indicating that myofibroblasts can be partially derived from mature adipocytes during wound healing.SCENIC TF analysis identified several WNT-related TFs as top enriched TFs in dFBs at w.d.][33][34][35] Co-staining of active β-catenin with DPP4 and/or ACTA2 revealed that at w.d. 3 the active β-catenin signal was barely detectable, but at w.d.7 β-catenin was robustly activated in the upper wound region, where ACTA2 + myofibroblasts were generated, and by w.d.14, active β-catenin was lost and myofibroblasts disappeared from the wound GT (Figures 6D, S6D, and S6E).Although nuclear β-catenin signal can be detected in cultured dFBs upon WNT3A stimulation (Figure S6F), it was difficult to distinguish β-catenin within nuclei from that within the cytosol in cells within whole wound tissue.
Gene plot analysis showed that both WNT and TGF-β signaling activities were prominently activated in the r7 myofibroblast cluster (Figure S6G), indicating that the crosstalk between TGFB and WNT signaling may promote adipocyte-to-myofibroblast transition during wound healing.
WNT activation in adipocytes promotes adipocyte dedifferentiation and myofibroblast formation
The TGF-β pathway plays a role in triggering lipolysis and dedifferentiation of mature adipocytes 36,37 ; moreover, physical stress was observed to trigger adipocyte dedifferentiation via WNT-β-catenin signaling. 38Here, we found that a combination of both lithium and TGF-β robustly promoted cell elongation and the breakdown of large lipid droplets into numerous smaller lipid droplets (Figures 6E and 6F), which are characteristic of dedifferentiating adipocytes. 38,39Furthermore, co-treatment with both lithium and TGF-β2 led to the most significant increase in ACTA2 + cell percentage (Figure S6H), induction of pro-fibrotic genes, and suppression of Cd36 expression (Figures 6G and S6I).
To lineage trace adipocyte dedifferentiation in vitro, adipocytes were differentiated from Adipoq-CreERT2;mTmG dFBs and labeled by GFP in the presence of TAM, then labeled cells were treated with lithium and TGF-β to trigger lipolysis (Figures 6H, S6J, and S6K).We found that, while lithium or TGF-β alone had some effects, co-treatment profoundly changed GFP + adipocytes from rounded to elongated morphology and promoted the internalization of GFP from membrane to cytosol and the breakdown of the PLIN1 + lipid droplets (Figures 6I and S6L).Furthermore, ACTA2 was induced along the peripheral membrane of the elongated GFP + cells upon co-treatment (Figures 6J and 6K), supporting the idea that ACTA2 + myofibroblasts can be generated de novo from dedifferentiating adipocytes.
Gsk3 deletion in adipocytes promotes myofibroblast formation during wound healing
To examine whether WNT activation in adipocytes promotes fibrogenesis in vivo, we genetically targeted both Gsk3a and Gsk3b, key inhibitory molecules for β-catenin, 40 from adipocytes by crossing Adipoq-CreERT2 mice with Gsk3a/b fl/fl mice (Gsk3 AD-KO ) (Figure 7A).Both GSK3 alleles must be deleted to achieve hyperactivation of the WNT-β-catenin signaling.41 We found that TAM application to Gsk3 AD-KO mice during wound healing led to specific depletion of GSK3 in PLIN + adipocytes, but not in other non-adipocyte cells (Figures 7A and 7B), indicating that targeted Gsk3 deletion in adipocytes was successful.Furthermore, targeted Gsk3 deletion in adipocytes not only increased activated β-catenin signaling in ATGL + (a critical lipolysis enzyme 42 ) adipocytes (Figures 7C and 7D), but also promoted myofibroblast formation (Figures 7E, 7F, and S7A-S7C) and increased CRABP1 expression (Figure S7D).However, Gsk3 deletion decreased the pool of LY6A + DPP4 − pAds (Figures 7E, 7G, and S7C) and inhibited Cxcl5 and Camp expression (Figures S7E and S7F) in skin wounds.These results show that WNT activation mediated by GSK3 deletion in adipocytes dampens wound-induced inflammatory and adipogenic responses and promotes adipocyte-to-myofibroblast transition during the wound-proliferative phase.
Human keloid is associated with sustained WNT activation and inhibition of dermal adipogenesis
Finally, to determine whether our observations in mice are also relevant to humans, we evaluated the changes in dWAT in human skin samples from normal wounds and keloids.IHC staining showed that numerous lobular structures enriched with COLIV + FABP4 + adipocytes were present in the granular tissue of normal wounded skin, whereas COLIV + FABP4 + adipocytes were largely absent in the keloid skin dermis (Figure 7H), suggesting that adipogenesis was blocked in keloids.In addition, immunostaining revealed a strongly activated β-catenin signal in both ACTA2 + myo-FBs and COLIV + cells in the keloid skin dermis (Figure 7I).Furthermore, reanalysis of published transcriptomic data of non-lesional skin or wounded skin from keloid-prone individuals and healthy matched subjects 43 showed that compared with normal wounds, keloid wounds expressed significantly lower levels of immune-or adipogenesis-related genes (IL1B, CEBPB, PLIN3), but higher levels of WNT-and/or TGF-β-related genes and ECM genes (Figures 7J and S7G).Together, these results indicate that unbalanced activation of WNT and immune pathways during wound healing may inhibit adipogenesis and promote fibrogenic development in keloid.
DISCUSSION
dWAT has been recognized as a functional skin layer with remarkable plasticity; it expands or regresses during development, wound healing, bacterial infection, and hair cycling. 3,44To trace the cellular and molecular changes in dWAT cells, we present here a compendium of skin development, maturation, and regeneration processes by scRNA-seq.First, we grouped the developing dFBs into distinct non-adipogenic and adipogenic cell states.Second, our data identified the WNT and IL-1 pathways as the key suppressing and activating signals, respectively, for dermal adipogenesis.Third, we found that skin injury triggered a sequence of events in wound GT, including rapid infiltration/activation of HI-APs and onset of adipogenesis during the wound-inflammatory phase, followed by adipocyte lipolysis/dedifferentiation and myofibroblast generation during the woundproliferative phase.Wound-induced adipogenesis was in part mediated by neutrophils and IL-1, and adipocyte dedifferentiation was in part mediated by activation of the WNT-βcatenin pathway.Finally, we showed that inhibition of dermal adipogenesis and sustained activation of dermal WNT signal was also associated with human keloid scar.Taken together, this study provides in-depth knowledge of the regulatory mechanisms of dWAT plasticity during skin development and wound regeneration at the single-cell level.
Defining the highly heterogeneous adipogenic and non-adipogenic dermal fibroblast subpopulations
][47] However, none of these studies focused on adipogenic cells, and most of these studies defined dWAT or fascia-related dFBs generally as only hypodermal or fascia dFBs, without further defining cell heterogeneity in the hypodermis.In this study, we defined several adipocyte-lineage cell subpopulations, including RET-APs, pAds, adipocytes, and three hypodermal populations (HI-pAds, Aregs, and HI-APs).In addition, we identified a distinct pool of CD24 hi perifollicular dFB clusters that were non-adipogenic, enriched in developing skin, and rapidly lost during skin maturation.
This study also identified several new dFB markers at both the transcript and the protein levels.We identified Trps1 as an exclusive marker for Ly6a + adipocyte-lineage cells.TRPS1 (both mRNA and protein) was found highly expressed in Cd24 hi developing or follicle-associated dFBs, at intermediate levels in Dpp4 + Ly6a − PAP dFBs, and almost undetectable in Ly6a + pAd/APs.We also identified Lgr5, a previously known hair follicle stem cell marker, 14 as a novel pAd marker in mouse skin and in vitro primary pAd culture (Figures 1 and 3).Furthermore, we found that a cluster of Fmo2 + Gdf10 + Areg fibroblasts accumulated in the skin during maturation.Emerging studies have unraveled how Areg cells negatively regulate adipogenesis by secreting GDF10 and/or RSPO2, 16,17,48 but whether the age-dependent increase in skin Areg contributes to the loss of the adipogenic potential of skin AP/pAds during aging needs further investigation.
Balance between the WNT-β-catenin and the IL-1-NF-κB pathways in controlling dermal adipogenesis
0][51] During skin development, WNT-β-catenin signaling is considered the earliest signaling event that instructs the migration of dFB progenitors from the somatic mesoderm to the dorsal skin dermis and the specification of these cells to fibroblasts. 52Inhibition of WNT-β-catenin signaling promotes the conversion of dermal-lineage cells to cartilage. 53Here, we identified the WNT-β-catenin pathway as one of the most differentially suppressed pathways in adipogenic dFBs compared with other non-adipogenic dFB subclusters.Consistent with our results, active WNT gene signatures have been shown to be preferentially associated with Lrig1 + PAP dFBs, but not with Ly6a + pAds. 54Furthermore, forced stabilization of β-catenin in Pdgfra + dFBs leads to a reduction in dWAT, followed by the development of prominent fibrosis. 55These data suggest that, while activation of WNT-β-catenin in mesodermal stem cells is required for stem cell commitment to the dFB lineage during early development, the subsequent inhibition of WNT signaling during later skin development may be required for the commitment of dFBs to adipogenic subpopulations.
Our results suggest that IL-1 may antagonize WNT activity during dermal adipogenesis.Mechanistically, we showed that IL-1β treatment not only triggered the activation of the pro-adipogenic CREB-CEBPβ pathway, but also promoted β-catenin degradation by inhibiting AKT phosphorylation.Consistently, several immune-related and inflammatory pathways have been found to be enriched and activated upon targeted deletion of β-catenin in adipocytes. 56Furthermore, IL-1β increases the expression/activation status of CREB and its target gene CEBPβ in a chondrocyte cell line. 57IL-1B signaling also activated CEBPβ in several other cell types, such as gastric epithelial cells 58 and myocytes, 59 suggesting that CREB-CEBPβ activation maybe a common downstream event of IL-1 receptor signaling.
In contrast, an inhibitory role for IL-1 has been observed in regulating adipogenesis in skeletal muscle tissues, 50,60 suggesting that a tissue-specific microenvironment may shape the differential response of AP to IL-1.
The crosstalk between WNT and TGF-β pathways in regulating adipocyte lipolysis and dedifferentiation
Using several independent approaches, we demonstrated that WNT cooperates with TGF-β signaling to promote adipocyte lipolysis and dedifferentiation of adipocytes to myofibroblasts during the wound-proliferative phase.In line with our findings, TGF-β signaling was shown to be the top enriched pathway activated by WNT3A treatment in adipocytes. 562][63] During the pathogenesis of idiopathic pulmonary fibrosis, myofibroblast differentiation is dependent on TGF-β-WNT crosstalk. 64Further studies are required to determine how signaling events downstream of the TGF-β-WNT axis differ between dermal development and wound regeneration.
Hypodermal interstitium, a newly recognized skin structure enriched with progenitors
The interstitium, a fluid-filled interstitial space enriched with fibroblasts and ECM proteins found in numerous tissues, has been recently recognized as an important anatomical structure in edema, fibrosis, and mechanical functioning of many organs. 12,65Our study defines a multipotent subpopulation of dFBs, the WNT2 + DPP4 + HI-APs, that may not only give rise to dermal adipocytes during development but also function as the origin of regenerating dFBs during wound healing.This HI-AP population is similar to the reported hypodermal fibroblasts, 66 fascia fibroblasts, 67 or interstitial APs, 12 which have a unique ECM production profile, multipotent differentiation potential, and ability to migrate to the wound in response to injury.These converging lines of evidence suggest that HI-APs may be key progenitor cells contributing to dermal cell regeneration.
Limitations of the study
A limitation of this study is that we did not use a genetic approach to ablate or lineage trace HI-APs to confirm the function of HI-APs in dermal adipogenesis.We found that WNT2 is a highly specific marker gene for HI-APs; therefore Wnt2-Cre mice can be used to genetically ablate or lineage trace these cells to validate their roles in dermal adipogenesis during skin development or wound regeneration.
Altogether, our data defined the cellular and molecular mechanisms through which adipocyte-lineage cells are regulated during skin development and wound regeneration.Future lines of investigation examining the crosstalk between immune cells, APs, and adipocytes under conditions of abnormal wound responses, such as keloid and diabetic wounds, and other skin fibrotic diseases such as scleroderma, could lead to breakthroughs in treating human skin diseases.
STAR★METHODS RESOURCE AVAILABILITY
Lead contact-Further information and requests for reagents and resources may be directed to and will be fulfilled by the lead contact, Ling-juan Zhang (lingjuan.zhang@xmu.edu.cn).
Materials availability-This study did not generate new unique reagents.
EXPERIMENTAL MODEL AND STUDY PARTICIPANT DETAILS
Animals and animal cares-All animal experiments were approved by the Xiamen University and/or the University of California San Diego (UCSD), Institutional Animal Care and Use Committee.C57BL/6 WT mice were purchased from Xiamen University Laboratory Animal Center.Adipoq-CreERT2 mice (Stock No:025124) and ROSA-mT/mG mice (Stock No: 07676) were originally purchased from Jackson laboratory.Gsk3a flox/flox Gsk3b flox/flox mice were generously provided by professor Wen-Hsien Liu. 68Adipocyte specific Gsk3 knockout mice were generated by breeding Gsk3a flox/flox Gsk3b flox/flox with Adipoq-CreERT2.Il1r1 −/− mice were generously provided by professor Jiahuai Han (Xiamen university, Xiamen, Fujian, China).All mice were breeded and maintained in standard pathogen free environment of the Laboratory Animal Center in Xiamen University, and all animal experiments were approved by the Animal Care and Use Committee of Xiamen University.
Human skin sample collection and analysis-Fresh adult human full thickness skin biopsies, from age and sex matched healthy, wound and keloid donors (female or male) were collected by the department of dermatology at the First Affiliated Hospital of Fujian Medical University.Keloid disorder was diagnosed based on their clinical appearance, history and treatment history.All keloid samples analyzed in this study were consistent with clinical and pathological criteria for keloid patients.Wound skin biopsies were collected from healthy individual who had no history of keloid formation and receiving wound debridement procedure.All sample acquisitions were approved and regulated by Medical Ethics Committee of the First Affiliated Hospital of Fujian Medical University (reference number No. 2020 [146]).The informed consent was obtained from all subjects prior to surgical procedures.Upon collection, these samples were directly fixed with PFA then proceed for paraffin embedding for histological or immunofluorescent analyses.
Skin maturation and aging model-For skin maturation and aging mouse model shown in Figure 3, dorsal skin biopsies were collected from C57BL/6 wildtype adult male mice with various ages (3W, 2 months and 1 year old).Similar to our previous report, 21 here we used male exclusively to study age-related changes because loss of dermal fat and adipogenic AP in females is relatively slower compared to male counterparts, but the overall trend to lose functional dWAT is similar between female and male.
Mouse wound healing model-7~9 weeks old C57BL/6 mice (male or female) were wounded during telogen phase of hair cycling.Mice were anesthetized using isoflurane, hair was removed by clipper and Nair, and one full-thickness biopsy (1.0 cm × 0.5 cm in size) was generated on each side of dorsal skin (two wounds per mouse).Animal were sacrificed at indicated intervals after injury and wound beds were collected for analysis.To deplete neutrophils, mice were intraperitoneally administrated with 100 ug anti-LY6G (Biolegend, 127649) or IgG2a isotype control antibody (Biolegend, 400565) one day prior to wounding and then were injected daily with 50ug antibodies from day 0 ~ day 2 post wounding, and mice were sacrificed at day 3 post wounding for sample collection and subsequent analysis.
Adipocyte lineage tracing mouse model-Adipoq-CreERT2;mTmG mice (male or female) were daily administrated intraperitoneally with tamoxifen (TAM) (Sigma,T5648) solution (20 mg/mL stock solution dissolved in corn oil (Selleck, s6701)) at 32.5mg/kg body weight for consequent 4~6 days during indicated time period of wound healing.To tracing the cell fate of mature adipocyte in vitro, primary dFBs were isolated from Adipoq-CreERT2;mTmG neonatal mice, cultured and differentiated into adipocytes in the presence of tamoxifen (20 μM) to label mature adipocyte by GFP.Measurement of PLIN1 + lipid droplet size or ACTA2-expressing in GFP + adipocytes were performed by ImageJ software.
METHOD DETAILS
Single cell RNA library preparation, sequencing and data process-For developmental analyses, dorsal skin biopsies were collected from neonatal (P1), male young adult (3 weeks) and male mature adult (2 month) C57BL/6 mice.To minimize batch effects due to non-biological sources, samples from all age groups were processed, sequenced, and analyzed together.Skin biopsies were minced and digested with collagenase D and DNase1 to isolate single skin cells as previously described (Zhang et al., 2019; Zhang et al., 2021).Dead cells were removed using DeadCell Removal kit (Miltenyi Biotic, 130-090-101) according to manufacturer's instruction.Live cells were counted using a hemocytometer and resuspended in 2% BSA at a concentration of 3,000 cells/uL.All samples were loaded on a 10x Genomics GemCode Single-cell instrument that generates single-cell Gel Bead-In-EMlusion (GEMs).Barcoded, full-length cDNAs were reverse-transcribed from polyadenylated mRNA.Silane magnetic beads were used to remove leftover biochemical reagents and primers from post GEM reaction mixture.Next, libraries were generated and sequenced from the cDNAs with Chromium Next GEM Single Cell 3' Reagent Kits v2.cDNA libraries were sequenced on an Illumina Novaseq6000 platform (Illumina).The raw sequencing data was demultiplexed and aligned to the reference genome mm10-1.2.0 using Cell Ranger v3.0.2 pipiline(10x Genomics).The generated raw gene expression matrix was converted into Seurat objects using the R package Seurat v2.0.To remove doublets and poor-quality cells, we utilized the following procedures to control for cell quality: >200 genes/cell, <5000 genes/cell; >25,000 unique molecular identifiers(U-MIs); and <8% mitochondrial gene expression.Low quality cells and outliers were discarded, and only ~24,409 viable cells were used for downstream analysis.These included ~7,483 cells -NB; ~8,235 cells -young (3weeks mice); ~8,691cells-adult (2month mice).
For mouse wound samples, skin biopsies were collected from wound day 2 and day 7 granular tissues or unwounded control skin.Single cells were isolated and subjected to single cell RNAseq procedures including libraries construction and data processing as described above.After low quality cells and outliers were discarded, 10233 cells from non-lesional skin, 7807 cells from wound beds at post-wounding day 2 (PWD2), and 4925 cells from wound tissue at PWD7 were used for downstream analysis.
Unsupervised clustering and gene expression were visualized with the Seurat 2.0 on R studio, and assignment of cell clusters was based on expression of validated marker genes.To investigate the developmental path of dermal fibroblasts, pseudotime analysis was performed on selected dFB subsets by Monocle 2.10.1 (Xiaojie Qiu et al., 2017) to infer the pseudotime trajectories of these cell, followed by scEpath to identify pseudotime-dependent gene expression changes and classify into distinct differentiation states.To further determine the cell fate of dFBs, RNA velocity was performed using the package velocyto (Gioele La Manno et al., 2018) with the default parameter, and the BAM files were used as inputs.
To analyze activity of transcriptional regulators in skin development or wound healing, SCENIC (Van de Sande et al., 2020) conjunction with the mm9 Rcis Target database was used and UMIs for transcriptionally defined fibroblasts as input to construct regulon networks between transcription factors and potential target genes and score the active value of regulon for each FB clusters.Single cell RNA libraries construction, sequencing and bioinformatic analysis was assisted by GENE DENOVO Inc (Guangzhou, China).Cellrank (v1.5.0) analysis was performed to calculate cell-cell transition probabilities according to literature. 30Briefly, scVelo (v0.2.4) was first performed on selected dFB clusters (r0 -r12) to estimate RNA velocities and velocity graphs.Secondly, transition probabilities were calculated in a weighted transition matrix with 80% RNA velocity and 20% similarity.The Generalized Perron Cluster Analysis(GPCCA) was used to compute fate probabilities.The terminal states were estimated from the so-called 'eigengap heuristic' of the spectrum of the transition matrix.Analysis was repeated for the initial states.Probability of each cell to transit to the terminal states was computed.Finally, directed PAGA was used to aggregate the individual fate maps into a cluster-level fate map.
Bulk RNA sequencing and bioinformatic analysis-Total cellular RNA were extracted using TRIzol reagent (Sigma, T9424) and RNAExpress Total RNA Kit (NCM, M050).RNA quality was analyzed by bioanalyzer and RNA samples with RIN value >7 were used for sequencing.Next generation sequencing library preparations were constructed according to the manufacturer's protocol (NEBNext Ultra RNA Library Prep Kit for Illumina).The poly(A) mRNA isolation was performed using NEBNext Poly(A) mRNA Magnetic Isolation Module (NEB).The mRNA fragmentation and priming were performed using NEBNext First Strand Synthesis Reaction Buffer and NEBNext Random Primers.First strand cDNA was synthesized using ProtoScript II Reverse Transcriptase and the second-strand cDNA was synthesized using Second Strand Synthesis Enzyme Mix.The purified double-stranded cDNA was then treated with End Prep Enzyme Mix to repair both ends and add a dA-tailing in one reaction, followed by a T-A ligation to add adaptors to both ends.Size selection of Adaptor-ligated DNA was then performed using AxyPrep Mag PCR Clean-up (Axygen), and fragments of ~420bp (with the approximate insert size of 300 bp) were recovered.Each sample was then amplified by PCR.The PCR products were cleaned up using AxyPrep Mag PCR Clean-up (Axygen), validated using an Agilent 2100 Bioanalyzer (Agilent Technologies), and quantified by Qubit 2.0 Fluorometer (Invitrogen).
Then libraries with different indexs were multiplexed and loaded on an Illumina Navoseq instrument for sequencing using a 2×150 paired-end (PE) configuration according to manufacturer's instructions.The sequences were processed and analyzed by GENEWIZ.Venn diagrams between gene sets were made by BioVenn.KEGG and gene ontology (GO) pathway analysis for differentially expressed genes and correlation analysis between single cell RNA-seq with bulk RNA-seq were performed by R package clusterProfile 3.12.0.
Pearson correlation analysis-Pearson correlation analysis was performed between the scRNA-seq datasets and bulk RNAseq datasets to determine the degree of correlation/ similarity between dFB clusters identified by scRNA-seq with skin samples at various ages or different dFB treatment states.The scRNA-seq and bulk RNAseq transcriptomic datasets were normalized and matrixed, and Pearson correlation coefficient of the matrix was calculated, and hierarchical clustering analysis was performed to generate correlation heatmap in R Studio software.
Cell-chat signaling network analysis-R package CellChat 1.3.0(Jin et al., 2021) was used to evaluate the potential cell-cell communication between dFB subclusters and other cell types, especially immune cells, during wound healing.The scRNA-seq data was imported to implement CellChat platform in R software.The process includes projecting the gene expression data onto protein-protein interaction (PPI) network, then assigning a probability value to infer biological cell-cell communication network, and calculating the centrality indicator of interacting network to identify the role / contribution of each cell population in distinct signaling pathways.The number and strength of identified cell-cell communication is displayed through hierarchical graphs, circle charts, heatmaps, etc. to visualize single or multiple signaling pathways.
Primary mouse dermal fibroblast isolation and in vitro adipocyte differentiation:
Primary mouse dermal fibroblasts were isolated from mouse skin as described previously (Zhang et al., 2019; Zhang et al., 2021).In brief, mouse dorsal skin was first into strips and digested with dispase overnight to remove epidermis.The dermis tissue was then cut into small pieces and digested with 2.5 mg/mL Collagenase D and 30 ng/mL DNAse1 for 2 hous at 37°C to release dermal fibroblasts.Cell mixture was then filtered through 30 μm filter and treated with red blood cell lysis buffer.Isolated dermal fibroblasts were cultured in growth medium (DMEM supplemented with 10% FBS and antibiotics/antimycotics) in a humidified incubator at 5% CO2 and 37°C under sterile conditions.Fresh medium was replenished daily to remove debris or dead cells.Primary cells were then trypsinized within 3 days and replated at 5 × 104/mL for in vitro assays, and only passage 1 cells were used for experiment.To induce adipocyte differentiation, two day post-confluent dFB were switched to adipocyte differentiation medium containing 2 μM Dexamethasone, 250 μM IBMX, 200 μM Indomethacin and 10 μg/mL recombinant human insulin.Fresh differentiation medium was changed at day 2 then medium was switched to maintenance medium (growth medium supplemented with 10 μg/mL recombinant human insulin) to promote maturation and hypertrophy of differentiated adipocytes.
Histology, collagen trichrome staining and immunohistochemistry (IHC)-To prepare paraffin sections, tissue biopsies were fixed with 4% PFA (Alfa Aesar, Shanghai, China) overnight then dehydrated and embedded in paraffin and sectioned at a thickness of 5-8 μm.For OCT embedding, fresh dorsal skin tissues were directly embedded in OCT then sectioned at a thickness of 15-20 μm, and frozen sections were fixed with 4% PFA for 15min prior to staining.Histology was assessed by Hematoxylin and Eosin (HE) staining using solutions from ZSGB-BIO (Beijing, China), and collagen was stained by the Masson's Trichrome Stain Kit (Solarbio, Beijing, China), and elastic fiber staining was performed by elastic fiber stain kit (Saint-Bio, Shanghai, China) according to manufacturer's instruction.Dermal cell density count shown in Figure S3C was performed manually by counting the number of nuclei per 1mm 2 dermal area.Lipid staining was performed on frozen sections using BODIPY (ThermoFisher Scientific, Waltham, MA, USA) and PHA (Cytoskeleton, Denver, CO, USA) dyes.For IHC, fixed sections were permeabilized with 0.1% saponin (Sigma-Aldrich, St Louis, MO) and blocked in 5% BSA (Bioforxx, Hessen, Germany), and blocked sections were incubated with primary antibodies at 4°C overnight followed by appropriate 488-, Cy5 or Cy3-coupled secondary antibodies (Jackson ImmunoResearch Laboratories, West Grove, Pennsylvania, USA) in the dark for 4 hours at 4°C.Finally, sections were mounted by ProLong Gold Antifade Mountant with DAPI (Thermo Scientific Inc., Rockford, IL, US).All images were taken with Aperio VERSA Brightfield, Fluorescence Digital Pathology Scanner or Leica TCS SP8 White Light Laser Confocal Microscope, and processed by photoshop and/or Aperio ImageScope software (Leica Biosystems, Nußloch, Germany).Quantification of the fluorescence integrated intensity for each fluorophore was performed using ImageJ software.
Quantitative reverse transcription-quantitative PCR (qRT-PCR) analyses-Total cellular RNA was extracted using the RNAExpress Total RNA Kit (NM, M050) and 500 ng of RNA was reverse transcribed to cDNA using HiScript II Q RT SuperMix kit (Vazyme, R222-01).Quantitative, realtime PCR was performed on the Qtower real time system (Analytikjena, Swavesey, Cambridge, UK) using SYBR Green Mix (Bimake, Houston, Texas, USA).All of the primers used with SYBR green were designed to span at least one exon to minimize the possibility of nonspecific amplification from the genomic DNA.The expression of Tbp gene (TATA-Box Binding Protein) was used as a housekeeping gene to normalize data for the expression of mouse genes.Specific primer sequences are shown in Table S4.
Cell extract preparation and western blotting analyses-Primary dFBs were lysed in a denaturing lysis buffer containing 20 mM HEPES pH 7.4, 250mM NaCl, 2 mM EDTA, and 1% SDS supplemented with completed proteinase inhibitor cocktail (apexbio, Houston, USA) and phosphatase inhibitor cocktail (Roche, Basel, Switzerland) to maximally preserve protein post-transational modifications as described previously (Zhang et al., 2016).Lysates were boiled for 3 mins and homogenized by sonication using digital sonifier FS-350T (Sxsonic, Shanghai, China) followed by centrifugation to remove DNA and cell debris.Protein concentrations were measured by BCA protein assay kit (Thermo Scientific Inc., Rockford, IL).For western blotting, 20ug of protein was mixed with same volume of 2x loading buffer with 20% βmercaptoethanol (Sigma-Aldrich, St Louis, MO) and boiled for 5 mins at 95°C Then samples were separated on a 10-20% Tris-Tricine precast gel (Thermo Scientific Inc., Rockford, IL), transferred to PVDF membrane (Roche, Basel, Switzerland), followed by immunoblotting using indicated primary antibodies followed by fluorescent secondary antibodies (LI-COR, Lincoln, Nebraskal) and imaging using fluorescent Odyssey System (LI-COR).All western blot images shown in the manuscript are representative of 3 independent experiments.
QUANTIFICATION AND STATISTICAL ANALYSIS
Experiments were repeated at least 3 times with similar results and were statistically analyzed by GraphPad Prism 8 software.Quantification analyses of immunostained tissue sections showing the fluorescence integrated intensity of indicated fluorophores were performed by ImageJ (version 1.53).Quantified intensity profiles showing signals from indicated fluorescent channels across skin sections (from top to bottom) is adapted from publications. 71,72Quantification analyses of Oil-red-O or alizarin staining (Figure 1) were performed by ImageJ software according to literature. 73To quantify lipid-droplet (LD)
•
Highly heterogeneous adipocyte lineage cells are defined in developing mouse skin
Figure 1 .
Figure 1.Characterization of heterogeneous dermal adipocyte lineage cells in developing neonatal skin (A) scRNA-seq analysis of cells isolated from neonatal skin.Pdgfra + dFBs were reclustered into 12 clusters.(B) Neonatal dFBs were grouped into three cell states along differentiation trajectories.(C) Violin plots of indicated genes.(D-G) Neonatal mouse skin sections were immunostained with antibodies against various dFB markers as indicated, and the nuclei were counterstained with DAPI in either blue or white.Scale bars, 50 μm.Abbreviations: p.c., panniculus carnosus muscle layer; p.f.,
Figure 2 .
Figure 2. Interplay between the WNT-β-catenin and IL-1-immune signaling pathways in regulating dermal adipogenesis (A) Signaling enrichment analysis of neonatal dFB clusters shown by heatmap.Color scale of red to blue indicates enrichment score.(B) Violin chart showing the expression of indicated genes.(C-E) Neonatal dFBs were pretreated with WNT3A and/or IL-1β and then subjected to adipocyte differentiation, and differentiated cells were stained with ORO (C).The percentages of ORO+ adipocytes was quantified (D) (n = 6/group).Western blotting analysis of CAMP and FABP4 in adipocyte conditioned medium (E).
Figure 3 .
Figure 3. Tracing dermal adipogenesis and fibrogenesis during skin maturation (A) BODIPY (green dye for lipid) and phalloidin (red dye for actin fiber) staining (top) and Gomori trichrome staining (bottom) of skin sections from mice at indicated ages.Scale bar, 100 μm (B) Schematic of scRNA-seq of dorsal skin cells isolated from NB, 3-week, and 2-month mice.(C) tSNE plots showing cell distribution by clusters indicated with unique color.(D) tSNE plots showing the expression of indicated marker genes.(E) tSNE plots showing cell distribution by age of NB (red), 3-week (green), and 2-month (blue) mice.(F) Bar chart showing age-related changes in the percentage of various cell types.
Figure 4 .
Figure 4. Tracing dermal adipogenesis and fibrogenesis during skin wound healing (A) Full-thickness mouse wounds were collected at indicated wound days (w.d.) for PHA (red) and BODIPY (green) staining.The dotted lines mark wound central granular tissues.Scale bar, 400 μm.(B) LY6A (red), DPP4 (blue), and ACTA2 (green) immunostaining.Dotted lines mark wound GT.Scale bar, 400 μm.(C) Quantified intensity profiles for (B), showing signals from all three fluorescence channels from wound top to bottom (representative of n = 3/group).(D-F) Wound day 2 and 7 and unwounded control skin samples were subjected to scRNAseq analysis.UMAP (uniform manifold approximation and projection) plot (D) showing cell
Figure 5 .
Figure 5. Wound-induced adipogenesis is regulated by neutrophils and the IL-1 signaling axis (A-C) FACS plots (A and B) and quantified bar graphs (C) showing the percentage of HI-APs (DPP4 + LY6A + ) and myofibroblasts (ACTA2 + LY6A − ) in control, w.d. 2, and w.d.7 skin samples (n = 3/group).(D) Heatmap showing the top enriched transcriptional factors in w.d. 2 cells compared with control and w.d.7 cells.(E) KEGG/GO pathway analysis comparing the r0 HI-AP cluster of control and w.d. 2 skin samples showing top enriched signaling pathways activated in HI-APs after wounding.(F) Violin plots showing the expression of indicated genes in the control and w.d. 2 samples.(G) Circle plot showing the inferred intercellular communication network for IL-1β signaling in control and w.d. 2 skin cells.
Figure 6 .
Figure 6.Activation of WNT and TGF-β signaling pathways in adipocyte-lineage cells during the wound-proliferative phase (A) qRT-PCR analysis showing the mRNA expression kinetics of listed genes during skin wound healing (n = 3 per group).(B) Adipoq-CreERT2;mTmG mice were administered tamoxifen (TAM) from day 1 to 6 during wound healing, and wound tissues were collected at w.d. 10 for immunostaining of ACTA2 (blue) and GFP (green).Scale bar, 400 μm.(C) Heatmap of SCENIC TF analysis showing the top enriched transcriptional factors.(D) Wound tissues were stained with active β-catenin, ACTA2, and/or DPP4 as indicated.Scale bar, 500 μm.Quantified intensity profiles showing signals from all three fluorescence channels from wound top to bottom (the lower right box) are shown (representative of n = 3/group). | 2023-06-20T06:17:09.543Z | 2023-06-01T00:00:00.000 | {
"year": 2023,
"sha1": "d621574fbd40919df7231198d8eee6fd6a083278",
"oa_license": "CCBYNCND",
"oa_url": "http://www.cell.com/article/S2211124723006587/pdf",
"oa_status": "GOLD",
"pdf_src": "PubMedCentral",
"pdf_hash": "2b021b1f2b8d5ff24e3f4c372b4b9b9f877a0b85",
"s2fieldsofstudy": [
"Biology",
"Medicine"
],
"extfieldsofstudy": [
"Medicine"
]
} |
40323936 | pes2o/s2orc | v3-fos-license | Evaluation of sodium sulfite and protein correction in analyses of fibrous compounds in tropical forages 1
The objective of this study was to evaluate the contents of fibrous compounds in tropical grasses and legumes according to utilization of sodium sulfite in the neutral detergent solution or using a procedure for contaminant protein correction. Samples of ten grasses and ten legumes were used. The contents of neutral detergent fiber were decreased when sodium sulfite was used; however, more prominent reductions were verified in legumes. Sodium sulfite decreased the acid detergent fiber content in both forage groups. The contents of neutral and acid detergent insoluble protein and lignin were reduced by sodium sulfite in legumes, but no effect was observed in grasses with regard to these variables. The decrease in fiber contents in legumes could be explained by the solubilization of lignin and decrease in insoluble nitrogen. However, the decreases in fiber in grasses could not be solely explained by the decrease in contaminant protein and solubilization of lignin, and loss of other fibrous compounds probably occurred. The utilization of sodium sulfite compromises the accuracy of the estimates of fibrous compounds contents in tropical forages. The precision of the estimates were not relevantly increased by sodium sulfite. The correction of insoluble fibrous compounds for protein is suggested instead of using sodium sulfite because there are no modifications on neutral detergent solution or undesirable solubilization of fibrous compounds.
Introduction
The content and the diversity of insoluble fibrous compounds present a main role in the evaluation of tropical forages in ruminant nutrition because they directly influence several characteristics of the diets, such as the rumen fill effect and the energy and nitrogenous compounds availability (Detmann et al., 2008).Nevertheless, the concepts of fiber applied in animal nutrition are essentially defined by the analytical method.Thereby, any variation in the analytical procedures may potentially produce results that will differ from the original proposition.As a consequence, this can compromise the rusticity and accuracy of the method and the comparison of results obtained in different experimental or productive conditions (Detmann, 2010).
Sodium sulfite has been suggested as a component of the solution used to analyze the neutral detergent insoluble fiber (NDF).The addition of sodium sulfite to neutral detergent solution was proposed aiming at the decrease of the contamination with protein in the gravimetrically measured residue (Mertens, 2002).However, its utilization was also associated with the undesirable solubilization of fibrous compounds, such as lignin (Hartley, 1972;Van Soest et al., 1991;Dorleans et al., 1996).From this, it could be assumed that sodium sulfite utilization in NDF analysis is controversial.
Nitrogenous compounds are supposed to be the main contaminant of gravimetrically measured fiber (Van Soest, 1994;Detmann & Valadares Filho, 2010).To avoid biases on insoluble fiber and non-fibrous carbohydrates estimates, it has been suggested that crude residue of NDF be corrected for contaminant protein and ash (Detmann & Valadares Filho, 2010;Detmann et al., 2010).Particularly, protein correction is an uncomplicated procedure because it is based on routine analyses such as the Kjeldahl technique.When compared with sodium sulfite, protein correction could present two probable analytical advantages.First, the analysis of crude residue of fiber for protein content would not promote the undesirable solubilization of any fibrous compounds as highlighted before for sodium sulfite utilization.Second, protein correction allows taking into account the total contaminant nitrogen in the fiber residue whereas sodium sulfite decreases, but not eliminate such contamination.However, an accurate comparison of the estimates of fibrous compounds considering the use of sodium sulfite or protein correction in tropical forages still needs to be established.
Thereby, the objective of this study was to evaluate the effects of using sodium sulfite in neutral detergent solution and protein correction on estimates of insoluble fibrous compounds in tropical grasses and legumes.
Material and Methods
The experiment was conducted at the Laboratório de Nutrição Animal of the Departamento de Zootecnia of the Universidade Federal de Viçosa, Viçosa, Minas Gerais, Brazil.
Samples were oven-dried at 60°C and processed in a knife mill (1-mm).After that, the dry matter (DM, index no.934.01) contents of the samples were analyzed according to the methods of the AOAC (1990).
For the NDF analysis, twelve aliquots of each forage (1 g) were put in 120-mL polyethylene pots, and 100 mL of neutral detergent were added (Mertens, 2002).Sodium sulfite was added in six pots (0.5 g/100 mL).Heat-stable alpha amylase was used in all aliquots (Termamyl 2X, Novozymes).After sealing, pots were autoclaved at 105 ºC for 1 h (Pell & Schofield, 1993).The neutral-detergent-insoluble residue was retained by vacuum filtration in filter crucibles, washed sequentially with hot distilled water and acetone, ovendried at 105 ºC for 16 h, put in a dissecator and weighed.Four aliquots (two with sodium sulfite and two without sodium sulfite) were separated and analyzed for neutral detergent insoluble protein (NDIP) contents (Licitra et al., 1996).
The eight crucibles remaining were conditioned in 120-mL polyethylene pots, and 100 mL of acid detergent were added (Van Soest & Robertson, 1985).After sealing, the pots were autoclaved at 105 ºC for 1 h (Pell & Schofield, 1993).The acid-detergent-insoluble residue was retained by vacuum filtration in the filter crucibles, washed sequentially with hot distilled water and acetone, and oven-dried and weighed as described before.Similarly, four aliquots (two with sodium sulfite and two without sodium sulfite) were separated and analyzed regarding acid detergent insoluble protein (ADIP) contents (Licitra et al., 1996).
Both NDIP and ADIP contents were obtained by multiplying the contents of insoluble nitrogen in NDF and ADF by 6.25 (Licitra et al., 1996).
The four crucibles remaining were used to quantify lignin contents.The filter crucibles were put in 120-mL polyethylene pots, and 30 mL of 12 M sulfuric acid were added and homogenized with a glass rod.Subsequently, 12 M sulfuric acid was added up to half the height of the crucible, and the samples were homogenized after 30 minutes (Van Soest & Robertson, 1985).After 3 h, the crucibles were subjected to vacuum filtration and washed with hot distilled water to completely remove the acid.The material was oven-dried at 105 ºC for 16 h and then weighed to obtain the mass of the residue composed of lignin and minerals.Then, the crucibles were transferred to a muffle furnace at 500 °C, where they remained for 3 h.They were weighed again, and the mass of lignin was calculated by the weight loss after incineration.
The contents of NDF, NDF corrected for protein (NDFp), acid detergent fiber (ADF), ADF corrected for protein (ADFp), lignin, NDIP, and ADIP obtained with or without using sodium sulfite were directly compared between the different species groups (grasses or legumes) according to the model: where μ = general constant; G i = effect of the species group i (grass or legume; fixed effect); S (i)j = effect of species j nested within group i (random effect); M k = effect of the method of analysis k (with or without sodium sulfite; fixed effect); GM ik = interaction effect of the species group i and the method k; and ε ijk = random error.The mean square of S (i)j was used to test the species group effect.On the other hand, the sodium sulfite and interaction effects were evaluated using random error mean square.Some relationships between variables evaluated with or without sodium sulfite were evaluated by adjusting a simple linear regression equation (Table 1).These statistical analyses were conducted independently for grasses and legumes under the null hypothesis: H 0 : β 0 = 0 and β 1 = 1 (2).Variables X and Y were considered to be similar when the null hypothesis was not rejected.
All statistical procedures were performed using the PROC MIXED and the PROC REG of SAS (Statistical Analysis System, version 9.1) adopting α = 0.05.
Results
Except for ADF and ADFp contents (P>0.05),there was interaction (P<0.05) of sodium sulfite utilization and forage group on the other variables (Table 2).
The ADF and ADFp contents were decreased (P<0.05) by using sodium sulfite (Table 2).The NDF and NDFp contents were decreased (P<0.05) by using sodium sulfite in both forage groups.However, the decrease was more prominent in legumes compared with grasses (Table 2).The NDIP, ADIP and lignin contents were decreased by sodium sulfite in legume samples (P<0.05),but such effect was not observed (P>0.05) in grass samples (Table 2).
The decreases in NDF and ADF contents caused by sodium sulfite in legumes were found to be equivalent (P>0.05) to the sum of solubilized lignin and NDIP, for NDF, and solubilized lignin and ADIP, for ADF (Table 3).As there was no decrease in lignin and insoluble nitrogen in grasses NDF = neutral detergent fiber; NDFp = NDF corrected for protein; ADF = acid detergent fiber; ADFp = ADF corrected for protein; NDIP = neutral detergent insoluble protein; ADIP = acid detergent insoluble protein; WSS = without sodium sulfite; SS = using sodium sulfite; SDap = standard deviation among plots; SDwp = standard deviation within plots; F = effect of forage group; S = effect of sodium sulfite; F × S = interaction of forage group and sodium sulfite.a, b: means in same row within grasses or legumes followed by different letters differ at P<0.05. 1 g/kg DM. 2 g/kg NDF. 3 g/kg ADF.
Table 2 -Estimates of fibrous compounds contents according to species groups and utilization of sodium sulfite (Table 2), such equivalence could not be observed in this forage group (P<0.05;Table 3).
Discussion
The recommendation for using sulfite in neutral detergent solution is based on its ability to decrease contaminant protein in the insoluble residue (Mertens, 2002).Those contaminant nitrogenous compounds can be originated from four potential sources: nitrogen that is naturally associated with the cell wall, nitrogen attached to artifacts (non-enzymatic products), nitrogen linked to tannins (proanthocyanidins), and keratins of animal origin (Van Soest, 1994).The last source is not applicable to the results of this study.
Part of the protein contamination could be attributed to the formation of artifacts by the non-enzymatic reactions.However, their formation occurs mainly during drying at temperatures of at least 65 ºC, which were avoided in this study (samples were dried at 60 ºC).The use of adequate temperatures and ventilated ovens reduces the formation of these artifacts by accelerating the removal of humidity from the material, which is necessary for non-enzymatic reactions to occur (Van Soest, 1994).
It has been speculated that protein contamination would be more prominent in tannin-rich material or samples with higher crude protein content (Hintz et al., 1996;Krueger et al., 1999;Pagán et al., 2009).Considering this, it will be expected that legumes show higher protein contamination than grasses due to their greater tannin content, which would result in the formation of insoluble complexes with the protein components of forages (Van Soest, 1994;Krueger et al., 1999).Such statement seems to support the results obtained in this study, where sodium sulfite caused more prominent decrease in NDF and ADF contents in legumes when compared with grasses (Table 2; Figure 1).
On the other hand, the utilization of sodium sulfite has been associated with a partial solubilization of lignin (Hartley, 1972;Van Soest et al., 1991), which was observed for legume samples (Table 2).Such pattern indicates that lignin estimates can be less reliable in this type of forage when sodium sulfite is used.However, the decreases of NDF and ADF contents in grasses could not be solely explained by the decrease in contaminant protein and solubilization of lignin (Tables 2 and 3).
The decreases in NDF and ADF contents caused by protein correction or sodium sulfite were approximately equivalent to each other in legume samples (Figure 1).Despite this, those decreases present different components.The utilization of sodium sulfite in legumes decreased, but did not obliterate the contaminant protein (Table 2; Figure 1), which allowed verifying an additional decrease in fiber contents when protein correction was applied to samples previously treated with sodium sulfite (Figure 1).Thereby, NDF and ADF decreases were at least partially caused by the solubilized portion of lignin (Tables 2 and 3).In this way, despite providing results similar to sodium sulfite (Figure 1), it can be assumed that protein correction provides more accurate estimates of fibrous compounds in legumes under a chemical point of view.
Actually, the decreases in NDF and ADF contents caused by sodium sulfite or protein correction were approximately additive to each other in grass samples (Figure 1).This can be affirmed because sodium sulfite did not promote any significant decrease in the insoluble protein included in fiber residues (Table 2).Moreover, the sodium sulfite did Figure 1 -Decrease of fiber contents in relation of analytical procedure without using sodium sulfite or protein correction (s = using sodium sulfite; p = considering protein correction).NDF = neutral detergent fiber; ADF = acid detergent fiber.
not cause significant decrease in lignin content in grasses (Table 2).In this context, the actual cause of the decreases in NDF and ADF contents (Figure 1) could not be entirely explained by decrease in insoluble nitrogen or solubilization of lignin (Table 3).Such pattern stresses that sodium sulfite can solubilize other components of the cell wall (Hintz et al., 1996).When compared with the control method (without sodium sulfite or protein correction), about 26 and 49% of the decreases caused by sodium sulfite in NDF and ADF could not be identified from results obtained in this study (Figure 2), which implicates lack of accuracy on fiber content estimates.
Considering the results obtained with grass samples, the protein correction could be assumed as an alternative to avoid the bias caused by sodium sulfite.This statement is based on the fact that protein correction does not demand any modification in neutral detergent solution and avoids the undesirable loss of known or unknown fibrous compounds.Moreover, considering the results obtained with legume samples, the partial solubilization of insoluble nitrogen and lignin could compromise the estimates of these compounds when they are imputed in feed evaluation models.
Oven-drying procedures of forages can cause the formation of nitrogen-containing artifacts, which seem to be originated from oxidative polymerization of tannins and protein (Pagán et al., 2009) or through non-enzymatic reactions between sugars and free amino acids (Van Soest, 1994).Those compounds can noticeably increase the protein contamination of neutral detergent insoluble residue, but they would be not necessarily eliminated by sodium sulfite.Such pattern could be more relevant by considering that different oven-drying procedures could cause different increases in insoluble nitrogen contents (Pagán et al., 2009;Pelletier et al., 2010), which would obviously decrease the reproducibility of the analytical method.Nevertheless, Terrill et al. (1994) affirmed that protein correction is able to eliminate the variation associated with drying procedure on insoluble fibrous residue.
A possible problem associated with protein correction procedure is the conversion of insoluble nitrogen contents into equivalent protein.According to Hintz et al. (1996), the conversion factor of nitrogen into protein of a simple Maillard artifact (amino acid plus sugar) would be around half of that one applied for true protein (6.25).The probable bias caused by uncorrected conversion of nitrogen into equivalent protein could be increased in digestion assays because little true protein is found in feces.A great part of fecal nitrogenous compounds presents nitrogen content around 70-110 g/kg (Van Soest, 1994).This would demand a conversion factor varying from 14.3 to 9.1.Thereby, despite of the 6.25 factor being recommended in the evaluation of nitrogenous compounds associated with fiber (Licitra et al., 1996), its utilization could underestimate the actual mass of contaminant nitrogenous compounds and overestimate the mass of insoluble fiber.However, evaluations with regard to the true nitrogen content in the nitrogenous compounds associated with insoluble fiber remains to be performed.Despite this, it must be emphasized that bias observed with protein correction would be less relevant when compared with effective loss of nitrogenous and fibrous compounds caused by sodium sulfite utilization (Table 2; Figures 1 and 2).
According to Hintz et al. (1996), the sodium sulfite utilization could be advantageous because it would increase the precision of fiber estimates.The benefit of using sodium sulfite in NDF analysis is that it would make some samples easier to filter, especially those that are high in protein or those that have been heated or fermented.In these cases, sulfite would greatly reduce variability among replicates and improve repeatability as well.
However, using sodium sulfite caused higher precision only on lignin contents (Figure 3).It probably occurred due to decrease in the contaminant protein of lignin residue.The lignin estimated by solubilization of cellulose in sulfuric acid presents significant contamination by nitrogenous compounds (Henriques et al., 2007;Gomes et al., 2011).Nonetheless, the increased precision (Figure 3) may be associated with decreased accuracy (Table 2), which would not be a good analytical advantage.
Considering the NDF contents, the sodium sulfite utilization produced results with lower precision when compared with control method or protein correction.If the filtration problems are more prominent in NDF analysis than in ADF or lignin analyses, the analytical advantage pointed out by Hintz et al. (1996) for using sodium sulfite seems not to be relevant for tropical forages analyses.
Conclusions
The utilization of sodium sulfite compromises the accuracy of the estimates of fibrous compounds contents in tropical forages.The procedure for contaminant protein correction is suggested as alternative to sodium sulfite because it does not encompass any modification in neutral detergent solution or cause any undesirable solubilization of fibrous compounds.
Figure 2 -
Figure 2 -Descriptive evaluation of the average decrease in neutral detergent fiber (NDF) and acid detergent fiber (ADF) contents caused by sodium sulfite in grass samples (g/kg DM).
Figure 3 -
Figure3-Relative standard deviation (RSD) for estimates of fiber compounds according to utilization of sodium sulfite or protein correction (w = without sodium sulfite or protein correction; s = using sodium sulfite; p = considering protein correction).NDF = neutral getergent fiber; ADF = acid detergent fiber; LIG = lignin.
Table 1 -
Description of variables evaluated in the equality test using linear regression analysis 1 See details of variables in Table1.2Estimate±standarderror.3Seeequation 2 for details.
Table 3 -
Estimates of regression parameters for different relationships according to utilization of sodium sulfite | 2017-10-03T15:59:03.054Z | 2012-01-01T00:00:00.000 | {
"year": 2012,
"sha1": "1fec10ff2f20e8e359bf748808d65742587cab7d",
"oa_license": "CCBY",
"oa_url": "https://www.scielo.br/j/rbz/a/zcXmszjTyR93QDHfStmTkRM/?format=pdf&lang=en",
"oa_status": "GOLD",
"pdf_src": "Anansi",
"pdf_hash": "1fec10ff2f20e8e359bf748808d65742587cab7d",
"s2fieldsofstudy": [
"Agricultural And Food Sciences"
],
"extfieldsofstudy": [
"Chemistry"
]
} |
234598457 | pes2o/s2orc | v3-fos-license | Metabonomic Profiling Analyses Reveal ANS Upregulation to Enhance the Flavonoid Pathway of Purple-Fleshed Sweet Potato Storage Root in Response to Deep Shading
Intercropping, as a common worldwide cultivation pattern, provides opportunities for sustainable agriculture with fuller use of light, temperature and land resources and greater yield per unit of land. The intercropping impact on crop quality is a current focus. This study found that shading cultivation of purple-fleshed sweet potato can improve the storage root pigment accumulation by more than 20% to increase economic benefits. We performed gas chromatography and mass spectrometry analysis of storage roots of the anthocyanin-enriched cultivar Jihei-1 under 60% shading and nonshaded treatments. A total of 224 differential metabolites were identified, among which N-acetyl-5-hydroxytryptamine, 1-monopalmitin, 4-pyridoxic acid, dodecano, arbutin, tryptophan, citrulline and phenylalanine were significantly upregulated under shading with a more than 10-fold change. Furthermore, metabolic pathway enrichment maps were based on the biological processes and stratification level selected. These metabolites mainly influenced the pathways of phenylpropanoid biosynthesis, the citrate cycle, organic acid biosynthesis and metabolism and amino acid metabolism. Through tissue-specific dynamic changes in amino acids, soluble sugars, starch and anthocyanins during storage root development, we proposed a variety-specific strategy of purplefleshed sweet potato in response to prolonged deep shading, that is, utilizing and enhancing broad aboveground-tissue photosynthesis and transferring photosynthates into roots in advance, leading to a rapid increase in storage root anthocyanin synthesis. With comprehensive qPCR, western blot and enzyme activity analyses, we identified three key enzymes, CHS, ANS and 3GT, in purple-fleshed sweet potato storage roots in response to shading, which affect the root anthocyanin content by influencing the flavonoid metabolism pathway. This study provides a theoretical basis for revealing the regulation of anthocyanin synthesis in crops and a guidance for high-quality sweet potato cultivation and nutritional improvement using shade facilities.
Introduction
Purple-fleshed sweet potato (PSP) is an important resource with high anthocyanin levels in storage roots. In recent years, it has become popular in international markets. Considering its broad prospects, it is necessary to study ways to maintain or improve the high anthocyanin contents in sweet potatoes. Owing to their potential health benefits, anthocyanin has been investigated extensively. These compounds are a final product of flavonoid metabolic pathways. The pathways have been elucidated in plants, and many structural genes have been successfully identified. The process consists of three stages. For the upstream stage, p-coumaroyl-CoA is produced in the general phenylpropanoid pathway. The upstream stage is also a common synthetic pathway of flavonoids, isoflavones, and flavonols. There are three enzymes in the pathway: phenylalanine ammonialyase (PAL), 4-coumarate-CoA ligase (4CL) and cinnamate 4-hydroxylase (C4H). As the first flavonoid product of the pathway, chalcone is generated by chalcone synthase (CHS) at the intermediate stage. The pigment is synthesized by anthocyanidin synthase (ANS) at the downstream stage [1,2]. Then, anthocyanin is formed through glycosylation by glycosyltransferase (GT). Finally, anthocyanin is transported and stored in vacuoles [3][4][5]. However, the key enzymes of the flavonoid pathway always show differential expression and complicated regulation in a developmental stage-specific, tissue-specific and/or interspecific manner in plants [6][7][8].
The external environment significantly influences the flavonoid metabolic pathway. CHS is a light-regulating enzyme. Both the intensity and wavelength of light can regulate its gene expression and activity, thereby affecting pigment synthesis in plants. Studies have shown that ultraviolet light (UV light) can regulate the specific tissue expression of CHS in Arabidopsis [9]. UV light is an environmental signal perceived by plants that affects the flavonoid pathway and influences the levels of anthocyanins, flavonols, and proanthocyanidins. Recent studies have reported that low UV light induces downregulated changes in the gene expression of not only the key enzyme but also the activator of anthocyanin biosynthesis, MYB, such as MYB10and MYB22-activating transcription of flavonol synthase (FLS) in apple and MYB gene TT2 comprised of MYB-bHLH-WD regulating the late biosynthetic genes in Arabidopsis thaliana, to regulate anthocyanin biosynthesis [10,11]. In addition, key enzymes of the anthocyanin pathway are sensitive to temperature. Intense light and low temperature can significantly accelerate flavonoid pathway in different plant tissues, mainly by inducing PAL expression [12][13][14]. There is a trade-off between plant primary and secondary metabolites. For example, starch degradation can promote the accumulation of pigment in PSP roots. Moreover, it has been reported that sucrose acts as a signal molecule to regulate the expression of ANS and 3GT in the flavonoid pathway [15][16][17] and fruit ripening [18]. The flavonoid pathway is closely linked to fertility and stress resistance. Proteomic analysis of photoperiodinduced male sterility was carried out in a previous study. OsCHS is a significantly differential protein between long and short light conditions. The photoperiod regulates the expression of OsCHS and affects the N/C metabolic network, resulting in a change in pollen fertility [19]. Most studies on anthocyanin regulation have already been reported in fruits and ornamental plants, but such reports are limited for PSP [7,[20][21][22].
With urbanization and competition for land among the limited areas for cultivation, intercropping is becoming a common cultivation pattern worldwide to provide opportunities for sustainable agriculture with greater yield per unit land [23][24][25]. Sweet potato, as a creeping plant with broad leaves, is a promising plant for intercropping with other tall-stemmed crop species [26,27]. In southwestern regions of China, the three-crop intercropping system of "wheat/corn/potato" is predominantly used, that is, simultaneously growing two or more crops with strip-intercropping arrangement for each crop. However, not all crops can provide benefits in terms of yields or quality from the intercropping systems. For example, short-and tall-stemmed mixtures often give lower biomass or yields than when they are independent, in particular for short-stemmed crops with limited solar radiation resources due to shading from neighbor interactions [28]. Improvement of the intercropping systems for the future production region of sweet potato requires a clear understanding of the physical and biological effects of shading on these crops [28]. Solar energy, which is the basis for biomass production, is important for plant yield [29][30][31], but limited information is available on pigment quality. Jihei-1 is widely cultivated in China due to its high yield and anthocyanin-rich characteristics. Taking shading as an example, the effects of shading on the process of storage root anthocyanin synthesis and accumulation were investigated in this study. This study provides a theoretical basis for revealing the regulation of anthocyanin synthesis in crops and a basis for high-quality sweet potato cultivation and nutritional improvement. In addition, these findings provide a reference for the trait of pigment accumulation in other root crops.
Plant Materials and Growth Conditions
The popular PSP cultivar Jihei-1 was used in this experiment, and was compared to a nonpigmented cultivar, the yellow-fleshed sweet potato cultivar (SP). Both Jihei-1 and yellow-fleshed SP were generously provided by Wuhan Academy of Agriculture Sciences (Wuhan City, China). Among the two, Jihei-1 was widely cultivated in China with high-yield and anthocyanin-enriched characteristics, and the yellow-fleshed sweet potato cultivar was a testing nonpigmented one also with high-yield characteristics. They had the same reproductive period. A pot experiment was performed at Huazhong Agricultural University in Wuhan (30 • Fifty days after planting (DAP), plants were subjected to three levels of 100% (control), 70% and 40% natural irradiance. A nonshaded group was used as a control and subjected to 100% natural light. Two treated groups shaded 30% and 60% of light via cultivation in iron frames (length of 2.0 m, width of 1.2 m, and height of 1.2 m) were covered with plastic shading nets from 50 DAP to 130 DAP, respectively. In each treatment, we selected 60 plants with more than two storage roots per plant (n ≥ 120) and then immediately froze them in liquid nitrogen for physiological and biochemical determination, molecular validation and metabolite content evaluation.
Untargeted Metabolomics Analysis of Storage Roots
Samples of ripe storage roots (130 DAP) were prepared before gas chromatographymass spectrometry (GC-MS) analysis. With an internal standard of 0.3 mg mL −1 2-chlorol-phenylalanine added, 60 mg of the powder samples were ground with small steel balls in cold methanol. The mixtures were ultrasonicated for 30 min. After centrifugation and vacuum drying, a quality control (QC) sample was prepared, and 80 µL of 15 mg mL −1 methoxylamine hydrochloride was subsequently added to pyridine. The mixture was vortexed and incubated for 90 min. Eighty microlitres of BSTFA (with 1% TMCS) and 20 µL of n-hexane were added to the mixture and derivatized at 70 • C for 60 min.
The samples were analyzed on an Agilent 7890B gas chromatography system coupled to a 5977A MSD system (Agilent, Guangzhou, China) according to Lai et al. [32]. The metabolites were annotated using the Fiehn or NIST database [33]. A DB-5 MS fused-silica capillary column (30 m × 0.25 mm × 0.25 µm) was utilized to separate the derivatives. Helium was used as the carrier gas at a constant flow rate of 1 mL/min through the column. The parameters were as follows: injector temperature of 260 • C and injection volume of 1 µL (split ratio is 10:1). Mass data were acquired in full-scan mode (50-450 m/z) with the solvent delay time set to 5 min. Eight independent replicates for each group were determined (n = 8). Three QCs were injected throughout the analytical run.
The resulting GC−MS data were normalized to the total peak area of each sample and log2 transformed by Excel software (Microsoft Corp., Redmond, WA, USA) then imported into SIMCA software (version 14.0, Umetrics, Umeå, Sweden), where principal component analysis (PCA), partial least-squares discriminant analysis (PLS-DA) and orthogonal partial least-squares discriminant analysis (OPLS-DA) were performed. The differential metabolites were selected on the basis of the combination of statistically significant thresholds including the variable importance in the projection (VIP > 1.0) obtained from the OPLS-DA model and p values (p < 0.05) from a two-tailed Student's t-test of the normalized peak areas.
Measurement of Soluble Sugar, Starch, and Anthocyanin Contents in Sweet Potato
The soluble sugar and starch contents of the samples were measured according to the sulfuric acid-anthrone method [34]. First, sample powder was ground with 80% (v/v) ethyl alcohol and transferred into a centrifuge tube that was then placed in a water bath for 30 min at 80 • C. After cooling, the homogenate was centrifuged at 4000 rpm for 5 min. The supernatant was a soluble sugar solution gathered in a 100-mL volumetric flask. Second, the residue was gathered into another 100-mL volumetric flask, 20 mL hot dH 2 O was added to create a boiling water bath for 15 min and 2 mL of 9.2 M perchloric acid was added under the boiling water bath for another 15 min. After cooling, the mixture was centrifuged to a constant volume, and the supernatant was a starch extract solution. Extract solutions were diluted 5-folds. Then, the absorbance of the solutions was evaluated at 620 nm using a fluorescence microplate reader (Infinite 200 NanoQuant, Tecan, Gr dig, Austria). Three independent replicates for each group were determined (n = 3).
Anthocyanin was extracted as described by Jing et al. [35]. The sample was ground under citric acid into sodium hydrogen phosphate buffer solution (pH 3.0). The homogenate was transferred to a 100-mL volumetric flask, brought to a constant volume and rested in the dark for 30 min. After centrifugation at 6000 rpm for 10 min, the supernatant was collected. The anthocyanin extract solution was read at 525 nm by a fluorescence microplate reader. For soluble sugar, starch and anthocyanin analysis, three independent replicates for each group were determined.
Measurement of Amino Acid Contents in Developing Storage Roots
Samples of developing storage roots at 80, 100 and 130 DAP were prepared. Total amino acid contents were determined by an HPLC method [36] with three replicates (n = 3). The samples were hydrolyzed with 6 N HCl at 110 • C for 22 h. The mixtures were evaporated and dissolved in 1 mL of 0.02 N HCl. The samples were filtered and analyzed in three replicates via an Agilent 1260 system (Agilent, Guangzhou, China) with the C18 column (4.6 mm × 250 mm × 5 µm).
Measurement of CHS, ANS, and UFGT Enzyme Activities
The key enzymes of CHS, ANS and UFGT of the flavonoid metabolic pathway were selected and determined in PSP storage roots at 60, 80 and 100 DAP. First, a crude enzyme sample was extracted with cold extraction buffer (pH 8.2) comprising 50 mM HEPES-KOH. After 20 min of chilling, the mixture was centrifuged at 12,000 rpm for 15 min at 4 • C. The supernatant was assayed at a wavelength of 450 nm using enzyme linked immunosorbent assay (ELISA) kits (Jiangsu KeJing Biological Technology Co., Ltd., Nanjing, China) with specific antibodies against CHS, ANS, and UFGT and HRP-conjugate reagent with three independent replicates for each group (n = 3).
qPCR Verification of CHS, ANS, and UFGT Gene Expression
The primers for candidate genes designed by EMBL-EBI and NCBI primers are shown in Appendix A (Table A1). Actin (https://www.ebi.ac.uk/, accessed on 25 February 2021, Accession EU250003) was used as the reference gene. Total RNA of storage roots was extracted using the TIANGEN RNAprep Pure Plant Kit as described by the supplier. First-strand cDNA was synthesized in a 20 µL reaction solution with oligo(dT) primers using a ReverTra Ace-a kit (TOYOBO). Quantitative real-time PCR (qPCR) was performed in a 10 µL volume using SYBR Green SuperMix (Bio-Rad) on a QuantStudio 6 Flex instrument (Applied Biosystems) [38]. Each sample was represented by three biological replicates (n = 3). Analysis of the relative gene expression was carried out by the relative quantitative method.
Data Analysis
The acquired MS data from GC−MS were analyzed by ChromaTOF software (v 4.34, LECO, St Joseph, MI, USA). The metabolites were identified by the NIST and Fiehn databases using ChromaTOF software. The resulting data were normalized to the total peak area of each sample and transformed by log2 in Excel software (Microsoft Corp., Redmond, WA, USA) and imported into SIMCA (version 14.0, Umetrics, Umeå, Sweden). Sequence alignment of proteins was analyzed using online tools (http://www.UniProt. org/help/uniprotkb, accessed on 25 February 2021).
Statistical analysis of the soluble sugars, starch and protein contents and activity values were performed with analysis of variance, and significant difference (p < 0.05) analysis was performed using Excel software. For protein quantitative analysis with western blotting, two replicated membranes were prepared, and the band grey values were analysed with β-actin as a reference with Quantity One software (Bio-Rad, Hercules, CA, USA).
Effects of Shading on Storage Root Yield and Anthocyanin Content in PSP
For PSP, three levels of natural irradiance that influenced the yield of Jihei-1 were sorted in descending order, as follows: nonshaded group > 30% shading group > 60% shading group ( Figure 1A, Table 1). The average weight of a single storage root of the 30% shading group was consistent with the control value, and that of the 60% shading group decreased by 9%. The average number of storage roots per plant was reduced in the shaded groups. Accordingly, the total root yields decreased by approximately 26-40% under shading. However, pigment outputs increased by approximately 25% under shading. In particular, the anthocyanin contents of the storage roots increased by 70-105% under the shading treatment. Thus, it was concluded that shading produces a negative effect on storage root yields but may have a positive effect on PSP anthocyanin outputs.
Significant characteristics related to anthocyanin enrichment were detected in Jihei-1 storage roots compared to those of the yellow-fleshed sweet potato cultivar (SP) ( Figure 1B). For 60% shading, the anthocyanin contents in the developing storage roots of Jihei-1 were significantly influenced, that is, the anthocyanin content was 118% that of the control at the ripe stage at 130 DAP ( Figure 1B, Table 1). However, the shading condition slightly affected anthocyanin contents in the yellow-fleshed SP ( Figure 1C), which might be due to the low background levels of storage root anthocyanins in this cultivar. It was suggested that the shading treatment had an interspecific influence on root anthocyanins, notably specific to PSP; shading could improve or stabilize the anthocyanin accumulation at the mid-late stage of storage root development during 100-130 DAP. The differences in anthocyanin content observed between the two varieties were likely a result of the cumulative effect of various environmental factors that affect storage root anthocyanin synthesis and outputs. the mid-late stage of storage root development during 100-130 DAP. The differences in anthocyanin content observed between the two varieties were likely a result of the cumulative effect of various environmental factors that affect storage root anthocyanin synthesis and outputs. The AC contents of SP storage roots under 60%-shading and non-shading treatments are marked with green-background or blue-border bars, respectively. Data represent the mean ± SD from three individual plants with main storage roots mixed per plant (three independent biological replicates, n = 3). The asterisks represent significant differences (*, p < 0.05; **, p < 0.01). Sweet potato, SP; anthocyanin, AC; days after planting, DAP. Storage Root FW: average weight of single sweet potato root (g); Storage Root Num.: average number of storage roots of single plant; The data of storage root yield and anthocyanin outputs have been obtained according to potted plant data determined. Data of storage root FW and outputs per plant are shown as means ± SD from 60 individual plants with more than two storage roots per plant (n = 120) in each group during 2017-2019. Data of anthocyanin content per plant are shown as means ± SD from nine individual plants with main storage roots mixed per plant (nine biological replicates, n = 9). The asterisk represents a significant difference compared to non-shading group (*, p < 0.05; **, p < 0.01). PSP, purple-fleshed sweet potato.
Tissue-Specific and Cultivar-Specific Effects on Soluble Sugar Contents and Starch Accumulation by Shading Treatment
Shading influenced tissue soluble sugar contents in both PSP and PS but with different changing patterns. For PSP Jihei-1, the soluble sugar contents in all tissues, such as the leaf, petiole, stem and storage root tissues, were significantly increased under 60% shading conditions, especially with a higher growth rate of 76-92% at 80 DAP ( Figure 2A). There were no significant differences in the soluble sugar contents of each tissue during the ripe period of 130 DAP between the control and shaded groups. For SP, some tissue- The AC contents of SP storage roots under 60%-shading and non-shading treatments are marked with green-background or blue-border bars, respectively. Data represent the mean ± SD from three individual plants with main storage roots mixed per plant (three independent biological replicates, n = 3). The asterisks represent significant differences (*, p < 0.05; **, p < 0.01). Sweet potato, SP; anthocyanin, AC; days after planting, DAP. Storage Root FW: average weight of single sweet potato root (g); Storage Root Num.: average number of storage roots of single plant; The data of storage root yield and anthocyanin outputs have been obtained according to potted plant data determined. Data of storage root FW and outputs per plant are shown as means ± SD from 60 individual plants with more than two storage roots per plant (n = 120) in each group during 2017-2019. Data of anthocyanin content per plant are shown as means ± SD from nine individual plants with main storage roots mixed per plant (nine biological replicates, n = 9). The asterisk represents a significant difference compared to non-shading group (*, p < 0.05; **, p < 0.01). PSP, purple-fleshed sweet potato.
Tissue-Specific and Cultivar-Specific Effects on Soluble Sugar Contents and Starch Accumulation by Shading Treatment
Shading influenced tissue soluble sugar contents in both PSP and PS but with different changing patterns. For PSP Jihei-1, the soluble sugar contents in all tissues, such as the leaf, petiole, stem and storage root tissues, were significantly increased under 60% shading conditions, especially with a higher growth rate of 76-92% at 80 DAP ( Figure 2A). There were no significant differences in the soluble sugar contents of each tissue during the ripe period of 130 DAP between the control and shaded groups. For SP, some tissue-specific soluble sugar contents (leaf-, petiole-and stem-) were significantly increased under shading conditions at 130 DAP ( Figure 2A). contents of developing (at 80 DAP) and ripe storage roots (at 130 DAP) increased by 26% and 13%, respectively, while at the same time, the leaf starch contents decreased markedly ( Figure 2B). However, there were also significant differences, but at lower levels, in tissue starch contents from yellow-fleshed SP, except for the leaf starch content that showed a 2-fold increase during the ripe period at 130 DAP under shading ( Figure 2B).
Overall, the shading treatment had a cultivar-specific influence on sweet potato soluble sugar content and starch accumulation during storage root development. That is, for dealing with deep and prolonged shading, one strategy was to mobilize petioles to participate in photosynthesis to accumulate more starch in PSP during the whole root development period, and another was to maintain leaf photosynthesis in yellow-fleshed SP even in the ripe period. Notably, shading improved the soluble sugar level in all PSP tissues at the early root development period of 80 DAP to prematurely stabilize storage root starch accumulation.
To explore the metabolic changes under shading, the differential metabolites of storage roots between the shaded and control groups were grouped into two primary clusters by hierarchical cluster analysis ( Figure 4). Deep insight into these metabolites revealed many primary metabolites, including organic acids, soluble sugars and amino acids (Appendix A, Figure A2). There were several differential metabolites that were intermediate metabolites of the citrate cycle that increased under shading. Among them, five organic acids, succinic acid, L-malic acid, citric acid, pyruvic acid and isocitric acid, were significantly downregulated with FCs of 0.2-0.5 (Table 2). For soluble sugars, sucrose accounted for the largest component of the total soluble sugar content, accounting for more than 50%. It was downregulated with an FC of 0.7 with shading. In addition, most amino acids were upregulated. Among them, four significantly differential metabolites were Phe, Glu, Gly and Ser (Table 2).
In plants, primary and secondary metabolites influence each other. Phenylpropanoid biosynthesis is a common upstream pathway of several secondary metabolites. In the upstream pathway, one differential metabolite of 3,4-dihydroxycinnamic acid was upregulated with an FC of 1.8 under shading. Despite the limited anthocyanins identified by GC-MS analysis, the acyl ligands of 4-hydroxybenzoic acid and caffeic acid were upregulated under shading with FC values of 1.3 and 1.2, respectively ( Table 2). Anthocyanins should be acylated and glycosylated to exist in a stable form for accumulation in plants.
For the PSP cultivar Jihei-1, caffeic acid could be one of the main acyl ligands of anthocyanins [39,40]. The results demonstrated that shading accelerated flavonoid biosynthesis as an essential route of phenylpropanoid biosynthesis and increased the acyl ligands required for anthocyanin accumulation. Figure 2, that is carbohydrates and carbohydrate conjugates, organic acids and derivatives, amino acids, peptides, and analogues. 2 The differential metabolites were analyzed on the basis of the combination of a statistically significant threshold of variable influence on variable importance in the projection (VIP > 1.0) obtained from the OPLS-DA model ( Figure A1) and p values (p < 0.05) from a two-tailed Student's t test on the normalized peak areas (n = 8, eight individual biological samples). 3 FC: fold change. PSP, purple-fleshed sweet potato.
Changes in the Amino Acid Contents of PSP Storage Roots in Response to Shading
Amino acid analysis showed the nutritional characteristics of PSP storage roots ( Figure 5). In the ripe storage roots, the ascending sort of the essential amino acid content was His (0.1 g kg −1 ), Thr (0.4 g kg −1 ), Lys (0.4 g kg −1 ), Ile (0.5 g kg −1 ), Phe (0.5 g kg −1 ), Met (0.5 g kg −1 ), Val (0.6 g kg −1 ) and Leu (0.6 g kg −1 ). Among the essential amino acids, the contents of Thr and Met increased by 28% and 8%, respectively, under shading treatment, while the contents of His, Val and Ile decreased by approximately 10%. However, almost all the amino acid levels increased in the growing storage roots under shading, with an increase ranging from 12-159% during the rapid growth periods from 80 DAP to 100 DAP. In particular, the storage root amino acid levels of Cys, Asp and Thr under shading were two-fold higher than those of the control at 100 DAP ( Figure 5). These findings suggest that the amino acid contents were highly related to the anthocyanin content in PSP storage roots.
test on the normalized peak areas (n = 8, eight individual biological samples). 3 FC: fold change. PSP, purple-fleshed sweet potato.
Changes in the Amino Acid Contents of PSP Storage Roots in Response to Shading
Amino acid analysis showed the nutritional characteristics of PSP storage roots (Figure 5). In the ripe storage roots, the ascending sort of the essential amino acid content was His (0.1 g kg −1 ), Thr (0.4 g kg −1 ), Lys (0.4 g kg −1 ), Ile (0.5 g kg −1 ), Phe (0.5 g kg −1 ), Met (0.5 g kg −1 ), Val (0.6 g kg −1 ) and Leu (0.6 g kg −1 ). Among the essential amino acids, the contents of Thr and Met increased by 28% and 8%, respectively, under shading treatment, while the contents of His, Val and Ile decreased by approximately 10%. However, almost all the amino acid levels increased in the growing storage roots under shading, with an increase ranging from 12-159% during the rapid growth periods from 80 DAP to 100 DAP. In particular, the storage root amino acid levels of Cys, Asp and Thr under shading were twofold higher than those of the control at 100 DAP ( Figure 5). These findings suggest that the amino acid contents were highly related to the anthocyanin content in PSP storage roots. Figure 5. The contents of amino acids in developing storage roots of purple-fleshed sweet potato under shaded conditions. Data represent the mean ± SD from three individual plants (three biological replicates, n = 3) with more than two storage roots mixed per plant. The asterisks represent significant differences revealed by Student's t-test at p < 0.05 (*) and p < 0.01 (**), respectively. The columns of amino acid contents from PSP storage roots under 60% shading treatment (60%) are represented by a black background and those under the nonshaded condition (CK) are represented by a grey background.
Effects of Shading on Three Key Anthocyanin Metabolism Enzymes, CHS, ANS, and 3GT, in Treated PSP Storage Roots
The ELISA results showed that ANS activity increased by 37.1-43.4% during the rapid growth periods from 80 DAP to 100 DAP (Figure 6). At the same time, the CHS and 3GT activities decreased with different degrees of decline, ranging from 8.5-17.0% ( Figure 6). By western blot analysis, the ANS contents were consistent with enzyme activity, which increased with an increase of 17-26% during the growth periods from 80-100 DAP under shading (Figure 7). In the shading group, the 3GT contents increased during growth periods, with an increase of 14% at 60 DAP (Figure 7). Data represent the mean ± SD from three individual plants (three biological replicates, n = 3) with more than two storage roots mixed per plant. The asterisks represent significant differences revealed by Student's t-test at p < 0.05 (*) and p < 0.01 (**), respectively. The columns of amino acid contents from PSP storage roots under 60% shading treatment (60%) are represented by a black background and those under the nonshaded condition (CK) are represented by a grey background.
Effects of Shading on Three Key Anthocyanin Metabolism Enzymes, CHS, ANS, and 3GT, in Treated PSP Storage Roots
The ELISA results showed that ANS activity increased by 37.1-43.4% during the rapid growth periods from 80 DAP to 100 DAP (Figure 6). At the same time, the CHS and 3GT activities decreased with different degrees of decline, ranging from 8.5-17.0% ( Figure 6). By western blot analysis, the ANS contents were consistent with enzyme activity, which increased with an increase of 17-26% during the growth periods from 80-100 DAP under shading (Figure 7). In the shading group, the 3GT contents increased during growth periods, with an increase of 14% at 60 DAP (Figure 7).
Based on sequence analysis, the functions and sequences of CHSs and ANSs were highly conserved in sweet potato (Ipomoea batatas L.), with a high homology of 73-74% to Arabidopsis ANS and CHS (UniProt ID: Q96323 and P13114). In addition, six sequences of ANSs from sweet potato already identified were 95% homogenous amino acid sequences (Appendix A, Figure A3). Although sweet potato CHSs with seven family members probably exhibit functional redundancy, thirteen sequences contained the CHS family in sweet potato with 95% homogeneity of highly conserved sequences (Appendix A, Figure A4). Accordingly, western blot analysis showed representative changes in the whole protein family from sweet potato in response to shading. ANS belongs to the 2-oxoglutaratedependent dioxygenase (2-ODD) superfamily, which encodes a key enzyme for the catalytic reaction of pigment metabolites from non-colored to colored [41]. Obviously, shading can enhance the background ANS at the protein content and enzyme activity levels during 80-100 DAP, thereby leading to increased anthocyanin accumulation via the flavonoid pathway (Figures 6 and 7). Root and leaf 3GT (UniProt ID: A0A0G2QMF5, Gene Accession: JN258961) and CHS1 (UniProt ID: Q9MB33, Gene Accession: AB037680) expression levels in sweet potato were upregulated under shading at 60 DAP; notably, leaf CHS1 were upregulated under shading during the storage root growth periods (Figure 7). groups ( Figure 8). As shown, the gene expression levels were upregulated significantly during early storage root development at 60-80 DAP, coinciding with the protein content analysis. The lowest ANS expression occurred during the storage root growth period at 80-100 DAP, showing at least a 60% decrease compared to the control levels at 60 DAP, suggesting primary enzymes of the glycometabolic pathway are involved in rapid starch accumulation in the middle tuber growth stage (Figure 8). The expression of three key genes in the flavonoid pathway was also detected by qPCR assay (Appendix A, Table A1). The relative transcript abundance of CHS1, ANS and 3GT in the control group was set as 1 for the convenience of comparison with the shading groups ( Figure 8). As shown, the gene expression levels were upregulated significantly during early storage root development at 60-80 DAP, coinciding with the protein content analysis. The lowest ANS expression occurred during the storage root growth period at 80-100 DAP, showing at least a 60% decrease compared to the control levels at 60 DAP, suggesting primary enzymes of the glycometabolic pathway are involved in rapid starch accumulation in the middle tuber growth stage (Figure 8).
noid pathway in PSP storage roots of 60 DAP, 80 DAP and 100 DAP under shaded conditions. Data represent the mean ± SD of three individual plants (n = 3) with main storage roots mixed in each plant. The asterisks represent significant differences by Student's t-test at p < 0.01 (**), respectively. The columns of enzyme activities from PSP storage roots under 60% shading treatment are shown with green filling and those under nonshaded conditions are shown with yellow filling. chalcone synthase, CHS; anthocyanidin synthase, ANS; glycosyltransferase, GT; purple-fleshed sweet potato, PSP.
Effect of Shading Treatment on Storage Root Development in Sweet Potato and Species-Specific Strategy
Solar energy is the basis for biomass production [29,30]. During plant production, overshadowing from intercropping plants impacts crop yields, particularly for sweet potato as a creeping plant. The storage root yield in the pot experiments of three years showed deep decreases with increasing shade levels, while shading increased the PSP pigment concentrations and outputs to a noteworthy degree (Table 1). We focused on the establishment of pigment quality during PSP storage root development in response to shading; that is, anthocyanins accumulate richly and especially increase in the later period of storage root development (Figure 1). The benefits of intercropping the PSP cultivar Jihei-1 under shading with respect to solar energy use for quality formation, particularly storage root anthocyanin contents, are presented. The shading treatments reduced its root yield by decreasing the storage root number per plant. However, it is worth noting that shading also resulted in a significant increase in total pigment output with an increase of more than 20% (Table 1). Apparently, PSP cultivation under mild shading can influence yield in exchange for the improvement of storage root pigment accumulation, possibly increasing economic benefits.
Plants show two different strategies of tolerance and/or avoidance under shading [42,43]. This study showed a variety-specific strategy of sweet potato in response to prolonged deep shading. Comparing the two varieties of anthocyanin-enriched purplefleshed and yellow-fleshed sweet potatoes, a notable effect on tissue soluble sugar and starch contents under shading treatment was not present, but there were different changing patterns. For the PSP Jihei-1 under shading, the soluble sugar contents in all tissues, such as leaf, petiole, stem and storage root tissues, were significantly increased at 80 DAP, and the storage root starch content also increased in the corresponding period (Figure 2A). For the yellow-fleshed SP, the aboveground-tissue soluble sugar contents (leaves, petioles
Effect of Shading Treatment on Storage Root Development in Sweet Potato and Species-Specific Strategy
Solar energy is the basis for biomass production [29,30]. During plant production, overshadowing from intercropping plants impacts crop yields, particularly for sweet potato as a creeping plant. The storage root yield in the pot experiments of three years showed deep decreases with increasing shade levels, while shading increased the PSP pigment concentrations and outputs to a noteworthy degree (Table 1). We focused on the establishment of pigment quality during PSP storage root development in response to shading; that is, anthocyanins accumulate richly and especially increase in the later period of storage root development (Figure 1). The benefits of intercropping the PSP cultivar Jihei-1 under shading with respect to solar energy use for quality formation, particularly storage root anthocyanin contents, are presented. The shading treatments reduced its root yield by decreasing the storage root number per plant. However, it is worth noting that shading also resulted in a significant increase in total pigment output with an increase of more than 20% (Table 1). Apparently, PSP cultivation under mild shading can influence yield in exchange for the improvement of storage root pigment accumulation, possibly increasing economic benefits.
Plants show two different strategies of tolerance and/or avoidance under shading [42,43]. This study showed a variety-specific strategy of sweet potato in response to prolonged deep shading. Comparing the two varieties of anthocyanin-enriched purplefleshed and yellow-fleshed sweet potatoes, a notable effect on tissue soluble sugar and starch contents under shading treatment was not present, but there were different changing patterns. For the PSP Jihei-1 under shading, the soluble sugar contents in all tissues, such as leaf, petiole, stem and storage root tissues, were significantly increased at 80 DAP, and the storage root starch content also increased in the corresponding period (Figure 2A). For the yellow-fleshed SP, the aboveground-tissue soluble sugar contents (leaves, petioles and stems) were significantly increased under shading at 130 DAP, while the leaf starch content increased during the ripe period (Figure 2A). In response to shading, photosynthesis in all green tissues, particularly the petiole, was noted; this was required to feed the developing storage roots of SPS in the premature expansion period. On the other hand, green tissue sugar was mobilized and transferred into storage roots at 80 DAP, twenty days ahead of the process in SP. Another strategy is to maintain and enhance leaf photosynthesis in yellowfleshed SP even during the maturation period. Plant starch production is largely dependent on current photosynthesis. Light intensity is one of the most critical environmental factors for crop yield and the physiological and biochemical processes related to growth and development. According to past studies, the dry matter and photosynthetic rate of different tissues and whole plants decreased under low light conditions [44]. Some studies suggest that a decrease in light intensity leads to marked increases in leaf photosynthesis characteristics, such as leaf morphology and structure, leaf angle orientation and movement in crop plants [45,46]. However, information about sweet potato photosynthetic characteristics in response to shading during intercropping is limited. Recent studies suggest that the SP net photosynthetic rate can be improved in cases of compatible high-density intercropping under walnut saplings [47]. Here, we propose a specific strategy by which SPS respond to continuous and extensive shading treatment with three points: (I) the utilization of photosynthesis in broad aboveground tissues with creeping characteristics, (II) the advance (by 20 days) mobilization and transfer of photosynthates into storage roots, and (III) the maintenance and enhancement of aboveground tissue photosynthesis in the maturation period, for example, in the leaf petiole, leading to a rapid increase in physiological process rates and demands while storage root yield decreases.
Effect on Key Enzyme Expression of Flavonoid Pathways to Promote PSP Storage Root Anthocyanin Biosynthesis in Response to Shading Treatment
Anthocyanins are plant secondary metabolites that play a key role as pigments, in response to nutrient availability, in male fertility of some species, in the modulation of auxin transport, and in UV protection [48,49]. Light intensity can affect both flavonoid and anthocyanin contents in plants. The anthocyanin contents of SPS storage roots increased significantly at 130 DAP under the shading treatments in this study. As mentioned above, shading affects carbohydrate transport from photosynthetic organs to storage roots. Upregulation of the expression and enzyme activity of several anthocyanin biosynthesis genes/proteins was observed in the shaded SPS groups, which accumulated high levels of soluble sugars at 80 DAP in the early period of storage root development (Figures 6-8). Obviously, in the early expansion period, the upregulated expression of storage root CHS, ANS and 3GT genes in SPS could promote the flavonoid pathway, leading to an increase in anthocyanin biosynthesis ( Figure 8) and ultimately to a notable increase in storage root pigment outputs and anthocyanin contents. However, ANS expression was not detected in yellow-fleshed SP but was weakly detected in other SPS tissues except storage roots (data not shown). ANSs, as key enzymes downstream of the pathway, are functionally conserved with highly homologous sequences ( Figure A3). Accordingly, the changing patterns of these enzymes upstream from the flavonoid pathway could represent the strengthening of the whole pathway and the increased pigment outputs under shading in this study.
The synthesis of plant anthocyanins shares common steps with the flavonoid pathway. However, the enzymes from the pathway may be variety-/time-/tissue-specific and regulated by multigene family members in addition to showing species-specific chemical diversity of the anthocyanins ultimately produced in these plants. For the shading effect on anthocyanin accumulation, it is unclear which signals are involved in transfer to underground organs. Plant growth is centered around the production and utilization of sugars that serve as the primary supplies of energy as well as signals to guide development and adaptation [50]. It has been reported that sucrose can affect the anthocyanin content and the flavonoid pathway with a sugar-dependent upregulation of enzyme expression in plants [51][52][53][54][55]. For sweet potato, the interrelationships between the developmental, shading and metabolic signals of sugar control the accumulation of flavonoids (Figure 8). With limited knowledge of anthocyanin synthesis and regulation in sweet potato, it is difficult to determine the contribution of specific regulatory sites or factors responsible for storage root anthocyanin biosynthesis. We thus speculate that the soluble sugar level influenced anthocyanin accumulation via an upregulation in gene expression and the activity of the key enzyme ANS in the flavonoid pathway. Certainly, additional studies are required to confirm this hypothesis to guide the development of intercropping production.
Nitrogen/Carbon Balance and Sugar/Hormone Signalling to Regulate Storage Root Flavonoid Pathways under Shading
For crop plants, a long-time and low-level light intensity could affect their carbon balance because of mismatching between the carbohydrate demand increases for physiological processes and the photosynthetic production reduction [56]. Shading affects not only crop yield but also crop quality. During shading, valuable compounds are remobilized and accumulate in storage roots. Our data essentially reveal a balanced and trade-off relationship between primary metabolites and secondary metabolites in sweet potato. On the one hand, the main soluble sugar, sucrose, which accounts for more than half of the total amount, decreased by 26% in the ripe storage roots ( Table 2). The intermediate metabolites of the central pathway of the citrate cycle were also reduced with FC values of 0.2-0.6 ( Figure 9). On the other hand, most of the amino acids in the ripe storage roots were upregulated under shading. The significant differential expression of Phe was upregulated with an FC of 18.6 ( Figure 9). Based on metabolic pathway enrichment maps, the top biological process was the biosynthesis of phenylpropanoids with an initial substrate of Phe, which is also the upstream pathway of pigment synthesis. Amino acids provide initial carbon skeletons for the biosynthesis of specific secondary metabolites. The first limited amino acid of Lys and a healthy functional 4-aminobutyric acid were also significantly upregulated. In addition, the significantly differential Cys, Thr, Asp and Glu, which are the polar amino acids distributed in the enzyme activity center and have important catalytic functions, showed a two-fold increase (Figure 9). Recent studies have also suggested that numerous pathways associated with amino acid metabolism of tea altered in a Chinese chestnut and tea intercropping system greatly influence tea quality [57].
Nutrient signaling is the most ancient and fundamental mechanism to regulate and sustain life [58]. As mentioned above, a specific strategy for SPS in response to continuous and extensive shading treatment is proposed, that is, a rapid increase in physiological process rates. The nutritional levels of the edible storage roots, consisting of the richness of carbohydrates, polyphenolic compounds and peptides/amino acids, also significantly changed under shading (Table 2). Anthocyanins are plant secondary metabolites that play a key role as pigments in responses related to nutrient availability. Genes coding for dihydroflavonols 4-reductase (DFR) and ANS were upregulated, and the accumulation of anthocyanins was strongly increased by sucrose in grapes [15]. Recent studies have suggested that structural genes of tea flavonoid biosynthesis altered throughout shading greatly enhanced tea quality [59]. In Arabidopsis, the pho3 mutant, which has a defective copy of the sucrose transporter gene, can lead to soluble sugar, starch and anthocyanin accumulation, possibly because sugars are in vivo triggers of anthocyanin biosynthesis [54,60]. The sugar-regulating mechanism operating in sweet potato is poorly studied. Due to the outstanding accumulation of sugars during storage root development (Figure 2), it is tempting to speculate that sugars are endogenous signals modulating the expression of anthocyanin biosynthesis genes (Figures 7 and 8). Under shading conditions, the absolute amount of sucrose in mature storage roots of SPS was downregulated, but the ratio of sucrose to total soluble sugar was upregulated by metabolomics analysis. Sucrose is the transport form of photosynthetic products in sweet potato. Combined with tissue-specific soluble sugar contents, it is speculated that the sucrose proportion influences the flavonoid pathway and anthocyanin accumulation. In addition to sucrose, trehalose showed a significant 2.5-fold increase in SPS storage roots under shading through metabolomics data ( Table 2). In plants, trehalose is an important metabolic signal that regulates plant development, abiotic stress tolerance and anthocyanin synthesis pathways [61][62][63]. Nutrient signaling is the most ancient and fundamental mechanism to regulate and sustain life [58]. As mentioned above, a specific strategy for SPS in response to continuous and extensive shading treatment is proposed, that is, a rapid increase in physiological process rates. The nutritional levels of the edible storage roots, consisting of the richness of carbohydrates, polyphenolic compounds and peptides/amino acids, also significantly changed under shading (Table 2). Anthocyanins are plant secondary metabolites that play a key role as pigments in responses related to nutrient availability. Genes coding for dihydroflavonols 4-reductase (DFR) and ANS were upregulated, and the accumulation of anthocyanins was strongly increased by sucrose in grapes [15]. Recent studies have suggested that structural genes of tea flavonoid biosynthesis altered throughout shading greatly enhanced tea quality [59]. In Arabidopsis, the pho3 mutant, which has a defective copy of the sucrose transporter gene, can lead to soluble sugar, starch and anthocyanin accumulation, possibly because sugars are in vivo triggers of anthocyanin biosynthesis [54,60]. The sugar-regulating mechanism operating in sweet potato is poorly studied. Due to the outstanding accumulation of sugars during storage root development (Figure 2), it is tempting to speculate that sugars are endogenous signals modulating the expression of anthocyanin biosynthesis genes (Figures 7 and 8). Under shading conditions, the absolute amount of sucrose in mature storage roots of SPS was downregulated, but the ratio of sucrose to total soluble sugar was upregulated by metabolomics analysis. Sucrose is the transport form of photosynthetic products in sweet potato. Combined with tissue-specific soluble sugar contents, it is speculated that the sucrose proportion influences the flavonoid pathway and anthocyanin accumulation. In addition to sucrose, trehalose showed a significant 2.5-fold increase in SPS storage roots under shading through metabolomics
Conclusions
The shade cultivation of PSP can improve pigment outputs by an extra 20% to increase economic benefits. This report described a comprehensive investigation of sugar, anthocyanin and amino acid contents in PSP storage roots under shading by metabolomics and biochemical characterization. Accordingly, we proposed a variety-specific and tissuespecific strategy of PSP in response to shading, leading to a reduction in yield but a rapid increase in storage root anthocyanin synthesis. With molecular validation data, we elucidated the regulation of genes involved in the flavonoid pathways by shading treatment; that is, shading significantly impacts the key enzyme ANS upregulation at the transcriptional level and enzymatic activity, leading to enhancement of storage root anthocyanin biosynthesis and pigment outputs. These results indicate that intercropping cultivation patterns provide opportunities for sustainable agriculture with greater yield per unit land but also nutritional improvement of sweet potato.
Patents
There is one patent authorized resulting from the work, the antigenic peptides of Anthocyanin synthase and the anti-ANS application (number 2019111228892.2).
Informed Consent Statement: Not applicable.
Acknowledgments: Plant material of Jihei-1 and yellow-fleshed SP were generously provided by Aihua Wang and Ping Wang researchers in Wuhan Academy of Agriculture Sciences (Wuhan City, China). Guidance on purple potato cultivation were generously provided by Xinsun Yang researchers in Hubei Academy of Agricultural Sciences (Wuhan City, China).
Conflicts of Interest:
All authors declare that they have no competing interests. Table A1. A Primer information of three candidate genes related to flavonoid biosynthesis used for qPCR.
Agronomy 2021, 11, 737 20 of 25 Figure A1. Multivariate statistical analysis of differential metabolites from PSP storage roots under 60% shading treatment and control, including PCA, OPLS-DA and PLS-DA. Green and blue circles and red triangles reflected the 60% shading and non-shading samples and three QC sample (n = 3), respectively. Analysis represents with eight independent replicates (n = 8). principal component analysis, PCA, orthogonal partial least-squares discriminant analysis, OPLS-DA, partial leastsquares discriminant analysis, PLS-DA. Figure A1. Multivariate statistical analysis of differential metabolites from PSP storage roots under 60% shading treatment and control, including PCA, OPLS-DA and PLS-DA. Green and blue circles and red triangles reflected the 60% shading and non-shading samples and three QC sample (n = 3), respectively. Analysis represents with eight independent replicates (n = 8). principal component analysis, PCA, orthogonal partial least-squares discriminant analysis, OPLS-DA, partial least-squares discriminant analysis, PLS-DA. Figure A1. Multivariate statistical analysis of differential metabolites from PSP storage roots under 60% shading treatment and control, including PCA, OPLS-DA and PLS-DA. Green and blue circles and red triangles reflected the 60% shading and non-shading samples and three QC sample (n = 3), respectively. Analysis represents with eight independent replicates (n = 8). principal component analysis, PCA, orthogonal partial least-squares discriminant analysis, OPLS-DA, partial leastsquares discriminant analysis, PLS-DA. Figure A2. Classification of differential metabolites of PSP storage roots under shading treatment. | 2021-05-16T11:54:00.509Z | 2021-04-10T00:00:00.000 | {
"year": 2021,
"sha1": "76e31473ead9039927c5bcd2fed7b6ef4d648f43",
"oa_license": "CCBY",
"oa_url": "https://www.mdpi.com/2073-4395/11/4/737/pdf",
"oa_status": "GOLD",
"pdf_src": "MergedPDFExtraction",
"pdf_hash": "76e31473ead9039927c5bcd2fed7b6ef4d648f43",
"s2fieldsofstudy": [
"Environmental Science",
"Biology",
"Agricultural and Food Sciences"
],
"extfieldsofstudy": [
"Biology"
]
} |
247831678 | pes2o/s2orc | v3-fos-license | Autoxidation of 4-Hydrazinylquinolin-2(1H)-one; Synthesis of Pyridazino[4,3-c:5,6-c′]diquinoline-6,7(5H,8H)-diones
An efficient synthesis of a series of pyridazino[4,3-c:5,6-c′]diquinolines was achieved via the autoxidation of 4-hydrazinylquinolin-2(1H)-ones. IR, NMR (1H and 13C), mass spectral data, and elemental analysis were used to fit and elucidate the structures of the newly synthesized compounds. X-ray structure analysis and theoretical calculations unequivocally proved the formation of the structure. The possible mechanism for the reaction is also discussed.
Pyridazine and its polycyclic structures have still played an interesting role in organic chemistry because of their remarkable properties in forming supramolecular assembly [15][16][17][18]. Pyridazine molecules are important heterocycle scaffolds that reveal diverse biological activities in medicine [19][20][21][22][23][24][25] and agriculture [26]. For example, Pyridazomycin is an antifungal and antibiotic compound, the first pyridazine derivative isolated from a natural source [27]. In contrast, Pyridaben is widely used as an acaricide with a long residual action, whereas Chloridazone has a long history of use as an herbicide [28]. Minaprine is a psychotropic drug that has effectively treated various depressive states [29] (Figure 1).
Hydrazines have shown basic and reducing characteristics that enable their utility in many industrial and medical applications. Accordingly, hydrazines have served as rocket fuel, antioxidants, oxygen scavengers, and as intermediates for the production of explosives, propellants, and pesticides [32].
It has been demonstrated that oxygen makes hydrazine solutions unstable, especially under alkaline or neutral conditions. However, hydrazines are stable under strongly acidic conditions or without oxygen [33].
Wagnerova et al. [34] have reported that cobalt-tetrasulphophthalocyanines enhance the autoxidation process of hydrazines [34]. On the other hand, Ichiro Okura et al. [35] reported that the autoxidation of hydrazine occurred with manganese (III)-hematoporphyrin Functionalized pyridazines have high electron-deficient properties, encouraging their utilization as electrochromic materials and metal-organic frameworks [30,31].
Hydrazines have shown basic and reducing characteristics that enable their utility in many industrial and medical applications. Accordingly, hydrazines have served as rocket fuel, antioxidants, oxygen scavengers, and as intermediates for the production of explosives, propellants, and pesticides [32].
It has been demonstrated that oxygen makes hydrazine solutions unstable, especially under alkaline or neutral conditions. However, hydrazines are stable under strongly acidic conditions or without oxygen [33].
Hydrazines have shown basic and reducing characteristics that enable their uti many industrial and medical applications. Accordingly, hydrazines have served as fuel, antioxidants, oxygen scavengers, and as intermediates for the production of sives, propellants, and pesticides [32].
It has been demonstrated that oxygen makes hydrazine solutions unstable, espe under alkaline or neutral conditions. However, hydrazines are stable under str acidic conditions or without oxygen [33].
Previously, it was reported [38] that 1-ethyl-4-hydroxyquinolin-2(1H)-one (1) re with hydrazine hydrate, in 1,2-dichlorobenzene (o-DCB), to give a mixture of two pounds. These compounds were separated using fractional recrystallization to give olinylhydrazine 2a in 19% yield and diquinopyridazine 3a in 41% yield (Scheme 1). A convenient microwave-assisted, one-pot, four-component synthetic approach was developed as a route to functionalized benzo[a]pyridazino [3,4-c]phenazine derivatives starting from 2-hydroxy-1,4-naphthoquinone, aromatic aldehydes, methyl hydrazine, and o-phenylenediamine. Compounds of a similar pentacyclic structure such as bisanthranilate showed an intramolecular electrophilic cyclization and afforded an angular cis-quinacridone compound, which condensed with hydrazine to give a phthalazine derivative [39]. The biological profiles of some of the compounds mentioned above exhibited good cytotoxic activities against KB, HepG2, Lu1, and MCF7 human cancer cell lines. In addition, a compound of the derivatives exhibited promising antimicrobial activities toward Staphylococcus aureus and Bacillus subtilis bacterial strains with IC 50 < 6 µM [40].
Recently, it has been reported that hydrazines can be used as catalysts for removing oxygen in the closing carbonyl-olefin metathesis process [41]. We have also found that prolongated reflux during the formylation process of 2-quinolones via a DMF/Et 3 N mixture caused dimerization to occur, and unexpected 3,3 -methylenebis(4-hydroxyquinolin-2(1H)-ones) were obtained [42].
The above-mentioned findings encouraged us to generalize the method of preparation for heteroannulated pentacyclic compounds with the structure of this interesting molecule.
tion, a compound of the derivatives exhibited promising antimicrobial activities toward Staphylococcus aureus and Bacillus subtilis bacterial strains with IC50 < 6 μM [40].
Recently, it has been reported that hydrazines can be used as catalysts for removing oxygen in the closing carbonyl-olefin metathesis process [41]. We have also found that prolongated reflux during the formylation process of 2-quinolones via a DMF/Et3N mixture caused dimerization to occur, and unexpected 3,3′-methylenebis(4-hydroxyquinolin-2(1H)-ones) were obtained [42].
The above-mentioned findings encouraged us to generalize the method of preparation for heteroannulated pentacyclic compounds with the structure of this interesting molecule.
Results and Discussion
The strategy started with the preparation of derivatives of compounds 1, 2, 4, and 5 according to reported methods, and their structures were confirmed by matching their spectral data with those reported [43,44,45]. The key intermediates, hydrazine quinolones 2a-g, were prepared by refluxing compounds 4a-g with hydrazine hydrate (Scheme 2) [46]. During the heating of 4-hydrazinylquinolin-2(1H)-ones 2a-g in pyridine, we observed the abnormal formation, in good yields, of pyridazino[4,3-c:5,6-c′]diquinoline-6,7-(5H,8H)-diones 3a-g. As had been suggested, compounds 2a-g underwent an autoxidation reaction. The structures of the products 3a-g were proved from their elemental analyses and IR, 1 H NMR, and 13 C NMR spectra. For example, the mass spectrum and elemental analysis of 3a established its molecular formula as C 22 H 18 N 4 O 2 . The 1 H NMR spectrum of 3a exhibited the ethyl protons as a triplet at δ H = 1.22 (J = 7.6 Hz) for CH 3 and a quartet at δ H = 4.39 ppm (J = 7.6 Hz) for CH 2 . The eight aromatic protons appeared as three multiplets at δ H = 7.36-7.40 for 2H, 7.68-7.78 for 4H, and 8.06-8.08 ppm for 2H. The reported spectroscopic data for the 13 C NMR spectrum of compound 3a showed the carbonyl-quinolone, 2NCH 2, and CH 3 carbon signals at δ C = 165.72, 39.11, and 14.11 ppm, respectively. Similar spectroscopic results of compound 3a were also reported [38]. The structure of 3a was unambiguously determined by a single crystal structure ( Figure 2). exhibited the ethyl protons as a triplet at δH = 1.22 (J = 7.6 Hz) for CH3 and a quartet at δH = 4.39 ppm (J = 7.6 Hz) for CH2. The eight aromatic protons appeared as three multiplets at δH = 7.36-7.40 for 2H, 7.68-7.78 for 4H, and 8.06-8.08 ppm for 2H. The reported spectroscopic data for the 13 C NMR spectrum of compound 3a showed the carbonyl-quinolone, 2NCH2, and CH3 carbon signals at δC = 165.72, 39.11, and 14.11 ppm, respectively. Similar spectroscopic results of compound 3a were also reported [38]. The structure of 3a was unambiguously determined by a single crystal structure ( Figure 2). We carried out the reaction in different conditions using compound 1a as an example with the optimized reaction conditions. In EtONa/EtOH (Method B, Table 1), it was found that the yield of 3a was decreased (74%). Refluxing of 1a in toluene/Et3N (Method C, Table 1) did not increase the yield (60%), and the time taken to obtain 3a was increased (2d). Furthermore, adding a few drops of Et3N to DMF (Method D, Table 1) improved the yield of 3a compared with methods B and C, but it was still lower compared with our method A. Using Na/toluene, the oxidation of 1a occurred satisfactorily; however, it was lower compared with method A. In our trial of an acidic medium using HCl/EtOH mixture, the reaction failed. Thus, the best condition to obtain high yields and a short reaction time of 3a-g is reflux in dry pyridine (Method A, Table 1). The formation of pyridazino[4,3-c:5,6-c′]diquinoline-6,7(5H,8H)-diones 3a-g can be rationalized as depicted in Scheme 3. It is clear from the suggested mechanism (Scheme 3) that it constitutes several steps of nucleophilic substitution, dimerization, autoxidation, and electrocyclic reactions in a one-pot process leading to the pentacyclic final products 3a-g. The mechanism starts with a proton shift of compound 2 to its isomer 2′ (Scheme 3). We carried out the reaction in different conditions using compound 1a as an example with the optimized reaction conditions. In EtONa/EtOH (Method B, Table 1), it was found that the yield of 3a was decreased (74%). Refluxing of 1a in toluene/Et 3 N (Method C, Table 1) did not increase the yield (60%), and the time taken to obtain 3a was increased (2d). Furthermore, adding a few drops of Et 3 N to DMF (Method D, Table 1) improved the yield of 3a compared with methods B and C, but it was still lower compared with our method A. Using Na/toluene, the oxidation of 1a occurred satisfactorily; however, it was lower compared with method A. In our trial of an acidic medium using HCl/EtOH mixture, the reaction failed. Thus, the best condition to obtain high yields and a short reaction time of 3a-g is reflux in dry pyridine (Method A, Table 1). The formation of pyridazino[4,3-c:5,6-c ]diquinoline-6,7(5H,8H)-diones 3a-g can be rationalized as depicted in Scheme 3. It is clear from the suggested mechanism (Scheme 3) that it constitutes several steps of nucleophilic substitution, dimerization, autoxidation, and electrocyclic reactions in a one-pot process leading to the pentacyclic final products 3a-g. The mechanism starts with a proton shift of compound 2 to its isomer 2 (Scheme 3). Then, the starting molecule of 2 reacts with its isomer 2 to give 6 (Scheme 3). The elimination of a hydrazine molecule in 6 would give the dimerized hydrazone 7, which undergoes another proton shift to give the intermediate 7 . Because the reaction did not proceed under an inert argon atmosphere (i.e., under argon atmosphere, the starting quinolinyl-hydrazines 2a-g were recovered), we proposed that the intermediate 7 undergo an aerial oxidation NH-NH group to give the intermediate 8. oxidation NH-NH group to give the intermediate 8. After that, the intermediate 8 would undergo internal electrocyclization to give 9. Finally, another mode of aerial oxidation of 9 would produce compound 3 (Scheme 3).
The preceding literature supports the mechanism [47] describing aryl hydrazine chlorides' aerial oxidation into diazines. Accordingly, it supports the steps of transformations of 7′ into 8 and 9 into 3. Moreover, aerial catalytic oxidation in pyridine transformed hydrazones into diazo compounds [48]. ..
Scheme 3.
The suggested mechanism describes the formation of compounds 3a-g.
Firstly, the stability of compound 3a (Figure 3), as an example, was described. Therefore, the quantum mechanical calculations were performed for compound 3a. The investigated compound was first optimized using the DFT method (see the Methods section for details). The optimized structure was then subjected to vibrational frequency and singlepoint energy calculations. The quantum theory of atoms in molecules (QTAIM) was invoked to achieve an in-depth insight into the topological features of compound 3a [49]. In the context of QTAIM, the (3,-1) bond critical points (BCPs) and bond paths (BPs) were generated, and the electron density was computed. Moreover, noncovalent interaction (NCI) index analysis was executed to pictorially elucidate the origin and nature of intramolecular interactions within compound 3a [50]. According to the results, no imaginary frequencies were observed for the investigated structure of compound 3a, confirming that this conformer is a true minimum. Based on the QTAIM results presented in Figure 4a, the occurrence of intramolecular bonds within the inspected compound was revealed by the existence of BPs and BCPs. Chalcogen•••chalcogen intramolecular interaction was also noticed in compound 3a via the BP and BCP between the two oxygen atoms (O•••O). The Scheme 3. The suggested mechanism describes the formation of compounds 3a-g.
The preceding literature supports the mechanism [47] describing aryl hydrazine chlorides' aerial oxidation into diazines. Accordingly, it supports the steps of transformations of 7 into 8 and 9 into 3. Moreover, aerial catalytic oxidation in pyridine transformed hydrazones into diazo compounds [48].
Firstly, the stability of compound 3a (Figure 3), as an example, was described. Therefore, the quantum mechanical calculations were performed for compound 3a. The investigated compound was first optimized using the DFT method (see the Methods section for details). The optimized structure was then subjected to vibrational frequency and single-point energy calculations. The quantum theory of atoms in molecules (QTAIM) was invoked to achieve an in-depth insight into the topological features of compound 3a [49]. In the context of QTAIM, the (3,-1) bond critical points (BCPs) and bond paths (BPs) were generated, and the electron density was computed. Moreover, noncovalent interaction (NCI) index analysis was executed to pictorially elucidate the origin and nature of intramolecular interactions within compound 3a [50]. According to the results, no imaginary frequencies were observed for the investigated structure of compound 3a, confirming that this conformer is a true minimum. Based on the QTAIM results presented in Figure 4a, the occurrence of intramolecular bonds within the inspected compound was revealed by the existence of BPs and BCPs. Chalcogen···chalcogen intramolecular interaction was also noticed in compound 3a via the BP and BCP between the two oxygen atoms (O···O). The BCP at the BP O···O within compound 3a exhibited electron density with a value of 0.0144 au.
The stability of compound 3a might also be interpreted as a consequence of the aromatic planarity, which could be detected from Figure 4a via dihedral angles (Φ) with a value of 1.83 • . Notably, the difference between the dihedral angle of the optimized geometry of compound 3a and the X-ray data was nearly 0.36 • .
As shown in Figure 4b, the NCI results (green isosurfaces) occurred at the interatomic space between the interacting atoms, asserting the occurrence of the intramolecular interactions towards the investigated compound. Large, green, round domains within the The stability of compound 3a might also be interpreted as a consequence of the aromatic planarity, which could be detected from Figure 4a via dihedral angles (Φ) with a value of 1.83°. Notably, the difference between the dihedral angle of the optimized geometry of compound 3a and the X-ray data was nearly 0.36°.
As shown in Figure 4b, the NCI results (green isosurfaces) occurred at the interatomic space between the interacting atoms, asserting the occurrence of the intramolecular interactions towards the investigated compound. Large, green, round domains within the intramolecular forces N13•••HC12 and N14•••HC1 of compound 3a were crucially denoted, reflecting the favorable contribution of such intramolecular forces to the further stability of compound 3a.
Chemistry
The IR spectra were recorded using the ATR technique (ATR = Attenuated Total Reflection) with an FT device (FT-IR Bruker IFS 88), Institute of Organic Chemistry, Karlsruhe University, Karlsruhe, Germany. The NMR spectra were measured in DMSO-d6 on a Bruker AV-400 spectrometer, 400 MHz for 1 The stability of compound 3a might also be interpreted as a consequence of matic planarity, which could be detected from Figure 4a via dihedral angles (Φ value of 1.83°. Notably, the difference between the dihedral angle of the optimized etry of compound 3a and the X-ray data was nearly 0.36°. As shown in Figure 4b, the NCI results (green isosurfaces) occurred at the inte space between the interacting atoms, asserting the occurrence of the intramolecul actions towards the investigated compound. Large, green, round domains within tramolecular forces N13•••HC12 and N14•••HC1 of compound 3a were crucially deno flecting the favorable contribution of such intramolecular forces to the further sta compound 3a.
Chemistry
The IR spectra were recorded using the ATR technique (ATR = Attenuated T flection) with an FT device (FT-IR Bruker IFS 88), Institute of Organic Chemistry ruhe University, Karlsruhe, Germany. The NMR spectra were measured in DMS a Bruker AV-400 spectrometer, 400 MHz for 1 The isosurfaces were generated with a reduced density gradient value of 0.50 au and colored from blue to red according to sign (λ 2 ), ρ ranging from −0.035 (blue) to 0.020 (red) au.
Conclusions
The unprecedented dimerization and oxidation cascade of 4-hydrazinylquinolin-2(1H)ones delivered pyridazino[4,3-c:5,6-c ]diquinoline-6,7(5H,8H)-diones in good yields. The synthesis of the obtained pyridazino-diquinolones was achieved in different conditions. Heating 4-hydrazinylquinolin-2(1H)-ones in pyridine was the best condition in which to obtain the corresponding products. Quantum mechanical calculations were also performed using the DFT method to prove the stability of the formed products. The method above can be used as a general method for the preparation of various classes of pentacyclic heterocycles from compounds with structural features similar to 4-hydroxy-2-quinolones. Importantly, the biological activity of the obtained products could assist in the development of new drugs.
Chemistry
The IR spectra were recorded using the ATR technique (ATR = Attenuated Total Reflection) with an FT device (FT-IR Bruker IFS 88), Institute of Organic Chemistry, Karlsruhe University, Karlsruhe, Germany. The NMR spectra were measured in DMSO-d 6 on a Bruker AV-400 spectrometer, 400 MHz for 1 H, and 100 MHz for 13 C; the chemical shifts are expressed in δ (ppm), versus internal tetramethylsilane (TMS) = 0 for 1 H and 13 C, and external liquid ammonia = 0. The description of signals includes: s = singlet, d = doublet, t = triplet, q = quartet, m = multiplet, dd = doublet of doublet, and m = multiplet. Mass spectra were recorded on a FAB (fast atom bombardment) Thermo Finnigan Mat 95 (70 eV). Elemental analyses were carried out at the Microanalytical Center, Cairo University, Egypt. | 2022-03-31T16:22:37.906Z | 2022-03-25T00:00:00.000 | {
"year": 2022,
"sha1": "170af6cce28ceb3b836854077d071479b815c970",
"oa_license": "CCBY",
"oa_url": "https://www.mdpi.com/1420-3049/27/7/2125/pdf",
"oa_status": "GOLD",
"pdf_src": "PubMedCentral",
"pdf_hash": "adb434fbc2309e5805d56007e1dcb175d7481198",
"s2fieldsofstudy": [
"Chemistry"
],
"extfieldsofstudy": [
"Medicine"
]
} |
255840788 | pes2o/s2orc | v3-fos-license | Neoadjuvant nivolumab + T-VEC combination therapy for resectable early stage or metastatic (IIIB-IVM1a) melanoma with injectable disease: study protocol of the NIVEC trial
Trials investigating neoadjuvant treatment with immune checkpoint inhibitors (ICI) in patients with melanoma have shown high clinical and pathologic response rates. Treatment with talimogene laherparepvec (T-VEC), a modified herpes simplex virus type-1 (HSV-1), is approved for patients with unresectable stage IIIB-IVM1a melanoma and has the potential to make tumors more susceptible for ICI. Combination ICI and intralesional T-VEC has already been investigated in patients with unresectable stage IIIB-IV disease, however, no data is available yet on the potential benefit of this combination therapy in neoadjuvant setting. This single center, single arm, phase II study aims to show an improved major pathologic complete response (pCR) rate, either pCR or near-pCR, up to 45% in 24 patients with resectable stage IIIB-IVM1a melanoma upon neoadjuvant combination treatment with intralesional T-VEC and systemic nivolumab (anti-PD-1 antibody). Patients will receive four courses of T-VEC up to 4 mL (first dose as seroconversion dose) and three doses of nivolumab (240 mg flatdose) every 2 weeks, followed by surgical resection in week nine. The primary endpoint of this trial is pathologic response rate. Secondary endpoints are safety, the rate of delay of surgery and event-free survival. Additionally, prognostic and predictive biomarker research and health-related quality of life evaluation will be performed. Intralesional T-VEC has the capacity to heighten the immune response and to elicit an abscopal effect in melanoma in combination with ICI. However, the potential clinical benefit of T-VEC plus ICI in the neoadjuvant setting remains unknown. This is the first trial investigating the efficacy and safety of neoadjuvant treatment of T-VEC and nivolumab followed by surgical resection in patients with stage IIIB-IVM1a melanoma, with the potential of high pathologic response rates and acceptable toxicity. This trial was registered in the European Union Drug Regulating Authorities Clinical Trials Database (EudraCT- number: 2019–001911-22) and the Central Committee on Research Involving Human Subjects (NL71866.000.19) on 4th June 2020. Secondary identifying number: NCT04330430.
Background
The worldwide incidence of melanoma is still increasing and accounted for more than 57.000 deaths in 2020 [1]. The prognosis of patients is clearly correlated with disease stage, but since the introduction of the current standard systemic treatments comprising targeted therapies and immune checkpoint inhibitors (ICI), 5-year overall survival (OS) rates up to 50% have been reached in patients with unresectable stage IIIC-IV melanoma in clinical trials [2,3]. For patients with high-risk stage IIIB, IIIC, IIID (AJCC 8th edition) melanoma, the prognosis is slightly better, with 5-year OS rates of 83, 69 and 32% respectively, although it must be noted that this is prior to effective adjuvant therapy becoming available [4]. Adjuvant targeted therapy or ICI has shown major improvements in relapse-free survival (RFS) in patients with resectable stage III or IV melanoma [5][6][7][8] and is currently standard of care.
Next to the currently available systemic therapies for patients with advanced melanoma, local oncolytic viral immunotherapy with talimogene laherparepvec (T-VEC) has been approved for the treatment of patients with unresectable stage IIIB-IVM1a melanoma with cutaneous, subcutaneous or nodal metastases [9]. T-VEC, a modified herpes simplex virus type-1 (HSV-1), is administered intralesionally and resulted in high and durable response rates with a mild toxicity profile, consisting mostly of fatigue and influenza-like symptoms, confirmed in real-world setting [10][11][12]. However, there is still a group of patients that has no (durable) benefit upon these currently approved systemic and local treatment options.
A possible way to overcome this resistance to therapy is combination of treatments. Although single agent T-VEC has already proven its efficacy, evidence is also emerging that combination of T-VEC with ICI could have an additive anti-tumor effect. This is demonstrated by two clinical trials treating patients with unresectable stage IIIB-IV melanoma with T-VEC in combination with either ipilimumab, an anti-CTLA-4 antibody, or pembrolizumab, an anti-PD-1 antibody, reaching objective response rates of 39% [13] and 62% [14] respectively. In both trials, the responses were not limited to the injected lesions, suggesting an abscopal effect of T-VEC in combination with ICI. In the phase Ib trial by Ribas et al. [14], 21 patients with unresectable stage IIIB-IV cutaneous melanoma were treated with intralesional T-VEC and systemic pembrolizumab. The majority of patients showed increases in circulating CD4 + and CD8 + T cells after T-VEC alone, with no significant further increase after pembrolizumab administration. Additionally, increases in CD8 + T cells, IFN-γ gene and PD-L1 protein expression were seen within the tumors of responding patients treated with T-VEC and pembrolizumab compared to baseline. These changes in the tumor microenvironment have also been observed by multiple other preclinical and clinical trials [15][16][17], indicating that T-VEC is able to heighten the immune response and turn an immune desolate "cold" tumor into an immunogenic "hot" tumor, making the tumor more susceptible to ICI treatment. Although the clinical benefit of the combination of T-VEC and pembrolizumab compared to single agent pembrolizumab in patients with unresectable stage IIIB-IVc was not confirmed in the recent phase III trial by Ribas et al. (2021), progression-free survival did seem to be improved in the combination treatment arm and patients with few sites of disease tended to have a better response upon treatment [18], still suggesting a heightened immune response in this patient population. In the previous trials no doselimiting or novel adverse events (AE) were observed, indicating that combination treatment with T-VEC and ICI can be administered safely to possibly overcome tumor resistance.
Aside from the type of treatment, there is increasing evidence that the timing of the given therapies also plays a key role in the chances of a tumor response [19]. Neoadjuvant therapy, administered prior to surgery, could result in a broader tumor-specific T cell response compared to adjuvant treatment, downsizing of the tumor leading to less extensive surgery, and usage of the pathologic response for fitted (adjuvant) therapy [19,20]. The benefit of neoadjuvant ICI has been investigated in multiple clinical trials, resulting in high pathologic complete response (pCR) rates of 25-80% [21][22][23][24]. Recently, the role of neoadjuvant T-VEC in resectable stage IIIB-IVM1a melanoma has been investigated by Dummer et al. [25] in a randomized study of single agent T-VEC followed by surgery, compared to surgery alone. This trial Based on the recent data of neoadjuvant single agent nivolumab and pembrolizumab shown by Amaria et al. [23] and Huang et al. [24], with pCR rates of 25 and 20% respectively, neoadjuvant single agent T-VEC shown by Dummer et al. [25] and the abscopal effect of T-VEC in combination with ICI, we hypothesized that neoadjuvant T-VEC in combination with nivolumab can improve these pCR or pathologic near complete response (near-pCR) rates further. For this, we designed a single arm phase II trial treating patients with stage IIIB-IVM1a melanoma with resectable (sub)cutaneous satellite or intransit metastases and/or tumor positive lymph nodes with neoadjuvant nivolumab and T-VEC (NIVEC trial). The aim of this trial is to show an improvement in pCR (or near-pCR) rate of 25% compared to the pCR rate observed by Amaria et al. [23] and Huang et al. [24], reaching a pCR rate of 45% in this patient population. In total, 24 patients will be included and evaluated for efficacy and safety. This is the first trial evaluating neoadjuvant T-VEC and ICI combination therapy and may benefit a large group of future melanoma patients.
Study design
The NIVEC trial is an investigator initiated, single-center, single-arm, open-label, phase II study investigating the efficacy and safety of neoadjuvant combination of T-VEC plus nivolumab in patients with resectable stage IIIB-IVM1a melanoma. Patients will be enrolled and treated at the departments of Medical and Surgical Oncology at the Netherlands Cancer Institute (NKI), Amsterdam, The Netherlands, after signing a written informed consent form. A Data and Safety Monitoring Board (DSMB) has been established to monitor the safety of the patients throughout the study.
Primary objective
The primary objective of this trial is to investigate the major pathologic complete response rate, either pCR or near-pCR, of neoadjuvant combination of T-VEC and nivolumab, in patients with resectable stage IIIB/C/D/ IVM1a melanoma, with the aim to achieve a major pathologic complete response rate of 45%.
Exploratory objectives
-To assess radiologic response rates according to RECIST 1.1 after neoadjuvant T-VEC and nivolumab at week eight; -To evaluate health-related quality of life.
Participants
Patients of ≥18 years with treatment naïve resectable stage IIIB/C/D/IV M1a melanoma according to the American Joint Committee on Cancer (AJCC) 8th edition, a good clinical performance score of 0-1 and < 2 times elevated lactate dehydrogenase levels will be eligible for this trial. Patients must have measurable disease according to RECIST 1.1 [26] and must be a candidate for intralesional therapy with at least one injectable cutaneous, subcutaneous or nodal melanoma lesion (≥ 10 mm in longest diameter) or with multiple injectable lesions that in aggregate have a longest diameter of ≥10 mm. Patients with visceral or brain metastases, prior systemic cancer therapies or a history of other malignancies or autoimmune/infectious diseases will not be eligible. For a full overview of all in-and exclusion criteria, see Table 1.
Study procedures and intervention
Prior to enrollment, all patients will be adequately informed by the investigator(s) about participation in the trial. Participation is voluntary and patients are allowed to withdraw from the trial at any time. Following signing of the informed consent form for screening and enrollment into the study, the remainder of the screening procedures and tests will be completed and evaluated before start of treatment. If the screening shows that patients do not fulfill the eligibility criteria anymore, patients will be withdrawn from the study and will be replaced by another patient. See Table 2 for the full schedule of screening assessments and study procedures.
The total neoadjuvant treatment duration comprises 7 weeks and is followed by surgery in week nine. The treatment schedule is based on four courses of T-VEC and three courses of nivolumab: -T-VEC: Up to 4 ml T-VEC (first dose 10 6 plaqueforming units (PFU) per mL as a seroconversion dose, subsequent doses at 10 8 PFU per mL) in week 0, 3, 5 and 7. The volume of T-VEC to be injected into the tumor depends on the size of the tumor(s) and will be determined based on the longest diameter of each lesion according to manufacturers' protocol [27]; -Nivolumab: Flatdose of 240 mg nivolumab intravenously every 2 weeks after the first intralesional T-VEC injections (starting at the second T-VEC dose at week 3, followed by the next doses at week 5 and 7).
T-VEC will be administered first, in order to achieve the best synergistic effect with influx of CD8 + T cells [14]. Patients will be offered adjuvant treatment as standard of care following surgery. See Fig. 1 for a full overview of the course of the trial.
Evaluation and follow-up of participants
Prior to each treatment course, physical examination, laboratory analyses and evaluation of toxicity will be performed in each patient. Additionally, photos of cutaneous lesions will be taken. At baseline, before first nivolumab administration, time of surgery and moment of potential relapse, blood-and tumor samples will be collected for translational research purposes. Health-related quality of life assessment will be performed at baseline, prior to surgery, at week 12 and every follow-up. 5 T-VEC seroconversion dose 10 6 PFU/mL. 6 T-VEC dose 10 8 PFU/mL. 7 AEs will be graded according to CTCAE v5.0 from moment of signing informed consent. New occurring AEs and any SAEs will reported up to 100 days post last treatment. 8 Only registration of medication that is used for treatment of immune-related AEs from moment of signing informed consent. 9 Toxicity from prior treatment will be evaluated. 10 100 ml heparinized blood (for PBMC). 11 Only at week 3 prior to first nivolumab. 12 At relapse. 13 20 ml EDTA blood (for isolation of plasma for ctDNA). 14 Three biopsies taken with a 14G needle of thru-cut and will be stored as fresh frozen and formalin-fixed paraffin-embedded material for translational research purposes. At baseline pathological confirmation of melanoma will be performed. 15 If possible, surgical material should be preserved as fresh frozen and formalin-fixed paraffin-embedded material Prior to surgery, patients will undergo physical examination, laboratory analyses and a Computed Tomography (CT) scan. Following surgery, patients will be evaluated every 12 weeks during the first 2 years after the end of adjuvant treatment by physical examination, laboratory analyses, CT or PET-CT (according to center's discretion). Subsequent structured follow-up will be according to the current national melanoma guidelines. In case of progression to unresectable stage III or stage IV melanoma, follow-up data will be collected until first systemic treatment dose. After initiation of systemic treatment, only the OS data will be collected. See Fig. 1 and Table 2 for all specific time points and study assessments.
Study endpoints Primary endpoint
The primary endpoint of this trial is the pathologic response rate according to central revision by pathology of NKI, determined after resection following neoadjuvant T-VEC and nivolumab treatment.
Secondary endpoints
-Rate of delay of surgery > 14 days and rate of failure to perform surgery defined as no surgery at all; -EFS, defined as time from randomization to any of the following events: progression of disease that precludes surgery, local or distant recurrence, or death due to any cause; -Safety of neoadjuvant combination of T-VEC and nivolumab; -Description of possible prognostic and predictive biomarkers, for example CD8, IFN-γ and PD-L1, and exploration of expansion of tumor-resident T cells.
Exploratory endpoints
-Response rate according to RECIST 1.1 at week eight; -Quality of life as measured by EORTC QLQ-C30 and the Melanoma Surgery Subscale of the FACT-M.
Evaluation of efficacy
All patients included in the trial who received at least one cycle of therapy will be assessed for pathologic and radiologic response to treatment. Each patient will be assigned one of the following categories for pathologic response based on the resection specimen at week nine: pCR, near-pCR (< 10% vital tumor remaining), pathologic partial response (pPR; < 50% vital tumor remaining), pathologic non-response (pNR) or unknown (not assessable, insufficient data), according to guidelines of the International Neoadjuvant Melanoma Consortium (INMC) [28]. Furthermore, objective radiologic tumor response according to RECIST 1.1 will be measured with CT scans at baseline, week eight and every follow-up. Patients' response will be classified as "unknown" if insufficient data were collected to allow evaluation per these criteria.
Safety evaluation
All patients that have received at least one cycle of T-VEC + nivolumab will be evaluated for toxicity according to the National Cancer Institute's Common Terminology Criteria for Adverse Events version 5.0 (NCI-CTCAE v.5.0) from the moment of signing informed consent until 100 days after last study treatment. Both AEs related and unrelated to treatment will be followed. Additionally, surgical complications will be scored according to the Clavien-Dindo classification [29]. Serious adverse events (SAEs) will be reported to the sponsor and the collaborating party Amgen Inc. (providing T-VEC) within 24 hours. Suspected unexpected serious adverse reactions (SUSARs) will be reported to the Central Committee on Research Involving Human Subjects (CCMO). In addition to the expedited reporting of SUSARs, the sponsor will submit an annual safety report to the CCMO and competent authority. This safety report will also be provided to the subsidising party Amgen Inc. at the time of reporting to the CCMO.
A DSMB has been instituted to monitor the safety of the patients throughout the study. Interim analyses will include only safety information and will be reviewed by the DSMB. After inclusion and treatment of every six patients a report will be provided to the DSMB with an overview of all toxicity observed up until that moment. It is intended not to include any data on response and thus not to look at the primary endpoint before the end of the study.
Evaluation of health-related quality of life
Health-related quality of life will be assessed with the European Organization for Research and Treatment of Cancer Quality of Life Questionnaire Core 30 (EORTC QLQ-C30), a self-reported questionnaire specifically developed for patients with cancer who are receiving cancer treatment and is the most common quality of life instrument used in melanoma studies [30]. The guideline of Cocks et al. [31] will be used for the interpretation of changes in QLQ-C30 scores. Additionally, the Melanoma Surgery Subscale of the FACT-M [32] will be evaluated in all patients every 12 weeks during the first year and at year two and three.
Translational research
Tumor material (at baseline, during treatment and the resection specimen), peripheral blood mononuclear cells (PBMCs) and serum will be collected in all patients (see Fig. 1 and Table 2 for all time points) to perform in depth translational research into the mechanism of action of T-VEC in combination with nivolumab, both on the tumor and the tumor micro-environment. The planned analyses of the biopsies and PBMCs will primarily provide the information to underline our hypothesis about the synergistic effect of T-VEC and nivolumab.
Data collection, management and monitoring
All source data from the trial will be collected and managed in the electronic case report form (eCRF) developed by the datacenter of the NKI via ALEA (FormsVision BV, Abcoude, The Netherlands), a web-based software system. The completed eCRFs must be reviewed and signed by the principal investigator or sub-investigator. The handling of personal data complies with the Dutch Personal Data Protection Act and all data management activities will follow local standard operating procedures. Statistical analysis will be performed after a database lock, after all study data have been collected and cleaned.
According to the Dutch Federation of University Medical Centers, this trial was classified as a 'low risk' study. This implies at least one on-site source data verification of the eCRFs and check of the Investigator Study File documents by the clinical research monitor.
Statistical considerations
We suspect that neoadjuvant combination treatment of T-VEC with nivolumab will achieve a higher major pathologic complete response rate in patients with resectable stage IIIB/C/D-IVM1a melanoma compared to neoadjuvant treatment with either single agent T-VEC or nivolumab. Additionally, we expect that neoadjuvant T-VEC plus nivolumab will result in a higher response rate in our patient population compared to patients with unresectable disease, as the tumor burden is lower. The combination treatment will be considered successful if the major pathologic complete response rate lies significantly above the response rate of 20% obtained with neoadjuvant single agent ICI. For the current neoadjuvant combination study we thus expect a pCR (or near-pCR) rate of 45% for T-VEC combined with nivolumab. An exact binomial test with a nominal 10% two-sided significance level will have 82.7% power to detect the difference between the Null hypothesis proportion, π 0 , of 0.2 and the Alternative proportion, π 1 , of 0.45 when the sample size is 24.
Assuming a surgical success rate of 95%, defined as a complete dissection of the involved lymph nodes and/or all cutaneous/subcutaneous satellite/in-transit disease as planned, including 24 patients will have 88% power to exclude a lower level of 75% with 95% confidence (onesided). The combination needs to be reconsidered when surgery as planned can no longer be performed due to progressive disease or AEs in more than two patients. Considering the chosen scenario of 24 patients, the study is also powered to analyze this secondary endpoint.
Discussion
This manuscript describes the clinical trial protocol of the NIVEC trial, the first single-center, single-arm, phase II trial investigating the efficacy and safety of neoadjuvant nivolumab in combination with T-VEC in patients with resectable stage IIIB-IVM1a melanoma. To date, T-VEC in combination with ICI has only been evaluated in clinical trials with patients with unresectable stage IIIB-IVM1c melanoma [13,14,18]. It is known that T-VEC is able to modify the tumor microenvironment to make it more susceptible to concurrent ICI [14] and that neoadjuvant treatment has the ability to elicit a broader tumorspecific T cell response [19]. The design of the trial was based on the successful results of earlier neoadjuvant trials with single agent ICI and T-VEC [23][24][25] and is the first trial to administer this treatment combination in patients with resectable stage IIIB-IVM1a disease, creating a window of potential for improved response rates in this patient population.
In prior neoadjuvant trials in patients with resectable stage III melanoma, such as the OpACIN-neo trial [22], neoadjuvant treatment with three different dosing schedules of ICI with ipilimumab and nivolumab were administered during a period of 6 weeks prior to surgery. In the current trial, a similar neoadjuvant treatment period is followed, however, as the first T-VEC dose concerns a seroconversion dose and the next (first optimal) T-VEC dose can be administered 3 weeks thereafter, the surgery will take place 9 weeks after initiation of the neoadjuvant treatment. As T-VEC is administered every 2 weeks, we suggested the same dosing schedule for T-VEC and nivolumab every 2 weeks for the purpose of this trial.
This dosing schedule will result in a short delay for patients compared to standard treatment, as regular times to surgery in The Netherlands are 4-6 weeks. However, standard of care for this patient population would be surgery followed by adjuvant ICI. In this trial, patients will already have received three courses of ICI in combination with T-VEC prior to surgery. Considering the very high chance of relapse in this patient category (approximately 70-80% risk) [33], any neoadjuvant approach might reduce this risk of relapse, adding to the chance of a more durable response for these patients, comparable to the observed results from the earlier neoadjuvant OpACIN and OpACIN-neo studies [21,22]. Results from Dummer et al. [25], already demonstrated 25% less chance of recurrence in patients with resectable stage IIIB-IVM1a melanoma when treated with neoadjuvant T-VEC compared to surgery alone. For patients with subcutaneous or in-transit metastases, who are often excluded from ICI neoadjuvant trials [22] and who would otherwise receive locoregional management (followed by adjuvant therapy in case of surgery) or systemic therapy as standard of care, this neoadjuvant approach could be extra beneficial. Additionally, some patients may have the chance to undergo less extensive surgery, or no surgery at all, due to a good clinical response upon neoadjuvant treatment.
Earlier neoadjuvant trials with combination ICI have shown improved pCR rates compared to monotherapy ICI in neoadjuvant setting. However, this was often also associated with higher toxicity profiles [21][22][23][24]. Ways to improve neoadjuvant treatment by diminishing toxicity rates whilst maintaining these improved response rates are currently under investigation in multiple clinical trials, evaluating the safety and efficacy of different neoadjuvant combination treatments. In an ongoing phase II trial, the combination of neoadjuvant pembrolizumab and lenvatinib, a multiple kinase inhibitor, is being investigated in patients with resectable stage III/IV melanoma [34] and improved toxicity rates have recently already been reported with neoadjuvant relatlimab, an anti-LAG-3 antibody, in combination with nivolumab in patients with unresectable or metastatic melanoma [35]. Both neoadjuvant single agent nivolumab and T-VEC have shown mild toxicity rates, with grade 3-4 AEs occurring in 8% [23] and 5.5% [25] of patients respectively. We expect the toxicity profile of the combination of T-VEC and nivolumab to be acceptable, as T-VEC combined with pembrolizumab already showed no increase in toxicity rate compared to single agent ICI in patients with unresectable stage IIIB-IV melanoma [14,18].
In conclusion, this is the first trial to investigate neoadjuvant T-VEC and nivolumab in patients with resectable stage IIIB-IVM1a melanoma. It has the potential to show improved efficacy with acceptable toxicity profiles, compared to single agent or ICI combination neoadjuvant treatment, and could thus lead to a potential novel viable treatment option for this patient population.
Current trial status
The NIVEC trial was open for accrual on 7th July 2020 and a total of 13 of the planned 24 patients were enrolled in the trial on 31st May 2022. refuse further participation in the protocol whenever he/she wants. This will not prejudice the patient's subsequent care. Documented informed consent will be obtained for all patients included in the study before they are registered in the study. This will be done in accordance with the national and local regulatory requirements. The informed consent procedure will conform to the ICH guidelines on GCP. This implies that "the written informed consent form will be signed and personally dated by the patient or by the patient's legally acceptable representative". The patient material will be stored at the pathology department and the core facility molecular pathology and biobanking at the NKI. The PBMCs will be generated from peripheral blood according to local protocol and stored at the immunology division of the NKI. Material that is not used for current translational research will be stored for up to 15 years after end of study. | 2023-01-16T14:42:40.690Z | 2022-08-04T00:00:00.000 | {
"year": 2022,
"sha1": "a900abb0543ebb423d085be3e25bbe63a3d82685",
"oa_license": "CCBY",
"oa_url": "https://bmccancer.biomedcentral.com/counter/pdf/10.1186/s12885-022-09896-4",
"oa_status": "GOLD",
"pdf_src": "SpringerNature",
"pdf_hash": "a900abb0543ebb423d085be3e25bbe63a3d82685",
"s2fieldsofstudy": [
"Medicine"
],
"extfieldsofstudy": []
} |
246814232 | pes2o/s2orc | v3-fos-license | Plasma Membrane Phosphatidylinositol-4-Phosphate Is Not Necessary for Candida albicans Viability yet Is Key for Cell Wall Integrity and Systemic Infection
ABSTRACT Phosphatidylinositol phosphates are key phospholipids with a range of regulatory roles, including membrane trafficking and cell polarity. Phosphatidylinositol-4-phosphate [PI(4)P] at the Golgi apparatus is required for the budding-to-filamentous-growth transition in the human-pathogenic fungus Candida albicans; however, the role of plasma membrane PI(4)P is unclear. We have investigated the importance of this phospholipid in C. albicans growth, stress response, and virulence by generating mutant strains with decreased levels of plasma membrane PI(4)P, via deletion of components of the PI-4-kinase complex, i.e., Efr3, Ypp1, and Stt4. The amounts of plasma membrane PI(4)P in the efr3Δ/Δ and ypp1Δ/Δ mutants were ∼60% and ∼40%, respectively, of that in the wild-type strain, whereas it was nearly undetectable in the stt4Δ/Δ mutant. All three mutants had reduced plasma membrane phosphatidylserine (PS). Although these mutants had normal yeast-phase growth, they were defective in filamentous growth, exhibited defects in cell wall integrity, and had an increased exposure of cell wall β(1,3)-glucan, yet they induced a range of hyphal-specific genes. In a mouse model of hematogenously disseminated candidiasis, fungal plasma membrane PI(4)P levels directly correlated with virulence; the efr3Δ/Δ mutant had wild-type virulence, the ypp1Δ/Δ mutant had attenuated virulence, and the stt4Δ/Δ mutant caused no lethality. In the mouse model of oropharyngeal candidiasis, only the ypp1Δ/Δ mutant had reduced virulence, indicating that plasma membrane PI(4)P is less important for proliferation in the oropharynx. Collectively, these results demonstrate that plasma membrane PI(4)P levels play a central role in filamentation, cell wall integrity, and virulence in C. albicans.
medium containing serum, the cells elongated with protrusions that were roughly one-third the length of wild-type cells after 2 h at 37°C. However, under these repression conditions, the stt4 mutant still expressed STT4 and plasma membrane PI(4)P was still detected (14). We also generated a DAmP (decreased abundance by mRNA perturbation) allele (23) in C. albicans (24), by constructing strains in which one copy of STT4 was deleted and a dominant nourseothricin resistance marker (SAT1) was integrated just 39 of the STT4 stop codon. These DAmP mutants had between 2-and 4-fold reduction in STT4 transcript levels compared to a wild-type strain, yet filamentous growth was indistinguishable from that of the wild type in liquid and on solid media containing fetal calf serum (Fig. S1). More recently, an stt4 deletion mutant was isolated in a screen for mutants exhibiting hypersensitivity to Hsp90 inhibition via geldanamycin, with aberrant filamentation in the presence of geldanamycin or RPMI (17).
To determine if plasma membrane PI(4)P is essential, we attempted to delete this PI-4-kinase, as well as the two other putative components of the Stt4 complex, EFR3 and YPP1. To generate an stt4 deletion mutant, we removed the remaining STT4 copy in the DAmP mutant, by taking advantage of the 39 SAT1 marker to target homologous recombination. Southern blotting, as well as PCR of genomic DNA (gDNA), confirmed the absence of STT4 in this homozygous deletion strain ( Fig. S2A and B). In addition, we were able to generate homozygous EFR3 and YPP1 deletion mutants, which were verified by PCR (Fig. S3A). Reverse transcription-PCR (RT-PCR) revealed the complete absence of STT4 mRNA transcript, as well as that of EFR3 and YPP1 in the respective mutants ( Fig. S2C and S3B). Interestingly, the levels of mRNA transcripts of other PIand PIP-kinases and phosphatase, including PIK1, MSS4, and SAC1, were unaffected in the stt4 deletion mutant (Fig. S2C). These strains all grew with doubling times that were indistinguishable from that of the wild-type strain (81 6 6 min for the wild-type strain compared to 88 6 7 min for the stt4D/stt4D strain, 86 6 5 min for the stt4D/ stt4D1STT4 strain, 86 6 2 min for the efr3D/efr3D strain, and 89 6 1 min for the ypp1D/ypp1D strain), indicating that the PI-4-kinase complex is not necessary for viability or yeast-phase growth in C. albicans.
In the presence of serum, however, we observed a striking filamentous growth defect ( Fig. 1 and 2) in the efr3, ypp1, and stt4 deletion mutants. The mutants appeared to form short germ tubes but were unable to form longer hyphal filaments. Similarly, all three strains were completely defective in invasive growth on agar medium containing fetal calf serum ( Fig. 1C and 2C). These defects were complemented by the reintroduction of a copy of the respective genes, which was confirmed by PCR of gDNA and RT-PCR of mRNA transcripts ( Fig. S2B and C and S3A and B). An stt4 allele that is a hypomorph with reduced in vivo lipid kinase activity (25) was identified in S. cerevisiae in a genetic screen for aminophospholipid transport mutants (7). Hence, we also examined whether such a mutant, in which a highly conserved amino acid in the catalytic domain, Gly1782, was changed to Asp (in C. albicans G1810D), was critical for hyphal growth. Figure 1B shows that a strain expressing as a sole copy this mutated version of STT4 exhibited defects in filamentous growth that were intermediate between the stt4 deletion mutant and the complemented strain. Furthermore, compared to the wild-type and complemented strains, the efr3, ypp1, and stt4 deletion mutants did not grow in the presence of the cell wall perturbants, including the antifungal drug caspofungin, calcofluor white, and Congo red (Fig. 3). A lower concentration of caspofungin (50 ng/mL compared to 125 ng/mL) revealed that the stt4 deletion mutant exhibits the greatest sensitivity to this antifungal drug, with the ypp1 deletion mutant exhibiting intermediate sensitivity and the efr3 deletion mutant having the lowest sensitivity (Fig. S4A). These mutants were, however, not temperature sensitive and grew similarly to wild-type controls at 30°C and 37°C (Fig. S4B).
In contrast to what has been previously reported (26), they did not exhibit an increased sensitivity to fluconazole.
Given the effects of cell wall perturbants, we examined the cell wall thickness and composition in the stt4 homozygous deletion mutant. Figure 4A and B show that, in this PI-4-kinase deletion mutant, the cell wall was on average 50% thicker than that in wild-type cells. Flow cytometry was used to quantitate the exposed b(1,3)-glucan, as well as the total chitin, mannan, and glucan. A significant increase in exposed b(1,3)-glucan, mannan, and glucan content was observed in the stt4 deletion mutant compared to the wild-type strain (Fig. 4C). On average stt4 mutant cells had a 30% increase in exposed b(1,3)-glucan and a 60% increase in mannan and glucan content. This cell wall integrity defect and increase in cell surface exposure of b(1,3)-glucan are reminiscent of that observed in the cho1D/cho1D phosphatidylserine synthase mutant (27)(28)(29)(30). Together, our stt4D/D1STT4, PY5131) were incubated with serum at 37°C for 90 and 120 min. Bar, 5 mm. (B) Percentages of filamentous cells were determined from three independent experiments (n $ 120 in each) with the abovementioned strains in addition to PY5757, an stt4D/D1stt4* strain encoding Stt4[G1810D]. Cells were considered filamentous if germ tubes were twice length of the mother cell or longer. Error bars indicate standard deviations (SD). (C) Stt4 is required for invasive filamentous growth. Strains were incubated for 4 days at 30°C on serum agar plates. Similar results were observed in three independent experiments. results show that the PI-4-kinase complex is critical for filamentous growth and cell wall integrity in C. albicans, specifically masking cell wall b(1,3)-glucan.
The Stt4 PI-4-kinase complex is specifically required for plasma membrane PI(4)P. The defect in filamentous growth in mutants for all three components of the PI-4-kinase complex suggested that plasma membrane PI(4)P is critical for this process. Therefore, we examined the in vivo PI(4)P levels using a fluorescent reporter that binds preferentially this acidic phospholipid at the plasma membrane (13). This reporter can also bind PI(4)P at the Golgi apparatus, and we have previously shown that, upon a Role of Plasma Membrane PI4P in C. albicans ® reduction in plasma membrane PI(4)P, the reporter relocalizes to the Golgi apparatus (13). In wild-type cells, we observed this green fluorescent protein (GFP)-PH OSH2 -PH OSH2 -GFP reporter localize predominantly at the plasma membrane, yet in efr3, ypp1, and stt4 mutants, there was a decrease in plasma membrane PI(4)P, with a concomitant increase in intracellular Golgi PI(4)P signal (Fig. 5A). Quantification of the reporter fluorescence from these central z-sections, using the Matlab program Hyphal-Polarity (14), indicated that the ratio of mean plasma membrane signal to mean internal signal decreased progressively in the efr3, ypp1, and stt4 mutants, resulting from the decrease in plasma membrane PI(4)P together with the overall increase in internal PI(4)P levels ( Fig. 5B). Closer examination of stt4 mutant cells revealed little to no plasma membrane PI(4)P (Fig. 5A), and this was confirmed by quantification of the normalized fraction of total signal at the plasma membrane, i.e., the plasma membrane signal excluding the Golgi cisternae divided by the total cell signal excluding the Golgi cisternae (Fig. 5C). The average normalized plasma membrane PI(4)P in stt4 mutants was very close to 0 (0.078 6 0.016), with 60% of cells (n = 200) having undetectable plasma membrane PI (4)P. Together, these results indicate that C. albicans cells with undetectable plasma membrane PI(4)P are viable and suggest that this plasma membrane acidic phospholipid is specifically critical for filamentous growth.
Previously, we showed that a reduction in Golgi PI(4)P results in Golgi cisternae proliferation (13). Hence, we examined whether the reduction of plasma membrane PI(4)P observed in efr3, ypp1, and stt4 deletion mutants affected the Golgi cisternae. While there was a small decrease (20 to 25%) in the number of Golgi cisternae per cell in efr3 and ypp1 mutants compared to the wild-type (Fig. 6A), there was no difference in the number of Golgi cisternae per cell in the stt4 mutant (Fig. 6B). These results further confirm that Golgi apparatus and plasma membrane PI(4)P pools are functionally distinct. Our previous analyses of a STT4 repressible strain revealed that upon a 10-fold repression of STT4 transcript, there was a reduction of plasma membrane PI(4,5)P 2 (14). As a result, we examined plasma membrane PI(4,5)P 2 levels in the efr3, ypp1, and stt4 deletion mutants. Figure 7 shows that this phosphatidylinositol phosphate was observed at the plasma membrane in all three mutants. Compared to the wild-type strain, there was a reduction of 10 to 15% in the plasma membrane PI(4,5)P 2 levels to internal ratios in the efr3, ypp1, and stt4 mutants. Comparison of the fraction of plasma (B and C) Quantitation of plasma membrane and internal signals reveals little to no plasma membrane PI(4)P in the stt4 mutant. The ratio of plasma membrane to internal signal (normalized to the wild type) and the relative plasma membrane signal (normalized plasma membrane/total signal) is shown. Quantitation of plasma membrane and internal signals was carried out excluding Golgi cisternae. For the wild type, the mean ratio of plasma membrane to internal signal was 3.8 and the ratio of plasma membrane to total signal was 0.8. We were able to detect ;1.5% of wild-type plasma membrane PI(4)P levels. Smaller symbols are values from two experiments (6 to 15 cells per experiment), and larger symbols are means for each experiment, with bars indicating overall means and standard deviations. ****, P , 0.0001. membrane PI(4,5)P 2 signal revealed a small (10% or less) reduction in the stt4 mutants with respect to the wild-type strain, similar to our previous observations with a repressible strain (14). Together, these results demonstrate that the dramatic reduction of plasma membrane PI(4)P does not alter Golgi PI(4)P, nor does it substantially alter plasma membrane PI(4,5)P 2 .
Oxysterol-binding proteins, such as Osh6 and Osh7 in S. cerevisiae, are lipid transfer proteins that transfer phosphatidylserine (PS) from the ER to the plasma membrane concomitant with transfer of PI(4)P from the plasma membrane to the ER, where it is hydrolyzed by Sac1 (31)(32)(33). In C. albicans, as in S. cerevisiae, Sac1, which localizes to the ER and Golgi apparatus, is critical for regulating plasma membrane PI(4)P levels (13,34,35). Given that in the efr3, ypp1, and stt4 deletion mutants there was a reduction in PI(4)P levels, we examined whether PS plasma membrane levels were affected using a fluorescent reporter that preferentially binds this acidic phospholipid (36). In wild-type cells, we observed this LactC2-GFP reporter localized predominantly at the plasma membrane, with little to no internal signal observed (Fig. 8A). In contrast, in the efr3, ypp1 and stt4 mutants, a perinuclear signal was observed, characteristic of the ER, yet plasma membrane PS was still apparent in each of these mutants (Fig. 8A). Quantification of signals from central z-sections using the Matlab program Hyphal-Polarity (14) confirmed that the ratio of mean plasma membrane signal to mean internal signal decreased progressively in the efr3, ypp1, and stt4 mutants, resulting in part from a progressive increase in internal PS signal (Fig. 8B). Similarly, the mean plasma membrane PS fraction also decreased progressively in the efr3, ypp1, and stt4 mutants, with the stt4 deletion mutant exhibiting an approximately 50% reduction compared to wild-type cells. The stt4 hypomorph, Stt4[G1810D], had plasma membrane PS levels intermediate between those of the efr3 and ypp1 mutants. Together, these results suggest that sufficient PI(4)P is critical for the transport of PS from the ER to the plasma Role of Plasma Membrane PI4P in C. albicans ® membrane. We next examined if there was a correlation between the plasma membrane PI(4)P and PS levels. Figure 9 shows that there is a direct correlation between the levels of these two lipids in the different deletion mutants and the wild-type strain. Plasma membrane lipid levels in the Stt4[G1810D] mutant were also consistent with such a correlation. Note that while we were unable to detect plasma membrane PI(4)P in the stt4 deletion strain, approximately 50% of the plasma membrane PS was detectable in this mutant. Furthermore, plasma membrane PS levels in the efr3 mutant were not dramatically different from that of the wild-type cells. Together, our results suggest that the filamentation and cell wall integrity defects observed in the three Stt4 complex mutants are likely to be due to lack of plasma membrane PI(4)P and not PS. (B and C) Quantitation of plasma membrane and internal signals reveals that plasma membrane PI(4,5)P 2 is largely unaffected in the absence of Efr3, Ypp1, and Stt4. The ratio of plasma membrane to internal signal and the relative plasma membrane signal were determined as for Fig. 5B and C (15 to 20 cells; 2 experiments for the wild type [WT]). For the wild type, the mean ratio of plasma membrane to internal signal was 2.8 and the ratio of plasma membrane to total signal was 0.7. *, P , 0.02; **, P , 0.01; ***, P , 0.0005.
Stt4 PI-4-kinase complex localizes to cortical patches. To determine the distribution of Efr1, Ypp1, and Stt4, we generated 3Â-mScarlet fusions by tagging the chromosomal copy of the respective genes. These fusions were functional in that as a sole copy, they complemented the cell wall integrity defect of the respective mutants (Fig. S5). Despite the low abundance of these Stt4 PI-4-kinase complex subunits, we observed patches around the cortex of the mother cell and buds (Fig. 10A), which were also visible along the cortex of the germ tubes with reduced signals in the mother cell (Fig. 10B). In S. cerevisiae, YPP1 and EFR3 are critical for Stt4 membrane localization. Here, we observed that while Stt4 localization to the cortex is dependent (B and C) Quantitation of plasma membrane and internal signals reveals a progressive decrease in plasma membrane PS in efr3, ypp1, and stt4 strains. The ratio of plasma membrane to internal signal and the relative plasma membrane signal were determined as for Fig. 5B and C. For the wild type, the mean ratio of plasma membrane to internal signal was 4.5 and the ratio of plasma membrane to total signal was 0.8. Smaller symbols are values from two experiments (15 cells each); larger symbols are means from each experiment, with bars indicating overall means and standard deviations. ****, P , 0.0001.
on Ypp1 in C. albicans, in the efr3 mutant there were still some cells with cortex-localized Stt4 (Fig. 10C). Efr3 cortex localization depended upon Ypp1 and Stt4, with loss of cortex signal observed in either mutant. In contrast, there were some cells with YPP1 cortex signal in the efr3 mutant but not in the stt4 mutant. In this efr3 mutant, 40 to 50% of cells exhibited punctate localization of Stt4 and Ypp1. In the ypp1 mutant, we did not observe cells with either Stt4 or Efr3 localized. Finally, in the stt4 mutant, only ;10% of cells exhibited punctate localization of Ypp1. RT-PCR revealed that the mScarlet transcript levels in all mutants were similar to that of the wild type (Fig. S6). As these results suggested that Ypp1 and Stt4 were the most critical components of this PI-4-kinase complex, we examined whether these proteins colocalized to the same cortical patches. Figure S7 shows that, in a strain expressing Ypp1-mTurquoise and Stt4-3xmScarlet, there was only limited colocalization of these two proteins, suggesting that, if they form a complex, it is likely to be transient.
Plasma membrane PI(4)P is critical for virulence. To investigate the importance of plasma membrane PI(4)P in virulence, we examined the efr3, ypp1, and stt4 mutants in two murine infection models, HDC and oropharyngeal candidiasis (OPC). As C. albicans responds to cues, such as the presence of serum, by the induction of hypha-specific genes (HSGs), many of which are critical for virulence, we initially examined HSG levels after 30 and 120 min incubation with serum. Figure 11A shows that the stt4 deletion mutant induced a range of HSGs, including ECE1, HGC1, HWP1, ALS3, and SAP4 to -6, 10 3 -to 10 5 -fold (excluding SAP4 to -6), similar to the wild-type strain, with only a 5fold reduction in induced transcript levels observed after 30 min incubation. In the HDC model, 20% and 100% of mice infected with the ypp1 and stt4 mutants, respectively, survived 2 weeks after injection, while all of the mice infected with the efr3 mutant or wild-type strain died within 5 to 6 days (Fig. 11B). The virulence was significantly restored in complemented strains (Fig. 11B). Furthermore, we examined whether the stt4 deletion mutant could form filaments after long incubation times in serum or in kidney homogenate (37). After 6 h incubation with either serum or kidney homogenate, despite observing elongated cells, the stt4 mutant did not form hyphal filaments, compared to wild-type and complemented strains (Fig. S8). It is unlikely that the unmasking of cell surface b(1,3)-glucan and these virulence defects are due to altered plasma membrane PS levels, as a cho1D/cho1D::CHO1 strain with .50% reduction in PS levels had no increase in unmasked b(1,3)-glucan and exhibited full virulence in mouse models of systemic and oropharyngeal infection (27)(28)(29). In the OPC model, only the ypp1 mutant was substantially less virulent than the wild-type strain, with the oral fungal burden of the infected mice reduced by ;25-fold (Fig. 11C). Nonetheless, examination of the histopathology of tongue thin sections revealed that in infection lesions, all strains were able to form filaments (Fig. S9). Together, these data suggest that plasma membrane PI(4)P is required for virulence during hematogenously disseminated candidiasis, with a decrease in virulence observed upon a 60% reduction in plasma membrane PI(4)P and the lack of lethality observed in the absence of this phosphatidylinositol phosphate. Our data also indicate that only a small amount of plasma membrane PI(4)P is required for normal virulence during oropharyngeal candidiasis.
DISCUSSION
Our results show that plasma membrane PI(4)P is critical for the C. albicans yeast to filamentous growth transition and cell wall integrity. We show that all three members of the Stt4 PI-4-kinase complex are dispensable for viability yet are required for filamentous growth and cell wall integrity. Furthermore, quantitative analyses indicate that these mutants have decreasing levels of plasma membrane PI(4)P, from efr3 to ypp1 to stt4; a majority of the stt4 cells lack detectable PI(4)P at the plasma membrane. In addition, an stt4 hypomorph (25) had plasma membrane PI(4)P levels similar to those of the efr3 mutant. We observed little to no alteration in Golgi PI(4)P and plasma membrane PI(4,5)P 2 in these Stt4 PI-4-kinase complex mutants. Consistent with a link between plasma membrane PI(4)P and PS, there is a gradual increase in internal pools of PS in efr3, ypp1, and stt4 mutants, yet even in the stt4 mutant that lacks plasma membrane PI(4)P, PS is still detected at the plasma membrane. All three of these PI-4kinase complex proteins localize to cortical patches, but only Ypp1 and Stt4 appear to be critical for the complex formation. Furthermore, our results reveal that plasma membrane PI(4)P is important for masking cell surface b(1,3)-glucan but not for induction of a number of hyphal-specific genes. Plasma membrane PI(4)P is critical for pathogenicity during hematogenously disseminated candidiasis but less so for oropharyngeal candidiasis, suggesting that this lipid has different roles in distinct anatomic infection sites, which we attribute, in part, to host immune recognition via unmasked cell surface b(1,3)-glucan.
Using strains in which either the C. albicans PI-4-kinase, i.e., Pik1 at the Golgi apparatus or Stt4 at the plasma membrane, could be repressed, we previously showed that the Golgi PI-4-kinase is strictly required for invasive filamentous growth (13), whereas repression of the plasma membrane PI-4-kinase mutant resulted in cells that can still form short protrusions and invasive filaments (14). This repressible stt4 mutant had an ;10-fold reduction in STT4 transcript levels compared to a wild-type strain, yet PI(4)P was still detectable at the plasma membrane. To address the importance of plasma membrane PI(4)P in C. albicans, we generated homozygous deletion mutants in the PI-4-kinase complex, composed of Efr3, Ypp1, and Stt4. None of these PI-4-kinase components is essential for viability in C. albicans, in contrast to other fungi, specifically S. cerevisiae, where both Pik1 and Stt4 are essential (6,7,38,39). We speculate that the plasma membrane PI(4)P is required to maintain filamentous growth in C. albicans via contributions to cell polarity and membrane traffic. A. nidulans and C. neoformans appear to have only a Stt4 PI-4-kinase, which is essential for viability (4,5). In addition, although C. albicans stt4, ypp1, and efr3 mutants showed increased sensitivity to cell wall perturbants, they did not exhibit a temperature-sensitive growth defect, as was the case in S. cerevisiae, where a temperature-sensitive stt4 mutant lysed at the nonpermissive temperature (15). Our quantitative analyses revealed that the majority of C. albicans cells lacking Stt4 have undetectable levels of PI(4)P at the plasma membrane, suggesting that this lipid is dispensable for viability. Such C. albicans mutants with little to no plasma membrane PI(4)P are sensitive to cell wall perturbants and have thicker cell walls with increased levels of glucan and mannan, along with an increase in cell surface exposed b (1,3)-glucan, indicating that PI(4)P is critical for maintaining cell wall integrity. We speculate that plasma membrane glucan synthase activity is regulated by PI(4)P, via the Rho1 regulatory subunit (40). This raises the question of how PI(4,5)P 2 is generated in the stt4 deletion mutant, and we speculate that PI(4)P from the Golgi apparatus may be phosphorylated by Mss4. This could occur via Mss4 localization to the Golgi apparatus or secretory vesicles, although this kinase has not been detected in these compartments in C. albicans (14). Alternatively, a small amount of Golgi PI(4)P could reach the plasma membrane and be immediately phosphorylated.
Previous studies in S. cerevisiae indicated that the levels of PS and plasma membrane PI(4)P are linked. For example, an S. cerevisiae stt4 mutant with reduced levels of plasma membrane PI(4)P accumulates PS (7,25). Furthermore, an S. cerevisiae sac1 phosphatase mutant that results in a dramatic increase in PI(4)P (34,35) has decreased PS levels at the plasma membrane, with an increase in intracellular membranes (41). Similarly, in fibroblasts, PI-4-kinase IIIa knockouts have decreased plasma membrane PS (42). The S. cerevisiae oxysterol proteins Osh6 and Osh7 have been shown to exchange PS for PI(4)P in vitro, and in vivo, a sac1 mutation resulted in a redistribution of added lyso-PS, which is normally at the plasma membrane, to the ER (32). It was proposed that a PI(4)P gradient from the plasma membrane to the ER drives PS transport via Osh6/7 from the ER to the plasma membrane (32). Given that C. albicans cells are viable with little to no plasma membrane PI(4)P, it is likely that this so-called "phosphoinositide-motive force" is not essential (43). Nonetheless, we observed a progressive increase in intracellular PS in C. albicans mutants with decreasing plasma membrane PI (4)P, demonstrating the plasma membrane PI(4)P is critical for PS transport to the plasma membrane. This result indicates that Osh6/7 counter-transporters account for roughly half of the plasma membrane PS and suggests that the remaining PS may be delivered to the plasma membrane via vesicular traffic.
In S. cerevisiae, Stt4 and Ypp1 and Efr3 and Ypp1 colocalize in cortical patches (18), and the latter interaction has also been observed by bimolecular fluorescence complementation (44). In mammalian cells and fission yeast, Stt4, Ypp1, and Efr3 also form a complex observed by coimmunoprecipitation and colocalization (8,19,45,46). Here, we show that all three Stt4 PI-4-kinase complex proteins localize to cortical patches during budding and hyphal growth, yet we did not observe substantial colocalization between Ypp1 and Stt4 proteins, although each protein was critical for the cortical patch localization of the other protein. This suggests that Ypp1 is important for targeting and/or stabilization of the Stt4 PI-4-kinase, as has been observed in S. cerevisiae (18). Interestingly, although Efr3 is important for cortical patch localization of Ypp1 and Stt4 proteins, it is not absolutely required as in S. cerevisiae (18). These results suggest that Efr3 facilitates targeting and/or stabilization of the complex, but that plasma membrane PI-4-kinase activity is not strictly dependent on it.
Cell wall defects in mutants lacking the PS synthase Cho1, which have little to no PS, are in some respects similar to those of stt4 deletion mutant cells. For example, a C. albicans cho1 deletion mutant also had a thicker cell wall and exhibited increased sensitivity to the antifungal drug caspofungin (27). However, this cho1 mutant had a dramatic increase in cell wall chitin levels (27,30,47), in contrast to a mutant lacking plasma membrane PI(4)P. Interestingly, both stt4 and cho1 deletion mutants exhibited an increase in exposed cell wall b(1,3)-glucan, with the latter mutant having a roughly 10-fold-greater increase in this polysaccharide (30) than the stt4 mutant. However, given that a cho1D/ cho1D::CHO1 strain with .50% reduction in PS levels had no increase in unmasked b(1,3)-glucan (27,28), it is unlikely that the cell wall defects observed in the mutant lacking plasma membrane PI(4)P is due to the reduced PS levels.
The importance of lipids with respect to C. albicans virulence has been challenging to determine, as a number of lipids are essential for viability and cell growth. One lipid that has been shown to be critical for pathogenicity in a range of different fungi is the sphingolipid glucosylceramide (48)(49)(50)(51). Furthermore, mutants lacking either the PS synthase Cho1 or PS decarboxylases (Psd1 and Psd2) were avirulent in murine models of systemic candidiasis and oropharyngeal candidiasis (27,29). However, both of these mutants grew substantially more slowly than wild-type strains and exhibited an ;50% reduction in phosphatidylethanolamine (PE) levels (27). Expression of a heterologous serine decarboxylase revealed that the observed virulence defects, as well as b(1,3)glucan unmasking, were in fact due to the ethanolamine auxotrophy of these mutants (29). Using an in vitro assay for host pathogen interactions, an ;2-fold reduction in macrophage lysis was observed with an stt4 mutant (17), suggesting that this kinase may be important for virulence. Similarly, the filamentation defect and increased exposure of cell surface b(1,3)-glucan in the plasma membrane PI-4-kinase deletion mutant indicated that PI(4)P has a role in virulence.
Interestingly, a recent study by Dunker et al. showed that rapid proliferation can compensate for the absence of filamentation in a murine model of systemic candidiasis, directly challenging the idea that filamentation is strictly required for virulence (37). Of the three Stt4 PI-4-kinase complex mutants, stt4, and to a lesser extent ypp1, exhibited virulence defects in a murine model of systemic candidiasis. Given that all Stt4 PI-4-kinase complex mutants are defective in filamentation, this decrease in virulence is unlikely to be attributable to a defect in morphogenesis. Indeed, elongated stt4 mutant cells were observed after long incubation times with serum or kidney homogenate, and hyphal gene induction was not substantially different from the wild-type strain after 2 h incubation in serum. Furthermore, in a murine model for oropharyngeal candidiasis, all Stt4 PI-4-kinase complex mutants were able to filament. Our results, however, indicate a correlation between PI(4)P at the plasma membrane and systemic candidiasis, given that the ypp1 mutant and the stt4 mutant have a 3-fold or greater decrease in plasma membrane PI(4)P. As the Stt4 PI-4-kinase complex mutants all have altered cell wall integrity, substantial alterations in the level of plasma membrane PI(4)P lead to corresponding changes in the cell wall, in particular, exposure of masked b(1,3)-glucan, which is recognized by host immune cells, ultimately contributing to the reduction in virulence in the systemic candidiasis model. Consistent with this explanation, the stt4 deletion mutant exhibited little to no defect in an OPC infection assay, in which the host immune response is less critical. It will be interesting to further analyze the cell wall defects in these Stt4 PI-4-kinase complex mutants given their different levels of plasma membrane PI(4)P, which will be useful tools in dissecting how plasma PI(4)P regulates the cell wall during fungal infection.
MATERIALS AND METHODS
Strain and plasmid construction. Standard methods were used for C. albicans cell culture and for molecular and genetic manipulations. Strains were grown in yeast extract-peptone dextrose (YEPD) at 30°C unless otherwise indicated, and induction of filamentous growth was carried out with 50% serum at 37°C for 90 and/or 120 min. To determine doubling times, cells were grown in YEPD medium at 30°C and optical density was followed over 8 h of logarithmic growth. For induction with kidney homogenates, kidneys from male C57BL/6 mice were aseptically removed and Dounce homogenized with sterile phosphate-buffered saline (PBS; 0.4 g/mL), and this homogenate was diluted 1:1 with logarithmically growing C. albicans strains in YEPD. Serial dilutions of the different strains on YEPD plates containing Congo red (400 mg/mL), calcofluor white (25 mg/mL), caspofungin (50 and 125 ng/mL), and fluconazole (10 mg/mL) were examined after 2 to 3 days of incubation at 30°C (52). The strains and plasmids used are listed in Table S1A and B, respectively, and the oligonucleotides used are listed in Table S1C. All strains are based on the BWP17 background (53). The efr3D/efr3D and ypp1D/ypp1D strains were generated by homologous recombination. Each copy was replaced by either HIS1 or URA3 using knockout cassettes generated by amplification from pGemHIS1 and pGemURA3 (53) with the primers CaEfr3pKO/ CaEfr3mKO and CaYpp1pKO/CaYpp1mKO. In order to generate prototrophic strains, the pExpARG plasmid was linearized with StuI and integrated into the RPS1 locus. pExpARG-pEFR3-EFR3 and pExpARG-pYPP1-YPP1 plasmids were constructed by amplification of gDNA using primers with a unique XhoI site at the 59 ends and a unique NotI site at the 39 ends, with 1 kb upstream and downstream of the respective open reading frames (ORFs). These fragments were subsequently cloned in pExpARG, yielding pExpARG-pEFR3-EFR3 and pExpARG-pYPP1-YPP1, respectively. Finally, pExpARG-pEFR3-EFR3 and pExpARG-YPP1-YPP1 were integrated into the RPS1 locus, yielding the efr3D/efr3D RPS1::EFR3p-EFR3 and ypp1D/ypp1D RPS1::YPP1p-YPP1 recovery strains, respectively.
To generate mutants, we first generated an stt4-DAmP (decrease abundance by mRNA perturbance) allele by integration of the SAT1 gene (via amplification of pFASAT1 [54] with the primers CaSATDAmPpS1 and CaSATDAmPmS2) 59 of the STT4 stop codon. The remaining STT4 copy was replaced with URA3 using a knockout cassette generated from amplification of pGemURA3 (53) with primers CaStt4pKO and CaStt4mKO. As the stt4D/stt4DAmP strain behaved identically to the wild type, we next generated an stt4D/ Role of Plasma Membrane PI4P in C. albicans ® stt4D strain (PY4377) by replacing the allele stt4-DAmP with HIS1 using a knockout cassette amplified from pGemHIS1 (53) with the primers CaStt4pKO and CaSATDAmPmKO. The URA3 gene of the stt4D/stt4D strain (PY4377) was then replaced with SAT1, which was amplified from pGFP-Nat (55) using the primers CaURApSAT and CaURAmSAT, so that URA3 could be subsequently integrated at the RPS1 locus for murine HDC assays. To generate this URA 1 strain, pExpURA3 (56) was linearized and integrated in the RPS1 locus, yielding PY5040, which was subsequently rendered ARG 1 by transformation with linearized pExpARG plasmid, yielding PY5111. In order to reintegrate STT4 at the STT4 locus, we generated a pSTT4-STT4-STT4t cassette in which unique AscI and PmeI sites were inserted into the STT4 terminator (623 bp 39 of the stop codon) by site-directed mutagenesis using the primers CaStt4term_mAscIPmeI and CaStt4term_pAscIPmeI. Subsequently, ARG4 was amplified from pFAARG4 using the primers CaARG4AscI-S1 and CaARG4PmeI-S2, yielding plasmid pExpARG-STT4-STT4-STT4t::ARG4. To generate the recovered strain, the pSTT4-STT4-STT4t:: ARG4 fragment (digested by XhoI and NotI) from this plasmid was used to replace stt4::HIS1 in PY5040, resulting in stt4D/stt4D::STT4, PY5119. To generate a prototroph recovered strain, HIS1 was added back using linearized pGemHIS1, resulting in PY5131. To generate the hypomorph stt4 mutant, which encodes Stt4[G1810D], site-directed mutagenesis was carried out with pExpARG-pSTT4-STT4-STT4t::ARG4 using primers CaStt4G1810DpEcoRV and CaStt4G1810DmEcoRV, resulting in pExpARG-STT4p-STT4*-STT4t::ARG4, which was subsequently digested with XhoI and NotI and transformed into PY5040, resulting in PY5757.
Southern blot analyses, RT-PCR, qRT-PCR, and chitin staining. For Southern analysis, EcoRV-digested gDNA was separated on a 1% agarose gel, transferred to a nylon membrane, and fixed by UV cross-linking as described elsewhere (36). The hybridization probes were generated by PCR using primers (for STT4, CaStt4p5199 and CaStt4m5543; for URA3, CaUra3pXhoI and CaURA3m81) and an Amersham ECL Direct nucleic acid labeling and detection system kit (GE Healthcare UK Ltd., Little Chalfont, Buckinghamshire, UK) following the manufacturer's instructions. For RT-PCR and quantitative RT-PCR (qRT-PCR), primers used (Genep-TM and Genem-TM) are listed in Table S1C, and RNA extraction was carried out using a Master Pure yeast RNA extraction purification kit (Epicentre) from budding cells and cells incubated with serum. For RT-PCR actin amplification, 30 cycles were used, and 32 cycles were used to amplify EFR3, YPP1, STT4, PIK1a, MSS4, SAC1, and the mScarlet gene. qRT-PCR analyses were carried out as previously described (36) with indicated primers (Genep-TM and Genem-TM; Table S1C). For chitin staining exponentially growing cells were fixed, stained with 25 mg/mL calcofluor white solution, and imaged using a spinning disk confocal microscope (58).
Microscopic analyses. For colony morphology analysis plates were incubated for 3 to 6 days prior to imaging (36). mScarlet and mTurquoise fusions were imaged as described elsewhere (57, 58) with 17 0.5-mm z-sections. Quantitation of plasma membrane and internal mean signals was performed on central z-sections using the Matlab program Hyphal-Polarity (13). Ratios of plasma membrane to internal signals were normalized to the mean wild-type ratio. Plasma membrane fractions of PI(4)P, PI(4,5)P 2 , and PS were calculated as the ratio of plasma membrane signal to total signal, which was then normalized to the mean wild-type ratio. To represent these values between 1 and 0, 0.5 was subtracted from the normalized ratio (the Matlab program detects first signal going in from the region of interest [ROI] above background, which is the cytoplasm when there is no plasma membrane localization; hence, a value of 0.5 is the absence of plasma signal) and then multiplied by 2 so that the value for the wild-type plasma membrane fraction is 1 and the value for no plasma membrane signal is 0. Huygens professional software version 18.04 (Scientific-Volume Imaging) was used for deconvolution of image z-stacks using the appropriate settings for the microscope and excitation source. The signal-to-noise ratio was set to 10, and the background detection was set to auto, unless otherwise stated.
Virulence assays. HDC was induced in 10 BALB/c mice (Charles River, Italy) per group by injecting the lateral tail vein with an inoculum of 5 Â 10 5 cells (59). Animal body weight was monitored daily, and animals were sacrificed by cervical dislocation when they had lost more than 20% of their weight. OPC was induced in mice that had been immunosuppressed with cortisone acetate using 7 to 8 mice per strain as previously described (60). A Vectra Polaris slide scanner was used to scan histopathology of murine tongue thin sections, stained with periodic acid-Schiff stain.
Ethics statements. All OPC animal experiments were approved by the Institutional Animal Care and Use Committee (IACUC) of the Lundquist Institute at Harbor-UCLA Medical Center. All HDC animal procedures were approved by the Bioethical Committee and Animal Welfare of the Instituto de Salud Carlos III (CBA2014_PA51) and of the Comunidad de Madrid (PROEX 330/14) and followed the current Spanish legislation (Real Decreto 53/2013) along with Directive 2010/63/EU. Statistical analysis. Differences in mean signals, ratios, and percentages of filaments were analyzed by t test, and survival experiments with mice were analyzed by the Kaplan-Meier method (log-rank test) with GraphPad Prism 8 (GraphPad, La Jolla, CA).
Transmission electron microscopy. Budding cell samples were processed for electron microscopy and images were acquired as described elsewhere (61). Cell wall thickness was measured from electron micrographs at 3 or 4 locations around the mother cell and averaged.
SUPPLEMENTAL MATERIAL
Supplemental material is available online only. | 2022-02-15T14:14:39.003Z | 2022-02-15T00:00:00.000 | {
"year": 2022,
"sha1": "41a520e565c4a6be30fbcc6e39074a85c6819e5e",
"oa_license": null,
"oa_url": null,
"oa_status": null,
"pdf_src": "ASMUSA",
"pdf_hash": "41a520e565c4a6be30fbcc6e39074a85c6819e5e",
"s2fieldsofstudy": [
"Biology",
"Medicine"
],
"extfieldsofstudy": [
"Medicine"
]
} |
10673321 | pes2o/s2orc | v3-fos-license | Some Recollections of the American Association for the Study of Liver Diseases Transformative Decade 1970‐1979
For the inaugural issue of Hepatology Communications, the new Editor-in-Chief has asked me to write an article for the Members Corner to include recollections of personal experiences from my time of greatest activity in the American Association for the Study of Liver Diseases (AASLD). This is to be an informal message (no references are cited) aimed mainly at the newer members of this, our favorite, scientific organization who have not had an opportunity to know, as I have, some of the wonderful and remarkable hepatologists and other scientists who created this new discipline in medicine. My principal focus will be on the decade of the 1970s when I served on the Governing Board, a time when AASLD had become ripe for change. It had begun as a small private group of friends meeting in 1948 in the library of the Hektoen Institute for Medical Research of the Cook County Hospital in Chicago, hosted by Dr. Hans Popper. The Macy Conference on liver injury of 1943 stimulated a postwar series of informal exchange of ideas among scientists at meetings of the Central Society for Clinical Research at the Drake Hotel in Chicago. The issues concerned the problem of viral hepatitis in the armed forces in World War II and applying serum enzymology to diagnose liver injury. At a second Hektoen meeting held in November 1949, which was attended by 46 scientists, Popper led the group to agree on a formal organization to begin in 1950 as the Association for the Study of Liver Diseases (ASLD), with Leon Schiff its first president, Fred Hoffbauer vice-president, Robert Kark treasurer, and himself as secretary. Dr. Popper is shown in Fig. 1 at his favorite work as an hepatic pathologist. ASLD quickly grew to exceed the space in the Hektoen library, and later meetings were held elsewhere in Chicago. The organization in 1953 was renamed to add “American” to indicate clearly that it was a national society. Of the founders (Table 1), Hans Popper was the early driving force, but he remained as secretary for many years and only gave in to collegial pressure to become president in 1963. He kept minutes, appointed councilors, and generally ran the meetings behind the scenes, while a succession of his friends and trainees were ostensibly in the chair. He recounted his views of the early history of the formation and startup of the AASLD in Hepatology in
F or the inaugural issue of Hepatology Communications, the new Editor-in-Chief has asked me to write an article for the Members Corner to include recollections of personal experiences from my time of greatest activity in the American Association for the Study of Liver Diseases (AASLD). This is to be an informal message (no references are cited) aimed mainly at the newer members of this, our favorite, scientific organization who have not had an opportunity to know, as I have, some of the wonderful and remarkable hepatologists and other scientists who created this new discipline in medicine.
My principal focus will be on the decade of the 1970s when I served on the Governing Board, a time when AASLD had become ripe for change. It had begun as a small private group of friends meeting in 1948 in the library of the Hektoen Institute for Medical Research of the Cook County Hospital in Chicago, hosted by Dr. Hans Popper. The Macy Conference on liver injury of 1943 stimulated a postwar series of informal exchange of ideas among scientists at meetings of the Central Society for Clinical Research at the Drake Hotel in Chicago. The issues concerned the problem of viral hepatitis in the armed forces in World War II and applying serum enzymology to diagnose liver injury. At a second Hektoen meeting held in November 1949, which was attended by 46 scientists, Popper led the group to agree on a formal organization to begin in 1950 as the Association for the Study of Liver Diseases (ASLD), with Leon Schiff its first president, Fred Hoffbauer vice-president, Robert Kark treasurer, and himself as secretary. Dr. Popper is shown in Fig. 1 at his favorite work as an hepatic pathologist.
ASLD quickly grew to exceed the space in the Hektoen library, and later meetings were held elsewhere in Chicago. The organization in 1953 was renamed to add "American" to indicate clearly that it was a national society. Of the founders (Table 1), Hans Popper was the early driving force, but he remained as secretary for many years and only gave in to collegial pressure to become president in 1963. He kept minutes, appointed councilors, and generally ran the meetings behind the scenes, while a succession of his friends and trainees were ostensibly in the chair. He recounted his views of the early history of the formation and startup of the AASLD in Hepatology in December 1982, and details of his life were outlined in his obituary in that journal by Rudi Schmid and Steve Schenker in November 1989.
Each of these men deserves a special article for his work and interests, but it is notable that they worked in different disciplines when hepatology was still being thought of as a branch of gastroenterology. From the beginning, as reflected in the minutes of the Council (later called the Governing Board) meetings, they worked very closely together in discussing issues, planning meetings, and building interest in hepatology.
The work of the founders was followed by a period of development and further growth in the 1960s. I had started attending annual meetings in 1959 when working with Kurt Isselbacher at the Massachusetts General Hospital on the mechanisms of small intestinal absorption of fats. In 1966, I became official AASLD member #434 and was chosen (by Popper) to be a new councilor at the 1969 meeting. The Developers (Table 2) in the late 1960s solidified the principles and facilitated growth of the little club into an international scientific society. They formulated a new constitution specifying a small Governing Board, including a president, vice-president, secretary, treasurer, and three councilors, that was approved by the membership in 1972. Service on the Board was for a nominal 9 years: 3 years as a councilor, followed by 1 year each as vicepresident, president, and past-president, and 3 years on what was called the Scope Committee, formerly called the Steering Committee. The Board composition changed each year, usually with one person leaving and a new person coming in (Table 3). Consequently, members became very well acquainted with each other. I was closely acquainted on the Board with the four presidents from years 1966-1969, ten presidents from years 1970-1979, and four councilors elected for 1976-1979 (Jim Boyer, Steve Schenker, Willis Maddrey, Lee Forker). Of special note, Bill Summerskill, selected to serve the year after I served, died suddenly and unexpectedly in March 1977, and I was asked to fill his unexpired terms. Thus, I actually served 10 years on the Governing Board and worked on it with a total of 18 men (no women yet at that time) who at some time were presidents of the AASLD. I knew personally all 10 founding and next 10 developing presidents. This was an extraordinary experience indeed. The gradual progression from selection as a new councilor, through the presidency, and afterward on the Scope Committee allowed a full appreciation of trends and policy changes as they evolved (Table 4).
Around 1972, as we listened to the arguments and discussion at Board meetings, Bill Summerskill (Fig. 2) and I grew impatient to initiate change when our time came for authority to do so. For those who did not know him, Bill was an outstanding hepatologist chosen as a councilor for the Governing Board in 1970, and we quickly found that we agreed on what needed to be done. He had wide interests and was culturally diverse. He credited Shakespeare with the first description of hepatic encephalopathy in the alcoholic Sir Andrew Aguecheek, who believed his wits were harmed by eating too much beef. In 1959 he was invited to develop a new gastroenterology unit at the Mayo Clinic and subsequently built one of the best gastroenterology departments in the world. He continued in active research, particularly on chronic hepatitis and the treatment of cirrhosis, and was the author of some 130 original papers and many chapters in monographs and textbooks. It is perhaps paradoxical that one so remarkably English should have made his greatest mark in the Midwest of the United States. At the time of his death, aged only 51 years, Bill Summerskill was directing the gastroenterology unit as a professor of medicine at the Mayo Medical School, University of Minnesota. We planned to implement several much-needed changes in AASLD operations, beginning in 1974 when I was president and he the vice-president. This also happened to be the twenty-fifth anniversary year. The changes we co-conspired to envision were: Aim 1) At that time, there was no money available, and the annual budget when Dr. Harold Conn was president was only about $5,000. Space at the aging and crumbling Chicago Sheraton Hotel was not suitable. We arranged a move to the Ambassador East and West, also in Chicago, that had sufficient space and was glad to have a new organization reserve space for 2 years.
My inquiry into what it would cost to set up a CPC as the first course, obtain tissue blocks, make up glass slides, print clinical narratives, distribution, and advertising came to about $30,000. If we set a registration fee at $50, it would require 600 paid registrants. Brashly, I borrowed that amount from a local bank, and we embarked on setting it up in 1973. Our faculty for teaching was superb, including Hans Popper, Gerald Klatskin, Dame Sheila Sherlock, and others of our distinguished members. The course was a great success, with about 900 paid registrants and an income of $45,000 that paid off the loan and all our bills and left $15,000 as a nest egg for the second course by Bill Summerskill in 1975. These annual postgraduate courses have continued to the present, to the excellent program for 2016 organized by Anna Lok and Marc Ghany.
Aim 2) The second new objective was to establish a lay foundation for money raising to support research and patient education. A development committee chaired by Burt Combes (Fig. 3), who had been the AASLD president in 1972 and was serving as a junior member of the Scope Committee in 1974-1975, nominated him and he agreed to direct that aim.
Burt was born in New York City in 1927, went to college and medical school at Columbia, did his residency and a research fellowship at Presbyterian Hospital, and continued his initial scientific studies at University College Hospital Medical School in London. Burt joined the emerging University of Texas Southwestern Medical School faculty where he established and directed the liver section of the gastroenterology program. Burt was a superb clinician deeply devoted to the care of sick patients. He was characteristically soft spoken, erudite, logical, and very persuasive. His initial studies dealt with the metabolism of various forms of bromosulfophthalein as a marker of membrane transport. His more clinical studies were to define prognostic factors in acute liver failure. On the national scene, Burt was instrumental in developing the guiding principles for collaboration between the academic community and interested lay public in promoting research in liver disease; this led to starting the American Liver Foundation (ALF) in 1976. When he retired in 2009, control of the ALF had drifted away from the AASLD to an independent group now headquartered in New York City and a new foundation, the American Liver Foundation, was initiated. Its genesis may be described elsewhere or later in this new publication. Burt died November 23, 2013. Aim 3) A new journal, Hepatology, was to be developed under the dynamic leadership of Win Arias (Fig. 4). He chaired the publication committee and served as the first editor. As described by him in 2006 in his reflections on the first 5 years of Hepatology, 1981-1986, he outlined the process used to start a new journal from scratch and make it a success-which it has been. Ada Gelnick, his editorial assistant, quipped that "creating a new journal is probably the closest a man can get to parturition." Win is still very active in research as a Senior Scientist, National Institutes of Health, at age 90.
Others may judge the wisdom of the three transformative projects undertaken in the mid to late 1970s, but they appear to have been very successful. A final note is the logo (Fig 5), which was taken from a little diagram I scrawled on the back of an envelope some unknown date back then. | 2018-04-03T03:55:37.758Z | 2017-02-01T00:00:00.000 | {
"year": 2017,
"sha1": "b2c972d1c8fed6d4752757fefb3ef7c5c7806bf9",
"oa_license": "CCBYNCND",
"oa_url": "https://aasldpubs.onlinelibrary.wiley.com/doi/pdfdirect/10.1002/hep4.1016",
"oa_status": "GOLD",
"pdf_src": "PubMedCentral",
"pdf_hash": "b2c972d1c8fed6d4752757fefb3ef7c5c7806bf9",
"s2fieldsofstudy": [
"Medicine"
],
"extfieldsofstudy": [
"Medicine"
]
} |
247502785 | pes2o/s2orc | v3-fos-license | Iron Ore Tailings Dewatering: Measurement of Adhesion and Cohesion for Filter Press Operation
Globally, mining operators focus increasingly on tailings filtration to recover process water and store tailings more safely. Generally, required water contents below 20-w% are reached by using filter presses. To maintain high efficiency, complete cake detachment is needed because incomplete discharge reduces plant performance. However, filter cake discharge can occur in different ways, mainly influenced by adhesion of the filter cake to the filter cloth as well as by cohesion of the cake. Therefore, this study points out different major detachment behaviors and a theoretical approach to describe them. Furthermore, investigations on iron ore tailings filtrations were carried out to show the influences of different filter media, different filtration pressure and cake post-treatment on adhesion and cohesion.
Introduction
Due to the steadily growing demand for raw materials, the amount of mined material is also increasing [1]. Therefore, metal ores are extracted in large open pit mines around the world. Since only a small fraction of the mined rock consists of valuable product [2], an increasing amount of waste rock has to be handled, which is present as tailings at the end of the process. However, tailings management has been a major challenge for the industry for some time now [3]. Storage in tailings ponds has been increasingly replaced by dry storage (dry stacked tailings) due to process water loss and the risk of dam breaks. Increasing mechanical dewatering using thickeners and subsequent filtration can reduce the water content of the tailings, thus recovering a large part of the process water and enabling dry stacking [4]. Improved process water management and safer storage reduce costs and are an impossrtant aspect in terms of sustainable mining. For this reason, the number of filtered tailings solutions is steadily increasing [5] and larger and larger plants are being operated [6].
Due to the process-related characteristics of the tailings, their filtration is non-trivial and an existing challenge in solid-liquid separation. During the processing of valuable material, rock has to be crushed to a particle size below 100 µm. However, the particle size distribution is broad, and there is a significant clay content in the lower micrometer range [7]. These particles form a compressible filter cake [8] and influence the filtration process drastically. Therefore, high pressure difference in the form of mechanical pressure is crucial for dewatering. From an engineering point of view, chamber filter presses are suitable for this purpose [9]. These are operated in batch mode, i.e., they must be regenerated after each filtration. This happens by opening the individual press chambers, followed by detachment of the cakes from the filter medium caused by their weight and falling down on a conveyor belt placed below the filter press. Economical handling of
Theory
Analogous to the time sequences of the tailings filtration process in chamber filter presses, this section provides a brief insight into the relevant fundamentals and interrelationships of cake filtration. Furthermore, the properties of the network of solid particles after filtration are discussed. Finally, the prerequisites for cake detachment are outlined.
Filtration
Recessed plate filter presses operate on the principle of cake filtration. At the beginning of filtration, particle breakthrough occurs because the pores of the filter medium are usually selected to be larger than the particle diameter in order to reduce hydraulic resistance. If the particle concentration is sufficient, bridging over the pores occurs after a short time and afterwards the approaching particles are progressively deposited on the network of solid particles. Thus, the cake grows. Since the pressure drop within the cake is reciprocal to the pressure of the consolidated network of solid particles, mechanical support is provided behind the filter cloth by means of backing cloths or drainage structures.
The filter cake is a network of solid particles whose structure and properties are strongly dependent on the particle size distribution of the slurry. Clay-sized particles, for example, cause compressible behavior [8]. Normally, tailings have a relevant content of clay and corresponding filter cakes are therefore compressible [5,7,[12][13][14]. In order to ensure achieving crucial residual moisture contents, compaction of the network of solid particles by sufficient filtration pressure is necessary. For this reason, filter presses are used in a lot of applications [5,9,10,15,16]. Furthermore, mechanical dewatering by applying gas differential pressure can be implemented in filter presses with little constructive effort to reduce cake moisture by decreasing pore saturation even more. The adjusted differential pressure dewaters filter cake pores with a corresponding and lower capillary entry pressure [17,18].
The strength of a wet network of solid particles depends strongly on its saturation S (proportion of voids filled with liquid to entire void volume). In the saturation range between 0.3 and 0.9, liquid bridges are present, and capillaries are increasingly filled, i.e., capillary forces act; therefore, the largest tensile forces can be transmitted [19][20][21]. A maximum is to be expected for S = 0.8 to 0.9 [22,23].
Approach to Describe Cake Detachment
For the description of the detachment behavior of filter cakes, four stresses are relevant [21], which are shown in Figure 1. A distinction must be made between adhesion as the stress between two different systems (e.g., filter medium and cake) and the cohesion of the cake itself. In each case, a further subdivision can be made into shear stress τ and tensile stress σ. The volume on which the force acts is depicted in blue, whereas the surface associated with the stress is shown in orange. The sketched stresses and their abbreviations in this paper are as follows: (1) Shear adhesion (adhesion (shear)): τ Cloth .
(4) Tensile cohesion (cohesion (tensile)): σCake. Shear adhesion causes the sticking of filter cakes to filter media and must be overcome for cake detachment [21]. Corresponding to the apparatus used this can, e.g., be proceeded by compressed air blowing (vacuum disc filters), back washing (candle filters), filter media movement (tower filter presses) or gravity (chamber filter presses).
The driving force for cake detachment in tailings filtration in chamber filter presses is the weight force acting on the cake. The condition of complete detachment requires the following relationship in Equation (1), which assumes sufficiently high cohesion of the cake and an infinitely extended plate, i.e., neglecting support at the sealing edge and other chamber structures as well as detachment of the cake with subsequent wedging between two adjacent plates. Besides cake dimensions (thickness TCake, width WCake and height HCake), its porosity ε and saturation S, as well as the density of the solid ρSolid and the fluid ρFluid, are decisive for the weight force.
TCake · WCake · HCake · ((1 − ε) · ρSolid + ε · S · ρFluid) · g = FGravitation > FAdhesion (1) A common problem is an incomplete cake detachment (complete sticking or partial detachment). There are several cake detachment scenarios where the loading parameters mentioned in Figure 1 are crucial. Figure 2 shows these scenarios. It can be divided between the requested detachment (a) and the problematic cases (b-d). In detail, these can be described as follows: (a) The weight of the cake is sufficient. The cake falls off in one piece. Assumption: cohesion sufficient, no breaking. (b) The weight force is too low; the cake adheres. Assumption: cohesion sufficient, no breaking. (c) The tensile stresses transmitted during plate moving rupture the cake. If there are several fractures, partial falling may occur. Assumption: insufficient shear strength of the cake and low weight force. (d) A higher adhesion (tensile) than cohesion (tensile) splits the cake. If there are several fractures, partial falling may occur. Assumption: low weight force and local saturation/compaction differences. Shear adhesion causes the sticking of filter cakes to filter media and must be overcome for cake detachment [21]. Corresponding to the apparatus used this can, e.g., be proceeded by compressed air blowing (vacuum disc filters), back washing (candle filters), filter media movement (tower filter presses) or gravity (chamber filter presses).
The driving force for cake detachment in tailings filtration in chamber filter presses is the weight force acting on the cake. The condition of complete detachment requires the following relationship in Equation (1), which assumes sufficiently high cohesion of the cake and an infinitely extended plate, i.e., neglecting support at the sealing edge and other chamber structures as well as detachment of the cake with subsequent wedging between two adjacent plates. Besides cake dimensions (thickness T Cake , width W Cake and height H Cake ), its porosity ε and saturation S, as well as the density of the solid ρ Solid and the fluid ρ Fluid , are decisive for the weight force.
A common problem is an incomplete cake detachment (complete sticking or partial detachment). There are several cake detachment scenarios where the loading parameters mentioned in Figure 1 are crucial. Figure 2 shows these scenarios. It can be divided between the requested detachment (a) and the problematic cases (b-d). In detail, these can be described as follows: (a) The weight of the cake is sufficient. The cake falls off in one piece. Assumption: cohesion sufficient, no breaking. (b) The weight force is too low; the cake adheres. Assumption: cohesion sufficient, no breaking. (c) The tensile stresses transmitted during plate moving rupture the cake. If there are several fractures, partial falling may occur. Assumption: insufficient shear strength of the cake and low weight force. (d) A higher adhesion (tensile) than cohesion (tensile) splits the cake. If there are several fractures, partial falling may occur. Assumption: low weight force and local saturation/compaction differences.
It can be taken advantage of the interdependency of the four stresses mentioned. A relationship between cohesion (tensile) and adhesion (tensile) (σ Cake /σ Cloth ) can be derived from the theory of contact points. Using a bulk solids mechanics approach, the ratio between cohesion (shear) and cohesion (tensile) (τ Cake /σ Cake ) can be determined. Therefore, measurements of shear adhesion and shear cohesion allow the determination of all relevant stresses. It can be taken advantage of the interdependency of the four stresses mentioned. A relationship between cohesion (tensile) and adhesion (tensile) (σCake/σCloth) can be derived from the theory of contact points. Using a bulk solids mechanics approach, the ratio between cohesion (shear) and cohesion (tensile) (τCake/σCake) can be determined. Therefore, measurements of shear adhesion and shear cohesion allow the determination of all relevant stresses.
Theory of Contact Points
Using close-packed structure theory, it is obvious that the number of contacts to a wall is smaller than the number of contacts within a network of solid particles. Based on theoretical approaches combined with measurements of real polydisperse systems, the ratio of tensile strength in an undersaturated wet network of solid particles to tensile strength of the same network to a wall can be approximated with 1.15 according to Douglas [24]. Translated into the application and the nomenclature of this investigation, it is referred to as the ratio between the transmissible tensile cohesion inside the cake and tensile adhesion between cake and cloth (σCake/σCloth) in the following Equation (2): However, it should be noted that a network of solid particles deviates from an ideal packing structure if it is compressible (e.g., tailings). Here, the state of compression (e.g., corresponding to filtration pressure) plays an important role. For compressible structures, a higher filtration pressure leads to an increase in the number of contact points, and it becomes closer to the approximation of ideal packings.
Theory of Contact Points
Using close-packed structure theory, it is obvious that the number of contacts to a wall is smaller than the number of contacts within a network of solid particles. Based on theoretical approaches combined with measurements of real polydisperse systems, the ratio of tensile strength in an undersaturated wet network of solid particles to tensile strength of the same network to a wall can be approximated with 1.15 according to Douglas [24]. Translated into the application and the nomenclature of this investigation, it is referred to as the ratio between the transmissible tensile cohesion inside the cake and tensile adhesion between cake and cloth (σ Cake /σ Cloth ) in the following Equation (2): However, it should be noted that a network of solid particles deviates from an ideal packing structure if it is compressible (e.g., tailings). Here, the state of compression (e.g., corresponding to filtration pressure) plays an important role. For compressible structures, a higher filtration pressure leads to an increase in the number of contact points, and it becomes closer to the approximation of ideal packings.
Bulk Solid Mechanics
In contrast to rheology describing stress states of flowing systems, bulk solid mechanics characterizes the flow behavior of dry or undersaturated wet networks of solid particles [25]. After consolidation, they deform or rearrange at certain yield stresses due to internal friction caused by particle contacts. The flow properties, especially of initial flow, are depending on several parameters, for example, solid volume fraction, applied normal stress and stress at preshearing [26].
Relationships and derivable quantities can be depicted in a plot out of experimental shear measurement data, which are referred to as the yield locus and schematically illustrated in Figure 3. Consolidated bulk material starts to deform or rearrange under stresses above the yield locus. Such networks (e.g., filter cakes) have a positive shear stress of σ = 0, which is referred to as cohesion (shear) in this paper. The tensile strength of cohesive networks at τ = 0 occurs for negative normal tensions, which is referred to cohesion (tensile) in this paper. The yield locus for tensile fracture ends vertical to the abscissa [27].
In contrast to rheology describing stress states of flowing systems, bulk solid mechanics characterizes the flow behavior of dry or undersaturated wet networks of solid particles [25]. After consolidation, they deform or rearrange at certain yield stresses due to internal friction caused by particle contacts. The flow properties, especially of initial flow, are depending on several parameters, for example, solid volume fraction, applied normal stress and stress at preshearing [26].
Relationships and derivable quantities can be depicted in a plot out of experimental shear measurement data, which are referred to as the yield locus and schematically illustrated in Figure 3. Consolidated bulk material starts to deform or rearrange under stresses above the yield locus. Such networks (e.g., filter cakes) have a positive shear stress of σ = 0, which is referred to as cohesion (shear) in this paper. The tensile strength of cohesive networks at τ = 0 occurs for negative normal tensions, which is referred to cohesion (tensile) in this paper. The yield locus for tensile fracture ends vertical to the abscissa [27]. The ratio of τCake/σCake describes the brittleness of the network of solid particles in simplified terms. As can be seen in the literature, a prediction of τCake/σCake is not possible. However, it can be stated that τCake/σCake is in general between one and five, but even ratios below one and up to 25 are reported [21]. Regarding undersaturated limestone particle networks, Rumpf mentioned τCake/σCake > 2 leads to a brittle breaking behavior [28]. Stitchproof-to-brittle properties are stated to be crucial for cake detachment [21], as well as the fact that cake texture changes in a small range of residual moisture, as reported by Tittel [29]. Generally, τCake/σCake increases due to the progressive decreasing of saturation of the network of solid particles [28]. However, slurries at concentrations of the gel point or higher are also able to transfer forces, e.g., wet filter cakes after filtration [30]. Since they theoretically have a saturation of one, this work extends the method of the yield locus for this specific case. This assumption is justified since in the literature brittle behavior is also mentioned for full-saturated networks of solid particles [21].
Shear Breaking
It is important to note that the acting forces are always proportional to the area where they are acting. This is to be exemplified in Table 1 by scenario c using realistic chamber dimensions (2 m · 2 m · 0.05 m) and a cohesion/adhesion ratio (σCake/σCloth) of 1.15 (see 2.3). If the shear strength of the filter cake is too low, the cake becomes ruptured by shearing due to the adhesion of the cake parts sticking to the cloths. The area ratio of the attaching cake part in relation to the shear area is important and is therefore defining a critical ratio of τCake/σCake needed for the avoidance of rupture by shearing. If τCake/σCake is sufficient (over 17.4), no rupture of the cake is possible. The ratio of τ Cake /σ Cake describes the brittleness of the network of solid particles in simplified terms. As can be seen in the literature, a prediction of τ Cake /σ Cake is not possible. However, it can be stated that τ Cake /σ Cake is in general between one and five, but even ratios below one and up to 25 are reported [21]. Regarding undersaturated limestone particle networks, Rumpf mentioned τ Cake /σ Cake > 2 leads to a brittle breaking behavior [28]. Stitch-proof-to-brittle properties are stated to be crucial for cake detachment [21], as well as the fact that cake texture changes in a small range of residual moisture, as reported by Tittel [29]. Generally, τ Cake /σ Cake increases due to the progressive decreasing of saturation of the network of solid particles [28]. However, slurries at concentrations of the gel point or higher are also able to transfer forces, e.g., wet filter cakes after filtration [30]. Since they theoretically have a saturation of one, this work extends the method of the yield locus for this specific case. This assumption is justified since in the literature brittle behavior is also mentioned for full-saturated networks of solid particles [21].
Shear Breaking
It is important to note that the acting forces are always proportional to the area where they are acting. This is to be exemplified in Table 1 by scenario c using realistic chamber dimensions (2 m · 2 m · 0.05 m) and a cohesion/adhesion ratio (σ Cake /σ Cloth ) of 1.15 (see Section 2.3). If the shear strength of the filter cake is too low, the cake becomes ruptured by shearing due to the adhesion of the cake parts sticking to the cloths. The area ratio of the attaching cake part in relation to the shear area is important and is therefore defining a critical ratio of τ Cake /σ Cake needed for the avoidance of rupture by shearing. If τ Cake /σ Cake is sufficient (over 17.4), no rupture of the cake is possible.
If incomplete detachment occurs, in addition to manual cleaning, there are some ways developed over time to remove the cake or remnants of it. Weigert lists some patents for this, using the following ideas: stretching medium, plate tilting, vibration, shaking, scrapers and nozzles [21]. However, it would be optimal if an autonomous detachment of the complete cake could be achieved by systematic process parameter control. Therefore, it is obvious that not only filtration tests with regard to separation efficiency and hydraulic properties of the filter media are necessary for the design of filter presses. In addition, knowledge of the stresses and the parameters influencing them is of enormous importance. Therefore, beside presentation of a suitable, application-related measurement method for relevant variables, this paper aims to show the impact of various process parameters (e.g., filtration pressure and dewatering). Table 1. Exemplary calculation of critical τ Cake /σ Cake ratio whose undercutting causes shear breaking.
Materials and Methods
Test equipment and procedures used are described in this section. Furthermore, investigated process parameters are discussed.
Properties of the Tailings
The study considered iron ore tailings having a size distribution and a mass concentration typical for tailings. Detailed properties are listed in Table 2. As referred to in Section 2, a high fraction of fine particles can be stated. The elemental composition measured by angle dispersive XRF can be found in a previous publication [31].
Procedure
It would be ideal if a characterization of the stresses could be realized with the least possible effort while at the same time providing sufficient information. In the following, a possibility is therefore presented. The idea is to combine filtration tests and shear tests using one apparatus (a cylindric one-chamber frame filter press) and, in addition, commercial standard equipment with minimal adjustments (a tensile testing machine).
The tests in this paper used a laboratory frame filter press provided by FLSmidth, also used in previous investigations [31]. It consists of a cylindrical frame element (inner diameter 10 cm, height 4 cm) with a suspension inlet at the top and pressed by a spindle between two end plates. These end plates have drainage channels and four filtrate outlets, one at the top and at the bottom (Figure 4a). The four filtrate outlets are equipped with valves. Closing the lower filtrate outlet on one side and the upper filtrate outlet on the other side and introducing compressed air at the open upper filtrate outlet thus enables gas differential pressure dewatering of the filter cake (Figure 4b). Filling of the chamber is carried out using a stirred pressure vessel with a riser pipe overlaid with compressed air. After filtration, it is possible to remove the cylinder together with cake and adhere filter media from the press. This allows subsequent positioning in a commercial tensile testing machine (velocity 10 mm·min −1 ) with minimal modification ( Figure 5). First, the adhesion (shear) of the filter media was measured by a crescent-shaped clamp, as proposed by Ginisty [32], by pulling of the fabric. Then, the cake was pushed halfway out of the cylinder using a disc spacer and afterwards the top half was sheared off with a lid to determine the cohesion (shear) of the cake analogues to a Jenike shear cell [33]. The maximum of the force-displacement curve related to the shear area represents the respective measured stress value. If the filtration is considered as preconsolidation and thus as preshearing of the network of solid particles, each shearing test value describes one point of the yield point, depending on the associated pair of values from normal stress and resulting shear stress. Different normal stresses can be realized by weight disks on the lid. In this way, a yield locus is obtained for each pressure and each cake post-treatment [26].
Sustainability 2022, 14, x FOR PEER REVIEW 7 of 15 gas differential pressure dewatering of the filter cake ( Figure 4b). Filling of the chamber is carried out using a stirred pressure vessel with a riser pipe overlaid with compressed air. After filtration, it is possible to remove the cylinder together with cake and adhere filter media from the press. This allows subsequent positioning in a commercial tensile testing machine (velocity 10 mm·min −1 ) with minimal modification ( Figure 5). First, the adhesion (shear) of the filter media was measured by a crescent-shaped clamp, as proposed by Ginisty [32], by pulling of the fabric. Then, the cake was pushed halfway out of the cylinder using a disc spacer and afterwards the top half was sheared off with a lid to determine the cohesion (shear) of the cake analogues to a Jenike shear cell [33]. The maximum of the force-displacement curve related to the shear area represents the respective measured stress value. If the filtration is considered as preconsolidation and thus as preshearing of the network of solid particles, each shearing test value describes one point of the yield point, depending on the associated pair of values from normal stress and resulting shear stress. Different normal stresses can be realized by weight disks on the lid. In this way, a yield locus is obtained for each pressure and each cake post-treatment [26].
Process Parameters
Filtration tests were carried out with three filter media typical for tailings filtration application. Two cloths of polypropylene (PP) and nylon (NY) as well as a PP felt media After filtration, it is possible to remove the cylinder together with cake and adhere filter media from the press. This allows subsequent positioning in a commercial tensile testing machine (velocity 10 mm·min −1 ) with minimal modification ( Figure 5). First, the adhesion (shear) of the filter media was measured by a crescent-shaped clamp, as proposed by Ginisty [32], by pulling of the fabric. Then, the cake was pushed halfway out of the cylinder using a disc spacer and afterwards the top half was sheared off with a lid to determine the cohesion (shear) of the cake analogues to a Jenike shear cell [33]. The maximum of the force-displacement curve related to the shear area represents the respective measured stress value. If the filtration is considered as preconsolidation and thus as preshearing of the network of solid particles, each shearing test value describes one point of the yield point, depending on the associated pair of values from normal stress and resulting shear stress. Different normal stresses can be realized by weight disks on the lid. In this way, a yield locus is obtained for each pressure and each cake post-treatment [26].
Process Parameters
Filtration tests were carried out with three filter media typical for tailings filtration application. Two cloths of polypropylene (PP) and nylon (NY) as well as a PP felt media
Process Parameters
Filtration tests were carried out with three filter media typical for tailings filtration application. Two cloths of polypropylene (PP) and nylon (NY) as well as a PP felt media were compared. These were chosen to be able to compare two fiber materials and a cloth to a felt media. Their properties are listed in Table 3. Filter media resistance measurements were carried out in a pressurized filter cell according to VDI guideline 2762 [17].
To investigate the influence of compaction, filtrations were performed at 250 kPa and 1250 kPa. For each pressure, there were tests with filtration only and tests with a dewatering cake post-treatment, as can be seen in Figure 6. This was a gas differential pressure dewatering at 250 kPa for filtrations at 250 kPa and at 550 kPa for filtrations at 1250 kPa. A pressure of 550 kPa is an industrially used value for gas differential pressure dewatering. However, gas differential pressure is limited by the filtration pressure (danger of cake back flow). So, 250 kPa was the maximum pressure usable for 250 kPa filtration pressure. were compared. These were chosen to be able to compare two fiber materials and a cloth to a felt media. Their properties are listed in Table 3. Filter media resistance measurements were carried out in a pressurized filter cell according to VDI guideline 2762 [17]. To investigate the influence of compaction, filtrations were performed at 250 kPa and 1250 kPa. For each pressure, there were tests with filtration only and tests with a dewatering cake post-treatment, as can be seen in Figure 6. This was a gas differential pressure dewatering at 250 kPa for filtrations at 250 kPa and at 550 kPa for filtrations at 1250 kPa. A pressure of 550 kPa is an industrially used value for gas differential pressure dewatering. However, gas differential pressure is limited by the filtration pressure (danger of cake back flow). So, 250 kPa was the maximum pressure usable for 250 kPa filtration pressure.
Normally, a tailings filtration fabric is used for a thousand or more filtration cycles [34]. During this process, changes occur in the fabrics due to blinding and mechanical abrasion. This is not considered in this work. A new set of fabrics (two pieces) was used for each pressure level. In order to still be able to observe a steady state (e.g., turbidity impact constant), six filtrations were carried out previously with the filter media to soil them before a fivefold determination of each measuring point was made. No post-treatment and post-treatment measurements were carried out alternating.
Validation
To validate the theoretical description of the cake detachment behavior, various chambers for a laboratory recessed plate filter press (Simex Mini Mobil, Simex Filterpressen GmbH & Co. KG, (Calw, Germany)) were additively manufactured in order to be able to carry out tests with 5, 10 and 15 mm thick cakes. Three filter plates were printed in were compared. These were chosen to be able to compare two fiber materials and a cloth to a felt media. Their properties are listed in Table 3. Filter media resistance measurements were carried out in a pressurized filter cell according to VDI guideline 2762 [17]. To investigate the influence of compaction, filtrations were performed at 250 kPa and 1250 kPa. For each pressure, there were tests with filtration only and tests with a dewatering cake post-treatment, as can be seen in Figure 6. This was a gas differential pressure dewatering at 250 kPa for filtrations at 250 kPa and at 550 kPa for filtrations at 1250 kPa. A pressure of 550 kPa is an industrially used value for gas differential pressure dewatering. However, gas differential pressure is limited by the filtration pressure (danger of cake back flow). So, 250 kPa was the maximum pressure usable for 250 kPa filtration pressure.
Normally, a tailings filtration fabric is used for a thousand or more filtration cycles [34]. During this process, changes occur in the fabrics due to blinding and mechanical abrasion. This is not considered in this work. A new set of fabrics (two pieces) was used for each pressure level. In order to still be able to observe a steady state (e.g., turbidity impact constant), six filtrations were carried out previously with the filter media to soil them before a fivefold determination of each measuring point was made. No post-treatment and post-treatment measurements were carried out alternating.
Validation
To validate the theoretical description of the cake detachment behavior, various chambers for a laboratory recessed plate filter press (Simex Mini Mobil, Simex Filterpressen GmbH & Co. KG, (Calw, Germany)) were additively manufactured in order to be able to carry out tests with 5, 10 and 15 mm thick cakes. Three filter plates were printed in were compared. These were chosen to be able to compare two fiber materials and a cloth to a felt media. Their properties are listed in Table 3. Filter media resistance measurements were carried out in a pressurized filter cell according to VDI guideline 2762 [17]. To investigate the influence of compaction, filtrations were performed at 250 kPa and 1250 kPa. For each pressure, there were tests with filtration only and tests with a dewatering cake post-treatment, as can be seen in Figure 6. This was a gas differential pressure dewatering at 250 kPa for filtrations at 250 kPa and at 550 kPa for filtrations at 1250 kPa. A pressure of 550 kPa is an industrially used value for gas differential pressure dewatering. However, gas differential pressure is limited by the filtration pressure (danger of cake back flow). So, 250 kPa was the maximum pressure usable for 250 kPa filtration pressure.
Normally, a tailings filtration fabric is used for a thousand or more filtration cycles [34]. During this process, changes occur in the fabrics due to blinding and mechanical abrasion. This is not considered in this work. A new set of fabrics (two pieces) was used for each pressure level. In order to still be able to observe a steady state (e.g., turbidity impact constant), six filtrations were carried out previously with the filter media to soil them before a fivefold determination of each measuring point was made. No post-treatment and post-treatment measurements were carried out alternating.
Validation
To validate the theoretical description of the cake detachment behavior, various chambers for a laboratory recessed plate filter press (Simex Mini Mobil, Simex Filterpressen GmbH & Co. KG, (Calw, Germany)) were additively manufactured in order to be able to carry out tests with 5, 10 and 15 mm thick cakes. Three filter plates were printed in To investigate the influence of compaction, filtrations were performed at 250 kPa and 1250 kPa. For each pressure, there were tests with filtration only and tests with a dewatering cake post-treatment, as can be seen in Figure 6. This was a gas differential pressure dewatering at 250 kPa for filtrations at 250 kPa and at 550 kPa for filtrations at 1250 kPa. A pressure of 550 kPa is an industrially used value for gas differential pressure dewatering. However, gas differential pressure is limited by the filtration pressure (danger of cake back flow). So, 250 kPa was the maximum pressure usable for 250 kPa filtration pressure.
Normally, a tailings filtration fabric is used for a thousand or more filtration cycles [34]. During this process, changes occur in the fabrics due to blinding and mechanical abrasion. This is not considered in this work. A new set of fabrics (two pieces) was used for each pressure level. In order to still be able to observe a steady state (e.g., turbidity impact constant), six filtrations were carried out previously with the filter media to soil them before a fivefold determination of each measuring point was made. No post-treatment and post-treatment measurements were carried out alternating.
Validation
To validate the theoretical description of the cake detachment behavior, various chambers for a laboratory recessed plate filter press (Simex Mini Mobil, Simex Filterpressen GmbH & Co. KG, (Calw, Germany)) were additively manufactured in order to be able to carry out tests with 5, 10 and 15 mm thick cakes. Three filter plates were printed in Normally, a tailings filtration fabric is used for a thousand or more filtration cycles [34]. During this process, changes occur in the fabrics due to blinding and mechanical abrasion. This is not considered in this work. A new set of fabrics (two pieces) was used for each pressure level. In order to still be able to observe a steady state (e.g., turbidity impact constant), six filtrations were carried out previously with the filter media to soil them before a fivefold determination of each measuring point was made. No post-treatment and post-treatment measurements were carried out alternating.
Validation
To validate the theoretical description of the cake detachment behavior, various chambers for a laboratory recessed plate filter press (Simex Mini Mobil, Simex Filterpressen GmbH & Co. KG, (Calw, Germany)) were additively manufactured in order to be able to carry out tests with 5, 10 and 15 mm thick cakes. Three filter plates were printed in each case, i.e., two end plates and one intermediate plate with an edge length of 150 mm and a cake dimension of approx. 120 mm in width and height. For this investigation, the PP cloth was used. One set of cloth was used for each pressure level (250 kPa and 1250 kPa). At first, for each pressure level, six filtrations were made to ensure basic contamination of the cloth. At this number, the particle penetration occurring at the beginning of each filtration had decreased to an approximately constant amount. Then, five filtrations were made for each cake thickness.
Adhesion Measurements to Determine Required Cake Thickness for Detachment
First, the shear adhesion values of the different fabrics for two filtration pressures and two cake post-treatments are presented. A plot of the shear stress to be applied for the individual fabrics is given in Figure 7, including the standard errors of the means. The black data series represents the measured values of saturated cakes and the white Sustainability 2022, 14, 3424 9 of 15 data series those of dewatered (undersaturated) cakes. The lowest adhesion is found for the lower filtration pressure (250 kPa) and a fully saturated cake (S = 1) for all fabrics. Dewatering after filtration at 250 kPa increases adhesion for all fabrics. The same applies to an increased filtration pressure (1250 kPa). For example, the adhesion of the PP cloth is approximately twice as high after a filtration at 1250 kPa without post-treatment than at 250 kPa without post-treatment, i.e., an almost twice as thick cake would be required to fulfill the detachment condition. Dewatering of the cakes formed at higher filtration pressure further increases adhesion for the PP cloth at 250 kPa and 1250 kPa, the NY cloth at 250 kPa and 1250 kPa and the PP felt at 250 kPa, comparing measurements at the same filtration pressure level. These findings are consistent with theoretical considerations of fewer contact points and literature data on saturation influence. Only the felt filter media for filtrations at 1250 kPa show an exception to this. Adhesion is slightly decreasing after dewatering. This results from the fact of a structure with lower permeability compared to the cloths reducing dewatering performance. Therefore, dewatering after 1250 kPa filtration has no further adhesion-increasing effect. For the iron ore tailings, 5 kN·m −2 corresponds approximately to 25 mm cake thickness by assuming a residual moisture of 20 w-%, ρ Solid of 3050 kg·m −3 , ρ Fluid of 1000 kg·m −3 , a full saturated cake (S = 1) and no sealing edge.
Adhesion Measurements to Determine Required Cake Thickness for Detachment
First, the shear adhesion values of the different fabrics for two filtration pressures and two cake post-treatments are presented. A plot of the shear stress to be applied for the individual fabrics is given in Figure 7, including the standard errors of the means. The black data series represents the measured values of saturated cakes and the white data series those of dewatered (undersaturated) cakes. The lowest adhesion is found for the lower filtration pressure (250 kPa) and a fully saturated cake (S = 1) for all fabrics. Dewatering after filtration at 250 kPa increases adhesion for all fabrics. The same applies to an increased filtration pressure (1250 kPa). For example, the adhesion of the PP cloth is approximately twice as high after a filtration at 1250 kPa without post-treatment than at 250 kPa without post-treatment, i.e., an almost twice as thick cake would be required to fulfill the detachment condition. Dewatering of the cakes formed at higher filtration pressure further increases adhesion for the PP cloth at 250 kPa and 1250 kPa, the NY cloth at 250 kPa and 1250 kPa and the PP felt at 250 kPa, comparing measurements at the same filtration pressure level. These findings are consistent with theoretical considerations of fewer contact points and literature data on saturation influence. Only the felt filter media for filtrations at 1250 kPa show an exception to this. Adhesion is slightly decreasing after dewatering. This results from the fact of a structure with lower permeability compared to the cloths reducing dewatering performance. Therefore, dewatering after 1250 kPa filtration has no further adhesion-increasing effect. For the iron ore tailings, 5 kN·m −2 corresponds approximately to 25 mm cake thickness by assuming a residual moisture of 20 w-%, ρSolid of 3050 kg·m −3 , ρFluid of 1000 kg·m −3 , a full saturated cake (S = 1) and no sealing edge. In general, the residual moisture of the filter cake is the key parameter in filter press operation. It allows direct conclusions to be drawn about the throughput and is partly In general, the residual moisture of the filter cake is the key parameter in filter press operation. It allows direct conclusions to be drawn about the throughput and is partly used as a target parameter for successful discharge. If only the adhesion of the cake to the fabric is considered, it is obvious that the residual moisture has a decisive influence on adhesion (Figure 8). Even a small change in residual moisture changes adhesion significantly. Considering all data points of filtrations at different filtration pressure levels, with and without cake post-treatment by dewatering, the assumption of a quasi-linear increase in adhesion when decreasing residual moisture is justified. Furthermore, standard error of the mean increases for decreasing moisture, especially for woven filter media.
However, detachment behavior cannot be simply described by only using the adhesion of the cake to the fabric. This is obvious regarding the possible detachment cases mentioned above and the underlying interactions of the different stresses. Further differentiation is necessary, and the structure of the cake must be considered. First, the understanding of the adhesion increase with decreasing residual moisture is useful. In fact, the residual cake moisture is resulting out of two different processes. One is compacting, and the other is dewatering. Therefore, if only the residual moisture is considered, information becomes lost. The effect of the different influences is visible, for example, in the representation of the adhesion referred to in the saturation of the network of solid particles (Figure 9). The networks are completely saturated (S = 1) for all filtrations without cake post-treatment of gas differential pressure dewatering. Depending on the available gas differential pressure (250 kPa or 550 kPa), further undersaturation and even higher adhesions can be achieved. used as a target parameter for successful discharge. If only the adhesion of the cake to the fabric is considered, it is obvious that the residual moisture has a decisive influence on adhesion (Figure 8). Even a small change in residual moisture changes adhesion significantly. Considering all data points of filtrations at different filtration pressure levels, with and without cake post-treatment by dewatering, the assumption of a quasi-linear increase in adhesion when decreasing residual moisture is justified. Furthermore, standard error of the mean increases for decreasing moisture, especially for woven filter media. However, detachment behavior cannot be simply described by only using the adhesion of the cake to the fabric. This is obvious regarding the possible detachment cases mentioned above and the underlying interactions of the different stresses. Further differentiation is necessary, and the structure of the cake must be considered. First, the understanding of the adhesion increase with decreasing residual moisture is useful. In fact, the residual cake moisture is resulting out of two different processes. One is compacting, and the other is dewatering. Therefore, if only the residual moisture is considered, information becomes lost. The effect of the different influences is visible, for example, in the representation of the adhesion referred to in the saturation of the network of solid particles ( Figure 9). The networks are completely saturated (S = 1) for all filtrations without cake post-treatment of gas differential pressure dewatering. Depending on the available gas differential pressure (250 kPa or 550 kPa), further undersaturation and even higher adhesions can be achieved.
However, it should be noted that an increased filtration pressure acts as an antagonist. For compressible cakes and resulting compaction, the pores become smaller; thus, a higher capillary inlet pressure is required for dewatering.
Cohesion Measurements to Evaluate Shear Breaking Risk
The determination of the adhesion of the cake to the filter cloth helps to determine the required cake thickness for detachment (See Figure 2a,b). However, no statement can be made about the occurrence of the other possible scenarios (See Figure 2c,d). For these, the other stress states on and in the filter cake as well as their relationship to each other play an important role, e.g., the ratio of τCake/σCake, which indicates the brittleness of the network of solid particles in simplified terms. A first step is to determine the shear cohesion, i.e., the shear strength of the filter cake, using the procedure shown in Figure 5. Figure 10 shows the results of these cohesion measurements for the PP felt media over residual moisture. Analogous to shear adhesion, an approximately linear relationship between shear cohesion and residual moisture content is evident, regardless of whether the lower residual moisture content results from increased compaction (due to a higher filtration pressure) or undersaturation (gas differential pressure dewatering) However, it should be noted that an increased filtration pressure acts as an antagonist. For compressible cakes and resulting compaction, the pores become smaller; thus, a higher capillary inlet pressure is required for dewatering.
Cohesion Measurements to Evaluate Shear Breaking Risk
The determination of the adhesion of the cake to the filter cloth helps to determine the required cake thickness for detachment (See Figure 2a,b). However, no statement can be made about the occurrence of the other possible scenarios (See Figure 2c,d). For these, the other stress states on and in the filter cake as well as their relationship to each other play an important role, e.g., the ratio of τ Cake /σ Cake , which indicates the brittleness of the network of solid particles in simplified terms. A first step is to determine the shear cohesion, i.e., the shear strength of the filter cake, using the procedure shown in Figure 5. Figure 10 shows the results of these cohesion measurements for the PP felt media over residual moisture. Analogous to shear adhesion, an approximately linear relationship between shear cohesion and residual moisture content is evident, regardless of whether the lower residual moisture content results from increased compaction (due to a higher filtration pressure) or undersaturation (gas differential pressure dewatering) cles.
Cohesion Measurements to Evaluate Shear Breaking Risk
The determination of the adhesion of the cake to the filter cloth helps to determine the required cake thickness for detachment (See Figure 2a,b). However, no statement can be made about the occurrence of the other possible scenarios (See Figure 2c,d). For these, the other stress states on and in the filter cake as well as their relationship to each other play an important role, e.g., the ratio of τCake/σCake, which indicates the brittleness of the network of solid particles in simplified terms. A first step is to determine the shear cohesion, i.e., the shear strength of the filter cake, using the procedure shown in Figure 5. Figure 10 shows the results of these cohesion measurements for the PP felt media over residual moisture. Analogous to shear adhesion, an approximately linear relationship between shear cohesion and residual moisture content is evident, regardless of whether the lower residual moisture content results from increased compaction (due to a higher filtration pressure) or undersaturation (gas differential pressure dewatering) Furthermore, the determination of the ratio of τ Cake /σ Cake can be carried out by determining the yield loci and expanding them into negative normal stresses. How these curves are to be expanded is discussed controversially [21]. Linear extrapolation of the yield locus slope at σ = 0 is a common simplified way to describe the relationship between τ Cake and σ Cake . However, this overestimates the tensile strength and underestimates τ Cake /σ Cake , respectively. Nevertheless, a statement about the detachment behavior can already be derived from the ratio of the slopes. This is discussed using the example of the yield loci for filtration at 250 kPa and 1250 kPa using the PP felt media for no post-treatment of the cake and gas differential pressure dewatering ( Figure 11). When looking at the linear fit, a higher slope, which is similar to the ratio of τ Cake /σ Cake at a higher filtration pressure, can be observed. Qualitatively, scenario c is thus less probable with filtration at 1250 kPa. A slight impact by cake post-treatment can be stated as well.
spectively. Nevertheless, a statement about the detachment behavior can already be derived from the ratio of the slopes. This is discussed using the example of the yield loci for filtration at 250 kPa and 1250 kPa using the PP felt media for no post-treatment of the cake and gas differential pressure dewatering ( Figure 11). When looking at the linear fit, a higher slope, which is similar to the ratio of τCake/σCake at a higher filtration pressure, can be observed. Qualitatively, scenario c is thus less probable with filtration at 1250 kPa. A slight impact by cake post-treatment can be stated as well.
Validation
The presented different scenarios a-d of cake detachments can be observed in laboratory tests. Figure 12 shows images of each scenario after filtration of the iron ore tailings using PP cloths in the laboratory recessed plate filter press. The first image shows scenario a, which is a complete detached cake after a filtration with 5 mm chambers at 250 kPa filtration pressure without cake post-treatment. In the next image, scenario b, a complete sticking cake can be seen after a filtration with 5 mm chambers at 250 kPa filtration pressure and without cake post-treatment. The third image shows a shear breaking (scenario c) after a filtration with a 5 mm cake at 250 kPa filtration pressure and without cake posttreatment. Scenario d is a breaking parallel to the cloth, which could often be observed after filtration with cake dewatering as shown in the image on the right side. This was also taken after a filtration at 250 kPa with 5 mm chambers and air blow. Figure 11. Yield locus excerpt of different filtration process parameters for PP felt media.
Validation
The presented different scenarios a-d of cake detachments can be observed in laboratory tests. Figure 12 shows images of each scenario after filtration of the iron ore tailings using PP cloths in the laboratory recessed plate filter press. The first image shows scenario a, which is a complete detached cake after a filtration with 5 mm chambers at 250 kPa filtration pressure without cake post-treatment. In the next image, scenario b, a complete sticking cake can be seen after a filtration with 5 mm chambers at 250 kPa filtration pressure and without cake post-treatment. The third image shows a shear breaking (scenario c) after a filtration with a 5 mm cake at 250 kPa filtration pressure and without cake post-treatment. Scenario d is a breaking parallel to the cloth, which could often be observed after filtration with cake dewatering as shown in the image on the right side. This was also taken after a filtration at 250 kPa with 5 mm chambers and air blow. Figure 12 shows different scenarios observed for filtrations that do not differ in their chamber thickness. This is due to the fact that each detachment behavior has a certain probability for the respective process conditions. This stochastic distribution results, for example, from local inhomogeneities of the cake due to the broad particle size distribution. For example, shear breaking was observed for seven out of ten filter cakes having a thickness of 5 mm using PP cloths in the laboratory recessed plate filter press (see Table 4). This is the process parameter condition resulting in the highest residual moisture. A five-hold determination of each combination using this two-chamber filter press gave ten detachments that can be observed. Shear breaking did not occur for thicker cakes at filtrations with 250 kPa and without post-treatment or at any other combination. Figure 12 shows different scenarios observed for filtrations that do not differ in their chamber thickness. This is due to the fact that each detachment behavior has a certain probability for the respective process conditions. This stochastic distribution results, for example, from local inhomogeneities of the cake due to the broad particle size distribution. For example, shear breaking was observed for seven out of ten filter cakes having a thickness of 5 mm using PP cloths in the laboratory recessed plate filter press (see Table 4). This is the process parameter condition resulting in the highest residual moisture. A five-hold determination of each combination using this two-chamber filter press gave ten detachments that can be observed. Shear breaking did not occur for thicker cakes at filtrations with 250 kPa and without post-treatment or at any other combination. Table 4. Cake shear breaking probability of filtrations in the laboratory recessed plate filter press using the PP cloths.
Filtration Pressure
Cake Post-Treatment Shear Breaking
Discussion
In this study, several points are considered in a simplified way. Determination of filter media influences is limited since long-term behavior of the different filter media cannot be studied at the laboratory scale to an application-related extent. In addition, filter media having higher pressure losses reduces available gas differential pressure for dewatering and therefore hampers adhesion and cohesion comparisons. Generally, extensive field data acquisition and comparison between field and laboratory data would be suggested.
In this study, only iron ore tailings are considered. It would be interesting for future work to compare tailings from different mines but also from the same mine at different exploration times. Changes in rock composition are a major challenge for the entire process chain but also explicitly for solid-liquid separation. Larger data sets would then also allow predictive control of this process.
Conclusions
First, a detailed view on cake detachment cases as well as underlying forces and tensions was depicted. Afterwards, measurement procedures combining filtration tests and cake-to-cloth shear adhesion as well as cake shear cohesion were presented, and their suitability for iron ore tailings was proven. Thereby, filtration process parameter influences on shear adhesion and shear cohesion could be shown. Increasing compaction of the cake by applying a higher filtration pressure leads to a higher shear adhesion, requiring a thicker cake to reach sufficient cake weight to fulfil the detachment condition as well as to a higher shear cohesion. Furthermore, decreasing cake saturation by cake post-treatment (gas differential pressure dewatering) also increases shear adhesion and shear cohesion. In summary, an approximately linear increase in shear adhesion as well as shear cohesion with decreasing residual moisture can be observed, regardless of whether the decrease results from compaction (higher filtration pressure) or undersaturation (gas differential pressure dewatering). For the regarded amount of filtration cycles (relatively low compared to industrial use), no significant differences between cloth and felt media, as well as PP and NY, can be seen. Acknowledgments: The authors would like to thank all students and colleagues who have contributed to the successful completion of this work. Special thanks for support go to Scott Reddick, Dave Hanfland, Paul McCurdie, Todd Wisdom, James Chaponnel, Brent Stokes and Steve Ware from FLSmidth. The regular meetings and critical discussions have contributed significantly to the success of the work. This work was sponsored by FLSmidth. We acknowledge support by the KIT-Publication Fund of the Karlsruhe Institute of Technology.
Conflicts of Interest:
The funders had no role in the design of the study, in the collection, analyses or interpretation of data, in the writing of the manuscript or in the decision to publish the results. | 2022-03-18T15:30:21.152Z | 2022-03-15T00:00:00.000 | {
"year": 2022,
"sha1": "69b99f2343c46178a3a0a1e8712f3348c3e58ce7",
"oa_license": "CCBY",
"oa_url": "https://www.mdpi.com/2071-1050/14/6/3424/pdf",
"oa_status": "GOLD",
"pdf_src": "MergedPDFExtraction",
"pdf_hash": "900cab127eb3c0ada97f68cf114548500043f095",
"s2fieldsofstudy": [
"Materials Science"
],
"extfieldsofstudy": []
} |
259259595 | pes2o/s2orc | v3-fos-license | Comparative Study of Thermal and Plasma-Enhanced Atomic Layer Deposition of Iron Oxide Using Bis(N,N′-di-butylacetamidinato)iron(II)
Only a few iron precursors that can be used in the atomic layer deposition (ALD) of iron oxides have been examined thus far. This study aimed to compare the various properties of FeOx thin films deposited using thermal ALD and plasma-enhanced ALD (PEALD) and to evaluate the advantages and disadvantages of using bis(N,N′-di-butylacetamidinato)iron(II) as an Fe precursor in FeOx ALD. The PEALD of FeOx films using iron bisamidinate has not yet been reported. Compared with thermal ALD films, PEALD films exhibited improved properties in terms of surface roughness, film density, and crystallinity after they were annealed in air at 500 °C. The annealed films, which had thicknesses exceeding ~ 9 nm, exhibited hematite crystal structures. Additionally, the conformality of the ALD-grown films was examined using trench-structured wafers with different aspect ratios.
The objective of this study was to evaluate the viability of using bis(N,N -dibutylacetamidinato)iron(II) (FeAMD) as an iron precursor in the preparation of FeO x thin films. To this end, the properties of FeO x thin films deposited using thermal ALD and plasma-enhanced ALD (PEALD) with FeAMD as the precursor were compared. Avila et al. first reported the thermal ALD of FeO x thin films using FeAMD [41]; however, no further research has been reported on the use of FeAMD in FeO x ALD. The novelty of this study is that it dealt with the practical aspects of using FeAMD as an ALD precursor by considering the vapor pressure during deposition along with the thermal stability and chemical/physical properties of the deposited films. Moreover, this paper is the first to report a FeO x PEALD process using FeAMD. In this study, both thermal ALD and PEALD films were comprehensively characterized using spectroscopic ellipsometry (SE), atomic force microscopy (AFM), grazing incidence X-ray diffraction (GIXRD), X-ray photoelectron spectroscopy (XPS), and scanning electron microscopy (SEM).
Materials and Methods
A PEALD reactor system with an 8-inch wafer chuck was used for both thermal ALD and PEALD processes. The PEALD reactor included an inductively coupled plasma system with a remote plasma geometry; the plasma shower head was located 89 mm away from the sample plate. The reactor had a cross-flow design in which the precursor vapors (FeAMD or H 2 O) flowed across the substrate surface; the precursor delivery line was separated from the plasma gas line. For the ALD process, FeAMD (product number: 26-0145, Strem Chemicals, Inc., Newburyport, MA, USA) was used as the Fe precursor, and either H 2 O or O 2 plasma was used as the oxidizing coreactant. The temperatures of the FeAMD canister, gas line, and substrate were 150, 200, and 200 • C, respectively. During the entire ALD process, Ar carrier gas at 5 SCCM was continuously provided along the two gas lines delivering the precursor (FeAMD or H 2 O) vapor and plasma gas. During the oxygen plasma step, oxygen was provided along with Ar carrier gas at an increased flow rate (50 SCCM). The duration of a single dose of FeAMD was maintained at 1 s. The dose durations of H 2 O (0.1-2 s) and oxygen plasma (5-120 s) were varied as described in Section 3. In the FeAMD half cycles, a purge time of 300 s was chosen to allow sufficient time for FeAMD precursor evaporation. A Si wafer (p type, 1-10 Ωcm) with an approximately 2 nm native oxide layer was used as the substrate after being cleaned via sonication with ethanol and isopropanol.
The film thicknesses were measured at four wavelengths, namely 463, 523, 600, and 637 nm, using SE (FS-1, Film Sense LLC, Lincoln, NE, USA). The SE data were fitted using three parameters: refractive index, extinction coefficient, and film thickness. AFM (XE7, Park Systems Corp., Suwon, Republic of Korea) was used to measure the surface roughness of the films in the contact mode with a scan size of 5 µm × 5 µm and at a scan rate of 1 Hz. XPS (K-alpha, Thermo Fisher Scientific Inc., Waltham, MA, USA) with an Al K-α source of 1 keV was used to determine the chemical composition of the films. The XPS data were collected after 5 s of etching to enable surface contamination. The cross-sectional images of the films deposited on the trench-structured substrate were analyzed using SEM (Verios 5 UC, Thermo Fisher Scientific Inc., Waltham, MA, USA). An X-ray diffractometer (Smart-Lab, RIGAKU Co., Ltd., Tokyo, Japan) was used to collect the GIXRD data in the two-theta scan mode at a scan speed of 1.5 • /min and with a step size of 0.02 • .
Results and Discussion
A conventional ALD cycle consists of two half cycles with each cycle comprising a metal precursor (or coreactant) dosing step followed by a metal precursor (or coreactant) purging step. Multiple doses of the precursor may be administered within a single half cycle to ensure the saturation of the surface reactions between the surface functional groups and precursor, particularly when the vapor pressure of the precursor is insufficient. In this study, the number of mini doses within a single-half cycle of FeAMD was varied from one to five, while the oxidant half cycle consisted of a single oxidant dose. We employed the multiple dose scheme to evaluate the necessary FeAMD exposure amount for achieving the saturation of precursor surface coverage within a half cycle, as discussed in this work (vide infra). Figure 1 shows the growth per cycle (GPC) of the FeO x film grown using 100 ALD cycles, as a function of the average partial pressure of FeAMD during a "half" cycle (not with a mini dose). An example of a partial pressure measurement is shown in Figure S1. The vertical error bars in the GPC plot were measured using five samples located on the 8-inch wafer chuck at a considerable distance from one another. The horizontal error bars related to the partial pressure were calculated using ten representative ALD cycles under thermal ALD and PEALD conditions. The GPC values under both conditions tended to increase with increasing partial pressure until they reached approximately 80 mTorr. The saturated GPC values were within the range of 1.7-1.9 Å/cy at high partial pressures (>100 mTorr). We obtained the high partial pressure when using five mini doses of FeAMD, indicating that five mini doses are suggested as the optimal number. The GPC values measured in this study were greater than those measured using FeO x ALD processes that used other Fe precursors, such as Fe(thd) 3 , (0.14 Å/cy) [38], N,N-(dimethylaminomethyl)ferrocene (0.26 Å/cy) [37], and ferrocene (1.4 Å/cy) [33]. The GPC values were three to four times higher than those reported in a previous paper regarding the same precursor (FeAMD) [41]. This discrepancy could be attributed to the low thermal stability of the FeAMD precursor. The decomposition of FeAMD started at approximately 160 • C [42]. In this study, FeAMD was heated to 150 • C to obtain a sufficient vapor pressure while minimizing precursor decomposition. Even with a long purge (300 s) between two subsequent mini doses, the FeAMD partial pressure tended to decrease gradually during the ALD runs, probably due to the agglomeration of FeAMD powders subsequent to prolonged heating. To maintain a constant vapor pressure, the agglomerated FeAMD powders had to be broken down into small pieces two or three times during the 100 ALD cycles ( Figure S2). The GPC values of the PEALD films were lower than those of the thermal ALD films owing to the higher density of the PEALD films (approximately 4.9 g/cm 3 ) compared with those of the thermal ALD films (approximately 4.0 g/cm 3 ) measured using X-ray reflection spectroscopy ( Figure S3). Furthermore, the saturation behaviors of FeO x films at different oxidant doses (H 2 O and oxygen plasma) were confirmed ( Figure S4). ALD experiments were also performed with only the FeAMD half cycle (FeAMD partial pressure:~96 mTorr)-that is, without the coreactant half cycle-to study the potential contribution of the thermal decomposition of FeAMD to the growth of FeO x films. The grown film had a GPC of approximately 1 Å/cy, which was significantly lower than that of either thermal ALD or PEALD films (approximately 1.6-1.7 Å/cy) at the similar FeAMD partial pressures. These results suggest that the contribution made by the thermal decomposition of FeAMD to the film deposition cannot be disregarded. An ALD-like component in GPC measures approximately 0.6-0.7 Å/cy, which closely aligns with the GPC value observed by Avila et al. [41]. The presence of the non-negligible CVD component is a major disadvantage of FeAMD because well-saturated GPC curves have been reported when using FeO x ALD with FeCl 3 [40]. Future research directions could include the rational design of the molecular structures of Fe precursors to optimize their thermal stabilities and chemical reactivities toward oxidizing coreactants [43]. Additionally, technological developments in the design of precursor canisters and gas delivery systems may facilitate the efficient delivery of FeAMD vapors with reduced possibilities of FeAMD decomposition [44]. The GPC graph ( Figure 1) may be drawn as a function of the number of mini doses. However, the partial pressure can be a more representative parameter of the number of FeAMD molecules provided in each FeAMD half cycle than the number of mini doses because of the problems associated with powder agglomeration. Figure 2 shows the AFM images of the FeO x films grown using thermal ALD at different FeAMD partial pressures and using PEALD at different FeAMD partial pressures and oxygen plasma powers. The roughness values of the films were in the range of 0.2-0.7 nm. During the thermal ALD process, the surface roughness slightly improved as the FeAMD partial pressure increased from~24 to 53 mTorr, possibly because of the high surface coverage of the precursors at high partial pressures. The PEALD films exhibited a slightly lower surface roughness than the thermal ALD films grown at the similar FeAMD partial pressures. The difference between the surface roughness values of the two PEALD films prepared using 20 and 300 W oxygen plasmas was negligible. Overall, both thermal ALD and PEALD films with thicknesses < 12 nm exhibited excellent surface roughness on the subnanometer scale. The variation in the surface roughness with film thickness is discussed later in the paper. Figure 2 shows the AFM images of the FeOx films grown using thermal ALD at different FeAMD partial pressures and using PEALD at different FeAMD partial pressures and oxygen plasma powers. The roughness values of the films were in the range of 0.2-0.7 nm. During the thermal ALD process, the surface roughness slightly improved as the FeAMD partial pressure increased from ~24 to 53 mTorr, possibly because of the high surface coverage of the precursors at high partial pressures. The PEALD films exhibited a slightly lower surface roughness than the thermal ALD films grown at the similar FeAMD partial pressures. The difference between the surface roughness values of the two PEALD films prepared using 20 and 300 W oxygen plasmas was negligible. Overall, both thermal ALD and PEALD films with thicknesses < 12 nm exhibited excellent surface roughness on the subnanometer scale. The variation in the surface roughness with film thickness is discussed later in the paper.
High-resolution XPS (HRXPS) was performed to determine the chemical compositions of the films (Table 1). A marginal difference existed between the chemical compositions of the two thermal ALD films grown using different H2O dose durations (0.1 and 2 s). Both films contained C (<7 at. %) and N (~1 at. %) impurities and were slightly substoichiometric in nature (Fe2O2.5-2.6). The Si components originated from the Si substrates because the films deposited were thin (~9-20 nm). The PEALD film had a chemical composition (~6 at. % C and ~1 at. % N impurities) and an Fe-O stoichiometric nature (Fe2O2.6), similar to the thermal ALD films. The films deposited using only FeAMD half cycles (hereby denoted as "CVD-like film") contained higher concentrations of C (~14.5 at. %) and N (1.7 at. %) impurities than the thermal ALD and PEALD films. High-resolution XPS (HRXPS) was performed to determine the chemical compositions of the films (Table 1). A marginal difference existed between the chemical compositions of the two thermal ALD films grown using different H 2 O dose durations (0.1 and 2 s). Both films contained C (<7 at.%) and N (~1 at.%) impurities and were slightly substoichiometric in nature (Fe 2 O 2.5-2.6 ). The Si components originated from the Si substrates because the films deposited were thin (~9-20 nm). The PEALD film had a chemical composition (~6 at.% C and~1 at.% N impurities) and an Fe-O stoichiometric nature (Fe 2 O 2.6 ), similar to the thermal ALD films. The films deposited using only FeAMD half cycles (hereby denoted as "CVD-like film") contained higher concentrations of C (~14.5 at.%) and N (1.7 at.%) impurities than the thermal ALD and PEALD films.
The Fe 2p HRXPS spectra of the four samples ( Figure 3a) were deconvoluted into five peaks, namely an Fe 2+ 2p 3/2 peak at~709.9 eV, Fe 3+ 2p 3/2 peak at~711.6 eV, Fe 3+ 2p 3/2 satellite at~716.0 eV, Fe 2+ 2p 1/2 peak at~723.7 eV, and Fe 3+ 2p 1/2 peak at~728.6 eV. The four films contained a mixture of Fe 2+ and Fe 3+ components, and this composition agrees with the substoichiometric nature of the films. The component within the PEALD film that exhibited the highest binding energy (BE), marked by a black arrow, was positioned approximately 1.6 eV higher in BE, and this component displayed a broader full width at half maximum. This suggests the presence of an Fe 3+ 2p 1/2 satellite peak and indicates an increased contribution from the Fe 3+ component. The O 1s HRXPS spectra (Figure 3b The N 1s HRXPS spectra of the CVD-like films showed a higher intensity of Fe-N than the N 1s HRXPS spectra of the other samples ( Figure S5). Thus, the deposited FeO x films would be of poor quality because of the limited thermal stability of the FeAMD precursors. After the completion of the ALD process, the films deposited via a CVD-like reaction (composed mainly of surface-sorbed FeAMD molecules) were oxidized to form FeO x upon exposure to the O 2 in air. The Fe 2p HRXPS spectra of the four samples ( Figure 3a) were deconvoluted into five peaks, namely an Fe 2+ 2p3/2 peak at ~709.9 eV, Fe 3+ 2p3/2 peak at ~711.6 eV, Fe 3+ 2p3/2 satellite at ~716.0 eV, Fe 2+ 2p1/2 peak at ~723.7 eV, and Fe 3+ 2p1/2 peak at ~728.6 eV. The four films contained a mixture of Fe 2+ and Fe 3+ components, and this composition agrees with the substoichiometric nature of the films. The component within the PEALD film that exhibited the highest binding energy (BE), marked by a black arrow, was positioned approximately 1.6 eV higher in BE, and this component displayed a broader full width at half maximum. This suggests the presence of an Fe 3+ 2p1/2 satellite peak and indicates an increased contribution from the Fe 3+ component. The O 1s HRXPS spectra (Figure 3b Figure S5). Thus, the deposited FeOx films would be of poor quality because of the limited thermal stability of the FeAMD precursors. After the completion of the ALD process, the films deposited via a CVD-like reaction (composed mainly of surface-sorbed FeAMD molecules) were oxidized to form FeOx upon exposure to the O2 in air. Figure 4 compares the crystallinities of the films grown using thermal ALD and PEALD under different conditions and obtained using the GIXRD measurements of the samples. All samples were annealed in air at 500 • C for 2 h before taking the GIXRD measurements. The hematite phase was identified in most of the annealed samples. The films were grown using a fixed number of ALD cycles (100 cycles) and different FeAMD partial pressures (film thicknesses). A majority of the thermal ALD and PEALD films exhibited a polycrystalline nature, and their crystallinity increased with increasing film thickness. In addition, for the similar thicknesses and FeAMD partial pressures, the PEALD films exhibited better crystallinity than thermal ALD films. The formation of an amorphous phase in the asdeposited films is consistent with the results of previous studies on FeO x ALD using other precursors [33,41]. Crystalline phases were found in the films in as-deposited states only when the deposition temperature (>350 • C) [34] or the film thickness (>35 nm) [39,40] was significantly higher. Nanomaterials 2023, 13, x FOR PEER REVIEW 6 of 10 Figure 4 compares the crystallinities of the films grown using thermal ALD and PEALD under different conditions and obtained using the GIXRD measurements of the samples. All samples were annealed in air at 500 °C for 2 h before taking the GIXRD measurements. The hematite phase was identified in most of the annealed samples. The films were grown using a fixed number of ALD cycles (100 cycles) and different FeAMD partial pressures (film thicknesses). A majority of the thermal ALD and PEALD films exhibited a polycrystalline nature, and their crystallinity increased with increasing film thickness. In addition, for the similar thicknesses and FeAMD partial pressures, the PEALD films exhibited better crystallinity than thermal ALD films. The formation of an amorphous phase in the as-deposited films is consistent with the results of previous studies on FeOx ALD using other precursors [33,41]. Crystalline phases were found in the films in as-deposited states only when the deposition temperature (>350 °C) [34] or the film thickness (>35 nm) [39,40] was significantly higher.
Finally, thermal ALD was performed using two different types of wafers with trench structures, namely (1) a high aspect ratio (AR) trench structure (AR = 1:13) with a 65.0 μm trench depth (Figure 5a) and (2) low AR trench structures (AR = ~0.6, 2.2, and 4.6) with a trench depth of ~400 nm (Figure 5b). The step coverage of the high AR trench structure was ~ 49%, indicating the necessity for prolonged precursor exposure. The film deposited at the bottom of the trench structure was smoother than that deposited at the top, suggesting that the surface roughness had increased as film deposition continued. The surfaces of the thin films deposited using either thermal ALD or PEALD were significantly smooth ( Figure 2). The accurate measurement of the step coverage of the low AR structures was difficult because of their high surface roughness values. However, the rough surface morphology observed at the bottom of the structures implied that film growth was not limited to deep trench structures with low ARs. Finally, thermal ALD was performed using two different types of wafers with trench structures, namely (1) a high aspect ratio (AR) trench structure (AR = 1:13) with a 65.0 µm trench depth (Figure 5a) and (2) low AR trench structures (AR =~0.6, 2.2, and 4.6) with a trench depth of~400 nm (Figure 5b). The step coverage of the high AR trench structure was~49%, indicating the necessity for prolonged precursor exposure. The film deposited at the bottom of the trench structure was smoother than that deposited at the top, suggesting that the surface roughness had increased as film deposition continued. The surfaces of the thin films deposited using either thermal ALD or PEALD were significantly smooth ( Figure 2). The accurate measurement of the step coverage of the low AR structures was difficult because of their high surface roughness values. However, the rough surface morphology observed at the bottom of the structures implied that film growth was not limited to deep trench structures with low ARs. Grazing incidence X-ray diffraction patterns of the FeOx thin films deposited using thermal atomic layer deposition (ALD) and plasma-enhanced ALD. Peak assignment was performed using the reference ICDD card No. 00-001-1053, which is shown at the bottom of the graph. The film thickness and partial pressure of FeAMD used for the deposition are indicated. Figure 5a shows the schematic of a patterned wafer with a trench (width: 5.0 μm, depth: 65.0 μm, and pitch: 52.5 μm). The images on the left side of Figure 5b show the pattern height and space width of the patterned wafers with different ARs.
Conclusions
The properties of thermal and PEALD films were examined using various characterization methods, including SE, XPS, AFM, XRD, and SEM. The GPC values of the PEALD Figure 5. Cross-sectional SEM images of the FeO x thin films deposited on patterned wafers with (a) high AR and (b) low AR. The inset of Figure 5a shows the schematic of a patterned wafer with a trench (width: 5.0 µm, depth: 65.0 µm, and pitch: 52.5 µm). The images on the left side of Figure 5b show the pattern height and space width of the patterned wafers with different ARs.
Conclusions
The properties of thermal and PEALD films were examined using various characterization methods, including SE, XPS, AFM, XRD, and SEM. The GPC values of the PEALD films were~0.2 Å/cy lower than those of the thermal ALD films because of the higher density of the PEALD films. The thermal and PEALD films contained carbon and nitrogen impurities with similar atomic concentrations (C: <7 at.%, N: <1.1 at.%) and substoichiometric natures (FeO 2.5~2.6 ). The PEALD films presented a lower surface roughness (~0.2 nm) and better crystallinity when FeAMD with the similar partial pressure was provided for a single ALD cycle. A major advantage of FeAMD over other Fe ALD precursors identified in this study is that it enables the growth of FeO x films using both H 2 O and O 2 plasma as co-reactants with practical GPCs in the range of 1.6-1.7 Å/cy at a practical temperature (200 • C). However, the limited thermal stability and low vapor pressure of FeAMD are common to both processes. This problem could potentially be addressed by an advanced reactor design that enables efficient precursor delivery [45]. Despite this limitation, the formation of iron oxide films in the hematite phase with a subnanometer-scale roughness, demonstrated in this study, indicates the possibility of integrating ALD processes with various applications of iron oxides in the hematite phase.
Supplementary Materials: The following supporting information can be downloaded at: https: //www.mdpi.com/article/10.3390/nano13121858/s1, Figure S1: An example of the pressure log collected during the FeAMD half-cycle, in which the FeAMD dose was repeated five times. In this log, the partial pressure of FeAMD was recorded as 128 mTorr, Figure S2: Pictures of FeAMD precursor of (a) an as-received state, and (b) an agglomerated state; Figure S3: X-ray reflectivity spectra of (a) thermal ALD and (b) PEALD films; Figure S4: GPC of FeO x films as a function of (a) H 2 O dose time (thermal ALD) and (b) O 2 plasma time (PEALD). Figure S5: N 1s HRXPS of the thermal ALD, PEALD, and CVD-like films.
Data Availability Statement:
The data presented in this study are available on request from the corresponding author.
Conflicts of Interest:
The authors declare no conflict of interest. | 2023-06-28T06:17:26.514Z | 2023-06-01T00:00:00.000 | {
"year": 2023,
"sha1": "c711f7bd1a2e355daea61c4f5994b45053e628e3",
"oa_license": "CCBY",
"oa_url": null,
"oa_status": null,
"pdf_src": "PubMedCentral",
"pdf_hash": "a505602603271e067ea7a3895456e10e7f5d404e",
"s2fieldsofstudy": [
"Physics"
],
"extfieldsofstudy": [
"Medicine"
]
} |
18088113 | pes2o/s2orc | v3-fos-license | Optimal coloured perceptrons
Ashkin-Teller type perceptron models are introduced. Their maximal capacity per number of couplings is calculated within a first-step replica-symmetry-breaking Gardner approach. The results are compared with extensive numerical simulations using several algorithms.
I. INTRODUCTION
The perceptron which was first analyzed with statistical mechanics techniques in the seminal paper of Gardner [1] is by now a well-known and standard model in theoretical studies and practical applications in connection with learning and generalization [2][3][4][5]. A number of extensions of the perceptron model have been formulated, including many-state and graded-response perceptrons (e.g., [6][7][8][9][10][11]). Here we present some new extensions allowing for so-called coloured or Ashkin-Teller type neurons, i.e., different types of binary neurons at each site possibly having different functions.
The idea of looking at such a model is based upon our recent work on Ashkin-Teller recurrent neural networks [12,13]. There we showed that for this model with two types of binary neurons interacting through a fourneuron term and equipped with a Hebb learning rule, both the thermodynamic and dynamic properties suggest that such a model can be more efficient than a sum of two Hopfield models. For example, the quality of pattern retrieval is enhanced through a larger overlap at higher temperatures and the maximal capacity is increased. For more details and an underlying neurobiological motivation for the introduction of different types of neurons we refer to [13].
In the light of these results an interesting question is whether such a coloured perceptron can still be more efficient than the standard perceptron. In other words, can it have a larger maximal capacity than the one of a standard perceptron, which is known [1] to be α c = 2 (for random uncorrelated patterns). It has been suggested that this number is characteristic for all binary networks independent of the multiplicity of the neuron interactions. Thereby, the capacity is defined as the thermodynamic limit of the ratio of the total number of bits per (input) neuron to be stored and the total number of couplings per (output) neuron [8]. We remark that "input" and "output" refer specifically to the perceptron case.
In the sequel the maximal capacity of coloured perceptron models is studied using the Gardner approach [1,14]. First-step replica-symmetry-breaking effects are evaluated and the analytic results are compared with extensive numerical simulations using various learning algorithms.
The rest of this paper is organized as follows. In Sec. II we introduce two Ashkin-Teller type perceptron models. Section III contains the replica theory and determines the maximal capacity by calculating the available volume in the space of couplings both in the replica-symmetric (Sec. IIIA) and the first-step replica-symmetry-breaking approximation (Sec. IIIB). Section IV describes the results of numerical simulations with algorithms obtained by generalizing various algorithms for simple perceptrons. In Sec. V we present our conclusions. Finally, two appendices contain some technical details of the derivations.
II. THE MODEL
Let us first formulate the coloured perceptron models. We consider p input patterns ζ µ = {ζ µ i } = {ξ µ i , η µ i }, i = 1, . . . , N consisting out of two different types of patterns ξ µ = {ξ µ i } and η µ = {η µ i }, and a corresponding set of outputs ζ µ 0 = {ξ µ 0 , η µ 0 } µ = 1, . . . , p which are determined by where h r (r = 1, 2, 3) are the local fields acting on the patterns ξ, η and their product ξη respectively Both types of input patterns and their corresponding outputs are supposed to be independent identically distributed random variables (IIDRV) taking the values +1 or −1 with probability 1/2. The set of three equations (1)-(3) defines a mapping of the inputs ζ µ i onto the corresponding outputs ζ µ 0 . We call it model I. We remark that the specific form of the equations (1)-(3) is related to the transition probabilities for a spin-flip in the dynamics [12]. A second model, denoted by II, is defined by considering only the two equations (1) and (2). When |h 3 | > |h 1 | and |h 3 | > |h 2 | then the relations (1)-(2) are satisfied by two (out of the four possible) values of the output ζ 0 , otherwise model II gives the same output as model I. In other words, due to the presence of the η µ 0 and ξ µ 0 in the gain functions, model II contains more freedom and, strictly speaking, it is not a mapping. The sequential dynamics of these two models has been studied in the case of low loading with the Hebb rule and shown to lead to the same equilibrium behaviour [12]. However, this is not guaranteed here since we are concerned with optimal couplings maximizing the loading capacity. At this point we remark that when all J (3) i are equal to zero we find back two independent standard binary perceptron models. In the sequel we take the couplings to satisfy the spherical constraint n r = √ N .
III. REPLICA THEORY FOR THE MAXIMAL CAPACITY
The coloured perceptron is trained to store correctly p = 3 2 αN patterns with α the loading capacity. The factor 3/2 follows naturally from the definition of capacity given in the introduction. A pattern is stored correctly when the so-called aligning field [15] is bigger than a certain constant κ ≥ 0 whereby the latter indicates the stability. It is a measure for the size of the basin of attraction of that pattern. Specifically we require that i } denoting the configurations in the space of interactions. For κ ξ = κ η = κ ξη = 0 all patterns that satisfy equations (1)-(3) also satisfy (6)- (8). We remark that for model II the last inequality is superfluous.
The aim is then to determine the maximal value of the loading α for which couplings satisfying (6)-(8) can still be found. In particular, the question whether this model can be more efficient than the existing two-state models is relevant.
Following refs. [1,14] we formulate the problem as an energy minimization in the space of couplings with the formal energy function defined as We remark that for model II the third Θ-factor is absent. The quantity above counts the number of weakly embedded patterns, i.e., the patterns with stability less than κ ξ , κ η , κ ξη . Therefore, the minimal energy gives the minimal number of patterns that are stored incorrectly. This number is zero below a maximal storage capacity α c (κ ξ , κ η , κ ξη ).
The basic quantity to start from is the partition function .
with β the inverse temperature. As usual it is ln Z which is assumed to be a self-averaging extensive quantity [1,15]. The related free energy per site is equal, in the limit β → ∞, to which is the minimal fraction of wrong patterns (recall eq. (9)). In order to perform the average over the disorder in the input patterns ζ µ and the corresponding outputs ζ µ 0 we employ the replica method. The calculations proceed in a standard way although the technical details are much more complex. Introducing the order parameters q (r) , with r = 1, 2, 3 and γ, τ = 1, ..., n we write following [1] Z n (β) = r,γ,τ >γ dq (r) where ... denotes the average over the patterns, r ′ = 1, 2, 3 for model I and 1, 2 for model II. Because of the latter we remark that for model II the formula for G 0 can be simplified: the integrals with respect to λ 3γ and x 3γ are not present and thus x 3γ , x 3τ and λ 3γ have to be set to zero. Because of this simplification we only outline explicitly the calculations for model II in the sequel.
The corresponding formulas for model I can be found in Appendix B.
A. Replica symmetric anzatz
We continue by making the replica-symmetric (RS) anzatz q The latter is justified for model I because of the symmetry present in this model. Furthermore, since we are going to take all q (r) → 1 in the Gardner-Derrida analysis anyway, we keep this equality also for model II. Taking then the limits β → ∞, N → ∞ and n → 0 we arrive, in the case of where ν = 1, 2, D(s(y)) = ds exp(− 1 2y s 2 )/ √ 2πy is a modified Gaussian measure, and q takes those values that minimize v, the available volume in the space of couplings. For the corresponding expression in the case of model I we refer to Appendix B.
This maximal capacity as a function of κ is shown for both models in figs. 1 and 2 as a full line. For model I we obtain, e.g., α RS (κ = 0) = 1.92, a value that is smaller than the Gardner capacity for the simple perceptron. For model II however, we get the interesting result that α RS (κ = 0) = 2.74 > 2.
B. First-step replica symmetry breaking
It is straightforward to show geometrically that learning almost antiparallel patterns, i.e., patterns satisfying (ξ µ ξ µ 0 , η µ η µ 0 ) ≈ −(ξ ν ξ ν 0 , η ν η ν 0 ) results in a splitting of the space of couplings into disconnected regions. This suggests that RS is broken and, consequently, the results for α RS found in Sec. IIIA are only upperbounds for the true capacity. Therefore, we want to improve the RS results by applying the first step of Parisi's replica-symmetrybreaking (RSB) scheme (e.g., [21]). So, we assume that the q (r) γτ in equation (14) have the following matrix block structure where n is the size of the matrix q (r) γτ , m is the number of diagonal blocks and int(x) denotes the integer part of x.
For model II we take q γτ reflecting the symmetry of this model. For model I we repeat that all q (r) 's can be taken equal. We then consider the limits q = q 1 , remains finite. After a tedious calculation we arrive at the following expression for the RSB1 maximal capacity for model II and Dt i = dt i exp(− 1 2 t 2 i )/ √ 2π a Gaussian measure. The explicit form of the function ψ RSB1 (κ, t 1 , t 2 , q The results are presented in figs. 1 and 2 as full lines. As expected they lie below the RS results confirming the breaking of RS, e.g., α RSB1 (κ = 0) = 1.83 for model I and 2.28 for model II. We remark that the breaking for model II is stronger than for model I, the reason being that model II allows more freedom as explained in the introduction. Finally, on the basis of results in the literature for the simple perceptron [15], [16] we expect that the RSB1 results are very close to the exact ones. This is further examined by performing numerical simulations as described in the following section.
IV. NUMERICAL SIMULATIONS
The idea of these simulations is to train the network with a certain learning algorithm in order to learn as many random patterns as possible. The main technical difficulties are to find an efficient algorithm and prove its convergence.
We have tried to generalize various algorithms proposed for simple perceptrons [17][18][19][20]. The most effective ones appeared to be some particular generalization of the adaptive Gardner algorithm [18] and the Adatron algorithm [19]. In the sequel we only report on the results obtained with these two algorithms. We remark that we have chosen κ ξ = κ η = κ ξη = κ in all simulations.
One of the algorithms that has demonstrated its efficiency and for which convergence has been shown in the case of the standard perceptron is given in ref. [18]. It is an adaptive version of the original algorithm proposed by Gardner [1]. Using heuristic arguments presented in [18] we have constructed for the coloured perceptron model II the following analogous learning rule The form of the algorithm for model I is a bit different and given in Appendix B. This algorithm should be carried out sequentially over the patterns and sequentially or parallel over the couplings as long as one of the arguments of the Θ functions is positive. It appears to have the characteristics of the most efficient, non-linear algorithm discussed in [18]. Using this learning rule we have trained networks of sizes 50 ≤ N ≤ 1000 sites (depending on the value of κ) in order to store perfectly as many randomly chosen patterns as possible. For each value of κ we have calculated the maximal capacity for different N and extrapolated the results to N = ∞. Results for a given value of κ and N are averages over 1000 samples. As shown in figs. 1 and 2 this algorithm performs especially well for small values of κ for both the models I and II.
The second algorithm we report on is the Adatron algorithm [19] which works in a different way. Instead of searching the maximal capacity for a given stability it tries to find the maximal stability for a given capacity. The derivation of this algorithm and a proof of its convergence are based upon the assumption that the problem can be formulated as a quadratic optimization with linear constraints [19,7]. Such a formulation can not be given for the coloured perceptron model, because the three different types of couplings have to be normalized independently and because the stability conditions (6)-(7) are more complex. Hence, a straightforward generalization similar to the one for the Potts model [7] is not possible. Below we describe a learning rule that tries to incorporate the ideas of the Adatron approach. We assume that the couplings can be written in the form (cfr., [19] and references therein) where x µ r (r = 1, 2, 3) are the so called embedding strengths of pattern µ. Then, in the case of model II the couplings are updated by modifying x µ r with the following increments This is done sequentially over the patterns. We remark that again the algorithm for model I is somewhat different (see Appendix B). For each value of the capacity we have considered system sizes 50 ≤ N ≤ 500 and extrapolated the results to N = ∞. The best results were obtained for a learning rate γ ∈ (0, 2). Results for each size are averages over 1000 samples. For small values of the capacity the algorithm gives better results, both in the case of models I and II than the first algorithm we have discussed, as shown in figs. 1 and 2. For larger values of the capacity, however, it performs worse. The results for the Adatron algorithm are displayed only in the region where they are better than the results for the Gardner algorithm. We remark that the numerical simulations with the different algorithms give different results and that we have not shown their convergence analytically such that, in principle, the values for α c obtained here are lower bounds. Looking at figs. 1 and 2 in more detail we see that for the whole range of κ the values of the maximal capacity in model II are larger than those of a standard binary perceptron. For κ = 0, e.g., the simulations give α c = 2.26 ± 0.01, which is bigger than the maximal capacity of the binary perceptron model [1] and the binary many-neuron interaction model [8], both of which have α c = 2. For model I the maximal capacity at κ = 0 found by simulations is 1.78 ± 0.01.
V. CONCLUDING REMARKS
In this work we have calculated the maximal capacity per number of couplings for two coloured perceptron models. Compared with the standard perceptron these models have two neuronal variables per site and a local field that contains higher order neuron terms. The method used is a generalization of the Gardner approach and both the RS and RSB1 results have been discussed. We expect that the latter give very close upperbounds for the exact values.
Extensive numerical simulations have been performed for finite systems and extrapolated to N = ∞. The adaptive Gardner algorithm and the Adatron algorithm give the best, but different results. Hence, the results of the simulations can be considered only as lower bounds for the exact maximal capacity. Additional work looking for improved algorithms would be welcome.
Comparing both the RSB1 results and the results from numerical simulations we conclude that they are in good agreement. For bigger values of κ they even completely coincide. For model I we find that at κ = 0 the maximal capacity satisfies 1.78 ≤ α c ≤ 1.83. This suggests that it is equal to the maximal capacity of the Q = 4-Potts perceptron, i.e., α c = 1.83 (after appropriate rescaling of the latter [7]). This would parallel the situation for Hebb learning [13]. For model II we have for κ = 0 that 2.26 ≤ α c ≤ 2.28, which is larger than the maximal capacity of the standard binary perceptron. This is due to the fact that model II is not a strict mapping such that it allows for more freedom in the determination of the couplings.
with Ds a Gaussian measure and For the available space of couplings we get in the RS approximation (compare (15) where and q taking those values that minimizes v. Thus, for κ = κ ξ = κ η = κ ξη the maximal capacity in the RS approximation can be written as r D(s r (q))ψ RS (κ, κ, κ, s 1 , s 2 , s 3 , q) − ln 4 For the RSB1 approximation with the form of the order parameters given by (21) r Dt r ln ψ RSB1 (κ, t 1 , t 2 , t 3 , q 0 , M ) with ψ RSB1 (κ, t 1 , t 2 , t 3 , q 0 , M ) a linear combination of thirty-four, mostly double, integrals over error functions. An interested reader can find a complete formula for ψ RSB1 (κ, t 1 , t 2 , t 3 , q 0 , M ) in [22]. | 2014-10-01T00:00:00.000Z | 2000-04-11T00:00:00.000 | {
"year": 2000,
"sha1": "c373b7e2417dd973a2706333fb2ae7647bcbbd0c",
"oa_license": null,
"oa_url": "http://arxiv.org/pdf/cond-mat/0004173",
"oa_status": "GREEN",
"pdf_src": "Arxiv",
"pdf_hash": "c373b7e2417dd973a2706333fb2ae7647bcbbd0c",
"s2fieldsofstudy": [
"Computer Science"
],
"extfieldsofstudy": [
"Medicine",
"Physics",
"Mathematics"
]
} |
268743404 | pes2o/s2orc | v3-fos-license | Effect of Equisetum ramosissimum Desf. on body weight and Leptin/Ghrelin in standard and high-fat diet
on body
Introduction
In terms of known global pandemics, metabolic syndromes and cardiovascular diseases score highly in records of clinically distressed patients.The principal and most well-known metabolic diseases include, but are not limited to, insulin resistance and glucose intolerance, abdominal obesity, hypertension, low high-density cholesterol, and hypertriglyceridemia (Cӑtoi et al. 2018).The pathophysiology of these metabolic syndromes is often influenced by adipokine hormones, specifically leptin and ghrelin.Adipokine hormones are well-known to modulate energy expenditure, as well as glucose and lipid metabolism, and are therefore claimed as potential predictive clinical markers for metabolic diseases (Ghadge and Khaire 2019).Studies of the correlation between adipokine concentrations and the calculated body mass index (BMI) of an individual have documented that levels of leptin and ghrelin are directly related to BMI.Meanwhile, not only is obesity directly affected by leptin and ghrelin disturbances but so also is the progression of diabetes.Thus, concentrations of adipokine hormones offer a basis for early intervention in diabetic and obese patients (Sitar-Taut et al. 2021).
Leptin is a hormone released by the adipose tissue and indirectly functions as a suppressor of appetite through signalling feedback concerning food intake and energy disbursement.In addition, leptin is reported to influence glucose homeostasis, immune response, pathogenesis of hypertension, atherosclerosis, and cancer (Arabi et al. 2019).On the other hand, ghrelin, also known as a hunger hormone, is secreted in several organs including the stomach and functions as a potent appetite stimulator.Not only does it increase in fasting periods but it also promotes weight gain.Nevertheless, ghrelin has also been reported to possess anti-inflammatory properties (Lyra Jr et al. 2019).Furthermore, decreased ghrelin levels have been reported in different pathophysiological conditions including obesity, type 2 diabetes, and other conditions with metabolic disturbances (Pulkkinen et al. 2010).Leptin and ghrelin are hormones with opposite effects on energy homeostasis and fitness.Therefore, the 'leptin over ghrelin ratio' has been suggested to be a marker and predictor in energy restriction treatment (Labayen et al. 2011).A recent study investigated the importance of the leptin/ghrelin ratio as a biomarker in dietary-induced hyperlipidemia in female mice, and found that animals fed with an excess fructose and cholesterol diet had significantly lower leptin levels and a subsequent drop in the leptin/ghrelin ratio.In addition, the animals showed low total body fat deposition.On the other hand, in another study model, specifically for dyslipidaemia, the levels of ghrelin were found generally higher than leptin (Riger et al. 2018).The level of the leptin/ghrelin ratio is reported to be altered in animals in a fasting state, in comparison to the levels after intake of varying macronutrient contents (Adamska-Patruno et al. 2018).
The interest in identifying natural products or chemicals that enhance the body to modulate the levels of either leptin, ghrelin or both is increasing.Chemical compounds which have been obtained from natural sources, including saponins, tannins, alkaloids, alkenyl phenols, glycol-alkaloids, flavonoids, sesquiterpenes lactones, terpenoids and phorbol esters, are plant constituents that have diverse medicinal properties (Talib et al. 2019;Savaya et al. 2020;Ubaydee et al. 2022).Recently, a study showed that white grape juice extracts were reported to reduce fat accumulation through the modulation of ghrelin and leptin expres-sion in an in vivo model of overfed zebrafish, indicating that natural compounds could offer a potential insight into regulation of these hormones in metabolic diseases (Montalbano et al. 2021).
Equisetum ramosissimum Desf. is a native and rare plant known as "branched horse-tail".A recent study reported high prevalence of phenolic and flavonoid contents in its airborne parts (Savaya et al. 2020), rendering it as a phytochemical product with potential antioxidant properties.The plant species E. ramosissimum belongs to the family Equisetaceae, which is documented to possess a very potent anti-diabetic and antihyperlipidemic action (Safiyeh et al. 2007;Soleimani et al. 2007).Moreover, the plant was also reported to reduce oxidative stress in the context of some diseases, including atherosclerosis, ischemic cardiac disease, aging, and even infectious diseases (Boeing et al. 2021).
The E. ramosissimum plant has been used in folk medicine for treatment of various conditions, including reducing body weight and controlled some metabolomics diseases.The use of the extract is widely communicated between local herbalists for the treatment of different conditions and as an ingredient in herbal mixtures used for diabetes, lipidaemia and obesity.Therefore, the current study was conducted to answer whether E. ramosissimum plant extract would affect serum levels of leptin and ghrelin hormones and their ratios in an in vivo high-fat diet (HD) rat model.To our knowledge, it is a first to investigate the effect of this extract on metabolic hormones concerning body weight.
Preparation and extraction of E. ramosissimum
The species of E. ramosissimum was collected from Mujib Biosphere Reserve (84 Km from Amman -FH4V+6PQ, Dead sea road, Sweimeh, Jordan) after authentication by a professional taxonomist at the Nature Conservation Monitoring Centre (Amman, Jordan) (RCSN herbarium no.E.r-5/7/2017).The shoot (ariel) part of the plant was dried under shade and stored thereafter at room temperature.On demand, the dried plant was crushed using a commercial blender (Philips HL7756/00 Mixer Grinder, 750W, USA) to obtain an acceptable particle size for further processing.A maceration method using an ethanolic medium (100% EtOH) was used for phytochemical extraction based on an in-house validated method.Briefly, 0.3% (w/v) of plant powder was soaked in ethanol for 24 h with constant agitation at room temperature.The mixture was then paper-filtered and evaporated at 60 °C using a rotary evaporator (R-300, Buchi, USA) at 90 rpm producing an extraction yield of 40.3% (w/w dry weight) for ethanolic extract.Extracts were stored dry conditions at room temperature 22 °C.
Phytochemical analysis of Phenols and Flavonoids
In order to determine the total phenol content within the extract, the Folin-Ciocalteu method was used as described earlier (Zahra et al. 2015).Briefly, plant extract was dissolved in methanol (0.002% w/v) and then Na 2 CO 3 solution (5% v/v in distilled water) and Folin reagent (20% v/v in distilled water) were added to initiate the reaction medium.Reaction was maintained covered under aluminium foil for 1 h at room temperature.Then, the solution was quantified against gallic acid as a reference standard through determining the UV spectroscopy (Hitachi U-1800, UK) readings at 760 nm.
For the determination of total flavonoid content, a colorimetric method, based on the formation of a complex flavonoid-aluminium was employed, as described previously (Pękal and Pyrzynska 2014).Briefly AlCl3 solution (0.5 mL, 2 %, w/v) was added to 1 mL of the extract sample, and subsequently 0.5 mL of water was added.Then, the mixture was mixed and incubated for 10 mins at room temperature prior to spectral analysis.The solution was quantified against quercetin as a reference standard through determining the UV spectroscopy readings at 510 nm.
In vivo experimental design
Eight-week old male Wistar rats were housed and acclimated at the animal house of Applied Science University, Amman Jordan.Handling and treatment procedures were conducted after acquisition of ethical approval from the Institutional Review Board approval no.2021-PHA-40 (Date: 5/12/2021), and in accordance with the guidelines of its Institutional Animal Ethical Committee.Animals were maintained under conditions of 12 h light/ dark cycle, ambient room temperature (25 ± 2 °C), and with ad libitum access to water.
In our previous published work, an in vivo study on healthy standard diet-fed animals and an induced hyperlipidaemia model, it was concluded that the extract alone and in combination with Atorvastatin had a significant (P<0.05)reducing effect on serum lipid profile (Al-Bayati et al. 2023).These findings revealed the potential advantages of the extract alone and suggested further exploration of its mechanisms.Therefore, the current investigation focused on the action of extract on body weight variation and modulation of Leptin/Ghrelin concentrations post treatment, in both standard and high fat diet.Being a crucial element of the study, diet was served under specific parameters as described previously (Irudayaraj et al. 2013;Riger et al. 2018).Animals were randomized into 4 groups (n = 6) housed as 4 animals per cages and provided with corresponding diets for 14 days prior to treatments, specifically 2 groups receiving standard diet (SD) and 2 receiving high-fat diet (HD).The HD, containing 30% of lamb fat (50 g/kg/day) mixed with standard ro-dent chow, is considered rich in cholesterol and capable of inducing body weight gain.Then, after confirmation of the obesity model with at least 20-30% increase in weight, groups were treated once daily with either plant extracts (500 mg/kg) or vehicle (water) using oral intra-gastric tube for another 14 days (total study period of 28 days) (Alebous et al. 2016).Based on our previous study a lower dose of plant extract was employed (200 mg/kg) and was found to be effective in lowering the lipid profile of animals on high-fat diet, therefore in order to induce a prominent effect, a higher safe dose was used in the current study (Al-Bayati et al. 2023).Study timeline described in Scheme 1.
Serum concentration of Leptin and Ghrelin
In order to determine the concentrations of leptin and ghrelin in treated animals, serum samples were collected from all rats at the termination of the study.On the 28 th day of the study, animals were fasted overnight and prepared for sampling under 2% isoflurane anaesthesia.Blood samples were collected from the orbital plexus using heparinized capillary tubes into plain mini-collect tubes.Animals were sacrificed by cervical dislocation after completion of sampling.Serum was separated after centrifuging the blood tubes at 8,000×g for 10 mins at ambient temperature.Serum samples were quantified for leptin and ghrelin concentrations using colorimetric assays and in accordance with the manufacturer's instructions for each kit, Abcam (ab100773, UK) and MyBiosource (MBS731169, US), respectively.
Statistical analysis
The statistical analysis of data was done using SPSS, version 27.0 (Chicago, IL, USA).One-way ANOVA test using Tukey's post hoc was used to obtain statistical significance between all study groups, and student t-test was made to compare data of treated and non-treated groups of the same diet.Data are presented as mean values with standard deviation (SD).All experiments were run in triplicates unless stated otherwise.
Phytochemical analysis
Methanol extract of E. ramosissimum was examined for phenol and flavonoid content.The extract presented a yield of 0.032 ± 0.01 mg/mg phenolic content in dry extract, calculated as gallic acid equivalence.As for the flavonoid content, a yield of 0.44 ± 0.03 mg was calculated in the dry extract as quercetin equivalence, as shown in Fig. 1.
Percent change in Body Weight
After confirming the weight-gain animal model, animals were treated with E. ramosissimum extract and weights were recorded after 14 days of treatment.As shown in Fig. 2, a decrease in weight was observed in animals treated with the plant extract.The drop in body weight after treatment with plant extract was considered statistically significant in animals receiving a standard diet (p-value<0.001).Despite the statistical insignificance, a decrease in body weight was also noticed in animals that received the high fat diet.
Serum Leptin and Ghrelin concentrations
As shown in Fig. 3, the differences in serum ghrelin concentrations are marked between animals treated with high-fat diet, as well as all animals treated with the plant.The increase in ghrelin levels was considered statistically significant between the SD and SDP groups, which indicates a direct effect of the plant extract.On the other hand, a similar increase was also noticed in animals receiving a high-fat diet (p<0.05),regardless of whether they were treated with the plant extract or not.However, and although not considered statistically significant, animals on the high-fat diet had lower levels of ghrelin in comparison to those on a standard-diet which had been receiving plant extract.
Correlation of body weight change and Leptin/Ghrelin ratio
As shown in Table 1, a marked statistical correlation between the BW and the evaluated biomarkers of serum levels for leptin, ghrelin and their ratios was observed.The correlation of body weight change and Leptin/Ghrelin ratio was made based on calculating the correlation factor (R), the coefficient determination (R 2 ), and significant p-values (<0.05).Despite animals treated with plant extract after consuming standard-diet having showed strong correlation between change of body weight and leptin/ ghrelin concentration, based on high R and R 2 values, animals fed with a high-fat diet showed a stronger correlation between changes of body weight and levels and ratios of leptin and ghrelin.Nevertheless, animals treated with the plant extract after being on the high-fat diet also showed a higher significant correlation between the leptin/ghrelin concentration and change of body weight, in comparison to all study groups.
Discussion
The natural collaboration between leptin and ghrelin in maintaining appetite, weight control and energy homeostasis remains an important topic that needs further understanding.Adiposity and appetite regulating hormones, including leptin, ghrelin, insulin and others, are key biotargets in the fields of nutraceutics, herbal medicines and general physical health.The investigation of nutraceuticals has great potential value for limiting or preventing excess weight gain and its accompanying metabolic syndrome.
Extracts collected from different species of Equisetum have been reported to be rich sources of phenolic compounds and flavonoids that have pharmacological properties (Boeing et al. 2021).In addition, alkaloids, phytosterols, tannins, and triterpenoids of the Equisetum genus are among its most well-known phytochemical constituents (Mimica-Dukic et al. 2008).Horse tail species have been reported to have haemostatic, diuretic, antifungal, antiviral, antibacterial, antioxidant, and anticancer properties (Boeing et al. 2021).Hypoglycaemic effects were also reported in some species of horse tail plants such as E. arvense, E. myriochaetum, and E. giganteum (Angel et al. 2020;Hegedűs et al. 2020;Vieira et al. 2020).The prevalence of high phenolic and flavonoid content in the study species, aligns with a suggestion of its potential in controlling diabetes as well as for lowering cholesterol levels and body weight (Hegedűs et al. 2020).Several compounds such as kaempferol, luteolin, myricetin, quercetin, rutin, caffeic acid derivative, tannins, ferulic acid, linoleic acid, saponin, and phytosterol, which have been isolated from an alcoholic extract of E. rasmossessium, are well-documented to possess positive effects on metabolic diseases (Revilla et al. 2002;Afifi and Kasabri 2013;Batir-Marin et al. 2021).
Introducing the use of E. rasmossessium extract as a potential candidate in regulating targets involved in metabolic diseases has not previously been studied.Therefore, the current study focused on observing the potential of the extract on animals of high weight, and its capacity in stimulating body weight loss.Identifying the suspected underlying mechanism, we focused on evaluating the correlation of serum levels of leptin, ghlerin and their calculated ratios.Despite the levels were considerably lower than treated animals fed on SD, the direct effect of the E. rasmossessium extract on leptin levels in animals fed on HD was statistically insignificant, which is in accordance with previously published results (Schmid et al. 2005).Ghrelin appears to be inversely related to body weight (increases in standard diet rats that lose weight with treatment, Fig. 2) but directly related to body weight in rats that gain weight with high fat diet and show elevated ghrelin regardless of treatment (increased leptin with increased body weight with high fat diet).Therefore, it could be suggested that levels of ghrelin are increased in animals treated with the plant extract after being on a standard diet and are slightly decreased in others on a high-fat diet.Despite the fluctuating levels of ghrelin in HD animals, they remain higher than in animals of the same diet that were treated with the plant extract.Therefore, in order to ascertain the effect of plant extract on lowering ghrelin in animals on HD, a larger sample population study will be conducted.The contrary data of having the plant extract affect ghrelin levels based on the nature of the diet suggests that the plant extract could have a neutralizing influence on levels ghrelin.In line with these results, higher baseline ghrelin levels have been shown to be accompanied by weight loss (Garcia et al. 2006;Rosenbaum et al. 2019), and fasting ghrelin levels correlate inversely with food intake (Salbe et al. 2004).
Conclusions on leptin levels and its correlation with body weight loss could be completed through calculating and understanding its ratio in comparison to levels of ghrelin.Findings of the study revealed a direct effect of E. rasmossessium extract on body weight, regardless of whether exposed to a standard or high-fat diet.Nevertheless, its potential for reducing the body weight of high weight animals was also observed.These data highlighted the differences in the correlations between body weight and the measured biomarkers within the two sets of groups: standard and high-fat diet fed animals.Treatment with the plant extract showed that leptin levels are strongly correlated to body weight in animals on a standard diet.
On the other hand, the correlation was considered statistically weak when levels of leptin were compared in animals fed with a high-fat diet.Several studies have investigated the role of leptin on the changes in energy metabolism that occur during weight loss.In obesity, the existence of an endogenous leptin-resistance mechanism limits its impact on weight, interpreted as an energy-sparing mechanism operating in obese animals (Labayen et al. 2011;de Git et al. 2018;Barham et al. 2021).The reverse correlation between body weight and serum leptin levels is now well recognized in human and animal studies (Labayen et al. 2011;Arabi et al. 2019;Montalbano et al. 2021).In the same context, the reduction of leptin and leptin/ghrelin ratio reflects an improvement in leptin resistance that accompanies loss of body weight which has been considered as a key factor in improving insulin resistance (Pulkkinen et al. 2010;Riger et al. 2018).
Conclusion
E. rasmossessium extract shows promising characteristics of being introduced in dietary supplements, weight control regimens and some medical interventions.The current study presents the influence of E. rasmossessium extract in decreasing body weight in animals receiving standard diet and limiting weight gain in animals on high-fat diet.Further preclinical studies are warranted to reveal the clincial outcomes of E. rasmossessium extract and its vital influence on adipokines, specifically leptin and ghrelin.
Scheme 1 .
Scheme 1. Experimental in vivo study design.
Figure 1 .
Figure 1.Total Phenol and Flavonoid content in E. ramosissimum extract.Extracts are quantified against equivalent quantities of reference standards, specifically Gallic acid and quercetin, respectively.
Figure 2 .
Figure 2. Percent Body Weight change in healthy and obese animals treated with E. ramosissimum extract.Comparison of weight change was made between: Standard Diet (SD); SD receiving Plant extract (SDP); High-fat Diet (HD); and HD receiving Plant extract (HDP).***: p<0.001 in comparison to SD, as calculated through t-test.
Figure 3 .
Figure 3. Serum concentrations of leptin and ghrelin and their corresponding ratios.Serum concentrations of leptin and ghrelin and their corresponding ratios were quantified for animals receiving Standard Diet (SD); SD receiving Plant extract (SDP); Highfat Diet (HD); and HD receiving Plant extract (HDP).Ratios of leptin/ghrelin were calculated and presented in normalized values to the standard body levels (right y-axis), specifically per animals receiving SD. **: p<0.01; *p<0.05 in comparison to SD.
Table 1 .
The difference in correlation factor, coefficient determination, and significance p-values between change in body weight and its relation to serum levels of leptin, ghrelin and their ratio in study groups (n = 6). | 2024-03-30T15:10:40.803Z | 2024-03-26T00:00:00.000 | {
"year": 2024,
"sha1": "41b0023e5e43eba8d53a2eb40e170db4c98038c0",
"oa_license": "CCBY",
"oa_url": "https://pharmacia.pensoft.net/article/117823/download/pdf/",
"oa_status": "GOLD",
"pdf_src": "ScienceParsePlus",
"pdf_hash": "fcbe4e8f98654819f147960da605a945e489524e",
"s2fieldsofstudy": [
"Medicine",
"Agricultural and Food Sciences"
],
"extfieldsofstudy": []
} |
250297654 | pes2o/s2orc | v3-fos-license | Enhancing the Interfacial Stability of High-Energy Si/Graphite j j LiNi 0.88 Co 0.09 Mn 0.03 O 2 Batteries Employing a Dual-Anion Ionic Liquid-based Electrolyte
The poorly flammable room-temperature ionic liquid-based electrolyte composed of lithium bis(trifluoromethanesulfonyl)imide (LiTFSI) and N-butyl-N-meth-ylpyrrolidinium bis(fluorosulfonyl)imide (Pyr 14 FSI) with fluoro-ethylene carbonate (FEC) as an additive is investigated towards its compatibility with the LiNi 0.88 Co 0.09 Mn 0.03 O 2 (NCM88) cathode and a high-capacity Si/graphite (SiG) anode, revealing a remarkably stable performance in lithium-ion cells. Interestingly, this dual-anion electrolyte with FEC additive forms a stable electrode-electrolyte interphase on both sides, which suppresses the morphological degradation of the electrode materials and continuous electrolyte decomposition. Consequently, lithium-ion cells using such dual-anion ionic liquid-based electrolyte display significantly improved cycling stability compared to conventional carbonate ester-based electrolyte, achieving a high specific energy of 385 Whkg (cid:0) 1 (based on both cathode and anode active materials weight) with a capacity retention of 74% after 200 cycles at 0.2 C, demonstrating the possibility to realize safe and high energy density LIBs.
Introduction
High energy density lithium-ion batteries (LIBs) are required to increase the driving range of electric vehicles.Thus, selecting advanced electrode materials with high specific (theoretical) capacity is the simplest approach to improve the energy density of LIBs. [1,2]At the negative electrode, to avoid the safety risks posed by using metallic lithium, [3] silicon is considered the best candidate to replace graphite.Silicon has an almost ten times higher specific capacity than graphite (3579 mAh g À 1 ), is less expensive and far more abundant in the Earth's crust.[6] However, the main challenge of Si anodes is their large volume expansion during lithiation, resulting in unstable and excessive solid electrolyte interphase (SEI) formation and particle pulverisation.This leads to a rapid capacity fading of pure Si anodes and hinders their practical implementation. [6,7]The main strategies to counter this are the modification of Si by designing nanostructures, [8] coating the material with carbon, [9,10] or using functional binders with high modulus to buffer the volume changes. [11,12]However, from a practical perspective, the incorporation of only small amounts (3-5 wt.% [13] ) of Si in composites with graphite has been considered the most efficient to increase the capacity and energy density, as well as retaining the high stability of graphite electrodes; this has been introduced for commercial LIBs. [14]or the positive electrode, Ni-rich layered oxide materials (LiNi x Mn y Co z O 2 , x � 0.6, x + y + z = 1, NCM) are currently the preferred choice for high-energy LIBs, following the trend towards increasingly high Ni contents to reduce the amount of critical and toxic cobalt and increase the specific capacity (� 170 mAh g À 1 ). [15,16]Nevertheless, very high nickel contents (x � 0.8) also introduce safety concerns and reduce the cycle life of the cells, because of severe parasitic side reactions with the electrolyte as well as structural and morphological degradation of the cathode material at high state of charge. [17,18]The oxidative decomposition of the electrolyte in combination with the highly reactive Ni 4 + present in the charged state lowers the thermal stability of the material.In addition, the similar ionic radius of Li + (0.076 nm) and Ni 2 + (0.069 nm) results in an intermixing of Ni and Li ions in the structure, promoting the phase transition from the layered to the electrochemically inactive rock-salt structure. [19,20]Additionally, the large variation of unit cell volume generates mechanical stress on the secondary particles.During repeated cycling, cracks are formed rapidly causing the penetration of electrolyte into the secondary particles, where the detrimental side-reactions are triggered, accelerating the structure deterioration and voltage decay. [21,22]Thus, the application of these materials in lithiumion cells remains challenging, especially because of the formation of unstable cathode electrolyte interphases (CEI) and thermal instability.
Recently, a safety improvement was achieved using room temperature, non-volatile and poorly flammable ionic liquid electrolytes (ILEs) specifically with respect to Ni-rich cathodes, achieving excellent long-term cycling stability. [23,24]Heist et al. [23] employed a bis(fluorosulfonyl) imide (FSI)-based ILE for Ni-rich cathodes (LiNi 0.8 Mn 0.1 Co 0.1 O 2 ) for the first time and obtained satisfactory electrochemical performance.In our previous work, a dual-anion ILE consisting of bis(fluorosulfonyl) imide (FSI) and bis(trifluoromethanesulfonyl)imide (TFSI) was successfully applied to enhance the stability of highly Ni-rich layered oxide cathodes (LiNi 0.88 Co 0.09 Mn 0.03 O 2 , NCM88), achieving an outstanding capacity retention of 88 % over 1,000 cycles in lithium-metal batteries (LMBs). [24]Although these ILEs reportedly inhibit surface degradation of Ni-rich NCM layered cathodes, they are mostly used in lithium-metal cells due to the frequently observed incompatibility of ILEs with graphite. [25]Using ILEs with Si/graphite (SiG) negative electrodes is similarly challenging because the organic cations may insert into graphite, partially leading to its decomposition due to exfoliation resulting in decreased battery performance and the formation of unstable SEI. [26,27]Herein, for the first time, we successfully realized the combination of SiG composite negative and Ni-rich NCM88 positive electrodes in lithium-ion cells employing a safe poorly-flammable dual-anion ionic liquid-based electrolyte.The comparative study of the two slightly different ILEs based on the Pyr 14 + cation and the FSI À anion as solvents, investigates LiTFSI and LiFSI as Li salt, with fluoroethylene carbonate (FEC) as additives to achieve stable cycling of SiG j j NCM88 lithiumion cells.Remarkably, choosing the right ILE, highly stable electrode-electrolyte interphases are formed on both electrodes (SEI and CEI) and the SiG j j NCM88 lithium-ion cell shows a high specific energy of 385 Wh kg À 1 (based on both cathode and anode active materials weights) with a capacity retention of 74 % after 200 cycles.
Results and Discussion
The morphology of the SiG (Figure S1) and the NCM88 (Figure S2) active materials was characterised by scanning electron microscopy (SEM).Most of the 5-10 μm sized Si particles are evenly distributed among the graphite, with only a few particles of significantly smaller size (< 1 μm) visible.The NCM88 is mostly composed of secondary particles of a spherical morphology with the particle size in the range of 2 to 15 μm.Higher magnification SEM image indicates that the primary particles have an irregular rod-like shape and are tightly packed together (see Figure S2b).Prior to lithium-ion cell assembly, the electrochemical performance of SiG negative electrodes and NCM88 positive electrodes has been evaluated in half-cells using a conventional organic carbonate ester-based electrolyte (1 M LiPF 6 in EC/DEC + 10 wt.% FEC + 1 wt.%VC, labelled as OR) as shown in Figure 1.The first dis-/charge capacity of SiG is 501.0/625.6 mAh g À 1 at 0.1 C (1 C = 600 mA g À 1 ), corresponding to an initial Coulombic efficiency (ICE) of 80 %.The relatively low ICE (compared to pure graphite) is due to the irreversibility of Li alloying with Si and the electrolyte decomposition forming the SEI. [28]Figure 1d shows the rate capability and cycling performance of SiG in half-cells at initially 10 cycles each at 0.1 C, 0.2 C, and 0.5 C, before continuous cycling at 0.2 C. The capacity retention is 88.2 % after 100 cycles with an average Coulombic efficiency (CE) of 99.6 % (not considering the ICE).The electrochemical performance of NCM88 in half-cells is shown in Figure 1(b and e).The first dis-/charge capacity is 213.8/243.9mAh g À 1 at 0.1 C (1 C = 200 mA g À 1 ) in a voltage range of 3.0-4.3V vs. Li + /Li.After three formation cycles at 0.1 C, the NCM88 half-cell is continuously cycled at 0.2 C for 100 cycles, showing a capacity retention of 85.9 % and an average CE of 99.7 %.To achieve stable and high energy SiG j j NCM88 lithium-ion cells, the voltage window was set to 2.6-4.1 V. Considering the formation of SEI and CEI on the electrodes during the first cycle and the excess consumption of Li ions from electrolyte and cathode, the SiG anode was pre-lithiated to 0.25 V (~60 mAh g À 1 ), and then assembled against the NCM88 positive electrode. [27]Figure S3 presents typical voltage profiles of the lithium-ion cell during initial cycles.The specific dis-/charge capacity of the first cycle is 195.2/220.6 mAh g À 1 , corresponding to an ICE of 88.5 %.The irreversible capacity loss may still be due to electrolyte decomposition and SEI/CEI formation on both electrodes.The specific energy of such cells tested in OR electrolyte is 458 Wh kg À 1 , based on the cathode and anode active material weights.Figure 1(c and f) shows the evolution of voltage profiles and cycling performance of the SiG j j NCM88 cell cycled at 0.5 C in the voltage range of 2.6-4.1 V.The SiG j j NCM88 cell delivers 86 % of the initial capacity after 100 cycles with an average CE of 99.9 %.However, the capacity fades rapidly during the following cycles indicating the Li-reservoir from pre-lithiation of the anode has been consumed in the early stages of cycling.After 200 cycles, the capacity retention is only 53 % and the average CE decreased to 99.7 %.In addition, the voltage profiles show increasingly high polarisation.The severe capacity fading during long-term cycling is mainly caused by the large volume expansion and particle pulverisation of Si during repeated lithiation, causing the continuous reaction with electrolyte and resulting in a thick SEI that increases cell resistance. [6,7]This is additionally amplified by the instability of the NCM88 CEI in organic carbonate ester-based electrolytes, and cathode material degradation at high state of charge. [24,29,30]o fully exploit the great potential of the individual electrode materials, and at the same time improve the safety and electrochemical performance of SiG j j NCM88 lithium-ion cells, we aimed to replace the OR electrolyte by poorly flammable ILEs [0.8 mol Pyr 14 FSI-0.2mol LiFSI + 10 wt.% FEC (ILE1) and 0.8 mol Pyr 14 FSI-0.2mol LiTFSI + 10 wt.% FEC (ILE2)], with FEC as an SEI stabilising additive for the SiG negative electrode. [31,32]The electrochemical stability window of the ILEs was determined by linear sweep voltammetry (LSV) shown in Figure 2(a).The ILE with exclusively FSI À anions (ILE1) displays a steeper rise of the anodic current than the dual-anion electrolyte (ILE2) with additional TFSI À anions, with a slightly higher anodic stability.The ionic conductivity of the two ILEs is displayed in Figure 2(b).ILE1 exhibits slightly higher ionic conductivity, from 18 mS cm À 1 at 80 °C to 0.5 mS cm À 1 at À 20 °C.Meanwhile the ionic conductivity values of ILE2 range from 15 mS cm À 1 at 80 °C to 0.3 mS cm À 1 at À 20 °C.The lower ionic conductivity of ILE2 is due to the large molecular size of TFSI À compared to FSI À , which increases the viscosity and glass transition, and thus decreases the conductivity of the electrolyte. [33]Nevertheless, the room temperature ionic con- ductivity of ILE1 and ILE2 is in the same order of magnitude as the organic electrolyte (7 × 10 À 3 -8 × 10 À 3 S cm À 1 [34] ), i. e., suitable for application.To provide further insight regarding the compatibility of the ILEs towards the anode and its SEI forming capability, cyclic voltammograms (CVs) were recorded for SiG electrodes in both ILEs as shown in Figure S4.The CV curves show the typical features related to alloying and de-alloying of Li with Si.The corresponding cathodic peak is observed at 0.2 V and the anodic peaks appear at 0.31 and 0.5 V. [35] Moreover, the pronounced anodic feature located at 0.2 V represents the Li extraction from graphite. [29]The CV results indicate that the ILEs and graphite have good compatibility, and no other cathodic features which would indicate Pyr 14 + cation intercalation into graphite are observed.Figure 2(c and d) shows the charge/ discharge curves of SiG half-cells in these two electrolytes, with very similar electrochemical performance as in OR electrolyte (Figure 1).The first specific dis-/charge (delithiation/lithiation) capacity in ILE1 is 496.8/604.3mAh g À 1 , which is slightly lower than that observed in ILE2 (535.9/662.7 mAh g À 1 ).The cycling stability of the SiG electrode has been assessed using the same procedure as in OR electrolyte.The results further confirm the good compatibility of both ILEs with graphite.After 100 cycles at 0.2 C, the capacity retention is 76 % in ILE1 and 82 % in ILE2, respectively (based on the first cycle reversible capacity at 0.05 C; Figure S5).The higher specific capacity and more stable cycling performance was obtained for the dual-anion electrolyte containing FSI À and TFSI À anions (ILE2).This might be ascribed to the well-maintained integrity of SiG particles cycled in ILE2 electrolyte, facilitated through a protective and stable SEI on the surface of Si and graphite.The decomposition mechanisms of FSI À and TFSI À upon electrochemical reduction differ, [33,36,37] resulting in different chemical composition and morphology of the IL-derived SEI.In relation to the interaction of ILEs with the positive electrode, Figure 2(e and f) compares the electrochemical performance of NCM88 half-cells in the different ILEs.In both cases, NCM88 has its typical discharge/ charge profile and similar specific capacity.Considering our previous work, this is not surprising as excellent long-term cycling performance can be achieved in these types of ILEs which is attributed to the interface-stabilising properties of ILE and the formation of a beneficial CEI on Ni-rich positive electrodes. [24]he electrochemical performance of lithium-ion cells using either of the two ILEs was further investigated.The full-cell were balanced with a Negative/Positive capacity ratio of ~1.2.To prevent Li plating on the negative electrode and extreme delithiation of the positive electrode, the voltage window was limited to 2.6-4.1 V.The voltage profiles in Figure 3 display the typical response of SiG j j NCM88 lithium-ion cells and are recorded at 0.2 C. In Figure 3(a) the polarisation gradually increases upon cycling in ILE1, which could be due to the unstable electrode-electrolyte interphases and internal resistance increase.On the contrary, the polarisation only slightly increases in ILE2.The corresponding differential capacity plots are shown in Figure S6.The anodic redox features appear to be reversible in ILE2, while a substantial loss of redox activity and a strong shift of the feature around 3.6 V is observed in ILE1, which might reflect the structural degradation of the active materials. [38]Although both electrolyte systems show a gradual capacity decay, the capacity retention after 200 cycles is much higher for the cell using ILE2 (51.7 % vs. 73.8%, respectively).The fading is likely associated to the relatively low CE, i. e., irreversible Li losses in repeated SEI formation on the Si (and graphite) surface after rupture due to the large volume expansion of the Si fraction and exfoliation of graphite in the composite. [39]Besides, the structural and morphological degra- dation of the positive electrode might also affect the full-cell performance.Figure 3d compares the average CE of both lithium-ion full-cells, which in the early stages is clearly lower (98.5 %) for ILE1, before it gradually increases to 99.0 %.This indicates more severe Li loss during the first few cycles and is reflected in the rapidly fading capacity.In ILE2, a notably higher average CE of 99.8 % was achieved yet from the initial cycles, which is not only higher than for ILE1, but also on a similar level as for the OR electrolyte with fine-tuned additives.Consequently, Figure 3(e) displays the average discharge voltage of the SiG j j NCM88 lithium-ion cell using ILE2, which is extremely stable and close to 3.6 V.In addition, the first discharge capacity is 166 mAh g À 1 at 0.2 C, corresponding to a high specific energy of 385 Wh kg À 1 , which is an excellent value for an ILE-based LIB using SiG negative and high Ni content positive electrode.More importantly, a high specific energy of 300 Wh kg À 1 could be maintained throughout 200 cycles, which confirms a good stability that could potentially meet the requirement for electric vehicle application. [40]Electrochemical impedance spectroscopy measurements further demonstrated the effect of ILE2 on the stability of the electrode-electrolyte interface, as shown in Figure S7, which includes the SEI/CEI film resistance (R f ) and the charge transfer resistance (R ct ) of the cathode and anode electrode.Upon cycling, i. e., after 10 and 20 cycles, the full-cell shows rather low and stable impedance.These results suggest that ILE2 can stabilise the electrodeelectrolyte interfaces of both the electrodes improving the long-term cycling performance of the high-energy lithium-ion cells.
To elucidate the mechanism of enhanced lithium-ion cell performance in ILEs, an in-depth SEM analysis was carried out to investigate the structural evolution of the active materials after 200 cycles.The SEM images of cycled SiG electrodes in both ILEs are shown in Figure 4, serious exfoliation of the graphite layers can be seen for ILE1, which might be attributed to an unstable SEI.The co-insertion of solvent molecules together with Li-ions exfoliates the graphite layer.In contrast, the graphite and Si particles of SiG electrodes cycled in ILE2 still show the original flake-like morphology and no pulverisation of Si (Figure S8).This highlights the favourable impact of the mixed anions of FSI À and TFSI À with high electrochemical stability during repeated de-/lithiation.
A comparably striking difference is observed for the positive electrodes recovered after cycling.Figure 5 depicts top view and cross-section SEM images after focused-ion beam (FIB) milling.From the top view (Figure 5a, d, g), all the samples have a similar morphology without any evident cracks or fracture of the secondary particles surrounded by carbon black.However, at the interior of the particles, the picture is completely different.The fresh electrode has a well-structured shape with no microcracks between the clearly visible primary particle grains.Whereas the secondary particles cycled in ILE1 have several cracks inside their core, due to the strong anisotropic strain of the crystal lattice when lithium ions are extracted during charging. [40]On the contrary, the microcracks in NCM88 cycled in ILE2 are significantly reduced, if not absent, suggesting that a stable CEI is formed that protects the secondary particles' structure.Taken together these findings highlight the importance of the electrolyte, which appears to dictate the evolution of the morphology of the active materials in both positive and negative electrode and thus strongly affects the electrochemical performance.
To explicate the nature of the different electrochemical performance and how the two ILEs affect the morphology of the active material particles, the surface chemistry compositions of cycled SiG and NCM88 electrodes were investigated by X-ray photoelectron spectroscopy (XPS).According to the literature, the Li + cation is preferentially coordinated by TFSI À instead of FSI À , which is expected to be true for the charge/ discharge process and therefore reflected in the chemistries of both the SEI and CEI. [42,43]Figure 6 compares the XPS spectra, before and after sputtering 3 and 10 min of SiG anodes after 200 cycles in either electrolyte recovered from the lithium-ion cells.For ILE1 (Figure 6a), the O 1s spectrum has two components at 532.4 and 531.3 eV, which correspond to À CÀ OÀ CÀ /À S=O and À C=O, respectively. [44]In the C 1s region, the peaks characteristic for À CO 3 2À (289.0 eV), À CÀ OÀ CÀ /À CÀ NÀ (286.3 eV) and À CÀ CÀ /À CÀ HÀ (284.8 eV) [45] are observed, which is consistent with the O 1s spectrum.Interestingly, the C 1s region does not show À CÀ F bonding from FEC at high binding energies (> 290 eV) [46] indicating the FEC reduction by F À release and ring-opening forming À CÀ OÀ CÀ polymeric species. [47,48]This agrees with the F 1s region, where two signals are observed, which correspond to FSI À and LiF inorganic species.The latter one formed from FSI À and FEC reduction. [49,50]his is supported by the N 1s spectrum, where FSI À contribution is observed at 399.5 eV, as well as Li 3 N from FSI À reduction. [51]In addition, Pyr 14 + traces are shown at 402.3 eV.Finally, the S 2p region provides more details about IL fragments, such as, FSI À , FNSO 2 À /SO 4 2À and SO 3 2À (Li 2 SO 3 ) at 169.8, 168.7, and 166.8 eV, respectively. [52,53]After 3 and 10 min of sputtering, the chemical species and their relative concentration remains practically unchanged, indicating that a significant amount of IL is trapped on the SEI, with a thick SEI being formed that is chemically homogeneous at higher depths.A thicker SEI may increase the resistance of the electrode and cause sluggish Li + transport kinetics contributing to capacity fading.
On the other hand, even though the SEI of the SiG electrode cycled in ILE2 (Figure 6b) is composed of similar species as in ILE1, such as À COCÀ , À C=O (Li 2 CO 3 ), LiF, Li 3 N, NFSO 2 À and Li 2 SO 3 , different concentrations are observed.In addition, ROCO 2 Li, Li 2 O, and Li x C y species are also formed in ILE2 SEI, as the O 1s and C 1s spectra indicated. [44,45]Moreover, the spectra after 3 and 10 min of the sputtering indicate that the SEI species distribution is not homogeneous as in ILE1 SEI.In fact, the oxygen-carbon species, such as À CÀ OÀ CÀ , and ROCO 2 Li, as well as low concentration of ILE2 traces (Pyr 14 + and TFSI À /FSI À ) are mainly observed on the outermost layer of the SEI, while the sublayer (close to SiG electrode) is composed of inorganic-rich species, i. e., Li 2 O, Li 2 CO 3 (À C=O), Li x C y , LiF and Li 3 N.Moreover, the ILE2 SEI is overall richer in inorganic species, which provides high ionic conductivity, superior Li ion diffusion and electrochemical performance. [54]2À (Li 2 CO 3 )/À CF 2 (PVdF, FEC and/or FSI/TFSI). [53]The comparison of the F 1s, N 1s and S 2p spectra of cycled NCM88 and SiG electrodes shows that the ILEs are decomposed to a lesser extent at cathode side than the anode side, not forming inorganic-rich CEIs.The ILE2 CEI exhibits low concentration of LiF, which may be due to less decomposition of FEC and FSI À /TFSI À anions.This is expected since TFSI À prefers to absorb at the electrode and hence displaces a fraction of FSI À from the surface, therefore, the relatively slower reaction of TFSI À helps to control the FSI À decomposition kinetics. [42]As a result, less LiF and other FSI À / TFSI À reduction products accumulate on the surface, leading to the formation of a more uniform and robust CEI. [42]n summary, single-anion ILE forms a thick SEI and CEI, which potentially leads to rapidly increasing cell impedance, accelerating the growth of cell polarisation as observed in Figure 3, and resulting in a rapid capacity decay.On the contrary, the dual anion electrolyte (ILE2) possesses an SEI composed of chemically different layers, i. e., organic-rich outer-most and inorganic-rich sublayer, while thinner CEI is formed with respect to ILE1, due to the lower decomposition of the FSI/TFSI and FEC on the cathode side.The different SEI and CEI properties on the ILE2 system may be optimal for a superior electrode protection, since NCM88 shows reduced crack formation, and SiG does not show graphite exfoliation and pulverisation of Si during cycling.
Conclusion
In this work, the stable cycling of high specific energy LIBs comprised of NCM88 (Ni-rich) positive electrodes and SiG negative electrodes were demonstrated using an IL-based electrolyte.Lithium-ion cells employing ILE2 (0.2 mol LiTFSI-0.8mol Pyr 14 FSI-FEC) delivered a high specific energy of 385 Wh kg À 1 based on both positive and negative active materials weight with a capacity retention of 74 % after 200 cycles.The enhanced performance is ascribed to the different electrode-electrolyte interphase properties elucidated for ILE2.The SEI is composed of an inorganic-rich sublayer and organic outermost layer while NCM88 is covered by thin CEI.Consequently, the structural degradation and particle cracking of the Ni-rich material as well as the pulverisation of the micrometre-sized Si could be effectively mitigated.Moreover, the graphite fraction displays high compatibility with ILE2 and does not exfoliate.Our findings highlight the potential use of such IL-based electrolyte to develop high energy density and safer LIBs making full use of state-of-the-art positive and negative electrode materials.
Figure 1 .
Figure 1.a-c) Selected voltage profiles and d-f) galvanostatic cycling of a, d) SiG and b, e) NCM88 in half-cells configuration as well as c, f) SiG j j NCM88 lithium-ion cells in organic carbonate ester-based electrolyte (1 M LiPF 6 in EC/DEC + 10 wt.% FEC + 1 wt.%VC, labelled as OR).The specific capacity values refer to the weight of the electrode material under investigation in half-cells (SiG or NCM88), but to the weight of NCM88 only for the full-cell tests.
Figure 2 .
Figure 2. a) Linear sweep voltammetry to probe the electrochemical stability window of ILE1 and ILE2 recorded at a scan rate of 0.1 mV s À 1 using a Ni foil as working electrode.b) Ionic conductivity of both ILEs.c, d) Selected voltage profiles of SiG half-cells in ILE1 and ILE2, respectively.Voltage profiles during the first five cycles of NCM88 half-cells at 0.1 C in e) ILE1 and f) ILE2.The specific capacity values refer to the weight of the electrode material under investigation in half-cells (SiG or NCM88).
Figure 3 .
Figure 3. Selected voltage profiles of SiG j j NCM88 lithium-ion cells at 0.2 C in a) ILE1 and b) ILE2.c) Capacity retention and d) corresponding CE of the lithium-ion cells tested in both ILEs.e) Specific energy and average discharge voltage of a SiG j j NCM88 lithium-ion cell in ILE2.The specific capacity refer to weight of NCM88 only and the specific energy values refer to the weight of NCM88 and SiG.
Figure 4 .
Figure 4. SEM images of a, b) as made and c-f) cycled SiG electrodes recovered from lithium-ion cells after 200 cycles in (c, d) ILE1 and (e, f) ILE2.
Figure 5 .
Figure 5. Top view SEM images and corresponding cross-sectional FIB-SEM images of a-c) pristine NCM88 electrodes and d-i) electrodes recovered from lithium ion-cells after 200 cycles d-f) in ILE1 and in g-i) in ILE2.
Figure 7
Figure 7 depicts the comparison of the CEI chemical composition on the NCM88 positive electrodes after cycling.The deconvolution of the O 1s spectra of the electrodes cycled in ILE1 and ILE2 show components at 529.5, 531.8, 532.5 and 533.8 eV, which correspond to MÀ O (M=Ni, Co, Mn), and À C=O, À S=O/À CÀ OÀ CÀ , À O*À C=O, Li 2 CO 3 , FSI/TFSI/ethers and RCO 2 Li, respectively.After sputtering (3 and 10 min) the concentration of carbonates (À C=O) increase in both ILE systems.Furthermore, the MÀ O peak, which corresponds to NCM88, is more pronounced in the ILE2 CEI, suggesting a thinner CEI.In addition, the C 1s region in both configurations shows the same trend as O 1s, displaying peaks which correspond to
Figure 6 .
Figure 6.High-resolution core level XPS of the O 1s, C 1s, F 1s, N 1s and S 2p region of SiG electrodes recovered from SiG j j NCM88 lithium-ion cells after 200 cycles in a) ILE1 and b) ILE2.The time scale on the left indicates times for Ar + sputtering to remove the outer layers of the SEI and electrode (0.8 nm min À 1 ) and probe the depth profile.
Figure 7 .
Figure 7. High-resolution core level XPS of the O 1s, C 1s, F 1s, N 1s and S 2p spectra of NCM88 electrodes recovered from SiG j j NCM88 lithium-ion cells after 200 cycles in a) ILE1 and b) ILE2.The time scale on the left indicates times for Ar + sputtering to remove the outer layers of the CEI and electrode (0.8 nm min À 1 ) and probe the depth profile. | 2022-07-06T15:10:12.834Z | 2022-07-04T00:00:00.000 | {
"year": 2022,
"sha1": "6c4dc3c4bce8d332e22161d87e4f2df5fee35432",
"oa_license": "CCBY",
"oa_url": "https://onlinelibrary.wiley.com/doi/pdfdirect/10.1002/batt.202200286",
"oa_status": "HYBRID",
"pdf_src": "ScienceParsePlus",
"pdf_hash": "744ab5544f2af2432d92f5fffdb2f1956cd0621c",
"s2fieldsofstudy": [
"Materials Science"
],
"extfieldsofstudy": []
} |
16995336 | pes2o/s2orc | v3-fos-license | MiR-221 promotes trastuzumab-resistance and metastasis in HER2-positive breast cancers by targeting PTEN
HER2-overexpressing breast cancers are characterized by frequent distant metastasis and often develop resistance after short-term effective treatment with the monoclonal antibody drug, trastuzumab. Here, we found that the oncogenic miRNA, miR-221, inhibited apoptosis, induced trastuzumab resistance and promoted metastasis of HER2-positive breast cancers. The tumor suppressor PTEN was identified as a miR-221 target; overexpression of PTEN abrogated the aforementioned miR-221-induced malignant phenotypes of the cells. These findings indicate that miR-221 may promote trastuzumab resistance and metastasis of HER2-positive breast cancers by targeting PTEN, suggesting its role as a potential biomarker for progression and poor prognosis, and as a novel target for trastuzumab-combined treatment of breast cancers. [BMB Reports 2014; 47(5): 268-273].
INTRODUCTION
Human epidermal growth factor receptor 2 (HER2) is a transmembrane receptor tyrosine kinase of the epidermal growth factor (EGF) receptor family, playing a key role in normal cell growth and differentiation. HER2 overexpression, which was found in 25-30% metastatic breast cancers (MBC) (1,2), leads to a more aggressive tumor phenotype featured by frequent distant metastasis and often acquired resistance to tumoricidal drugs during one year (3)(4)(5)(6)(7). However, the mechanism underlying these malig-nant phenotypes of HER2-positive breast cancers remains elusive (3,8,9). MicroRNAs (miRNAs) are a class of conservative 22-25 nucleotide RNA molecules that negatively regulate gene expression (10). By base-pairing with the complementary sites in the 3'untranslated region (3'UTR) of the mRNA, miRNAs control mRNA stability and translation efficiency. Frequent deregulation of miRNAs has been detected in breast cancers, and some are associated with breast cancer metastasis and poor prognosis, suggesting an important role of miRNAs in breast oncogenesis and cancer progression (11,12). The oncogenic miRNA, miR-221, is one of the few miRNAs that are consistently up-regulated in malignancies of various tissue origins, including the glioblastomas (13), prostate carcinoma (14), gastric carcinoma (15), melanoma (16), luminal breast cancer cells (17). Consistent with these reports, our previous study suggested that miR-221 was upregulated in breast cancer cells resistant to the therapeutic HER2 antibody, trastuzumab (Gene Expression Omnibus, assigned accession #: GSE47011). Here, we established that miR-221 promotes the invasiveness and trastuzumab resistance of HER2-positive breast cancers by targeting the tumor suppressor gene PTEN. The effect of restored PTEN expression on the malignant phenotypes of breast cancers was also addressed.
MiR-221 conferred trastuzumab resistance in HER2-positive cell line
We previously generated recombinant lentiviruses of a miR-221 precursor, which achieved efficient delivery and expression of pre-miR-221 in HER2-positive cell line-SK-BR-3 (18). Conversely, transfection of cells with a synthesized miR-221 inhibitor caused dramatic downregulation of miR-221 (Fig. 1A). Then MTT assay showed that overexpression of pre-miR-221 increased cell growth in the presence of trastuzumab; cells transfected with miR-221 inhibitors showed a significant decrease of surviving cells (Fig. 1B). We next used flow cytometry to test the role of miR-221 in apoptosis. MiR-221 suppression triggered massive apoptosis of SK-BR-3 cells when exposed to trastuzumab, whereas inhttp://bmbreports.org BMB Reports MTT assays for SK-BR-3 cells transfect with pre-miR-221 expressing lentivirus, scramble, control, or miR-221 inhibitor. All bars and error bars represent means ± SD (n = 3) (*P ＜ 0.05). (C) SK-BR-3 cells transfect with pre-miR-221 expressing lentivirus, scramble, control, or miR-221 inhibitor were treated with trastuzumab (5 μg/ml) for 24 h, and then stained with FITC-conjugated Annexin V and PE-labeled PI. Apoptosis was analyzed by flow cytometry. All bars and error bars represent means ± SD (n = 3) (*P ＜ 0.05 and **P ＜ 0.01). troduction of pre-miR-221 significantly decreased the number of cells undergoing apoptosis (Fig. 1C).
MiR-221 promotes cell motility and HER2-positive breast cancer metastasis
Enforced overexpression of pre-miR-221 promoted the mobility of breast cancer cells in a wound-healing assay and dramatically enhanced the invasion capacity of cells on a matrigel ( Fig. 2A and B) as compared with control cells or cells modified to express a scramble miRNA. In contrast, miR-221 suppression by transfection with a miR-221 inhibitor resulted in a pronounced decrease in cell motility and invasion as compared with control cells ( Fig. 2A and B). A metastatic tumor model was further generated by challenging the nude mice in the tail vein with SK-BR-3 cells modified to stably express pre-miR-221 or a scramble pre-miRNA. At a time point allowing for lung metastasis development in our pilot experiment (5 weeks), mice were sacrificed. Metastatic nodules were observed on the surface of the lung in mice inoculated with cells expressing pre-miR-221, but not in those challenged with cells modified to express a scramble miRNA (Fig. 2C). Taken to-gether, these results indicated that miR-221 could effectively promote the motility and invasion of cultured breast cancer cells, and expedite metastasis of breast cancers in vivo.
PTEN is a direct target gene of miR-221 in HER2-positive breast cancer
MicroRNAs posttranscriptionally silence specific genes via pairing to the target mRNAs. We next searched for potential targets by prediction using the PicTar, miRanda and TargetScan Database (data not shown), and identified PTEN, a widely expressed tumor suppressor, as a candidate for miR-221 targeting. tential miR-221 binding site was cloned for a firefly luciferase reporter assay (Fig. 3D). Luciferase activity was reduced by approximately 50% in miR-221-expressing cells compared with the control transfectants (Fig. 3E). In clinical breast cancer samples, the expression of miR-221 was found to correlate inversely with PTEN levels (Fig. 3C). These data suggest that PTEN is the direct target of miR-221 in breast cancer cells.
Overexpression of PTEN rescues miR-221-induced malignant phenotype in HER2-positive breast cancer cell
We next evaluated the role of PTEN overexpression in counteracting miR-221 to repress the invasiveness and trastuzumab resistance of HER2-positive breast cancer cells. A construct of miR-221-refractory PTEN mutant lacking the 3'-UTR, pcDNA3.1-PTEN-mut, was generated. Transfection of SK-BR-3 cells with pcDNA3.1-PTEN-mut dramatically increased cellular PTEN level, which was not affected by the co-introduction of pre-miR-221 recombinant letiviruses (Fig. 4A and B). Enforced overexpression of PTEN significantly suppressed the invasion of SK-BR-3 cells induced by pre-miR-221 lentivirus (Fig. 4D), and restored trastuzumab sensitivity to a level comparable to control SK-BR-3 cells (Fig. 4C). These results indicate that miR-221 maintains the malignant phenotypes of HER2-positive breast cancers, e.g. high invasiveness and trastuzumab resistance, mainly by targeting and silencing the tumor suppressor PTEN.
DISCUSSION
The past decades has witnessed the considerable progresses in understanding the molecular machinery regulating the occurrence and metastasis of breast cancers. In particular, mutations in genes that constitute the phosphatidylinositol 3-kinase (PI3K) pathway occur in ＞70% of breast cancers (19). The PI3K pathway is regulated by numerous molecules. Of note is the well-documented tumor suppressor PTEN, which dephosphorylates phosphatidylinositol-3,4,5-triphosphate (PIP3), thereby opposing the action of the phosphatidylinositol-3 kinase (PI3K) (20). Considering the relatively low incidence of PTEN gene mutation, e.g. no more than 7% in invasive breast cancers (21), the aberrant PTEN expression due to posttranscriptional gene silencing in breast cancers may be critically involved, which is consistent with recent findings of several miRNAs like miR-22 (22), miR-21 (23), miR-153 (24) that targeting PTEN. Here, we demonstrated that PTEN was directly targeted and inhibited by miR-221, leading to elevated motility and invasiveness of HER2-positive breast cancer cells. Suppression of miR-221 or restoration of PTEN expression reversed the malignant phenotypes of HER2-positive breast cancer, suggestive of a pivotal role of key miRNA(s) in regulating HER2-positive breast cancer progression. Breast cancers overexpressing HER2 represent a pathological type with high metastasis incidence and poor prognosis (25,26). Despite the potent tumor suppressive effect of the monoclonal HER2 antibody, trastuzumab, the primary and acquired resistance has become the major obstacle of its clinical application. We found that silencing of PTEN by miR-221 conferred trastuzumab resistance of HER2-positive breast cancers. These results are also consistent with previous findings that miR-221 is involved in TRAIL resistance of liver and lung cancers (27). Unlike chemotherapeutics, resistance to antibody drugs is attributed to both behaviors of cancer cells and their interaction with the immune cells. In this regard, miR-221-mediated PTEN downregulation represents an autonomous mechanism of breast cancer cells, although a precise role of miR-221 in trastuzumab resistance of clinical breast cancers remains to be dissected. Whereas further investigations are needed to decipher a molecular link between miR-221 and HER2 signaling, combined HER2 and miR-221 targeting will be beneficial to improving the treatment of HER2-positive breast cancers. Distal metastasis and drug resistance are hallmarks of ad-vanced breast cancers and account for most morbidity of patients. Interestingly, these features can be concurrent and causative to each other. For instance, central nervous system metastasis of breast cancers is particularly common in patients receiving trastuzumab treatment (28). Although the molecular connection between these malignant phenotypes remains to be defined, they are reminiscent of the features of cancer stem cells. Therefore, it is worth further investigation whether miR-221 correlates with the expression of the established biomarkers of breast cancer-initiating cells. Collectively, this study revealed the critical involvement of miR-221 and the deregulation of PTEN in maintaining the malignant phenotype of HER2-positive breast cancers.
Clinical samples collection
Breast cancer samples were collected directly after surgical resection at Xijing Hospital affiliated to the Fourth Military Medical University, Xi'an, China. The tissues were immediately frozen in liquid nitrogen after surgical removal and stored at −80 o C. Sample collection was approved by the Ethics Committee of the Fourth Military Medical University.
Plasmid construction and preparation of lentivirus
Lentivirus packaging and infection were performed according to standard protocols as recommended by the provider's online protocol. Primers used for PCR amplification of pre-miR-221 coding sequence are as follows: 5'-GAATTCTAGGTAAGTC CCAGCAT-3' and 5'-CTCGAGTTTGGCTTCTATTTCTG-3'. The PCR fragment was cloned into the vector pMD-18T (Takara, Japan) and confirmed by DNA sequencing. The pre-miR-221 coding sequence was further subcloned into the lentivirus-based expression plasmid pLenti6/V5 (Invitrogen, USA), and virus packaging and infection were performed according to protocols as recommended by the manufacturer. Wild-type PTEN lacking the 3'UTR region was constructed into the pcDNA vector (pcDNA3.1-PTEN-mut) by Genesil Biotechnology Co. Ltd. (Wuhan, China). miR-221 mimics, miR-221 inhibitor and negative controls were purchased from Shanghai Genechem Inc. (Shanghai, China). Transfection with 50 nM miRNAs were performed using Lipofectamine 2000 (Invitrogen, Carlsbad, CA) according to the manufacturer's instructions.
Luciferase reporter assay
The intact 3'UTR of the PTEN and a mutant with replaced nucleotides in the putative binding site corresponding to the seed-sequence of miR-221 (Fig. 3C) were cloned downstream of the firefly luciferase gene in the pGL3 vector. Primers used for PCR amplification of PTEN 3'UTR were as follows: wild-type, 5'-TCTAGAGACTCTGATCCAGAGAATGAACC-3' and 5'-TCTA GAGTTGCCACAAGTGCAAAGGGGTAGGATGTG-3'; Mutant, 5'-GTTGAAAAAAGGTTGACATCGAGTGTCATGTATATAC-3' and 5'-TATATACATGACACTCGATGTCAACCTTTTTTCAAC-3'. SK-BR-3 cells were co-transfected with reporter constructs, an internal control vector (pGL4.73), and synthetic miR-221 mimics. Cells were rinsed in phosphate buffered saline (PBS) 48 h later, and luciferase activity was assessed using the Dual-Luciferase Reporter Assay System with a luminometer (Promega, USA). The luciferase activity of each lysate was measured and normalized to the activity of Renilla luciferase driven by the constitutively expressing promoter in the phRL vector. Basal promoter activity was measured as the fold change relative to the activity observed with the basic pGL3 vector alone.
Proliferation assay
Cells (3,000/well) were seeded in 96-well plates and treated with increasing concentrations of trastuzumab in complete medium after overnight serum starvation. Briefly, after transfection for 48 h, the transfection medium in each well was replaced by 100 μl fresh serum-free medium with 0.5 g/l MTT. After incubation at 37 o C for 4 h, the MTT medium was removed by aspiration and 50 μl DMSO was added to each well. After incubation at 37 o C for a further 10 min, the A490 values of each sample were measured with a plate reader.
Western blot analysis
Cells were washed in PBS twice before proteins were extracted, and proteins were separated on an SDS/PAGE gel, transferred onto a PVDF membrane and subjected to immunoblot analysis. Blotting was performed with antibodies against PTEN (Cell Signaling Technology, USA), GAPDH (Epitomics, USA). Goat anti-rabbit and goat anti-mouse immunoglobulin horseradish peroxidase-linked F(ab)2 fragments (ZB-2305, Zhong Shan Jin Qiao, China) were used as secondary antibodies.
Apoptosis assay
Cells were plated in 6-well plates (4 × 10 5 cells/well), and were transfected with pre-miRNAs or miRNA antisense using Lipofectamine 2000 (Invitrogen, USA). SK-BR-3 cells were treated with trastuzumab in a final concentration of 5 μg/ml, respectively. 24 hours after the treatment of trastuzumab, cells were stained with AnnexinV-FITC and PI, and flow cytometry was performed to detect apoptosis of the transfected cells.
Cell motility and invasion assay
For wound-healing assay, wound closure was observed by taking photos under a microscope at 0, 24 and 48 h after scratching. Invasion was performed with Matrigel (BD Biosciences, USA) following the manufacturer's instructions. Photographs of three randomly selected fields of the fixed cells were taken and cells were counted in high power fields (hpf) by light microscopy. Invasive potential was calculated as follows: % Invasion=(Mean cells invading through matrigel insert membrane) / (Mean cells migrating through control insert membrane) × 100%. Cell invasion rates were plotted as mean ± SD. Experiments were repeated three times independently.
Metastasis assay in nude mice
Briefly, 1 × 10 6 cells were injected through the tail vein into nude mice. 5 weeks later, the number of tumour nodules formed on the lung surfaces was counted. Lungs were excised and fixed in 10% formaldehyde, followed by preparation of 0.3 μm paraffin-embedded sections and subsequent haematoxylin and eosin (HE) staining.
Statistical analysis
Statistical analysis was carried out by SPSS 16.0 for Windows. Student's t test was used for analysing the results expressed as mean ± SD. The c2 test or Fisher's exact test was used to analyze the association between miR-221 and PTEN expressions. Differences were considered significant when the P value was less than 0.05. | 2016-05-12T22:15:10.714Z | 2014-05-01T00:00:00.000 | {
"year": 2014,
"sha1": "141071744428d33ce45458e60191aa6aa442d026",
"oa_license": "CCBYNC",
"oa_url": "http://society.kisti.re.kr/sv/SV_svpsbs03V.do?cn1=JAKO201416747605116&method=download",
"oa_status": "GOLD",
"pdf_src": "PubMedCentral",
"pdf_hash": "141071744428d33ce45458e60191aa6aa442d026",
"s2fieldsofstudy": [
"Biology",
"Medicine"
],
"extfieldsofstudy": [
"Medicine"
]
} |
16047360 | pes2o/s2orc | v3-fos-license | A lattice approach to the conformal $\OSp(2S+2|2S)$ supercoset sigma model. Part I: Algebraic structures in the spin chain. The Brauer algebra
We define and study a lattice model which we argue is in the universality class of the $OSp(2S+2|2S)$ supercoset sigma model for a large range of values of the coupling constant $g_\sigma^2$. In this first paper, we analyze in details the symmetries of this lattice model, in particular the decomposition of the space of the quantum spin chain $V^{\otimes L}$ as a bimodule over $OSp(2S+2|2S)$ and its commutant, the Brauer algebra $B_L(2)$. It turns out that $V^{\otimes L}$ is a nonsemisimple module for both $OSp(2S+2|2S)$ and $B_L(2)$. The results are used in the companion paper to elucidate the structure of the (boundary) conformal field theory.
Introduction
The solution of the AdS 5 × S 5 worldsheet string theory is one of the cornerstones of the AdS/CFT duality program. Despite continuous effort and progress on classical aspects in particular [1], and the generally accepted presence of both integrability and conformal invariance symmetries, most aspects of the quantum theory remain elusive.
It is natural to try to understand some aspects of this quantum theory by first tackling simpler models with similar properties. The so called OSp(2S + 2|2S ) coset model -specifically, a sigma model on the supersphere OSp(2S + 2|2S )/ OSp(2S + 1|2S ) -is a very attractive candidate for such an exercise: like the AdS 5 × S 5 worldsheet theory it is conformal invariant and its target space is a supergroup coset. Of course, it lacks other aspects such as the BRST structure of the string theory.
Apart from the string theory motivation, models such as the OSp(2S + 2|2S ) coset model are extremely interesting from the pure conformal field theory point of view. Indeed, they are sigma models which are massless without any kind of topological term, and for a large range of values of the coupling constant g 2 σ . To make things more precise let us briefly remind the reader of some generalities. Supersphere sigma models have target super space the supersphere S R−1,2S := OSp(R|2S )/ OSp(R−1|2S ) and can be viewed as a "supersymmetric" extension of the nonlinear O(N) sigma models (which differs of course from the usual O(N) "supersymmetric" models). Use as coordinates a real scalar field φ := (φ 1 , . . . , φ R+2S ) where the first R components are bosons, the last 2S ones fermions, and the invariant bilinear form where J is the orthosymplectic metric I denoting the identity. The unit supersphere is defined by the constraint The action of the sigma model (conventions are that the Boltzmann weight is e −S ) reads The perturbative β function depends only on R − 2S to all orders (see, e.g., Ref [2]), and is the same as the one of the O(N) model with N := R − 2S . Physics can be reliably understood from the first order beta function The model for g 2 σ positive flows to strong coupling for R − 2S > 2. Like in the ordinary sigma models case, the symmetry is restored at large length scales, and the field theory is massive. For R − 2S < 2 meanwhile, the model flows to weak coupling, and the symmetry is spontaneously broken. One expects this scenario to work for g 2 σ small enough, and the corresponding Goldstone phase to be separated from a non perturbative strong coupling phase by a critical point.
The case we are interested in here is R − 2S = 2, where the β function vanishes to all orders in perturbation theory, and the model is expected to be conformal invariant, at least for g 2 σ small enough, the Goldstone phase being replaced by a phase with continuously varying exponents not unlike the low temperature Kosterlitz Thouless phase. How the group symmetry combines with the (logarithmic) conformal symmetry in such models is largely unknown. It is an essential question to be solved before any serious attempts to understanding universality classes in non interacting disordered 2D electronic systems can be contemplated [3].
The OSp(2S + 2|2S ) coset model was considered in particular in two papers by Mann and Polchinski using the massless scattering and Bethe ansatz approaches. This is indeed a natural idea, since supersphere sigma models are in general integrable, and, when massive (ie R − 2S > 2) can be described by a scattering theory involving particles in the fundamental representation of the group. The S matrix is well knowň S (θ) = σ 1 (θ)E + σ 2 (θ)P + σ 3 (θ)I Here, I is the identity, P is the graded permutation operator, and E is proportional to the projector on the identity representation. For R, S arbitrary, factorizability requires that where N = R − 2S , while σ 2 itself is determined, up to CDD factors, by crossing symmetry and unitarity. One immediately observes that when N = 2, the amplitude σ 2 cancels out, leaving a scattering matrix with a simpler tensorial structure, since the P operator disappears. This corresponds to a particular point [4] on the sigma model critical line (where, among other things, the symmetry is enhanced to SU(2S + 2|2S )), the rest of which is not directly accessible by this construction. 1 The idea used in [5] is to consider an analytical continuation to R, S real, and an approach to R − 2S = 2 with proper scaling of the mass. Though interesting results were obtained, the emphasis in these papers was not on conformal properties. Another line of attack, more suited to the conformal aspects, was launched by Read and Saleur in 2001 [4], who proposed to use a lattice regularization to control the integrable features of the model. They obtained in this way the spectrum of critical exponents for several related sigma models on super target spaces, including the OSp(2S + 2|2S ) coset one at a particular (critical) value of the coupling g 2 σ . The results exhibited several mysterious features, including a pattern of large degeneracies, and a set of values of the exponents covering (modulo integers) all the rationals. In two subsequent papers [6,7], it was argued further that many algebraic properties of the conformal field theory could be obtained at the lattice level already. These include fusion, and the structure of conformal "towers" (see below for further details).
The work we present in this paper and its companion is an attempt at understanding the conformal field theoretic description of the OSp(2S + 2|2S ) model for all values of the coupling by using a lattice regularization. Foremost in the lattice approach is the understanding of the algebraic structure of the lattice model -the algebra defined by the local transfer matrices and its commutant. While in the cases discussed in [7] most necessary results were already available in the mathematical literature, the situation here is much more complicated: in a few words, we have to deal, instead of the Temperley Lieb algebra, with the Brauer algebra whose representation theory, in the non semi-simple case, is far from fully understood. An important part of our work has consisted in filling up the necessary gaps of the literature, sometimes rigorously, but sometimes at the price of some conjectures. This algebraic work is the subject of the first paper, which we realize might be a bit hard to read for a physics reader. We capitalize on the algebraic effort in the companion paper, where the boundary conformal field theory for the coset sigma model is analyzed thoroughly.
In the second section of this paper we discuss generalities about lattice regularizations of O(N) sigma models in 2 dimensions and define the model we shall be interested in. In section 3, the transfer matrix, the loop reformulation and the associated Brauer algebra are introduced and discussed. Section 4 is the main section, where the full decomposition of the Hilbert space of the lattice model under the action of OSp(2S + 2|2S ) and B L (2) is obtained. Our main result can be found in eqs. (4.36) and (4.37). Section 5 discusses aspects of the hamiltonian limit and section 6 contains conclusions. Technical aspects of representation theory are discussed further in the appendices.
For the reader's convenience, we provide here a list of notations used throughout the paper: • osp(R|2S ) is the Lie superalgebra of the supergroup OSp(R|2S ) • B L (N) is the Brauer algebra on L strings with fugacity for loops N = R − 2S • V R|2S = C R|2S is the mod 2 graded vector space C R ⊕ C 2S with even part V 0 = C R and odd part V 1 = C 2S . We shall often drop the indices R, 2S in V R|2S .
• V ⊗L is considered as a left osp(R|2S ) and right B L (N) bimodule • λ ⊢ L stands for "λ is a partition of L" and λ ′ is the partition λ transposed . This is however difficult to study technically, as the number of degrees of freedom on each site is infinite. A possible way to go is to discretize the target space, leading to various types of "cubic models" [8]. Another way which has proved especially fruitful in two dimensions has been to reformulate the problem of calculating the partition or correlation functions geometrically by using the techniques of high or low temperature expansions, thus obtaining graphs with complicated interaction rules and weights determined by properties of the underlying groups. The simplest of these formulations appeared in [9] where the authors studied the O(N) model on the honeycomb lattice in two dimensions, and replaced moreover the term <i j> e βJ S i . S j by its considerably simpler high temperature approximation <i j> (1 + K S i . S j ), K = βJ [10]. Expanding the brackets, in say the calculation of the partition function, one can draw graphs by putting a bond between neighboring sites i and j whenever the term S i . S j is picked up. The integral over spin variables leaves only loops, with a fugacity equal to N as there are N colors one can contract. Note that because of the very low coordination number of the honeycomb lattice, only self-avoiding loops are obtained. This leads to the well known self-avoiding loop gas partition function: where the sum is taken over all configurations G of self avoiding, mutually avoiding closed loops in number L, covering a total of E edges. Note that once an expression such as (2.1) is written down, it is possible to analytically continue the definition of the model for N an arbitrary real number. Barring the use of superalgebras, only N integer greater or equal to one has a well defined meaning as a spin model (the case N = 1 coincides with the Ising model 2 ). In two dimensions, the Mermin Wagner theorem prevents spontaneous symmetry breaking, so for N integer, critical behavior can only occur for N = 1, 2. Analysis of the same beta function suggests however that lattice models defined by suitable analytic continuation should have a Goldstone low temperature phase for all N < 2, though it says nothing about whether this phase might end by a second or first order phase transition. Model (2.1) lacks interaction terms which would appear with less drastic choices of the lattice and the interactions: these are the terms where the loops intersect, either by going over the same edge, or over the same vertex, maybe many times. It has often been argued that such terms are irrelevant for the study of the critical points of the O(N) models in two dimensions. Most of the interest has focused on such critical points for N ∈ [−2, 2], which have geometrical applications -in particular the case N = 0 is related with the physics of selfavoiding walks. It turns out however that intersection terms are crucial for the understanding of low temperature phases. Indeed, the model (2.1) does have a sort of Goldstone phase for N ∈ [−2, 2] called the dense phase, but its properties are not generic, and destroyed by the introduction of a small amount of intersections. A simple way to see that the dense phase is not generic is that the exponents at N = 2 are always those of the Kosterlitz Thouless transition point: model (2.1) does not allow one to enter the low temperature phase of the XY model. Also, model (2.1) has a first order transition for N < −2, which is not the behavior expected from the sigma model analysis.
It was suggested in [11] that model (2.1) can be repaired by allowing for some intersections. The minimal scenario one can imagine is to define a similar model on the square lattice, and allow for self intersections at vertices only, so either none, one or two loops go through the same vertex. The resulting objects are often called trails. This gives the new partition function where the sum is taken over all configurations G ′ of closed loops, which visit edges of the lattice at most once, in number L, covering a total of E edges, with I intersections.
The phase diagram of this model has not been entirely investigated. It is expected that at least for w small enough, the critical behavior obtained with K = K c , w = 0 is not changed (though K c is), while the low temperature behavior K > K c will be.
In [11] a yet slightly different version was considered corresponding, roughly, to the limit of very large K, where all the edges of the lattice are covered. The partition function of this fully packed trails model then depends on only two parameters: Note that expression (2.2) can be obtained very naturally if instead of putting the degrees of freedom on the vertices, one puts them on the edges of the lattice. In this case, the minimal form of interaction involves two edges crossing at one vertex. Invariance under the O(N) group allows for three invariant tensors as illustrated on the figure 8, while isotropy and invariance under an overall scale change of the Boltzmann weights leaves one with a single free parameter, the crossing weight w. Graphical representation of the contractions on the invariant tensors reproduces eq. (2.2), as will be discussed below.
For N < 2, model (2.2) flows to weak coupling in the IR, and therefore it is expected that the critical properties of the corresponding low temperature (Goldstone) phase do not depend on w, a fact checked numerically in [11]. The case N = 2 is expected to be different: as mentioned already in the introduction, the beta function of the corresponding sigma model is exactly zero so the coupling constant does not renormalize. It is indeed easy to see that the loop model (2.2) with N = 2 is equivalent to the 6 vertex model with a = b = 1 + w, c = 1. Consider the vertices of the 6 vertex model as represented on figure 2. We chose isotropic weights a = b, c. We can decide to split the vertices of a configuration into pieces of oriented loops as represented on figure 3. For each vertex, there are two possible splittings, and we assume that they are chosen with equal probability. The loops obtained by connecting all the pieces together provide a dense covering of the lattice, and come with two possible orientations, hence a fugacity of two once the orientations are summed over. The loops can intersect, with a weight w given from the obvious correspondence: 2 We note that there are indeed three invariant tensors for the case of O (2). The corresponding projectors are E, 1 2 (I − P), 1 2 (I − E + P). They project respectively on two one dimensional representations, and on a single two dimensional one. The parameter ∆ covers the interval [−1, 1] as w ∈ [0, ∞]. Changing ∆ is well known to change the exponents of 6 vertex model, and therefore eq. (2.2) for N = 2 exhibits a critical line, which is in fact in the universality class of the continuous XY (O(2)) model in the low temperature (Kosterlitz Thouless) phase.
All what was said so far can be easily generalized to the case of spins taking values on a supersphere OSp(R|2S )/ OSp(R − 1|2S ). The fugacity of loops is now equal to R − 2S : this combination is the number of bosonic minus the number of fermionic coordinates, and follows from the usual fact that when contracting fermions along a loop, a minus sign is generated 3 , see sec. 3.3. The loop model formulation therefore provides a convenient graphical representation of the discrete supersphere sigma models for all R − 2S , in particular R − 2S ≤ 2 where interesting physics is expected to occur. This physics was explored in [11], and the expected results were obtained for R − 2S < 2. The purpose of this paper is to explore the more challenging R − 2S = 2 case.
Of course, at the naive level of partition functions and without worrying about boundary conditions, it looks as if there is no difference between the O(N) spin model and its supersphere cousins provided R − 2S = N. The point is that the observables of the models are different or, at the very least, come with different multiplicities. Indeed, consider for instance correlation functions of spin variables. In the O(2) case, the spin has only two components S 1 , S 2 , so one cannot build a totally antisymmetric tensor on three indices. This means that the corresponding operator (which has a nice geometrical interpretation to be given in the next paper) will not be present in this case, though it will be in the OSp(2S + 2|2S ) model when S > 0. Note that in general, correlators involving spins within the first R bosonic and the first 2S fermionic labels will be the same for any choice of group OSp(R ′ |2S ′ ) with R ′ − 2S ′ = R − 2S and R ′ ≥ R. 4 This is immediately proved by performing a graphical expansion of the correlator: variables outside of the set of the first R bosonic and the first 2S fermionic labels are not getting contracted with the spins in the correlators, and cancel against each other in the loop contractions.
A standard trick to extract the full operator content of a model is to study the partition function with different boundary conditions. Consider for instance the spin model on an annulus with some symmetry preserving boundary conditions in the space direction. With what we will call periodic boundary conditions (corresponding to taking the supertrace of the evolution operator) in the time direction, representations of OSp(2S + 2|2S ) will always be counted with their superdimension, and the partition function will be identical with the one of the O(2) case. But if we take antiperiodic boundary conditions, we will get a modified partition function (in the sense of [4]) which is a trace over the Hilbert space instead of a supertrace, counts all observables with the multiplicities (not supermultiplicities), and will turn out to be a very complex object.
A good algebraic understanding of the lattice model will be essential to make further progress, and, since the area is largely unexplored, this will occupy us for most of the rest of this first paper.
Transfer matrices
As discussed briefly in the introduction, the OSp(R|2S ) spin model we consider is most easily defined on a square lattice with degrees of freedom (states) on the edges and interactions taking place at vertices. The set of states on every edge is a copy of the base space V of the fundamental OSp(R|2S ) representation. Interactions at a vertex can be encoded in a local transfer matrix t acting on V ⊗2 and commuting with the OSp(R|2S ) supergroup action. We call t an intertwiner and write t ∈ End OSp(R|2S ) V ⊗2 .
The Boltzmann weights of the model are components of the transfer matrix along a basis of intertwiners. A natural choice of basis are the projectors onto OSp(R|2S ) irreducible representations appearing in the decomposition of the tensor product of two fundamental OSp(R|2S ) representations. To find them one can apply the same (anti)symmetrization and trace substraction techniques used for reducing O(N) tensor representations. If e 1 , . . . , e R+2S is a mod 2 graded set of basis vectors in V with grading g, the decomposition of V ⊗2 will read Here J i j is the OSp(R|2S ) invariant tensor, J i j = (J −1 ) i j , and g(i) = 1 (resp. g(i) = 0) if i is fermionic (resp. bosonic). Each of the three terms on the l.h.s. of (3.1) transforms according to an irreps of OSp(R|2S ), or, in other words, belongs to a simple OSp(R|2S ) module. Introduce the identity I, the graded permutation operator P (also known as braid operator), and E the Temperley Lieb operator (proportional to the projector on the trivial representation), In terms of projectors onto irreducible OSp(R|2S ) modules, eq. (3.1) may be written in a more elegant way as Let P denote as usual the inversion of space, T the inversion of time and C the charge conjugation with the matrix J. One can check directly from definition (3.2) that P is C 12 , P and T 12 invariant, while E is P 12 and C 12 T 12 invariant. Moreover, E and I transform into each other under the π/2 rotation of the lattice R, while E and P are related by the crossing symmetry C 1 T 1 Take I, E and P as basis of intertwiners in End OSp(R|2S ) V ⊗2 . The local transfer matrix generally depends on three independent weights w I , w E and w P . However, on a homogeneous and isotropic lattice one can normalize w I = w E = 1 and leave only the weight w = w P . Finally, the local transfer matrix takes the form On a diagonal lattice with open boundaries represented in fig. 4 choose the time in vertical direction. The notation of sites at a fixed time is such that the left edge i and right edge i + 1 meet at vertex i. Let t i (w) ∈ End OSp(R|2S ) V ⊗L denote a transfer matrix acting nontrivially only at vertex i according to eq. (3.3). From the figure it is clear that odd and even times are inequivalent. The transfer matrix T , propagating one step forward at equivalent times, may be written as a product T = YX of one layer transfer matrices schematically shown in fig. 4. The simplest way to define a partition function that depends on the whole spectrum of the transfer matrix T is by taking the trace of T at a certain power β. Selecting other boundary conditions with some nontrivial symmetry generally amounts to restricting the whole space of states of the model to a subspace compatible with the symmetry of chosen boundary conditions. What exactly we mean by "symmetry of boundary conditions" will be explained later in sec. 3.3. For the moment let us just say that it is convenient to consider a more general class of boundary conditions, called quasiperiodic, in which T β is "twisted" by the action of an element D of the supergroup. Define the quasiperiodic partition function to be (3.5) We must take the supertrace in eq. (3.5) if we want the quasiperiodic partition function to be well defined. For instance, when D = J 2 we get the usual trace partition function and when D equals to the identity matrix we get the supertrace partition function. Note that because D is a supermatrix, the tensor product in D ⊗L has to be graded, that is After inserting the local transfer matrix from eq. (3.3) in eq. (3.4) and expanding the transfer matrix T , the quasiperiodic partition function reads as a sum of weighted products of E i 's and P i 's. Such linearly independent products must be considered as words of a transfer matrix algebra, while intertwiners E i and P i are generators of this algebra. In the next section we identify this algebra as a representation of the Brauer algebra.
The Brauer algebra
For an abstract introduction to the Brauer algebra see ref. [13,14] while in the context of osp(R|2S ) centralizer algebra see ref. [15]. We collect in this section some well known facts about the Brauer algebra we shall use in the next sections.
Let E i and P i , i = 1, . . . , L act nontrivially as E and P in eq. (3.2) only at the sites V i ⊗ V i+1 of V ⊗L . One can check that for P i and E i so defined the following relations hold: In the second line of these relations i and j are supposed to be nonadjacent sites. Relations (3.6) (is one of the many ways to) define the B L (N) Brauer algebra (also denoted sometimes by the names of braid-monoid algebra or degenerate Birman-Wenzel-Murakami algebra [16,17,18]). Note that this algebra depends on a single, generally complex, parameter N, and contains the maybe more familiar Temperley Lieb algebra, generated by E i 's alone, and the symmetric group algebra, generated by P i alone.
For N fixed and L big enough, the OSp(R|2S ) spin models provide highly unfaithful representations of the Brauer algebra B L (N). This is because, in V ⊗L , the generators P i and E i satisfy additional higher order relations R on top of (3.6). 5 For a simple example, consider the O(2) spin model on a lattice of width 3. The projector Figure 5: The graphical representation of the word P 5 P 3 E 1 P 2 in B 6 (N). Figure 6: Graphical representation of generators I, E i and P i .
onto the antisymmetric tensor of rank 3 is zero, thus, R contains the additional relation 1 + P 1 P 2 + P 2 P 1 = P 1 + P 2 + P 1 P 2 P 1 . Our spin models in general provide representations of the quotient algebras B L (N)/R. The set of relations R can be explicitly described for S = 0, see [19,20] and references therein, and we have little to say about the case S > 0. The first step in understanding the spectrum of the transfer matrix T brings up the question of B L (N) irreducible representations, and of their multiplicities in T for a particular choice of R and S . This leads us to discussing some results about the representation theory of the Brauer algebra.
The most natural representation to begin with is the adjoint representation. It admits a diagrammatic representation in terms of graphs on 2L points in which every vertex has degree 1. Usually one orders the 2L points on two horizontal parallel lines as shown in fig An example of such a labeled graph is shown in fig. 7. If the labellings are omitted the resulting graph is called a partial diagram.
The labeling of the m free points of a labeled graph is a permutation π in the symmetric group Sym(m). The labeled graphs will provide a representation of the Brauer algebra, which is irreducible for generic values of N, if we take the labellings π in an irreducible representation of Sym(m). We call such representations generically irreducible. Let µ be a partition of m, which we write as µ ⊢ m. In a more algebraic language the definition of generically irreducible left modules translates to where S (µ) is an irreducible Sym(m) module. In view of later numerical analysis we give below a basis in ∆ L (µ) and describe the action of B L (N) on this basis. Let p ⊗ π denote the labeling of a partial diagram p with the permutation π, v 1 , . . . , v f µ be a set of basis vectors in S (µ) and ρ µ (σ) be the matrix of the permutation σ in the representation ρ µ . A natural basis in ∆ L (µ) is given by all pairs p ⊗ v i . The action of a diagram d ∈ B L (N) on a basis vector is where σ is the labeling of d * g and g is the partial diagram p labeled with the identity permutation. The In simple words, a generically irreducible module is a span on graphs on L points, obtained by choosing m points among L, pairing all the others (this gives the multiplicity (L − m − 1)!! since intersections are allowed), choosing for the m unpaired ones a representation of the permutation group and setting to zero the action of any Brauer diagram that reduces the number m of unpaired points.
The generically irreducible representations labeled by µ ⊢ L − 2k, k = 0, . . . , [L/2] appear in the decomposition of the adjoint representation with multiplicity given by their dimension d µ when B L (N) is semisimple.
Let us conclude with a few words about the reducibility of generically irreducible modules ∆ L (µ). For integer N and a number of strings L > N the Brauer algebra is not semisimple and, as a consequence, certain of the modules ∆ L (µ) become reducible, though they remain indecomposable. 6 The irreducible components appearing in such reducible modules ∆ L (µ) are far from being understood (the situation is much worse than in the case of the nonsemisimple Temperley Lieb algebra [21,22]). Numerical computations based on the diagonalization of the transfer matrix in the diagrammatic representation of B L (N) restricted to ∆ L (µ) decreases in efficiency very fast with increasing L, compared to the ideal case where the transfer matrix is restricted to an irreps of B L (N). This is because for big L and µ fixed the number of irreducible components in ∆ L (µ) "goes wild" and there are a lot of "accidental degeneracies" in the spectrum of the transfer matrix restricted to ∆ L (µ).
However, a significant progress in this direction has been recently made in [23,24]. Let us note that, as described in [23], the content of (at least some) ∆ L (µ)'s can be computed by repeated applications of Frobenius reciprocity applied to the short exact sequence of [14] describing the structure of the induced modules In the end we recall the basic results for the Temperley Lieb algebra, to allow a quick comparison with Brauer. Temperley Lieb algebra diagrams are a subset of Brauer algebra diagrams subject to the constraint that no intersections between edges are allowed. The dimension of the algebra is given by the Catalan numbers (2L)!/L!(L + 1)!. The main line of reasoning for finding generically irreducible modules follows the same way, except there is no available action of the symmetric group on vertical lines. Therefore, the analogue of the labeled graphs will be the partial diagrams, in which no free points may be trapped inside an edge. The number of such graphs is C n The presented facts about the Brauer algebra should be enough to understand the loop gas reformulation of OSp(R|2S ) spin model, which we give in the next section.
Loop reformulation of OSp(R|2S ) spin models: the algebraic point of view
The emergence of dense intersecting loops becomes transparent if we take the local transfer matrices in the adjoint representation of the Brauer algebra. This simply amounts to replacing in eq. (3.3) the generators I, E i and P i defined by eq. (3.2) with the diagrams in fig. 6. The adjoint local transfer matrix is represented in fig. 8.
We now define a loop model on a diagonal lattice represented in fig. 4, with reflecting boundaries on the left and right (ie, free boundary conditions in the space direction) and identified boundaries on the top and . We call such loops bulk loops. Clearly the fugacity of bulk loops is fixed to N by the Brauer algebra. The second possibility is that the ends of the line close in the identified points of the top and bottom boundaries of the lattice. We call such loops cycles. The boundary condition we consider have an annulus geometry and, thus, a cycle can be either contractible or uncontractible. The fugacity of cycles is not fixed by the algebra. In fact, as we explain below, this is exactly the degree of freedom allowing for multiple mappings from the OSp(R|2S ) spin models with R − 2S = N fixed and the dense intersecting loop model with fugacity N for loops. We start by evaluating the trace tr V ⊗L d of a diagram d in the spin representation and then we generalize the result for quasiperiodic boundary conditions given by str V ⊗L D ⊗L d. We follow the same line of reasoning as in [13].
A cycle in a diagram d is the subgraph on the set of points belonging to a loop if we identify its top and bottom vertices. By an abuse of language we call the corresponding loop also cycle. If we put a diagram d 1 to the left of a diagram d 2 we get a new diagram which we denote d 1 ⊗ d 2 . Let c 1 , . . . , c l be the cycles in d. We can separate them by permuting the top and bottom vertices of d with the same permutation π π * d * π −1 = c 1 ⊗ · · · ⊗ c l .
Thus the trace of a diagram depends only on the weights of cycles More than that, the weight of a cycle depends only on how many times it winds the annulus.
Indeed, if a cycle on 2m points has no horizontal lines, then, by applying the same permutation to the top and bottom vertices we can bring it to the cycle P 1 . . . P m−1 . This is because permutations with one cycle are conjugate in Sym(m).
If a cycle c has a horizontal edge between the first and the second vertex in the top then it has the same weight as a certain cycle c ′ on four points less then c If we compare the c on the left with c ′ on the right it is clear that, in the end of the iterative application of eq. (3.9), the final cycle can be interpreted as being the initial cycle c maximally contracted on the annulus.
In the end, the only weights we need to compute explicitly are that of the cycles E and P 1 . . . P m−1 For boundary conditions twisted by the matrix D ∈ OSp(R|2S ) the generalized weights are computed to be In the fundamental representation, every supermatrix D is diagonalizable. The diagonal form of D ∈ OSp + (R|2S ) in the fundamental representation is determined by exponentiating elementary weights ǫ i and δ j introduced in sec. A.1. Thus, D restricted to V 0 has eigenvalues The braces in (x r+1 ) mean that x r+1 appears for odd R only. Eq. (3.10) can now be rewritten For D ∈ OSp − (R|2S ) only the eigenvalues in V 0 change with respect to the previous case. There are of the form To summarize the basic results in this section, let G be a dense loop covering of the lattice, I be the number of intersections, B be the number of bulk loops, C be the number of contractible loops (cycles) and E(O) be the number of loops winding the annulus an even(odd) number of times.
On the annulus the trace partition function (which would correspond to antiperiodic boundary conditions in the (imaginary) time direction) of the OSp(R|2S ) spin model may be reformulated as a dense intersecting loop model in the following way We see that it does depend on R, S separately and not only on N.
Meanwhile the supertrace partition function (which would correspond to periodic couplings) reads The only difference between the OSp(R|2S ) and OSp(R−2|2S −2) quasiperiodic partition functions is the weight of uncontractible cycles. For D ∈ OSp(R|2S ) a matrix with eigenvalues x i = 1 and y j = −1, except y 1 = 1, the weight of uncontractible cycles is, according to eq. (3.11) either N or R + 2S − 4. Notice that these are exactly the weights of uncontractible cycles in the trace partition function for the OSp(R − 2|2S − 2) spin model, which proves eq. (3.13). We will use the inclusion property (3.13) in the next section to derive some information about the indecomposable representations of OSp(R|2S ) appearing in the decomposition of V ⊗L .
General results
Assume the transfer matrix be a generic element of B L (N). The action of B L (N) on the tensor space V ⊗L was defined in the beginning of sec. 3.2. The complete picture of the reducibility of the transfer matrix can be conveniently encoded in the decomposition of V ⊗L into a direct sum of B L (N) indecomposable modules where m λ denotes the multiplicity of isomorphic indecomposable B L (N) modules IB L (λ) (it does not depend on L), and the set Y L (S ) is defined implicitly by the formula, and will be defined explicitly below. We remind the reader that V ⊗L is not necessarily a semisimple B L (N) module if L > N, so the modules IB L (λ) appearing on the rhs of eq. (4.1) can be reducible. The question of computing degeneracies of eigenvalues of the spin transfer matrix is easier to treat by looking at the centralizer Z := End B L (N) V ⊗L , which acts on V ⊗L from the left if one consider B L (N) acting from the right. The dimension of indecomposable modules IG(µ) in the decomposition of V ⊗L as a Z-module will give the desired degeneracies. This is due to the fact that n µ L are dimensions of simple B L (N) modules B L (µ) appearing as constituents of IB L (λ) in eq.
Because the action of osp(R|2S ) commutes with B L (N) we have that osp(R|2S ) ⊂ Z. However, when V ⊗L is semisimple it follows from the Wedderburn decomposition theorem that Z ≃ Z 2 × osp(R|2S ). In the following we suppose that there is still a Schur duality between osp(R|2S ) and the quotient of B L (N) faithfully represented on V ⊗L . This allows us to give an algorithm to compute the lhs of eqs. [20]. Under the homomorphism ρ : is a tensor representation and can be generated by trace substraction and symmetrization as where µ ⊢ L − 2k, T L−2k ∈ B L (N) extracts all the traces from the tensor space V ⊗L−2k and e µ acts nontrivially only on V ⊗L−2k as a Young symmetrizer. The double sided ideal J(S ) is completely characterized by the set of weights It is useful to notice that not all of these conditions are independent and as one can see from eq. (4.3) As discussed in sec. A.1 and A.2, the osp(R|2S ) irreducible components of IG(µ) are indexed (up to an equivalence under the action of the outer automorphism τ induced by the symmetry of the Dynkin diagram of osp(R|2S ) when R even) by the set H L (S ) = { λ ∈ X L (S ) | λ r+1 ≤ S } of hook shape partitions. Representing the supergroup as a semidirect product OSp(R|2S ) = Z 2 × OSp + (R|2S ), the elements of Y L (S ) naturally acquire the structure of a couple of the form 1 × λ or ε × λ if λ S < r and τ × λ if λ S ≥ r, where 1, ε, τ are the trivial, alternating (superdeterminant) and two dimensional representations of Z 2 = OSp(R|2S )/ OSp + (R|2S ). Thus, every λ ∈ H L (S ) gives rise to two OSp(R|2S ) inequivalent irreps with highest weights λ := 1 × λ and the associate λ * := ε × λ if λ S < r and a single self-associate irreps of highest weight λ = λ * := τ × λ if λ S ≥ r. For typical λ ∈ H L (S ), one can realize the OSp(R|2S ) irreps λ, λ * on tensors T L (λ), T L (λ * ) and describe their symmetry by some Young tableaux. As discussed in details in sec. B, the Young tableau corresponding to T L (λ * ) can be constructed by adding a border strip to the Young tableau of shape λ corresponding to T L (λ). Ultimately, this is justified by the fact that eq. (4.5,4.6) gives the right characters for T L (λ), T L (λ * ) and that they coincide up to sdet D. Although atypical representations cannot be realized as tensor representations we represent the associate weight λ * of an atypical weight λ by a Young tableau such that λ * /λ is a skew partition described in sec. B and sec. C.
The idea is to exploit the fact that the characters of indecomposable modules ∆ L (µ), given in [13], do not depend on the semisimplicity of B L (N). This and some properties of generalized Schur functions, which are summarized in [15], can be used to prove that is true even for all L. Here χ ′ µ (d) is the character of d ∈ B L (N) in the representation provided by ∆ L (µ). The functions sc µ (D) are polynomials in the eigenvalues of D ∈ OSp(R|2S ), which where introduced for the first time by Bars in [25] in an early attempt to describe the supercharacters of OSp(R|2S ). They can be defined recursively as and with sc µ being actual characters of tensor irreducible modules G(µ) as shown in [15]. For R, S such that L > N, the polynomials sc µ cannot generally be interpreted as the character of some OSp(R|2S ) representation. As we have seen in sec. 3.3, str V ⊗L D ⊗L d can be brought to the form N h m str V D m and eq. (4.4) is not more then a simple equality between two polynomials in eigenvalues of D. Moreover, the two eqs. (4.2,4.4) are still compatible, even if there are much more elements in X L then in X L (S ). This is possible because sc µ are not functionally independent when L > N. Then, for µ Y L (S ) the polynomials sc µ can be written in terms of functionally independent sc λ with λ ∈ Y L (S ) by means of modification rules for characters given in [26] and discussed in details in sec. C. The fundamental eq. (4.4) is useful for small widths L, when it is possible to compute the number b ′ (µ, ν) of irreducible components B L (ν) in ∆ L (µ) either by repeated applications Frobenius reciprocity, as explained in [23], or by numerically diagonalizing the transfer matrix of sec. (3.1) in the adjoint representation of B L (N) and detecting the "accidental degeneracies" in its spectrum. Indeed, from the explicit definition (3.7) it is clear how to restrict the adjoint transfer matrix to indecomposable modules ∆ L (µ). After we described in details the action of generators on the basis of ∆ L (µ) in sec. 3.2, the algorithm of a numerical diagonalization is straightforward.
The information about the structure of ∆ L (µ) and the modification rules in eq. (4.8) can now be used to bring eq. (4.4) to the form We see that µ ∈ X L (S ) iff 7 there is at least one λ ∈ Y L (S ) such that ν a(ν, λ)b(ν, µ) 0. To determine g(µ, λ) one has to decompose the factor of χ µ in eq. (4.9) as a sum of OSp(R|2S ) irreducible characters, which are explicitly known, as far as we know, only for OSp(3|2) and OSp(4|2). Given the huge order of the set of weights X L , it may seem that calculations according to eq. (4.9) are extremely cumbersome already for small L. The simplifying point is that a(ν, λ) (or b ′ (ν, µ)) is non zero only if both weights are in the same equivalence class of Y L (S ) (or X L (S )). The splitting of Y L (S ) (or X L (S )) into equivalence classes, called blocks and described in details in sec. B, is with respect to an equivalence relation between irreducible components of indecomposable OSp(R|2S ) (or B L (N)) modules. An important consequence of the fact that OSp(R|2S ) supertrace partition functions depends only on the O(N) part of the spectrum is the vanishing of the superdimension sdim IG(µ) = 0 for all indecomposable modules with µ X L (0). A more restrictive criterion for IG(µ) supercharacters deriving from the full inclusion sequence in eq. (3.13) can be derived by taking a matrix D with eigenvalues x 1 = y 1 and x i y j for i = 1 . . . , r, j = 1, . . . , S . Then, it can be seen from eqs. (3.11,3.12) or eqs. (4.5,4.6,4.4) that any OSp(R|2S ) quasiperiodic partition function will also be an OSp(R − 2|2S − 2) quasiperiodic partition function. As a consequence X L (0) ⊂ X L (1) ⊂ · · · ⊂ X L (S ) and the supercharacters of IG(µ) vanish when µ ∈ X L (S )/X L (S −1) and D can be embedded in OSp(R − 2|2S − 2).
For S = 0 the modules V ⊗L is semisimple. Therefore rad B L (L) ⊂ J(0). On the other hand, if S is big enough J(S ) = 0. It could be interesting to understand the relation between the sequence J(0) ⊃ J(1) ⊃ · · · ⊃ J(S ) = 0 and the cohomology of the radical rad B L (N) ⊃ rad 2 B L (N) ⊃ · · · ⊃ 0.
Observe that a filtration similar to that of X L (S ) is available on Y L (S ) by the degree of atypicality of its elements. In fact, we explain in sec. B of the appendix how the weights in a block of X L (S ) or Y L (S ) can be organized by the number of removable balanced continuous border strips in the corresponding Young tableau. This number can be interpreted as the degree of atypicality when the corresponding partition represents an OSp(R|2S ) weight.
O(2) spin model
Let V be a two dimensional vector space endowed with an action of O(2, R). The action of O(2) on the tensor B L (2) acts according to the following definition of generators E i , P i where P, E have the following representation on V ⊗2 (note that E differs in some essential way from the projection operator onto the singlet representation in the usual S U(2) basis). The decomposition of V ⊗L as a O(2)-B L (2)-bimodule is simply We are interested in giving an algebraic description of the 6 vertex model transfer matrix algebra. In other words we want to identify the Brauer algebra annihilator J := J(0) of V ⊗L and carry out the quotient B L (2)/J.
All the B L (2) weights λ ∈ X L such that µ k ⊂ λ satisfy either ν 0 = 1 3 ⊆ λ or ν 1 = 21 ⊆ λ. Thus, it is enough to consider L = 3 and impose the vanishing of the double sided ideal of the word W 0 , W 1 ∈ B L (2) projecting onto ∆ 3 (ν i ), i = 0, 1. As explained in the beginning of the previous section W i = T 3 e ν i , where T 3 extracts all the traces from V ⊗3 and e ν i are the Young symmetrizers corresponding to ν i . The projector T 3 can be found by looking at the form of an arbitrary tensor G i jk after extracting all of its traces which gives The Young symmetrizer e ν 0 is and e ν 1 = e T 1 + e T 2 , where are the projectors onto the standard Young tableau T 1 = [12, 3] and T 2 = [13,2]. The two orthogonal projectors e T 1 and e T 2 are independent only if we restrict to the right B L (2) action. In fact, the left ideal of the word W 1 = 0 is the same as the double sided ideal of the word T 3 e T 1 = 0. The condition W 0 = 0 gives the following restriction 1 + P 1 P 2 + P 2 P 1 = P 1 + P 2 + P 1 P 2 P 1 (4.13) on generators P 1 , P 2 . Putting P i = 1 − Q i , i = 1, 2 one can see that eq. (4.13) implies that Q i are Temperley Lieb operators with Q 2 i = 2Q i . There are no more restrictions that can be drawn from the conditions W 0 = 0, because W 0 is a one dimensional projector.
Before exploring the next vanishing condition let us revise the the defining relations of B L (2) given in eq. (3.6) which imply Observe that although the algebra has now two Temperley Lieb operators their role is not symmetric yet at this stage. Next, the condition T 3 e T 1 = 0 implies Multiplying eq. (4.19) by Q 2 on the right we get: which can be used to rewrite eq. (4.19) as Multiplying by E i , Q i on the left and on the right of eq. (4.21) and using only the relations between Q i , the relations between E i and eq. (4.14) one can get all the eqs. (4.15-4.20) and also which establish a complete symmetry between E i and Q i . The double sided ideal of T 3 e T 1 = 0 is composed of four linearly independent words -two generated by the left action and other two generated by the right action of B L (2). It is useful to note that after taking the quotient of B 3 (2) we are left with 10 independent words instead of 15, which is exactly what we need for the 6 vertex local transfer matrix.
We give the following abstract definition to the 6 vertex model transfer matrix algebra Thus, one can eliminate all of the generators Q i , i ≥ 2 and leave only Q 1 subject to satisfy Denote by d L the extension of the ordinary Temperley Lieb algebra, generated by E i , with the additional generator Q 1 satisfying eqs. (4.25). We see that d L and V L are isomorphic algebras. The graphical interpretation for the reduced words (products of generators of minimum length) of d L and its relation to the blob algebra and the Temperley Lieb algebra of type D is discussed [27]. The generators E i are diagrammatically represented as usual, whereas Q 1 is represented as E 1 with each of its horizontal edges marked by an involutive blob as shown in fig. 11. An unblobbed loop is identified with 2, while a blobbed loop with 0. Thus, we see that d L is a subalgebra of the blob algebra composed of all planar diagrams on 2L points with an even number of blobbed edges. The dimension of d L is, as explained in [27], half the dimension of the blob algebra, that is C L 2L /2. There are several important consequences arising from the isomorphism between V L and d L from the point of view of integrability. First of all, we check that indeed the solution to the Yang-Baxter equation provided by the algebra V 3 coincides with the well known XXZ spin chain R-matrix. For that, consider the ansatz R(u) = I + f (u)Q + g(u)E and plug it in eq. (4.26). Choosing as basis set in V 3 the 10 words 1, where with an arbitrary constant λ. Taking Q and E in the representation provided by the eq. (4.10) we find the famous XXZ spin chain R-matrix as expected.
Clearly, an integrable system in V L has to be related to an integrable system in d L because of the isomorphism of these two algebras. However, the ansatz R(u) = 1 + g(u)E plugged into the eq. (4.26) gives only the isotropic point (∆ = ±1) solution g(u) = u/(1 − u). The only possibility to give a richer content to the integrability in d L is by introducing nontrivial boundary conditions. This means that the anisotropy of the XXZ spin chain can be generated by introducing nontrivial boundary conditions at the isotropic points, an observation made earlier from a slightly different perspective in [28].
OSp(4|2) spin model
The representation theory of the superalgebra osp(4|2) is summarized in [29]. As we have already mentioned, all of osp(4|2) irreducible characters have been computed and indecomposable representations classified. We give a brief reminder of these results in sec.A.3 and make some remarks, based on the general discussion in sec. A.2, on the difference between the representation theory of the supergroup OSp(4|2) and its Lie superalgebra.
The tensor space V ⊗L , seen as a OSp(4|2) module, can be represented as a direct sum and a projective part where PG(λ) is the projective cover of G(λ). This decomposition can be proved by induction on L using two facts: • The tensor product between atypical irreducible representations with highest weights labeled by one row partitions (see bellow) and V decomposes to 8 This is proved by counting the dimensions on the right/left hand sides and, then, observing that G(k1) is typical and G(k ± 1), being in different blocks, cannot give rise to indecomposables.
• The tensor product of a projective module with any other module is projective, thus, decomposing to a direct sum of projectives.
In the following we use the fundamental eqs. (4.4,4.9) to decompose V ⊗L as a Z = End B L (2) V ⊗L module and verify the assumption that Z = Z 2 × osp(4|2) by comparing the result to eqs. (4.32,4.33).
The conditions of atypicality for a osp(4|2) weight λ are given in sec. A.3. In the partition notation we adopt, these are equivalent to λ ′ 1 = 1 or λ 1 + 1 = λ ′ 1 or λ 2 = λ ′ 1 . Typical weights satisfy none of atypicality conditions listed above. Note that typical representations are irreducible, have vanishing superdimension, and are simultaneously projective and injective. This means they cannot be a constituents of any other osp(4|2) representations without being a direct summand. One can say they are "their own blocks".
The atypical OSp(4|2) weights can be labeled by two integers k and l, where k denotes the isomorphism class, also called block.
We have just listed all the elements of Y L (1). Eq. (4.34) is a bijection between sch λ and sc λ . As a consequence, OSp(4|2) and B L (2) weights can be labeled by the same set Y L (1) = X L (1) in the partition notation we have adopted. This is supporting the assumption that there is some sort of exact equivalence between the category of OSp(4|2) and B L (2) modules on V ⊗L . Bellow all the weights are partitions and, to avoid confusion, we write λ ∈ Y L (1) if λ is considered as a OSp(4|2) weight and λ ∈ X L (1) if it is considered as a B L (2) weight.
Let us show that the terms in eq. (4.4) with λ Y L (1) do not actually contribute to str V ⊗L D ⊗L d. First note that if χ λ cancels out from eq. (4.4) then certainly δ L (λ) in eq. (4.3) is a trivial module. Therefore any module δ L (ν) will also be trivial if λ ⊂ ν. Second, if χ λ does not contribute to eq. (4.4) when λ ⊢ L then it does not contribute to it for any L. Thus, it is enough to prove for every k that the weights just greater (by inclusion) then λ k,l do not contribute to eq. (4.4) when the are allowed for the first time to appear.
Let λ ∈ Y L (1) be a typical (associate) weight. Then, as we show in sec. B, λ ∈ X L (1) is a minimal partition (with respect to the inclusion in its block). There will be a unique weight ν Y L (1) just greater then λ and, a priori, sc ν can modify to ±sc λ . It is proved by induction in sec. C that a positive sign would imply atypicality conditions on λ and, thus, sc ν = −sc λ . Moreover, from [23] we know that ∆ L (λ) has one composition factor B L (ν). Taking L = |ν|, we see that the contribution to eq. (4.4) of χ ν from ∆ L (λ) cancels out with the one from ∆ L (ν).
Before proceeding to nontrivial blocks we need to know the number of irreducible components B L (λ k,l ′ ) in ∆ L (λ k,l ). According to [23], the graph representing the partial ordering (by inclusion) of weights in a block k determines the required information about the content of modules ∆ L (λ k,l ). The ordering graph is represented in fig. 12. Now, let λ k,l ∈ Y L (1) be an atypical (associate) weight. Then, any weight ν X L (1) such that λ k,l ⊂ ν satisfies ν k ⊆ ν, with ν k represented by a white dot in fig. 12. The explicit form of ν k is ν 0 = 432 2 1, ν 0 * = 43 3 1, ν 1 = 43 2 21, ν 2 = 43 2 1 2 , ν 3 = 4 2 31 2 and ν k = k431, k ≥ 4. Next, one can check with the help of modification rules that sc ν k + sc λ k,1 + sc λ k,3 + sc λ k,4 = 0 (4.35) vanishes identically. Further, from fig. 12 each of the modules ∆ L (λ k,l ), l = 1, 3, 4 has a single irreducible component B L (ν k ). Finally, taking L = |ν k | one can see from eq. (4.35) that the contribution of χ ν k to eq. (4.4) cancels out. Let us introduce the compact notations B k,l := B L (λ k,l ) and G k,l := G(λ k,l ). Then, putting together eq. (4.34) and fig. 12 we get from eq. (4.4) the following content of indecomposable modules IG k,l appearing in eq. (4.2) where l = 3, . . . , m and λ k,m ⊢ L. The indecomposable modules IG k,l are represented below B k,l and it should be understood that they get "paired up" in the decomposition of V ⊗L as a OSp(4|2) × B L (2) bimodule. 10 Alternative, maybe more intuitive physically, representations of the blocks will be given in the next paper. The structure of modules IG k,l is in perfect agreement with eq. (4.32,4.33). We recognize in the first term IG k,0 = G k,0 of eq. (4.36) the contribution to V (0) , while the rest of the terms are exactly the projective modules appearing in V (1) , that is IG k,2 = PG k,0 , IG k,1 = PG k,1 and IG k,l = PG k,l−1 , l ≥ 2.
Observe that, as expected, only the modules B k,0 (which coincide with B L (λ k ) in eq. (4.11)) contribute to the supertrace str V ⊗L d. Indeed, typical modules modules G(λ) have superdimension 0. The same is true for projective modules. One can explicitly check from eq. (4.36) that sdim PG k,l = 0 if we take into account that only osp(4|2) fermionic generators connect irreducible components of indecomposable modules. For instance, As This results allows us to relate the decomposition of V ⊗L as a OSp(4|2) left module to the decomposition of V ⊗L as a B L (2) right module.
Collecting in a single indecomposable module IB k,l all factors B k,l ′ in eq. (4.36) which correspond to (happen to be above) an irreducible component G k,l we get 11 where l = 4, . . . , m − 1 and the content of IB k,l is represented below G k,l . Apart the last irreducible module IB k,m = B k,m , we recognize in the terms of eq. (4.37) the projective representations of the quiver E ∞ in fig. 12, which describes the homomorphisms between the B L (2) tensor modules δ L (λ) realized on V ⊗L .
The hamiltonian limit
It will turn out in our forthcoming analysis of conformal properties to be easier to study numerically the hamiltonian The expectation -which we will confirm in great details -is that this hamiltonian will be in the same universality class as the spin model we had started with.
The hamiltonian H ∆ is obviously local and has only nearest neighbour interactions if the E's and P's are taken in the spin representation provided by eq. (3.2). However, this is no longer true if we think of H ∆ as an element of the adjoint representation of B L (2).
The lowest eigenvalue of H ∆ belongs to the B L (2) irreducible representation labeled by µ = L mod 2.
For generic ∆ it is nondegenerate if L is even and has degeneracy dim V = 4S + 2 if L is odd. On the other hand, the highest eigenvalue belongs to the completely antisymmetric representation labeled by µ = 1 L . In this representation the P's act as -1 and the E's as 0.
The hamiltonian H ∆ is determined up to an arbitrary additive constant and multiplicative factor. For numerical diagonalization it is convenient to fix the additive constant such that the maximal eigenvalue of H ∆ be zero. The multiplicative factor is fixed by requiring with I, E, P as in eq. (3.2), J as in app. A.1 and H XXZ being the XXZ spin chain hamiltonian in its usual form The fact that the eigenvalues of the 6 vertex model appear as a subset of the eigenvalues of the transfer matrix for the OSp(2S + 2|2S ) model and S ≥ 1 carries over to a similar result for the hamiltonians. The velocity of sound for the massless excitations can thus be derived from its value for the XXZ subset, which is well known from [30] to be v s = π sin λ λ , ∆ = − cos λ.
The hamiltonian H ∆ is diagonalized numerically in the adjoint representation of the Brauer algebra by studying its action on the diagrams just like for the transfer matrices. Next, once the structure of indecomposable modules ∆ L (µ) is known, eq. (4.4) can be used as explained in sec. 4.1 to select the part of the spectrum which does indeed appear for a fixed S spin model. However, in the two special cases ∆ = ±1 the hamiltonian H ∆ greatly simplifies. In the following two subsections we discuss the behaviour of the spectrum of H ∆ in the two limits ∆ → ±1 ∓ .
The limit ∆ = 1
When ∆ = 1 the Temperley Lieb operators E i do not contribute to H ∆ and, thus, the hamiltonian is no longer a generic element of the Brauer algebra B L (2), but belongs instead to the subalgebra C Sym(L) ⊂ B L (2). This will translate to additional degeneracies in the spectrum of H ∆ at the point ∆ = 1 compared to other points in the range −1 ≤ ∆ < 1.
Hamiltonians of type − P i , with P's in the representation provided by eq. (3.2), are integrable and have been studied in [31] and [32]. Although the continuum limit of such spin chains is a gapless field theory, it fails to be conformal, because excitations have a L −2 scaling law in the thermodynamic limit. This can readily be seen from the vanishing of the sound velocity in eq. (5.1). We will not enter into the details here, but just mention that the different systems of Bethe ansatz equations are indexed by (2S + 2, 2S )-hook shape partitions λ ⊢ L. This is exactly the label of irreducible representations of the group algebra C Sym(L) realizing in the centralizer of the spin chain V ⊗L , with V being the fundamental representation of SU(2S + 2|2S ). We see that the symmetry of our spin model OSp(2S + 2|2S ) jumps to SU(2S + 2|2S ) at the point ∆ = 1.
The additional degeneracies in the spectrum of the OSp(2S + 2|2S ) spin model at the point ∆ = 1 can be understood by looking at the decomposition of B L (2) modules ∆ L (µ), into a direct sum of C Sym(L) irreducible modules S (λ). Let µ ⊢ L − 2k and λ ⊢ L, then it was shown in [33] that the multiplicity of S (λ) in the decomposition of ∆ L (µ) is m(µ, λ) = η⊢2k η even c λ µη , (5.2) where c λ µη are Littlewood-Richardson coefficients. Alternatively, m(µ, λ) is the number of tensors of rank L − 2k, with index symmetry of some fixed standard Young supertableau of shape µ, that can be obtained from a tensor of rank L, with index symmetry of some standard Young supertableau of shape λ, by contracting 2k indices in all the possible ways.
One can apply eq. (5.2) to understand the degeneracy of the lowest level of H ∆ at ∆ = 1. First, observe that − P i is minimized in the sector λ = L (where P's acts as 1). The only µ such that m(µ, λ) 0 are one row partitions. Thus, the lowest eigenvalues of H ∆ restricted to ∆ L (L−2k) for k = 0, . . . , [L/2] become all degenerate at ∆ = 1.
Arguments of this kind can be used to derive information about the critical exponents of the spin model in the limit ∆ → 1 − .
The limit ∆ = −1
The same reasoning can be applied to the point ∆ = −1. At this point, the hamiltonian H ∆ belongs to the Temperley Lieb subalgebra T L (1) ⊂ B L (2) and the model can be considered as a spin chain (V ⊗V) ⊗ L 2 where V,V are the fundamental representation of SU(2S + 2|2S ) and its conjugate. Additional degeneracies can be understood by looking at the decomposition of B L (2) modules ∆ L (µ) as a direct sum of standard irreducible Let us compute the multiplicity n L (µ, j) of irreducible modules D L ( j) in the decomposition of ∆ L (µ) with µ ⊢ L − 2k.
As explained in sec. 3.2, ∆ L (µ) has a natural basis composed of all possible pairings p⊗v i of partial diagrams p with m = L − 2k free points and basis vectors v 1 , . . . , v f µ of S (µ). We say that a horizontal line of a partial diagram p is intersected either if it intersects another horizontal line or if there is a free point in p between the two ends of the horizontal line. Let us associate to each partial diagram p the number of intersected horizontal lines l in p. It is not hard to see that the span on the basis vectors p ⊗ v i , with p's having at most l horizontal intersected lines, is a T L (1) submodule in ∆ L (µ). If we denote this submodule by ∆ l L (µ) there is an obvious . Consider the natural action of T L (1) on the quotient modules Q l L (µ) = ∆ l L (µ)/∆ l−1 L (µ). Observe that the action of T L (1) changes the labeling π ∈ Sym(m) of free points in a labeled graph p ⊗ π if and only if it also reduces the number of horizontal intersected lines. Therefore, Q l L (µ) is isomorphic to a direct sum of f µ modules Q l L (m). Obviously Q 0 L (m) ≃ D L (m) and, therefore, we get n L (µ, j) = 0 for j < m, n L (µ, j) = f µ n L (m, j) for m ≤ j ≤ L and finally n L (m, m) = 1.
Thus, our problem effectively reduces to understanding the action of T L (1) on the module ∆ L (m), which is composed of partial diagrams p on L points with m unlabeled free points.
At a closer look, one can see that the action of T L (1) on partial diagrams keeps the reciprocal configuration of intersected lines and free points intact. In other words, if ψ is a map that eliminates all the nonintersected horizontal lines from a partial diagram and acts as identity otherwise, then ψ defines an invariant of T L (1), that is ψ(E i · p) = ψ(p), i = 1, . . . , L.
To understand the meaning of this invariant let us define a local map φ between partial diagrams which sends intersected horizontal lines to free points as depicted in fig. 13 and acts as identity otherwise. The local map φ is applied repeatedly until there are no more horizontal intersected lines left. It is not hard to see that φ extends to a homomorphism of T L (1) modules φ : Q l L (m) → D L (2l + m). In fact, the role of the map φ is to show that Q l L (m) is composed of a direct sum of isomorphic D L (2l + m) modules, while that of the map ψ is to distinguish between these modules. The set of partial diagrams p in φ φ Figure 13: Illustration of the nontrivial local action of the map φ.
Q l L (m) splits into subsets of constant ψ(p) and each of these subsets is isomorphic to D L (2l + m) as a T L (1) module.
According to what was said before, we get that n L (m, j) equals to the number of graphs on j = m + 2l vertices and l intersected edges. It follows that n L (m, j) does not actually depend on L and we drop the index L in the following. This fact allows, in principle, for an iterative computation of n(m, j) by simply computing the dimensions of the left and right hand sides of the decomposition formula successively for L = 0, 2, . . . or L = 1, 3, . . . . One can give an explicit expression for n(m, j) with a little more combinatorial work. We call a horizontal line an empty cup if its ends are adjacent and simply a cup if there are separated by free points. Observe that all the lines in a partial diagram are intersected if and only if there is at least one free point in each cup. Thus, if the partial diagram has p cups with only one free point inside and a total of l edges then the remaining m − p free points can be added to the diagram in C 2l m−p+2l different ways in such a way that the resulting diagram has only intersected edges. Moreover, the number of diagrams on 2l points with p empty cups and a total of l edges is again n(p, 2l − p). This is because the condition of no cups in the connection of the remaining l − p edges is similar to the condition of composing a graph with p free points and l − p intersected edges. Putting everything together we get a new recurrence formula reducing the problem to the computation of n( j) := n(0, 2 j). Next, we want to find a recurrence relation for n( j) by looking at the connectivity of the first point in the partial diagrams on 2 j points with j intersected edges. The leftmost vertex in the partial diagram has to be connected to some other vertex at position k. The connectivity of the 2 j − 1 points to the left of the point at position 1 is equivalent to that in a partial diagram with j − 1 intersected edges and a free point except for the case where k = 2. Therefore we have that Now, eq. (5.4) yields n(1, 2 j − 1) = (2 j − 1)n( j − 1) + n(1, 2 j − 3). Using again eq. (5.5) for j − 1 we finally get that The solution of the recurrence eq. (5.6) with the initial conditions n(1) = 0 and n(2) = 1 is and coincides with the absolute value of Bessel polynomials y j (x)
Conclusion
Besides the careful definition of the spin model and its sectors, the main point of this first paper is the algebraic set up necessary to analyze its symmetries. This is a non trivial task since we are dealing with non semisimple algebras, and that the action of OSp(2S + 2|2S ) and B L (2) are meshed through a complex structure of indecomposable representations. The main results are the decomposition formulas (4.36,4.37) for V ⊗L 4|2 viewed as a OSp(4|2) and a B L (2) module. The decomposition in eq. (4.36) has been computed in two essentially different ways: first, by decomposing tensor products between OSp(4|2) representations and V without knowing anything about the Brauer algebra and, second, starting from eq. (4.4) with the assumption that the representations of OSp(4|2) and B L (2) on V ⊗L generate the full centralizers of each other (Schur duality). The fact that we arrive at the same result using both methods highly suggests that our assumption about the Schur duality between OSp(4|2) and B L (2) on V ⊗L is correct.
When the question of decomposing V ⊗L is addressed in sec. 4.3, the notion of block appears to be a particularly useful concept for organizing indecomposable representations. 12 These results will be applied to educated conjectures about the conformal field theory in the next paper.
Although there are many things left unclear about the representation theory of osp(2S + 2|2S ), S > 1, it is very tempting to speculate the form of the decomposition of V ⊗L 2S +2|2S . Before making the guess, observe that as a OSp(4|2) module V ⊗L 4|2 ≃ T ⊕P, where P is a direct sum of projectives organized in blocks, while T is a direct sum of simples indexed by the same Young tableau (in the partition notation for dominant weights) as the irreps of O(2). More than that, they appear with the same multiplicities as their partners in V ⊗L 2|0 . 13 Therefore, T and V ⊗L
2|0
are similar in all but the internal structure of their simple summands. The similarity between the two modules has to be understood in terms of their centralizers, because these are precisely the objects that do not "see" the internal structure of simples. 14 In conclusion, one should have End O(2) V ⊗L 2|0 ≃ End osp(4|2) T , which is quite natural once there is a Schur duality between OSp(4|2) and B L (2) on V ⊗L 2S +2|2S . It is tantalizing to speculate that as a OSp(2S + 2|2S ) module V ⊗L ≃ T ⊕ P, with P projective and End OSp(2S +2|2S ) T ≃ End OSp(2S |2S −2) V ⊗L 2S |2S −2 ≃ B L (2)/J(S − 1). Thus, the problem of the decomposition of V ⊗L 2S +2|2S as a OSp(2S + 2|2S ) module is reduced to understanding the projective representations of the supergroup, i.e. to finding the quiver diagram for each block. It has been suggested in [34] that the quiver diagram of blocks does not depend on S provided the degree of atypicality k and the action of the outer automorphism τ are fixed. 15 The discussion of sec. A.3 suggests that the two types of quivers for a block of osp(2S + 2|2S ) and a fixed k will give rise to the same quiver for the induced blocks in OSp(2S + 2|2S ).
We also succeeded in computing the multiplicity of Temperley Lieb representations in a standard B L (2)module ∆ L (µ). Finally, we gave a combinatorial description of B L (N) blocks as the set of minimal partitions dressed by balanced removable border strips and have shown that there is a similar description for osp(R|2S ) blocks. 12 Let us note that the blocks appear already in the representation theory of simple Lie algebras if infinite dimensional representations are allowed. They are precisely the orbits of the shifted action of the Weyl group on the weight lattice. 13 In is not hard to prove employing the methods we used in this paper and the results of [29] for osp(3|2) that the same phenomenon occurs for V ⊗L 3|2 . In this case T is the trivial representation. 14 By a corollary of the Schur lemma, if S is a simple module for the algebra A then End A S ≃ C. 15 τ can act in two ways: either leave invariant all the weights in the block or pairwise transform some of them. Figure 14: Distinguished Dynkin diagram for the Lie superalgebra osp(2r + 1|2S ) on the left and osp(2r|2S ) on the right. Figure 15: The so(R) × sp(2S ) representation to which belongs the highest weight state of a osp(R|2S ) representation.
The Lie superalgebra osp(R|2S ) is realized as a subset of gl(V, C) with elements T satisfying In terms of elementary matrices (e i j ) kl = δ ik δ jl the generators of osp(R|2S ) read The generators h i = T ii span the Cartan subalgebra H. Denote by ε i the basis in H * dual to h i . It can be easily checked that generators in eq. (A.4) correspond to roots of the type ε i − ε j , generators in eq. (A.5) correspond to roots of the type ε i + ε j and generators in eq. (A.6) correspond to roots of the type −ε i − ε j . The bilinear invariant form − 1 2 str(h i h j ) induces a scalar product on H * . The standard basis is recovered by putting ǫ i = ε i for i = 1, . . . , r and δ i = ε r+i for i = 1, . . . , S . Elementary weights δ i , ǫ j are orthogonal in H * and δ 2 i = −ǫ 2 i = 1. The first r + S − 1 simple roots are chosen to be α i = δ i − δ i+1 , α S = δ n − ǫ 1 , α S + j = ǫ j − ǫ j+1 for i = 1, . . . , S and j = 1, . . . , r − 1. The last simple root is α r+S = ǫ r for odd R and α r+S = ǫ r−1 + ǫ r . The roots ±δ i ± ǫ j are called odd and the rest -even.
The component of a weight Λ along the hidden simple sp(2S ) root 2δ S is R odd : b = a S − a S +1 − · · · − a S +r−1 − a S +r /2 (A.7) R even : b = a S − a S +1 − · · · − a S +r−2 − (a S +r−1 + a S +r )/2. (A.8) According to [35], an osp(R|2S ) highest weight is dominant iff it has integer Dinkyn labels a i S and integer b satisfying the following consistency conditions All irreducible finite dimensional representations are indexed by dominant weights Λ. Given a dominant weight Λ = ρ i δ i + σ j ǫ j in the standard basis, the first r + S − 1 Dynkin labels are a i = ρ i − ρ i+1 for i = 1, . . . , S , a S +i = σ i − σ i+1 for i = 1, . . . , r − 1. The last Dynkin label is a S +r = 2σ r for R odd and a S +r = σ r−1 + σ r for R even. From eq. (A.7) we also get b = ρ S .
The set of numbers ρ i , σ j define a partition, shown in fig. 15, provided that consistency conditions (A.9) plus some additional constraints depending on R are satisfied. These additional constraints require a S +r−1 < a S +r and a S +r−1 +a S +r to be even if R is even, and a S +r to be to be even if R is odd. The last two conditions define tensorial weights.
Partitions λ such that λ r+1 ≤ S are called hook shape. Let τ denote the outer automorphism induced by the symmetry of the osp(2r|2S ) Dynkin diagram under the exchange of the last two roots in fig. 14. This automorphism is extremely important in understanding the difference between the representation theory of the supergroup OSp(R|2S ) and its Lie superalgebra. Note that τ can be explicitly realized through the discrete transformation ρ exchanging the last two basis vectors in B 0 . Indeed, ρ(ǫ j ) = ǫ j for j = 1, . . . , r − 1 and ρ(ǫ r ) = −ǫ r because ǫ j are the duals of e j j − e j * j * . Therefore ρ(α S +r ) = ρ(ǫ r−1 + ǫ r ) = ǫ r−1 − ǫ r = α S +r−1 .
In the case of R odd, there is a bijective correspondence between hook shape partitions λ and dominant weights Λ. The same holds for R even, except for λ with σ r > 0 when λ represents both Λ and τ · Λ.
If there is a pair (i, j) such that at least one of the conditions below are satisfied the weight λ is called atypical. 16 See [39] for the origin of these conditions and note ref. [38], where these have been presented in the form (A.10,A.11). If none of these conditions is satisfied, the weight is called typical and, according to [39], the associated Kac moduleV(Λ) (which is a finite dimensional quotient of the corresponding highest weight module) is irreducible, its (super)character is given by the Weyl-Kac formula [40] and, in particular, its superdimension is zero.
A.2 OSp(R|2S ) supergroup
Let Γ = Γ 0 ⊕ Γ 1 be a Grassman algebra. The supergroup OSp(R|2S ) may be realized as a subset of even supermatrices with entries in A and D belonging to Γ 0 , and entries in B and C belonging to Γ 1 , which satisfies Equivalently, OSp(R|2S ) can be seen as the set of linear transformations leaving invariant the graded symmetric form where η α are arbitrary points in a superspace parametrized by coordinates b i α , b i * α ∈ Γ 0 and f j α , f j * α ∈ Γ 1 and α = 1, 2.
Representing M = I+ a α a T a with infinitesimal α a ∈ Γ 0 , Γ 1 and expanding eq. (A.12) one gets the definition (A.3) of the superalgebra osp(R|2S ). Thus, the subgroup of OSp(R|2S ) connected to identity is an exponential of osp(R|2S ). The representation theory of both is the same as long as we restrict to tensor representations which are the only ones appearing in the tensor space V ⊗L .
To see this, one can repeat the same reasoning typical of O(N) groups. Elementary transformations susceptible to change the sign of the superdeterminant belong to the discrete symmetry group W of the OSp(R|2S ) invariant form (A.13). The generators of W are read out from eq. (A.13) to be "reflections" ρ i : . For odd R there is also the reflection ρ r+1 : b r → −b r . The subgroup W is in fact the Weyl group of the root system of so(R) × sp(2S ). Denote by W ± the set of elements of W embedded in OSp ± (R|2S ). It is easy to see that all elements of W − are conjugate in W + to a single reflection ρ, which one can take ρ r if R is even and ρ r+1 if R is odd. Therefore, we see that indeed W/W + = Z 2 .
Note that the centralizer of B L (N) on V ⊗L is the direct product algebra Z 2 × osp(R|2S ) rather then osp(R|2S ). This algebra has the same tensor irreducible representations as the supergroup OSp(R|2S ).
The dimensions of g k,l := g(λ k,l ) can be computed with the help of character formulas given in [29].
The set of weights λ k,l with k fixed belong to the same block of osp(4|2). To (at least partially) see this one has to check that the second order Casimir invariant takes the same value k 2 on the whole block k. 18 The actual construction of the set of indecomposable modules providing the equivalence relation of sec. B between the weights of a block is done in [29].
We also claim that the quiver diagram of type A ∞ ∞ for the block k 0 of osp(4|2) will give rise to a single quiver diagram of type D ∞ , as shown in fig. 17, and a selfassociate block for the algebra Z 2 × osp(4|2), which we label also by k. As we shall sea bellow, the weights in the block k 0, 0 * are 1 × λ k,0 , ε × λ k,0 , τ × λ k,l , l ≥ 1.
B Blocks, minimality and atypicality
In this section we explain carefully the notion of block appearing in the representation theory of nonsemisimple algebras. We also look in details at the similarity between the blocks of osp(R|2S ) and B L (N).
In the representation theory of non semisimple algebras the block is an essential notion. The blocks are conjugacy classes of irreps with respect to the equivalence relation ≡ defined as follows. Let I be the category of indecomposable modules of the algebra. Write S 1 ≡ S 2 if there is an indecomposable module in I with simple summands S 1 , S 2 . Extend the relation ≡ by transitivity in order to get an equivalence. In a semisimple algebra the notion of block is irrelevant because indecomposable representation are irreducible and the congruence ≡ becomes an equality.
A relevant example is the Temperley Lieb algebra, with fugacity for loops N in its adjoint/diagrammatic representation. For generic values of N the algebra is semisimple and, thus, has only completely reducible representation. Restricting to subsets of planar diagrams, with the number of vertical lines fixed to m, and treating all the other diagrams as zero, we get all irreps, which are parametrized by m. However, at special points N = 2 cos πr ′ /r ′′ with coprime integers r ′ , r ′′ , the algebra becomes nonsemisimple, irreps labeled by m become reducible and m becomes a label for a whole block of the algebra, see [22].
The irreducible components B L (λ) of indecomposable modules ∆ L (µ), when the Brauer algebra B L (N) is nonsemisimple, where first studied by mathematicians Hanlon et al in [14]. Recently Martin et al gave a complete description for the blocks of the Brauer algebra in [23]. We introduce the same notation as in [23] to formulate their block result for B L (N). If the box ǫ is in the row i and column j of the Young tableau of a partition µ, then its content is c(ǫ) = j − i. Two boxes ǫ, ǫ ′ ∈ λ are called balanced if c(ǫ) + c(ǫ ′ ) = 1 − N. For two partitions µ ⊂ λ, the skew partition λ/µ is called balanced if it is composed of balanced pairs of boxes.
The necessary condition for ∆ L (µ) to contain B L (λ) is: i) µ ⊂ λ and λ/µ is balanced; ii) If N is even and the boxes of content 1 − N/2, −N/2 in λ/µ are configured as shown in case a fig. 18, then the number of columns in this configuration is even.
The given necessary criterion has the structure of a partial ordering. If µ ⊂ λ satisfy i) and ii) we write µ λ. The splitting of the set of weights X L into posets with respect to gives the blocks of B L (N). As shown in [23], there is a unique minimal partition in a block, which can serve as a label.
A sufficient criterion for the module ∆ L (µ) to contain B L (λ) was derived in [23] and requires λ to be the least weight λ µ.
We want to give a combinatorial description of the weights in a block. Consider the Young tableau of a partition λ in the block of the minimal partition µ. Let ǫ 1 (ǫ ′ 1 ) be the box with the highest (lowest) content in the a b Figure 18: Two possible configurations of boxes with content 1 − N/2, −N/2.
skew partition λ/µ. Let ǫ 2 (ǫ ′ 2 ) denote the box bellow (on the left of) ǫ 1 (ǫ ′ 1 ), if there is one, and the box on the left of (above) ǫ 1 (ǫ ′ 1 ) otherwise. Define by recurrence the balanced pairs By construction, the set of boxes {ǫ i , ǫ ′ i } l 1 belongs to a balanced removable border strip of width one or, simply, a balanced strip. One can repeat the same reasoning with the Young tableau of λ/{ǫ i , ǫ ′ i } l 1 (which is not necessarily in the same block as µ because of ii)). Thus, we clearly see that partitions λ in the same block can be constructed by dressing up with balanced strips a certain partition µ with no removable balanced strips. Denote by η the balanced strip of smallest length addable to µ. If N is odd denote byμ the minimal partitionμ/µ = η. If N is even denote byμ the minimal partitionμ/µ = η only if the two boxes with content −N/2, 1 − N/2 in η are disposed horizontally andμ = µ otherwise. Partitions which are of the form µ dressed up with an even (odd) number of balanced strips are in the same block as µ (μ). Note that it is irrelevant in what order the strips are dressed on µ. Also, there cannot be two balanced strips of the same length. Thus, a partition λ in the block µ (μ) is unambiguously specified by the length of balanced strips in the skew partition λ/µ.
We claim now and show bellow that a block of osp(R|2S ) is composed, in the partition notation of sec. A.1, of hook shaped partitions built up by dressing with balanced strips an atypical partition with no removable balanced strips. 20 For that we need to reformulate the original block result [34] for osp(R|2S ).
Let the degree of atypicality k of a dominant weight Λ, be the dimension of the subspace A of the root lattice orthogonal to Λ + ρ, where ρ is the Weyl vector of osp(R|2S ). Each atypicality condition in eq. (A.10,A.11) is, in fact, an orthogonality condition between an odd root δ i ± ǫ j , ǫ j 0 and Λ + ρ. Therefore, k is the number of odd roots orthogonal to each other and to Λ + ρ or, equivalently, the number of atypicality conditions labeled by couples (i, j) with distinct i and j. From the definition of the highest weight module V(Λ) it is clear that irreducible finite dimensional components of V(Λ) must have dominant weights of the form Λ − Nα, where the sum is over all odd positive roots α spanning A.
Consider a osp(R|2S ) weight λ, which, in the notation of app. A.1, has symplectic part ρ and orthogonal part σ. Suppose ρ n+1 , ρ m+1 are the first columns of λ satisfying ρ n+1 ≤ r − S + n and ρ m+1 ≤ m + R − S − ρ S − 1. Then, one can find rows i j , such that λ i j < S and the pairs (i j , j) satisfy the atypicality condition (A.11) for m < j < n if R is odd and m < j ≤ n if R is even and the atypicality condition (A.10) for n ≤ j. Indeed, from eq. (A.11) with σ i = 0 the condition m < j implies i j > ρ S and thus λ i j < S , while i j ≤ r implies j ≤ n if R is even and j < n is R is odd. From eq. (A.10) with σ i = 0 the condition n ≤ j implies i j ≤ r while i j > ρ S follows directly from ρ j ≥ ρ S .
Conversely, if ρ j , m < j satisfies an atypicality condition with σ i = 0, then λ i < S . As shown in fig. 19, n is the width of the foot of the narrowest hook with arm width r − S + n in which the Young tableau of λ can be drawn in.
Two atypicality conditions (i, j) and (i ′ , j ′ ) are called independent if i i ′ and j j ′ . Clearly, conditions (i j , j) are pairwise independent for m + 1 ≤ j < n and for n ≤ j. Let us show that an atypicality condition (i j , j), m + 1 ≤ j < n is independent of conditions (i ′ j ′ , j ′ ), n ≤ j ′ iff there is a row shorter then S such that the box ǫ at the end this row and the box ǫ ′ j at the end of column j are balanced. In order to do that it is useful to imagine the partition λ drawn on an infinite square lattice, as in fig. 19, with each square having its content written inside. The following cases are possible • Suppose first that there is no box with at the end of the row j. From j < n follows j ≤ S − r. Observe that the column 1 + S − r ≤ j ′ = 1 + S − i j is also empty. Therefore i ′ j ′ = i j and the atypicality conditions (i j , j) and (i ′ j ′ , j ′ ) are not independent. r S n r − S + n Figure 19: A partition λ drawn on a hook shaped square lattice and fitting exactly inside a hook with foot width n and arm width r − S + n. The black boxes represent the diagonal of squares with content c = S − r.
• Let ρ j 0 and let ǫ j be the box at the end of column j. No suppose that λ has a rightmost box ω with content c = 2 − N − c(ǫ ′ j ) and let j ′ be the column of that box. Condition ρ j ≤ m + R − S − ρ S − 1 gives c ≤ c(ǫ ′ S ) + 1 + m − j. The equality sign cannot hold because otherwise j = j ′ = S which contradicts j < n. Thus 1 + S − r ≤ c = 1 + S − i j < c(ǫ S ) and therefore S − r ≤ n ≤ j ′ < S . If j ′ − ρ j ′ < c then there is a box ǫ bellow ω, which is balanced with ǫ ′ j and has no box to the right, thus it is the end of a row shorter then S . If j ′ − ρ j ′ = c then comparing the i's from the two atypicality conditions we get i j = i ′ j ′ and the two atypicality conditions are not independent.
• Finally, if there is no box with content c then the column j ′ = c gives an atypicality condition (i ′ j ′ , j ′ ) with i j = i ′ j ′ . Next, by the definition of m, a column j ≤ m can satisfy an atypicality condition only with a row λ i ≥ S . After inserting σ i = λ i − S in eqs. (A.10,A.11) we get The lhs in eq. (B.1) is the hook length of the box in the row i and column j of λ, thus, always positive. On the other hand, eq. (B.2) requires the box ǫ ′ j at the foot of column j be balanced with the box ǫ i at the end of row i. In the end, we see that there are two sources for independent atypicality conditions satisfied by a weight λ. First, if n is the width of the foot of the narrowest hook with arm width r − S + n, in which the Young tableau of λ can be drawn in, then there are p := S − n atypicality conditions satisfied by the weight and we call them of type 1. Second, to each balanced pair of boxes ǫ, ǫ, such that ǫ is a box at the end a row and ǫ is a box at the end of a column, corresponds an atypicality condition of type 2. If λ satisfies q atypicality condition of type 2 then the degree of atypicality of the weight is k = p + q.
Let {ǫ i l , ǫ ′ j l } q 1 , j 1 <, . . . , < j q be the set of balanced pairs satisfying atypicality conditions of type 2. Applying the iterative construction explained above to the boxes ǫ i q , ǫ ′ j q one can see that there is a removable balanced strip η j q in λ q := λ, with its ends in ǫ j q , ǫ ′ j q . Clearly, by the same reasoning, one can identify a new balanced strip η q−1 removable in λ q−1 := λ q /η q . The end λ 0 of this iterative procedure has no more removable balanced strips. Note that λ 0 satisfies k atypicality conditions all of type 1 and the sequence of weights λ 0 , . . . , λ q has the same degree of atypicality k.
In order to complete the proof of the claim it remains to notice two things. First, if α 1 i is an odd root generating an atypicality condition of type 1, then Λ − p i=1 Nα 1 i is not dominant. Second, if α 2 = δ j + ǫ j is an odd root generating an atypicality condition of type 2, then Λ has a removable strip with its ends in the last box ǫ i of row i and ǫ ′ j of column j and Λ − α 2 i is dominant and can be represented by a partition of the form λ/{ǫ i , ǫ ′ j }, where λ is the Young tableau of Λ.
C Modification rules and OSp(R|2S ) associate weights
The explicit form of the characters of classical groups is easier derived in the limit of infinite rank of the corresponding Lie algebra. The inverse limit exists and is given by the modification rules for characters. The concepts of infinite rank and inverse limit are rigorously defined for the case of Schur symmetric functions, connected to the irreducible characters of GL(N), in [42]. Let us clarify this point. The characters of classical groups, evaluated on a group element, are polynomials in the eigenvalues of that element in the defining representation for the group. The infinite rank limit corresponds to considering polynomials depending on an infinite number of such variables. Irreducible characters are polynomials with a very specific symmetry, which is not obscured by the restriction of finite number of variables in the infinite rank limit. 21 These objects are known as symmetric functions. When the number of variables is set finite most symmetric functions become functionally dependent. Once an algebraically independent subset of symmetric functions is chosen, which is the actual set of characters in the case of classical groups, the modification rules "for characters" represent arbitrary symmetric functions along this basis.
One can introduce generalized symmetric functions sc µ for the supergroup OSp(R|2S ) according to eqs. (4.5, 4.6), see [25], [15]. The major difference with respect to classical groups is that functionally independent generalized symmetric functions are no longer irreducible characters of the supergroup. However, modification rules for sc µ exist and have been derived in [26]. We bring them bellow in the form of eq. (4.8) with the notations of our paper.
Suppose that λ is a typical osp(R|2S ) weight. Then, according to [26], only sc µ with µ of the form λ dressed by balanced strips η 1 , . . . , η m modify to sc λ sc µ = ε m w(µ/λ)sc λ , (C.1) here ε is the superdeterminant representation. We have also introduced the weight function w(µ/λ) = m i=1 (−1) c i −1 defined on skew partitions composed of balanced strips and c i is the number of columns in η i .
Consider now, the osp(R|2S ) block labeled by the weight ν with degree of atypicality k and no removable balanced strips. Then, any weight λ in the block B µ of µ is of the form ν dressed up by q λ ≤ k balanced strips. Then, according to [26], sc µ with µ of the form ν dressed up by m ≥ k + 1 balanced strips modifies to sc µ = λ∈B ν C m−q λ −1 m−k−1 w(µ/λ)(−1) k−q λ ε m−q λ sc λ . (C.2) For a typical weight λ we put λ * equal to λ dressed up by the balanced strip of minimal length η 1 if λ ′ S < r. In order to prove that sc λ * = εsc λ one has to show that η 1 runs over an odd number of columns.
Let us prove that η 1 runs over an odd number of columns c 1 if λ ′ S < r and an odd (even) number of columns if λ ′ S = r and R is odd (even). Indeed, each box in η 1 belongs either to a horizontal or a vertical part of the strip, except for the boxes at the corners of η 1 , which belong to both. We say the balanced pair ǫ, ǫ ′ ∈ η 1 has an allowable configuration if both boxes belong either to horizontal or vertical parts of η 1 , otherwise ǫ, ǫ ′ has a non allowable configuration. As discussed in app. B, to every non allowable configurations of ǫ, ǫ ′ with content c, c ′ corresponds a removable balanced strip in λ with its ends in the border boxes with content c − 1, c ′ + 1 or c + 1, c ′ − 1 depending on weather ǫ is on a horizontal or a vertical part of η. Because λ is a osp(R|2S ) typical weight and, thus, has no removable balanced strips, there are only allowable configuration of balanced pairs in η 1 . Thus, a balanced pair in η 1 , which is not in the same column, indexes either two different columns or none. There is at most one column containing the whole balanced pair and it appears always if N is odd and only for λ ′ S < r if N is even, as shown in fig. 20. Thus, c 1 is always odd for R odd and even only if λ ′ S = r for R even. Let η i denote the ith lowest length strip addable to λ and c i the number of columns in it. One can prove by the same method that η i+1 /η i has an even number of columns, one of which is already in η i and, consequently, c i+1 − c i is odd. Again by the same method it is possible to prove that η 1 +η 2 has an even number of columns. This is because η 2 contains a substrip η ′ 1 which can be obtained by moving down along the diagonal the strip η 1 . Applying what was said above about η i+1 /η i to η 2 /η ′ 1 we see that w(η 1 + η 2 ) = −1. If λ is typical and λ ′ S < r then µ = λ + η 1 + η 2 is the next partition in the block of λ, while if λ ′ S = r then µ = λ + η 1 is the next partition in the bloc of λ. Therefore we have just shown, as claimed in sec. 4.3 that sc µ = −sc λ .
We do not now how to explicitly define the associates of atypical weights for general osp(R|2S ). | 2008-01-02T20:34:41.000Z | 2008-01-02T00:00:00.000 | {
"year": 2008,
"sha1": "93bca30c7defdd7d1fce9ff847d1ecd93923e3bf",
"oa_license": null,
"oa_url": "http://arxiv.org/pdf/0801.0430",
"oa_status": "GREEN",
"pdf_src": "Arxiv",
"pdf_hash": "93bca30c7defdd7d1fce9ff847d1ecd93923e3bf",
"s2fieldsofstudy": [
"Mathematics"
],
"extfieldsofstudy": [
"Physics"
]
} |
13619929 | pes2o/s2orc | v3-fos-license | Dynamics of one-dimensional Bose liquids: Andreev-like reflection at Y-junctions and absence of the Aharonov-Bohm effect
We study one dimensional Bose liquids of interacting ultracold atoms in the Y-shaped potential when each branch is filled with atoms. We find that the excitation packet incident on a single Y-junction should experience a negative density reflection analogous to the Andreev reflection at normal-superconductor interfaces, although the present system does not contain fermions. In a ring interferometer type configuration, we find that the transport is completely insensitive to the (effective) flux contained in the ring, in contrast to the Aharonov-Bohm effect of a single particle in the same geometry.
Recently, guiding of atoms in low-dimensionally magnetic traps has been actively studied [1]. Such systems provide an ideal opportunity to study coherent quantum dynamics of interacting many-body systems. The theory of one-dimensionally trapped atoms in equilibrium [2,3,4,5], as well as questions related to coherent dynamics and nonequilibrium phenomena [6], has been vigorously studied. In this Letter we analyze a simple yet nontrivial example of the real-time dynamics of Bose-Einstein condensates (BEC) in the Y-shaped potentials (beam-splitters) [7], and in the related "ring interferometer" type geometry [8].
In contrast to previous works where the dynamics of wave packets in otherwise empty traps is studied, here we consider the situation where each one-dimensional branch of the Y-shaped potential is filled with interacting bosonic atoms, i.e. each branch contains one-dimensional Bose liquid. The Tomonaga-Luttinger (TL) liquid theory [3,9] is a powerful method to study this problem. The whole system in the Y-shaped potential may be regarded as three TL liquids connected at a junction, as in Fig. 1. While there are parallels with the corresponding electron problem [10,11], as we will show in the following, a quite different generic behavior is expected in the Bose liquid. In the present case, instead of the conductance, the real-time dynamics could be directly observed in experiments. Furthermore, as an application of the above analysis of the single Y-junction problem, we also consider the transport in a ring type interferometer geometry.
We have two main results in this Letter. The first is the prediction of the negative density reflection at a junction ( Fig. 1). In the discussion of Y junctions for systems of interacting electrons, such negative reflection has been interpreted as the Andreev reflection, which occurs in metal-superconductor interfaces due to Cooper pair formation. On the other hand, the negative reflection at the BEC Y-junction can be understood rather simply using an analogy with electromagnetic transmission line three-way junction. For an ideal three-way junction, there should be no voltage difference between the connected ends of the three lines, and there is conservation of the total charge in a pulse. These two conditions imply that when the amplitude of an incident signal is one, signals transmitted into the other two lines will have amplitudes of 2/3 and the reflected signal will have the amplitude of −1/3. As we discuss below the same phenomenon should appear in a BEC Y-junction.
The other main result is the absence of the Aharonov-Bohm (AB) effect in the ring type interferometer, which consists of two Y-shaped junctions as in Fig. 2. As a typical example of the AB effect, when the ring is empty, a single particle injected from one lead to the ring does not transmit to the other lead when the (effective) "magnetic flux" inside the ring is the half unit flux quantum. However, we find that the transport between the leads in our system is completely insensitive to the magnetic flux, in the low energy limit. These two findings are manifestation of the collective nature of the Bose liquid, rather than single-particle physics.
We start from the following one-dimensional Hamiltonian [12] with the low-energy scattering characterized by the low-energy s-wave scattering: where we take an unit ofh = 1. ψ j (x), ρ j (x) ≡ ψ † j (x)ψ j (x) are, respectively, the annihilation and the density operator of the boson with the mass m, on the j-th branch. (j = 1, 2, 3.) The effective interaction strength U > 0 is determined by the s-wave scattering length. We take x > 0, so the physics at the junction is represented by the boundary condition (b.c.) at x = 0. In case of the junction problem, the b.c. is a non-trivial problem and plays a crucial role. First of all, we have to identify what kind of b.c. describes the physics in the low-energy limit.
Any b.c. should satisfy the total current conservation where J j (x) is the current operator of the Bose liquid in the j-th branch. The simplest possible b.c. corresponds to an infinitely strong barrier at the junction, which prohibits any particle to be transmitted through the junction. It would give J j (t, x = 0) = 0 for j = 1, 2, 3. Naturally, this satisfies the current conservation (2). In practice, there would be a finite tunneling amplitude Γ between the different branches. The effect of the tunneling can be included by adding the perturbation at the boundary. We are interested in the low-energy behavior of the junction in the presence of the tunneling. In order to study the low-energy physics, it is convenient to use the "bosonization" technique [9]. In terms of the TL boson field θ j (x) and its dual ϕ j (x), the Bose atom annihilation operator and density operator on the j-th branch are, respectively, represented as where we have retained only the leading terms. ρ (0) represents the expectation value of the atom density in the ground state. The low-energy effective Hamiltonian for equation (1) is given by The fields θ, ϕ satisfy the commutation relation is the Heaviside step function. v is the velocity of the collective modes, and g is the so-called TL parameter, in which the interaction is essentially contained. The TL parameter, for the interacting fermions i.e. electrons, usually satisfies g < 1 due to the Coulomb repulsion. However, g > 1 occurs rather naturally in a Bose liquid with a short-range repulsive interaction. For example, for the Bose liquid with a δ-function interaction [13], the TL parameter g can be determined from the Bethe Ansatz exact solution. It turns out that g is always larger than 1 for this model, interpolating between g = ∞ in the non-interacting limit and g = 1 in the strongly interacting (or dilute) "Tonks gas" limit. [3,4,5] While g can also be less than 1 with other types of the interaction [14], g > 1 would be rather generic for the interacting Bose liquid.
In terms of the bosonization, the "disconnected" limit ψ j (0) = 0 corresponds to the Dirichlet boundary condition ∂ t θ j (0) = 0 for j = 1, 2, 3. It is equivalent to the Neumann b.c. on ϕ j . The tunneling perturbation (3) is bosonized as H B ∼ Γe −i(ϕj (0)−ϕj−1(0)) , where only the most relevant term is retained. The scaling dimension of this perturbation is given by g −1 , and it is relevant when g > 1. Namely, the effective tunneling amplitude Γ grows as the energy scale such as the temperature is lowered. It is expected that Γ eventually grows to infinity in the low-energy limit, when g > 1. [15] In the strong coupling limit, the ϕ fields should be pinned as ϕ 1 (t, 0) = ϕ 2 (t, 0) = ϕ 3 (t, 0), because the tunneling term acts as an infinitely strong potential on ϕ at the junction. This also implies ∂ t ϕ 1 = ∂ t ϕ 2 = ∂ t ϕ 3 . Translating it into the b.c.s on θ j , we find where the first line comes from the current conservation (2). The second line indicates that the density is continuous at the junction, reflecting that the junction is at the strong coupling limit. Using the boundary conformal field theory techniques, we can see that all the possible perturbations to the above b.c. are irrelevant if g > 3/4 [10,11] in the case of Bose liquid. This guarantees the stability of the "strong coupling" fixed point, supporting that Γ grows to infinity in the low-energy limit when g > 1. Now we discuss the dynamics of the system with the stable, strongly coupled b.c.. Let us suppose that the density is altered locally from the equilibrium. The variation of the density propagates in the system, and eventually would be transmitted to other branches.
We start from the Heisenberg equation of motion for the TL boson field θ j and dual field ϕ j (j = 1, 2, 3): x). Let us focus on the expectation values of the density and current. Instead of operators θ j and ϕ j , we then just need to determine the expectation values θ j and ϕ j , which obey the simple one-dimensional classical wave equation. The initial conditions are given as the expectation values of current and density at t = 0.
For simplicity, we consider the situation where a leftmoving packet is injected from one side (branch 1). Since the left movers go towards the boundary (x = 0), and the right movers go away from the boundary, the initial conditions may be given as ρ L x) /π is the scalar which gives the initial condition on the expectation value of the densities. Solving the classical wave equation with the initial conditions, we obtain: where η ± ≡ vt ± x. The second term in the rhs of the equation (7) represents the incident packet density, and the third term implies the (negative) reflection at the boundary. Namely, if a bump in the density moves through the branch 1 to the junction, a dip in the density is reflected back. (see Fig.1) Eq. (8) shows the transmission density from the branch 1. Defining the transmission tensor as the ratio of the incident current and transmission currents, the transmission tensor from the branch 1 to the branch 2 (branch 3), T 2,1 (T 3,1 ), are obtained as T 2,1 = T 3,1 = 2 3 . This means that the sum of the densities transmitted to branches 2 and 3 is greater than the incident density from the branch 1. The current conservation law is satisfied with the "negative reflection" current in the branch 1, which appears as the second term in equation (7). The time evolution of the density (7)-(8), including the remarkable negative reflection, could be observed in experiments, with present techniques on atomic BEC.
A similar behavior was found in the Y-junction of onedimensional electron systems, in terms of the conductance. It was argued as a consequence of Andreev reflection similar to that in a superconductor-normal interface. What is surprising here is that the Andreev-like negative reflection is also found for the boson systems. The system of bosons is usually not related to Cooper pair formation, which is the underlying mechanism of the Andreev reflection. These results may be also derived from a linearized mean-field theory which is applicable to the weakly interacting regime. While the mean-field approximation breaks down in the strongly interacting regime, our derivation based on the TL liquid theory are valid even in the presence of strong fluctuation. Thus our results represent universal feature of general repulsively interacting boson systems.
Let us extend our analysis to a "ring interferometer" type configuration, which consists of two Y-junctions. (see Fig.2) We take the TL fields on the upper and lower arc in the ring to θ u,l , respectively. Then the symmetric combination θ s = θ u +θ l is coupled with the right and left leads, while anti-symmetric combination θ a = θ u − θ l is decoupled. Here, the propagation of the atoms in the ring are symmetric so that anti-symmetric combination does not affect the propagation. As a result, we can obtain the inhomogeneous TL liquid [16] as the effective model of the double Y junction systems, where the TL parameter on the left and right lead is g, and that of the symmetric combination θ s is 2g. Following Ref. [17], we can investigate the transmission and the reflection at the junctions. The solution of the equation of motion naturally gives the contribution of multiple reflections. Interestingly, the effect of normal and Andreev-type reflections cancel out, so that the total transmission approaches 1 as the elapsed time approaches infinity. Experimentally, real-time observations of the density could resolve each reflection. Now let us consider the effect of a "magnetic flux" φ inside the ring. The present geometry is a typical setting to observe the AB effect. If we consider the transport of a single charged particle in the same geometry with an empty ring, the transmission probability from the left lead to the right vanishes when φ is the half unit flux quantum. This is because of the cancellation between the probability amplitudes coming from the two paths. Although a magnetic flux actually does not induce the AB effect for neutral atoms, several possible approaches for introducing gauge fields and AB type phase factors for neutral atoms have already been discussed in the literature [18]. A common ingredient of many methods is that of the Berry's phase arising from the orbital motion of atoms [19].
We find that, however, in our problem of filled onedimensional BEC, the transport between the leads is completely insensitive to the effective magnetic flux φ, in the low-energy limit. Namely, the AB effect is absent. This rather surprising conclusion follows quite naturally from the present formulation, as follows.
With a gauge transformation, the effect of the flux can be described by the boundary conditions ϕ u (−L) = ϕ l (−L) at the left junction and ϕ u (L) = ϕ l (L) + φ at the right one. They determine the boundary values of the antisymmetric combination as ϕ a (−L) = 0 and ϕ a (L) = φ. The current on the ring is just a superposition of the symmetric flow between the two leads described by ϕ s and the persistent current described by ϕ a . Only the latter depends on the flux φ. As it is only the symmetric combination ϕ s that couples with the leads, the flux φ has no effect on the transmission between the leads.
If we consider the system with fermions instead of bosons in the same geometry, the AB effect must be present, at least when the fermions are non-interacting. As the non-interacting fermions in one dimension corresponds to the TL liquid with g = 1, our result might appear in contradiction. However, there is actually no contradiction, because our analysis applies only to the "strongly coupled" b.c. (6). In the system of noninteracting electrons, the junction is described by a different b.c.. The "strongly coupled" b.c. is expected to be stable even in a fermionic system, when the interaction is sufficiently attractive (g > 9) [11]. For such a system, the AB effect should be absent as well.
Finally, we discuss the limitations of and possible corrections to the present results. First, the TL liquid description is only valid in the low-energy limit. This requires T, 2πv/L ≪ µ, where L is the system size (length of the branches), and the chemical potential µ = U ρ (0) gives the ultraviolet cutoff. [3] When a packet of extra particles is injected, the variation ∆ρ of the particle density corresponds to a variation of the local chemical potential ∆µ = U ∆ρ. Thus ∆ρ ≪ ρ (0) is required for the TL liquid description to apply. For a larger amplitude, the single-particle physics is expected to show up, instead of the collective nature characterized by the TL liquid.
The "strongly coupled" b.c. (6), which we assumed in the present Letter, is exact only in the low-energy limit, even when the bulk is completely described by the TL liquid. There are corrections due to finite energy scales present in a realistic system. The leading correction to the results based on the b.c. (6) can be related to, at a sufficiently low energy scale, the leading irrelevant operator with scaling dimension 4g/3. It would give, for example, a positive reflection proportional to ǫ 8g/3−2 on top of the universal negative reflection −1/3. Here ǫ denotes the largest among the possible infrared cutoff scales, such as T , 2πv/L, and U ∆ρ. When the interaction is weak, it is difficult to satisfy the conditions for the TL liquid theory to be applicable in the bulk. Interestingly, however, the corrections at the junction vanishes quickly because g is large in such a case. | 2017-09-07T01:27:22.205Z | 2007-03-23T00:00:00.000 | {
"year": 2007,
"sha1": "17de53c936348c232710b6c9f7396748d1c1b865",
"oa_license": null,
"oa_url": "http://arxiv.org/pdf/cond-mat/0703610",
"oa_status": "GREEN",
"pdf_src": "Arxiv",
"pdf_hash": "b3ca11142b16b6bd422713a4c9f800720920ee82",
"s2fieldsofstudy": [
"Physics"
],
"extfieldsofstudy": [
"Physics",
"Medicine"
]
} |
237434938 | pes2o/s2orc | v3-fos-license | A New Look at Vaccine Strategies Against PPRV Focused on Adenoviral Candidates
Peste des petits ruminants virus (PPRV) is a virus that mainly infects goats and sheep causing significant economic loss in Africa and Asia, but also posing a serious threat to Europe, as recent outbreaks in Georgia (2016) and Bulgaria (2018) have been reported. In order to carry out the eradication of PPRV, an objective set for 2030 by the Office International des Epizooties (OIE) and the Food and Agriculture Organization of the United Nations (FAO), close collaboration between governments, pharmaceutical companies, farmers and researchers, among others, is needed. Today, more than ever, as seen in the response to the SARS-CoV2 pandemic that we are currently experiencing, these goals are feasible. We summarize in this review the current vaccination approaches against PPRV in the field, discussing their advantages and shortfalls, as well as the development and generation of new vaccination strategies, focusing on the potential use of adenovirus as vaccine platform against PPRV and more broadly against other ruminant pathogens.
INTRODUCTION
Due to the risk of viral escape mutants from antiviral treatments as well as the excessive use of antibiotics that causes the appearance of bacterial resistances, vaccination continues to be one of the best measures to prevent infectious diseases. Since Jenner's time, vaccines have come a long way thanks to the development of knowledge and technology in molecular biology and immunology.
The relevance and impact of a pandemic due to a human pathogen is not the same as that of diseases in ruminants. But, precisely because we are living in a globalized world, in which pathogens jump more and more frequently from animals to humans, animal health should be a priority. This is framed within the One Health concept, in which animal health and human health are interdependent, and global strategies to prevent and control pathogens must be implemented.
Among the diseases of relevance in animal health, peste des petits ruminants (PPR) stands out. It is caused by the peste des petits ruminants virus (PPRV) and affects mainly small domestic ruminants (sheep and goats) as well as camels, with serious economic loss especially in many countries of Africa and Asia (1,2). Wild ruminants, such as gazelles, deer, roe deer, antelope can also be affected (3)(4)(5)(6)(7)(8)(9)(10)(11)(12), which consequently poses a further risk for the control and surveillance in vaccination programs.
PPRV belongs to the genus Morbillivirus among which are included the important human pathogen measles virus (MV), as well as veterinary pathogens such as canine distemper virus (CDV), feline morbillivirus, dolphin and porpoise morbillivirus (DMV, PMV), phocine distemper virus (PDV), morbilli-like bat or rodents virus, and the eradicated rinderpest virus (RPV) (13)(14)(15)(16)(17). Rinderpest virus (RPV) vaccination has shielded PPRV from visibility for years, as it provided partial protection against PPRV, but once RPV was eradicated and vaccination programs stopped in 2011 PPRV emergence became clearly evident. In 2014, the World Organization for Animal Health (OIE) considered PPRV the second animal pathogen candidate to be eradicated, establishing an eradication program aimed at 2030. To achieve this, synergies must be produced between governments, researchers, companies and farmers (18).
The vaccines currently used in the field against PPRV are live attenuated vaccines. Despite the fact that these vaccines generate protection, they present drawbacks that need addressing. Importantly, the eradication program would be greatly helped with the development of vaccines that allow differentiation of infected from vaccinated animals (socalled DIVA vaccines), which would facilitate the control and surveillance programs in the vaccinated areas. Likewise, vaccines that are independent of a cold chain for their preservation, one of the main drawbacks of live attenuated vaccines, would be advantageous since in most countries where PPRV is endemic, the maintenance of cold storage and transport facilities can be problematic and could lead to vaccine administration in poor immunization conditions. These are achievable goals as we have seen in these times of the Covid-19 pandemic, where the reaction capacity of pharmaceutical companies and the scientific community has successfully developed several vaccines, based on different technologies. Currently, several laboratories are working on different approaches, aimed at overcoming the aforementioned weak points of current vaccines, by developing a new generation of vaccines against PPRV. These alternative approaches include inactivated vaccines, DNA vaccines (19,20), recombinant subunit vaccines (21)(22)(23), virus-like-particles (VLPs) vaccines (24-27), reverse-genetic vaccines (28-30), and vectored vaccines (31-44) (Figure 1).
We summarize in this review the current vaccines available against PPRV in the field as well as the development and generation of new vaccination strategies, focusing on the potential use of adenovirus as vaccine platform against this pathogen.
PPRV CHARACTERISTICS
PPRV is an RNA virus classified into the order Mononegavirales, Paramyxoviridae family and genus Morbillivirus. It is a polymorphic enveloped negative single strand RNA virus with two external glycoproteins decorating the envelope, the fusion protein (F) and the hemagglutinin (H) (45). The viral particle size ranges from 400 to 500 nm (45). The non-segmented RNA molecule is packaged in a ribonucleoprotein complex (RNP) inside the envelope with the nucleoprotein (N), the phosphoprotein (P), and the RNA polymerase (L) (Figure 2A). The RNP adopts a helical structure and a single particle can incorporate more than one RNP, thus making PPRV polyploid (46). Associated with the inner surface of the plasma membrane and the cytoplasmic tails of F and H glycoproteins is the matrix protein (M). In addition to the six structural proteins mentioned (F, H, N, P, L, M), the single-stranded RNA molecule encodes two non-structural proteins, termed C and V, as well as a putative protein W ( Figure 2B).
PPRV is genetically grouped into four distinct lineages (I, II, III, and IV) on the basis of partial sequence analysis of fusion protein (F) gene or nucleoprotein (N), but only one serotype exists (47)(48)(49). Although there is cross-protection between lineages, it is interesting to classify them for control and epidemiological studies, thus allowing source tracing of outbreaks. All PPRV strains present in Asia belong to the genetic lineage IV (50), which was reported for the first time in Africa, during the Morocco outbreak in 2008 (51). All four lineages are prevalent in Africa. Lineages I and II have been reported in West Africa, whereas lineage III has been found in eastern Africa, Arabian Peninsula and southern India (48).
CURRENT VACCINES AGAINST PPRV IN THE FIELD
Live Attenuated Vaccines PPRV strains obtained from different circulating lineages and attenuated by several passages in tissue culture have been traditionally used in Africa, the Middle East and many countries in Asia as vaccines against PPR. Gilbert and Monnier were the first to adapt the virus to cell cultures, performing serial passages (12) in sheep embryo kidney epithelial cells and using for their first passage blood extracts from animals infected with PPRV (52). The Nigeria 75/1 virus of lineage II, together with the Sungri 96 of lineage IV, obtained after 63 or 75 successive passages in the Vero cell line, respectively, are the most frequently used commercial attenuated vaccines for PPRV in endemic countries (53)(54)(55)(56). The two other live attenuated vaccines currently available are Coimbatore 97 and Arasur 87, which are restricted to India. For a long time, the generation of a humoral immune response by a vaccine candidate was considered sufficient and the most important correlate of vaccination with protection. For some years now, the generation of a cellular response to PPRV has been considered almost as essential as the humoral one. Curiously, goats vaccinated with Nigeria 75/1 develop a greater antibody response than those vaccinated with Sungri 96. Conversely, Sungri 96 vaccination was more efficient at activating a cellular response with increase IFN-γ production and lymphocyte proliferation against PPRV and a higher number of CD4+ T lymphocytes (57). In spite of these differences in cellular and humoral immune responses, both vaccine strains are capable of protecting equally well-against PPRV challenge from the 4 different lineages (57).
These live attenuated vaccines generate a long-lasting immunity to PPRV that lasts for at least 3 years postvaccination (58,59). Thus, protection against the circulating lineage and cross-protection between lineages is obtained with these vaccines. The protection is based on potent humoral and cellular immune responses. However, these vaccines do not allow to differentiate vaccinated from infected animals and thus they are not DIVA vaccines. Moreover, although it is a rare event, virus reversion from attenuated to virulent can occur, which could potentially cause an outbreak. Therefore, from the point of view of epidemiological control and surveillance, live attenuated vaccines are not the most desirable despite their effectiveness, particularly for epizootic outbreaks. Additionally, the effectivity of these vaccines depends on the cold-chain preservation. Therefore, great efforts have placed into developing alternative to live attenuated vaccines to fulfill the need for DIVA and thermotolerant vaccines for PPRV.
Inactivated Vaccines
Inactivated virus vaccines offer the advantage of increased safety that comes nonetheless at the cost of loss of immunogenicity. As a result, addition of adjuvant is usually required for these inactivated vaccine formulations. In non-endemic regions inactivated vaccine is often preferred as it eliminates the risk of reversion and disease spreading of the live attenuated vaccines. An inactivated PPRV vaccine has been described based on the lineage IV Moroccan PPRV strain M/08 that was isolated from a deceased goat during the 2008 PPRV outbreak on Morocco (60). Inoculation of this binary ethyleneimine inactivated virus was safe in rats and goats and induced humoral responses (60). A transient seroconversion at day 9 until day 30 post first immunization was induced in goats, that after a booster immunization (day 36) was converted into a robust and persistent seroconversion with PPRV neutralizing antibody responses until, at least, day 110 post-booster (60,61). This inactivated PPRV vaccine protects the natural host against homologous virus challenge (61). As often is the case with inactivated virus vaccines, adjuvant addition was necessary to boost the immunogenicity of the formulation (60) (Supplementary Table 1). There are yet no data on the cellular immune response induced by this inactivated vaccine. Nevertheless, this approach remains attractive and is more readily acceptable for veterinary authorities particularly in nonendemic regions.
DNA Vaccines
DNA vaccines are often thought of as an alternative to conventional vaccines due to their relative ease of production and their stability at room temperature. A PPRV DNA vaccine candidate based on a Semliki Forest virus replicon expressing the PPRV-F or -H genes has been shown to induce cellular and humoral responses in a murine model (62,63). However, the immunogenicity of these constructs has however yet to be tested in the natural host. DNA vaccination using plasmids that express the anti-idiotypic determinants of PPRV-H protein as an antigen mimic has also been employed to induce immunity to PPRV (19,64). This DNA vaccination regime elicited cellular and neutralizing antibody responses in sheep although protection has to be addressed (19) (see Supplementary Table 1).
Recombinant Subunit Vaccines
Another approach to vaccination is the use of recombinant systems to express an antigenic viral protein that will be formulated for vaccination. Insect baculoviruses have been used as a display system for PPRV immunogenic proteins. Inoculation of recombinant baculovirus expressing PPRV-H in the envelope induces cellular immunity and PPRV neutralizing antibodies in goats (65). A recombinant Bombyx mori nucleopolyhedrovirus expressing the PPRV-F and RPV-H proteins was capable of eliciting neutralizing antibodies to both viruses when inoculated in mice (22). Other recombinant protein expression systems have also been studied. For instance, an alternative immunization strategy using recombinant PPRV-H protein expressed in transgenic peanut plants and fed orally to sheep produced anti-PPRV neutralizing antibodies and specific T cell responses to PPRV-H protein in sheep (23). Overall, although these PPRV antigen delivery systems can elicit immunity to the virus, their potency as vaccine has yet to be established (Summarized in Supplementary Table 1).
Virus-Like-Particles (VLPs) Vaccines
Another attractive strategy for vaccine design consists in immunizations with virus-like particles, i.e., providing the capsid antigens of the virus to the immune system for recognition without the viral genetic material. These vaccination systems are deemed extremely safe, as viral replication cannot occur in the absence of genetic material, but they often require the addition of an adjuvant to boost immunogenicity. PPRV VLP production has been described using different expression systems such as insect cells or mammalian Vero cells (25, 26). It appears that the matrix protein M is critical to the formation of PPRV VLPs (25), whereas inclusion of major neutralizing antibody determinants like the PPRV-H protein likely promote the immunogenicity of these VLPs. Some of these VLP constructs have proved to be immunogenic in mice eliciting humoral immunity (24). Immunogenicity in goats of PPRV VLPs has also been confirmed (26,66). In these studies VLP vaccination elicited neutralizing antibodies and cellular immune responses even in the absence of adjuvant in some reports (26). Recently, a PPRV VLP based on the virulent lineage IV Tibet/30 isolate was shown to induce stronger immune responses in goats and sheep than VLPs produced from the vaccine strain Nigeria 75/1 (27) indicating that this strategy could be applied to new virulent isolates. Overall, PPRV VLPs could be a promising vaccine candidate in spite of the likely necessity to supplement the formulation with adjuvant to boost immunogenicity. These VLPs have also the potential to be DIVA vaccines as they only express some of the viral gene products. Further studies are nonetheless required to demonstrate their protective efficacy against virulent PPRV challenge. Comparative VLPs vaccination details are summarized in Supplementary Table 1.
Reverse-Genetics-PPRV
Reverse genetic has been an important advance in virology. Through genetic engineering, this technique makes it possible to entirely obtain a recombinant virus from full-length complementary DNA copies (cDNA) of the viral RNA genome. Reverse genetic systems have provided the vaccine field with a powerful technology to generate, with a more rational approach, different types of vaccines that can be positively or negatively marked. Despite PPRV, like all morbillivirus, being an easy candidate and target for this technique, and having a minigenome described since 2007 (67), it was not possible to recover a recombinant PPRV based on reverse genetics until 2012 (28). Hu et al. generated a stable recombinant GFPexpressing PPRV virus that allowed for the development of high throughput fluorescence-based seroneutralization tests (28). Moreover, this genetic mark introduced into the vaccine makes it possible to differentiate between infected and vaccinated animals. Some years later, the PPRV vaccine strain Nigeria 75/1 was also modified by reverse genetics to eliminate a B cell epitope from PPRV-H protein that is recognized by a monoclonal antibody used in anti-H ELISA to attempt to produce a DIVA live-attenuated vaccine (30). The genetically modified recombinant vaccine strain showed similar vaccination potency as the PPRV Nigeria 75/1 vaccine both in terms of induction of humoral immunity and in providing protection against virulent PPRV challenge in goats. However, the mutations introduced in PPRV-H to avoid antibody recognition were not sufficient to differentiate infected from vaccinated goats using an anti-H ELISA kit (30). Reverse genetics can also be used to generate multivalent vaccines by expressing antigens from other diseases. Using the PPRV Nigeria 75/1 vaccine strain backbone, a recombinant PPRV vaccine expressing the VP1 structural protein from foot and mouth disease virus (FMDV) has been developed (29). This recombinant vaccine was capable of inducing neutralizing antibodies against both PPRV and FMDV. Moreover, it was able to provide partial protection against FMDV infection (29), indicating that this molecular strategy has the potential to develop bivalent vaccines for ruminant Frontiers in Veterinary Science | www.frontiersin.org diseases. Using reverse genetics, live attenuated PPRV vaccines could also be modified to remove additional virulence factors to further improve their safety. Reverse genetics recombinant PPRV vaccines share same advantages and disadvantages with live attenuated vaccines, i.e., they are likely to provide long-term PPRV immunity but still present the risk of reversion.
Vector Vaccines for PPRV
One of the most promising approaches to generate DIVA vaccines for PPRV is the use of recombinant viral vectors that express PPRV immunogenic proteins. Research aimed at generating vaccines based on different viral vectors is abundant. The H and F glycoproteins are the principal targets of neutralizing antibodies of the humoral immune response. These two glycoproteins have been considered the best to include in vaccine candidates and their genes have been cloned and expressed in several recombinant, replication-defective viral vectors: (i) Poxviruses (39,40), (ii), Bovine-Herpes viruses (43), (iii) Newcastle disease virus (44), and (iv) Adenoviruses (33, 34, 36, 37, 68).
Another strategy is to generate multivalent vaccines, i.e., a vaccine that can protect against several diseases. Some of the viral vectors that have been chosen as carriers of the PPRV H and /or F genes come from viruses that are also causative of disease in animals. For this reason, bivalent vaccines are desirable so that they generate an immune response in sheep or goats capable of protecting against PPRV and at the same time against the disease caused by the virus onto which the vaccine vector is based.
Poxvirus Vectors
Poxviral vectors can harbor large DNA inserts; do not integrate into the host genome due to their cytoplasmic replication, and importantly, induce cellular and humoral immunity to the inserted transgene. These characteristics have boosted the interest for these vectors in vaccinology (69,70). Several vectors derived from this family have been used for PPRV vaccination.
Vaccinia Vectors
Vaccinia vectors were the first recombinant vectors to show efficacy in vaccination against PPRV. Jones et al. showed in 1993 that a vaccinia virus vector based on the Wyeth strain and made to express the proteins F and H from RPV could protect goats against virulent PPRV challenge (71). Interestingly this protection occurred in spite of the vaccination failing to trigger detectable levels of neutralizing antibodies to PPRV. In a different study, goats were protected against a virulent Indian PPRV strain challenge when vaccinated with recombinant attenuated Modified Vaccinia Ankara (MVA) viruses expressing F or H PPRV genes (31). In this case vaccination correlated with neutralizing antibody induction. Cellular immunity induced by these MVA vaccines was not assessed in the study.
Fowl Pox Vectors
Fowl pox vectors expressing PPRV F or H protein have also been generated (37). Avian poxviruses present the safety advantage of being unable to replicate in mammalian cells, but are still able to infect and express the transgene of interest. Vaccination with these recombinant vectors triggered cellular immunity to PPRV-F or -H, but failed to induce significant neutralizing antibody levels (37).
Capripox Vectors
Capripoxvirus is the causal agents of the contagious diseases goatpox and sheeppox, the distribution of which often overlaps with regions where PPRV is endemic. Using attenuated capripox virus vaccine strains as the recombinant vector basis, bivalent vaccines that elicit protection against goatpox/sheeppox and PPRV have been developed. Introduction of H or F genes from PPRV conferred protective immunity against PPRV in goats (39,40,42). Indeed, even the expression of H or F genes from RPV conferred protection against PPRV infection in goats using this system (38). Some studies, nonetheless, highlighted a possible shortfall of this bivalent strategy: existing immunity to the capripoxvirus vector could limit the immunity induced to the expressed PPRV gene. Caufour et al., found that animals with pre-existing immunity to capripoxvirus developed only partial protection against PPRV (41). This was however not observed in another study indicating that this drawback can probably be overcome with a booster vaccination (42). Overall, poxvirusbased vaccine strategies appear promising. These vectors have often been described to work better in heterologous prime-boost strategies (with other recombinant vectors or with DNA for instance). These approaches have yet to be evaluated for PPRV.
Bovine-Herpes Viruses
Bovine Herpesvirus-4 (BoHV-4) can replicate in a broad range of host species, but only produces subclinical infections in cattle (72). Recombinant BoHV-4 vectors can also induce potent host immune responses (73), while generating low levels of neutralizing antibodies against the vector (74). A recombinant BoHV-4 expressing the PPRV H protein from Nigeria 75/1 strain induced a potent humoral and cellular immune response in mice (43), as well as in sheep, conferring protection against an heterologous virulent PPRV challenge (75). This recombinant vaccine could represent an attractive platform for PPRV vaccination.
Newcastle Disease Virus (NDV)
Recently a recombinant Newcastle disease virus expressing PPRV H protein was shown to induce protective immunity against virulent PPRV challenge in goats (44). NDV is a Paramyxovirus that produces diseases in poultry, but attenuated strains are used as vaccine. These viruses have a broad spectrum of infectivity but their replication is limited in mammalian host cells, thus, raising their safety profile when used as a recombinant vector in mammals. Dual injection of NDV expressing PPRV-H induced a similar degree of protection against challenge as vaccination with the conventional Nigeria 75/1 vaccine (44), indicating that NDV-based vector could be useful for PPRV control.
Adenovirus Vectors: the Vector of Choice for a PPRV DIVA Vaccine?
Vaccine developments against the SARS-CoV2 pandemic have put adenoviral vectors at the forefront for vaccine design. This is clearly exemplified by the approval by health authorities of several vaccine formulations against Covid-19 based on adenoviral vectors (76). Vaccination with adenoviral vectors expressing the immunogenic proteins F or H from PPRV have also shown promising results in protection studies in the natural hosts of PPR (35-37). In this section of the review we will describe in more details adenovirus vectors and discuss their advantages and limitations for vaccine development focusing on their veterinary use and more precisely on PPRV.
TYPES OF ADENOVIRUS VECTORS
Adenoviruses are 35-40 kb dsDNA genome, non-enveloped viruses (Figure 3). Infection with adenoviruses usually provokes common flu-like disease in humans and animals, during which the neutralizing antibody response controls the infection. They have demonstrated to be excellent candidates as vaccine delivery vehicles (77). Particles can be engineered as replicating or as defective in replication. They are safe, genetically stable and manufacturable in high amounts (78,79).
The type of adenoviral vectors that have been engineered can be summarized into three categories depending on the amount of adenovirus genome deleted to allow insertion of transgenes ( Figure 3B). The first generation of adenoviral vectors, in which the area of the adenoviral genome corresponding to E1 and E3 regions are deleted ( E1)( E3), renders them replicationdefective, thus able to infect host cells but unable to replicate ( E1) (80,81). Furthermore, since the E3 genes have been related to adenovirus immune evasion mechanisms, the deletion of the E3 region improves the immune response to the adenovirus (82,83). The second generation incorporates additional deletions or inactivated zones in the adenoviral genome, corresponding to E2 and E4 regions that code for proteins involved in viral replication in target cells (84)(85)(86), increasing vector safety by avoiding the generation of replication competent adenovirus by recombination (87). This comes also at the cost of diminishing vector immunogenicity (83,88). Finally, the third adenoviral vector generation eliminates the entire adenoviral genome except the ITRs and the packaging signal. These are called "gutless" or helper dependent vectors (89)(90)(91)(92). The "gutless" adenoviruses allow the immune response to be directed mainly against the transgene instead of the vector, but also causes a decrease in the adjuvant effect provided by the adenoviral vector itself. The second and third adenoviral generation vectors are more difficult to produce in high amounts.
The increase in deleted adenoviral genome regions increases the acceptance size of heterologous genes, from 4.5 kb for the first generation, to 10 kb for the second generation, and finally to 36 kb for the third generation. This provides the adenoviral vectors with the features of vehicle for delivery of antigens by expressing the transgenes in the target cells. Transgene expression is transitory and lasts for 2-3 weeks, as proved in different animal and human models. This generates a strong immune response consisting of CD4+ and CD8+ T cell directed against the transgene and also against adenoviral antigens (except in "gutless" vectors) (93-100). Replication-defective adenoviruses need to be obtained through transfection of HEK293 or Per.C cells which provide in trans the E1 and/or E2 and E4 functions . The proteins codified for the different transcripts are specified in the blue boxes for early genes and in orange circles for late genes. ψ, is the packaging signal; ITR, Internal terminal repeats; ∆, Deletions for the constructions of the different adenoviral vectors; first (∆E1 and ∆E3) and second generation (∆E1 and ∆E3 plus ∆E2 and ∆E4). In the case of "gutless" adenoviral vectors, the ITRs and the packaging signal (ψ) are the only adenoviral genome parts that remain. (101)(102)(103)(104)(105). The "gutless" vector additionally requires the presence of a helper adenovirus, rendering the production system more complex. For the second and third generation vectors, the complementation is not as efficient as desired in the producer cell lines (85,106), reducing the yields obtained. Although they have benefits, such as less immunogenicity and less cellular toxicity (107)(108)(109)(110)(111). Recombinant first generation adenoviruses are thermotolerant and not difficult to produce in large quantities and thus, they can be easily transported without losing their immunogenicity to endemic areas of PPRV, which coincide with hot climate areas in the world (Africa and Asia).
Adenoviruses can be engineered to be replication incompetent or to remain competent in replication, expressing in both cases a foreign gene. Both of them present advantages and disadvantages. Replication incompetent adenoviruses are elected primarily as vaccine candidates because the majority of the immune response they trigger is targeted to the transgene they expressed. The adenoviral protein expression is limited as it is overtaken by the transgene expression (112). They are also safer, as they cannot replicate and thus spread to a different host. In contrast, the competent replication adenovirus vectors enhance the immune response (113,114) against both the vector and the transgene, which could limit the vaccine efficacy due to vector neutralization. They can also cause serious issues in immunosuppressed individuals due to possible vector-derived pathologies, as well as escape of potentially virulent revertant viruses. For these reasons, it is more difficult to obtain approval by competent authorities to bring the replication-competent vectors to the market. However, in the veterinary field these replication competent vectors could have some applications. For instance, a competent replicative adenoviral vector expressing the rabies virus glycoprotein has been successfully delivered to wildlife through baiting for rabies control campaigns in Canada (115). This vaccine has proved to be safe in a number of species with minimal risk of horizontal transmission (116).
ADENOVIRAL VECTOR: IMMUNITY IN RESPONSE TO VIRAL VECTORS
Adenoviral vectors, like most viruses, display different pathogen associated molecular patterns (PAMPs) that are detected by cellular sensors called pathogen recognition receptors (PRRs). Typically, the activation of PRRs leads the activation of signaling cascades that induce the expression of type I IFN and proinflammatory cytokines (Figure 4). These first steps from the innate immune response allow the recruitment of different innate immune cells to the site of infection which in turn help to trigger a successful adaptive immune response (117)(118)(119)(120).
Innate Immunity to Adenovirus Vector: The Adjuvancy Effect
Besides the adaptive immunity to the transgene that is sought with recombinant viral vector vaccines, the innate host immune responses to the viral vector can enhance the immunogenicity of the insert. Indeed, innate immune recognition of the adenoviral vector itself and its own products probably provides an adjuvancy effect. Rapid physiological responses induced by systemic adenoviral vector delivery trigger the activation of innate immunity, with induction of cytokines, inflammation, transient liver toxicity and thrombocytopenia (121)(122)(123). Adenoviruses enter the cytoplasm of the cells through different receptors, such as the Coxackie adenovirus receptor (CAR), CD46, sialic acid, integrin ανβ5 heparin sulfate proteoglycans, etc..., depending on virus species (124)(125)(126)(127)(128). This entry process activates different pathways from the innate response involving toll-like receptors (TLRs), lectin receptors (LRs), autophagy, IFNs signaling, inflammasome signaling through AIM2-like receptors (ALRs), nucleotide-binding oligomerization domain (NOD)-like receptors (NLRs), and RIG-I receptors (RLRs) (Figure 4).
IFNs and anti-inflammatory cytokines activated via TLRsdependent and -independent pathways constitute the typical innate immune response to adenoviruses (129,130). TLR-2 and TLR-9 have been identified as the main activation pathways responsible for adenoviral recognition in vivo, leading to the production of some cytokines, such as MCP-1 and RANTES or IL6 in macrophages (130)(131)(132)(133). In TLR2 and TLR9 -deficient mouse models, inoculation of recombinant adenovirus vector results in reduced NF-kB activation, decreased neutralization Abs production against both the adenoviral vector and the transgene, and a reduction in pro-inflammatory response and IFNα levels. The immune response to the adenovirus vector was however not completely abolished in these murine models (131), suggesting that other TLR-independent pathways are also implicated in the activation of the innate immune response against adenoviruses (134)(135)(136) (Figure 4). The IL-1α, activated through the interaction between the RGD motif of adenoviral penton base protein and β3 integrins (137) during the viral entrance is one of them. IL-1α plays an important role in inflammatory adenoviral process, which diminished in mice treated with anti-ILα antibodies and in IL1R −/− mice (138).
The innate immune response induced by adenoviruses involves different receptors within huge network pathways. The adenovirus serotype, the DNA incorporated and the infection milieu determine the host response and the efficacy of the vaccination based on adenoviral-based vectors. Generally, in the case of using these vectors as antigen delivery vehicle, the innate immune response induced by the adenovirus backbone, that in other applications such as gene therapies could be The endocytosis process concludes with a vesicle in the cytoplasm that contains the virus inside, which will be sent to the endosome. The outer capsid of the virus disassembles with the acidification of the endosome, releasing the DNA-protein core. The viral core is liberated to the cytosol when the viral shedded spikes breach the endosomal membrane. The viral core then traffics to the nuclear pore where the genetic material is released and gene expression occurs. Systemic delivery of adenoviral vectors activates innate immune responses with secretion of pro-inflammatory cytokines and type I IFNs, through recognition of viral motifs by pattern recognition receptors (PRR). Several PRRs are involved in adenovirus recognition among these are included Toll-like receptors 2 and 9 (TLR2, TLR9), cyclic GMP-AMP synthase (cGAS) and retinoic-acid inducible gene-I (RIG-I). Adenovirus CpG DNA can be detected in the endosomes by TLR9 [that signals through myeloid differentiation primary response protein 88 (MyD88)]. Adenovirus dsDNA can be detected in the cytoplasm by the DNA sensor cGAS [that activates stimulator of IFN genes (STING)], while RNA sensor RIG-I can recognize adenovirus-associated RNAs. Recognition by PRRs triggers multiple signaling cascades (through inhibitor of nuclear factor (NF)-κB kinase ε (IKKε) and TANK binding kinase 1 (TBK-1) among others) that leads to the activation of transcription factors such as activator protein 1 (AP-1), IFN regulatory factor (IRF) 3, IRF7 or NF-κB, that promote the production of type I IFNs and pro-inflammatory cytokines. These factors promote the adjuvancy effect of the viral vector. As a result of the successive expression of exogenous and adenoviral backbone genes in the target cells, the adaptive immune response to the transgene can thus be triggered. disadvantageous, becomes a vaccine adjuvant that helps activate an efficient transgene-specific adaptive immune response.
Adaptive Immunity to Adenovirus Vector: A Limitation to Their Efficacy?
The adenoviral vector induces humoral and adaptive cellular immune responses. Humoral responses are mediated by neutralizing antibodies (nAb) directed against different epitopes in the hexon, fiber and penton adenoviral capsid proteins (148). The nAbs are mainly serotype-specific with no or minimal cross-neutralization capacity to other adenoviral serotypes. The serotype-specificity is mainly due to the high variability of epitopes in the hyper variable region (HVR) of the hexon protein and fiber knob among serotypes. In the cellular adaptive immune response against adenoviral vectors in humans, CD4+Th1 and CD8+ T cells against several structural adenoviral proteins have been detected. Dendritic cell (DC) infection also appears to play an important role in mounting adaptive immunity to the transgene (149). Indeed, DC infection and subsequent antigen presentation in lymph nodes is critical to establish CD8+ T cells responses.
Innate and adaptive immune response activation appears to be dose-dependent. The levels, amounts and duration of the transgene expression determine the immune responses. High transgene expression correlates with high antigen-specific cellular response (150). High and persistent transgene levels through human-or chimpanzee-adenoviral vectors in mice induce strong T cell responses but low innate immunity activation. By contrast, less potent T cell response is induced with a low transgene expression that induces a high innate immunity (150).
The magnitude of the immune response triggered by the adenoviral vector is important for the success of vaccination. The ideal situation is to induce an adjuvant response that is sufficient to mount an adaptive response against the transgene. Thus, a high transgene expression as well as high immunogenicity of the transgene product is also required to bias the adaptive immune response toward the antigen expressed by the adenoviral vector.
ADENOVIRAL VECTOR CHOICE TO OVERCOME PRE-EXISTING IMMUNITY
A crucial aspect in the field of recombinant adenoviral vectors is the choice of the adenovirus type. There are more than 60 serotypes in the Adenoviridae virus family. Many animal (sheep, cattle, swine, dogs, and monkeys) and human adenoviruses belong to the genus Mastadenovirus, one of the five genera (the others being Siadenovirus, Aviadenovirus, Ichtadenovirus, and Atadenovirus) included in the Adenoviridae family.
The adenoviral vectors most frequently used are based on human adenovirus type 5 (HAd5) and type 2 (HAd2). Their biology is well-understood and there are many commercial tools that facilitate their manipulation. One of the most important aspects to consider when choosing the virus-vector on which to base a potential vaccine is the pre-existing immunity against this virus in the host. In humans, Adenovirus serotypes 2 and 5 are the most prevalent (82%), but are nevertheless the most frequently used for vector development for clinical use. Due to the high seroprevalence of these serotypes, approaches have been developed to overcome the pre-existing immunity in humans to the backbone vector used.
The pre-existent humoral and cellular immunity against the vector reduce the expression-time of the transgene, and thereby its immunogenicity (112) due to the presence of vectorspecific neutralizing antibodies in the host. After adenoviral vector administration, most of the neutralizing antibodies induced are directed against the hyper-variable loops of the viral hexon protein. Antibodies directed against conserved regions of the viral particle and capable of cross-reacting with different serotypes are also generated (151). Interestingly, existence of pre-existing immunity to the vector is not always detrimental, for instance the induction of transgene-specific CD8+T cell after a passive antibody transfer was improved in presence of pre-existing antibodies (152). Nevertheless, as preexisting immunity remains a major throwback to the adenoviral vector development, different strategies have been developed to overcome it. The generation of chimeric adenoviral vector, replacing the HuAd5-HVR hexon sequences with the HVR from a different serotype, for instance, is one of these strategies employed to overcome pre-immunity in humans (148,(153)(154)(155)(156)(157). The most common strategy used to avoid seroprevalence problems is to vaccinate with a different adenoviral serotype from the seroprevalent one found in the host. Adenoviruses have broad tissue tropism but productive human infection with non-human adenoviruses is uncommon. Animal-derived adenoviruses can nonetheless infect certain human cell types and inversely, human adenoviruses are able to infect different animal organs. These cross-species infectivity characteristics of adenoviral vectors have been used for gene therapy and vaccine development. In human, different chimpanzee adenovirus have been developed as viral vectors to replace the classical HuAd5 as adenoviral vector (158)(159)(160)(161)(162)(163).
In animal health, the use of human adenovirus vectors can be advantageous as animals should not have immunity to these vectors. Indeed, a human adenovirus-based vector vaccine for FMDV has been approved for cases of emergency by the FDA (164). ) were efficiently protected against a virulent PPRV challenge after vaccination with adenoviral-based vaccine to PPRV. Moreover, these potential vaccines overcame the known T cell immunosuppression induced by PPRV during the first days of infection (35). Analysis of immune correlates with protection has shown that immunization with adenovirus vectors expressing PPRV protein F or H can elicit cellular immunity to epitopes generated during PPRV infections (166). These vaccines also elicit neutralizing antibodies (33, 35-37, 165), although neutralizing antibody titers typically increased after virulent PPRV challenge (35-37). Thus, adenoviral vector immunization can prime the humoral and cellular response against PPRV in a manner that allows recognition of the pathogen when exposure occurs. Importantly, Herbert et al. also showed that a single immunization with HuAd5 expressing PPRV-H could protect goats from virulent PPRV challenge 15 weeks after immunization, suggesting that immunization with these vectors generates memory immune responses (37). Work to establish the duration of the memory responses induced by these vaccination strategies will need to be performed to further characterize the protective potential of these formulations.
ADENOVIRUS-BASED VACCINATION FOR PPRV
Another aspect of immunization with recombinant adenoviruses is to choose the optimal antigen that induces protection. Wang et al. reported a slightly stronger cellmediated immune responses and VNT titers with an adenoviral construct expressing an F-H fusion protein than with adenoviral vectors expressing either of these proteins on their own (165). Vaccination with adenoviruses expressing F or H, or a combination of both vectors appear nonetheless to produce similar levels of protection (35-37). Delivery of both immunodominant immunogen F and H is nevertheless likely to provide a broader spectrum of protection. Larger study groups will be necessary to fully evaluate the most appropriate combination of antigens that induce protective immunity.
Overall, PPRV vaccinations based on adenoviral vectors have the potential to offer a DIVA vaccine solution to the field and help in disease eradication.
FUTURE PERSPECTIVES
The protective capacity in the natural PPRV hosts of adenovirusbased vaccines is now well-established in laboratory experiments. Adenoviruses are thermotolerant and induce potent immunity to the transgene. One of the main focuses for future research should be to establish DIVA diagnostic tests to accompany the adenovirus-based vaccines. This will help in the control and spread of the disease, but will also make these vaccines more desirable in non-endemic regions which are threatened by PPRV outbreaks. A comparative view of the experimental PPRV vaccines published in the area has been summarized in Supplementary Table 1, showing at a glance the advantages and disadvantages of these approaches. Vaccination with adenovirusbased vaccines represents a promising approach that could help combat this disease. Work nonetheless remains to be done to demonstrate efficacy in the farms and to establish the adequate protocol to provide protection to small ruminants from spreading the disease. Collaboration between the academic and the pharmaceutical sectors should be potentiated by institutions to bring to the field the advances made in PPRV vaccination based on adenoviral vectors.
AUTHOR CONTRIBUTIONS
JR and VM mainly wrote the manuscript. JR, NS, and VM wrote and reviewed the manuscript. All authors contributed to the article and approved the submitted version. | 2021-09-08T13:31:20.752Z | 2021-09-08T00:00:00.000 | {
"year": 2021,
"sha1": "029403cfb54b2c8e6e3ca174f709f9178a165930",
"oa_license": "CCBY",
"oa_url": "https://www.frontiersin.org/articles/10.3389/fvets.2021.729879/pdf",
"oa_status": "GOLD",
"pdf_src": "PubMedCentral",
"pdf_hash": "029403cfb54b2c8e6e3ca174f709f9178a165930",
"s2fieldsofstudy": [
"Medicine",
"Agricultural and Food Sciences"
],
"extfieldsofstudy": [
"Medicine"
]
} |
119413621 | pes2o/s2orc | v3-fos-license | Resonant parameters of the $Y(4220)$
The vector charmoniumlike state $Y(4220)$ was reported recently in the cross sections of $e^+e^-\to \omega \chi_{c0}$, $\pi^+\pi^-h_c$, $\pi^+\pi^- J/\psi$, and $D^0 D^{*-}\pi^+ + c.c.$ measured by the BESIII experiment. A combined fit is performed to the cross sections of these four final states to extract the resonant parameters of the $Y(4220)$. We determine a mass $M=(4219.6\pm 3.3\pm 5.1)$~MeV/$c^2$ and a total width $\Gamma=(56.0\pm 3.6\pm 6.9)$~MeV for the $Y(4220)$, where the first uncertainties are statistical and the second ones systematic. Assuming the $Y(4220)$ decays dominantly to the above four modes and their isospin symmetric modes, we also estimate its leptonic decay width and decay branching fractions. These information is essential for the understanding of the nature of this state.
I. INTRODUCTION
These Y states were observed at B factories with limited statistics since they are produced from initial state radiation processes with data collected at around 10.6 GeV in the bottomonium energy region [4,7,8]. The high precision cross section measurements and the study of these states in different final states in direct e + e − annihilation in the charmonium energy region from the BESIII experiment supply new insight into their properties.
In 2013, BESIII reported the cross section measurement of e + e − → π + π − h c at 13 centerof-mass (c.m.) energies from 3.9 to 4.2 GeV and found the magnitude of the cross sections is at the same order as that of e + e − → π + π − J/ψ but with a different line shape. Although no quantitative results were given, the resonant structure at around 4.22 GeV/c 2 is obvious [9]. A combined fit to the BESIII data together with the CLEO-c measurement at 4.17 GeV [10] results in a resonant structure, Y (4220), with a mass of (4216 ± 18) MeV/c 2 and a width of (39 ± 32) MeV [11], different from any of the known Y and excited ψ states in this mass region [12].
In 2014, BESIII reported the cross section measurement of e + e − → ωχ c0 at 9 c.m. energies from 4.21 to 4.42 GeV. By assuming the ωχ c0 signals come from a single resonance, BESIII reported a resonant structure with the mass and width of (4230 ± 8 ± 6) MeV/c 2 and (38 ± 12 ± 2) MeV, respectively, and the statistical significance is more than 9σ [13]. This structure is in good agreement with the Y (4220) observed in e + e − → π + π − h c [11], and combined fits assuming the structures at 4.22 GeV/c 2 are the same have been tried by the authors of Refs. [14,15].
BESIII updated the measurements with higher energy data up to 4.6 GeV included, in both e + e − → π + π − h c [16] and ωχ c0 [17] processes. In addition, more data points are added even at low energy, although with low integrated luminosity, to further constrain the line shape in e + e − → π + π − h c [16] process. While the structure in ωχ c0 mode was affected only slightly with the new measurements at high energies [17], in the e + e − → π + π − h c mode, the Y (4220) was observed with improved significance together with a new structure, the Y (4390). The resonant parameters are M = (4218.4±4.0±0.9) MeV/c 2 and Γ = (66.0±9.0±0.4) MeV for the Y (4220), and M = (4391.6 ± 6.3 ± 1.0) MeV/c 2 and Γ = (139.5 ± 16.1 ± 0.6) MeV for the Y (4390) [16]. The updated cross sections of e + e − → ωχ c0 and π + π − h c are shown in Fig. 1, where the measurements at energy points both with integrated luminosities larger than 40 pb −1 (referred to as 'XY Z data sample' hereafter) and with integrated luminosities smaller than 20 pb −1 (referred to as 'R-scan data sample' hereafter) are presented. The measured cross sections of e + e − → ωχ c0 , π + π − h c , π + π − J/ψ, and D 0 D * − π + + c.c. by the BESIII experiment. The dots are from the XY Z data sample and the triangles are from the R-scan data sample. The error bars are the sum in quadrature of the statistical and uncommon systematic errors. Here, for each process the correlated systematic uncertainties (13.3%, 14.8%, 5.8%, and 4.6% for ωχ c0 , π + π − h c , π + π − J/ψ, and D 0 D * − π + + c.c., respectively) are not shown.
The process e + e − → π + π − J/ψ at c.m. energies up to 5.0 GeV was first studied by the BABAR experiment, where the Y (4260) was observed [4]. Belle measured the cross sections of e + e − → π + π − J/ψ at c.m. energies between 3.8 and 5.0 GeV and reported that Y (4260) alone cannot describe the line shape satisfactorily [18]. Improved measurements with both BABAR [19] and Belle [5] full data samples confirmed the existence of non-Y (4260) component in e + e − → π + π − J/ψ but the line shape was parametrized with different models. Recently, BESIII reported a precise measurement of e + e − → π + π − J/ψ cross sections at c.m. energies from 3.77 to 4.60 GeV (as shown in Fig. 1) using data samples with an integrated luminosity of 9 fb −1 [20]. While the nature of the events at around 4 GeV is still ambiguous, the dominant resonant structure, the so called Y (4260), was found to have a mass of (4222.0±3.1±1.4) MeV/c 2 and a width of (44.1±4.3±2.0) MeV, in good agreement with the Y (4220) observed in e + e − → π + π − h c [16]. In addition, a new resonance with a mass of around 4.32 GeV/c 2 is needed to describe the high precision data.
BESIII also reported a measurement of the e + e − → D 0 D * − π + + c.c. cross sections at c.m. energies from 4.05 to 4.60 GeV with the same data samples [21], which is a significant improvement over the previous measurement at Belle [22]. Two resonant structures in good agreement with the Y (4220) and Y (4390) observed in π + π − h c [16] are identified over a smoothly increasing non-resonant term which can be parametrized with a three-body phase space amplitude. The cross sections of D 0 D * − π + + c.c. are also shown in Fig. 1.
An obvious feature in the above four channels from the BESIII measurements is that there is a common structure at around 4.22 GeV/c 2 , i.e., the Y (4220). As such a state is not observed in other open charm final states [23], these four final states are probably the dominant decay modes of the Y (4220). By applying constraints to the resonant parameters in a simultaneous fit to the cross sections of these four processes, we may obtain the best knowledge on the Y (4220), including resonant parameters (mass, width, coupling to lepton pair, and decay branching fractions), and thus a better understanding of its nature [1], especially whether it is an exotic state, such as a tetraquark state in the diquark-antidiquark model [24], a vector molecular state of DD 1 (2420) [25], a mixture of two hadrocharmonium states [26], an ωχ cJ molecule [27,28], or a charmonium-hybrid state [29].
II. THE DATA AND THE FIT FORMALISM
We use the measured cross sections of e + e − → ωχ c0 , π + π − h c , π + π − J/ψ, and D 0 D * − π + + c.c. processes [16,17,20,21] by BESIII experiment only to measure the parameters of the resonances presented. The data are shown in Fig. 1, where the dots with error bars are from the XY Z data sample and the triangles with error bars are from the R-scan data sample. Here, the error bars are the sum in quadrature of the statistical and uncorrelated systematic errors, and the correlated systematic uncertainties common to the energy points in each process are removed since they have no effect on the fitted resonant parameters.
We parametrize the cross section with the coherent sum of a few amplitudes, either resonance represented by a Breit-Wigner (BW) function or non-resonant production term parametrized with a phase space term. The BW function used in this article is where M is the mass of the resonance; Γ and Γ e + e − are the total width and partial width to e + e − , respectively; B f is the branching fraction of the resonance decays into final state f ; and P S n is the n−body decay phase space factor which increases smoothly from the mass threshold with the √ s [12].
We do a combined fit using a least squares method with minuit package in the CERN Program Library [30]. The χ 2 function is constructed as where σ data ij and σ f it ij are the measured and fitted cross sections of the ith energy point in the jth mode, δ ij is the corresponding total error with common systematic errors removed. The sum is performed over all the measured cross section points from the above mentioned four modes. The χ 2 is minimized to obtain the best estimation of the resonant parameters.
III. FIT RESULTS
We fit BESIII data on e + e − → ωχ c0 , π + π − h c , π + π − J/ψ, and D 0 D * − π + + c.c. cross sections simultaneously. Two solutions, four solutions, and four solutions with the same minimum values of χ 2 are found with the two, three, and three amplitudes interfering with each other for e + e − → π + π − h c , π + π − J/ψ, and D 0 D * − π + + c.c., respectively. The masses and the widths of the resonances are identical but the partial widths to e + e − and relative phases are different in different solutions for each process. There are no multiple solutions for e + e − → ωχ c0 since only one amplitude is used. Figure 2 shows the fit results with a goodness-of-the-fit of χ 2 /ndf = 241/273=0.9, where the solid curves show the projections from the best fit, the dashed curves show the fitted resonance components from different solutions, and the corresponding mass, width, and the product of the branching fraction to specific mode and the e + e − partial width for each resonance are listed in Table I.
From the fit we obtain M = (4219.6 ± 3.3) MeV/c 2 and Γ = (56.0 ± 3.6) MeV for the Y (4220) where the errors are combined statistical and uncorrelated systematic uncertainties. We can find that the resonant parameters are significantly different from those of the Y (4260) determined from low statistics experiments BABAR [19] and Belle [5], although they are obviously the same resonant structure.
IV. SYSTEMATIC ERRORS
The systematic uncertainties in the resonant parameters in the combined fit to the cross sections of e + e − → ωχ c0 , π + π − h c , π + π − J/ψ, and D 0 D * − π + + c.c. are mainly from the absolute c.m. energy measurement, the c.m. energy spread, cross section measurements, parametrization of the resonances and background shape.
The systematic uncertainties on the mass and width from the absolute c.m. energy measurement and the c.m. energy spread are taken from the original BESIII publications [16,17,20,21], where we take the largest values conservatively when they are different in different modes. The uncertainty from the cross section measurement in each mode is divided into two categories, correlated and uncorrelated systematic uncertainties. For the uncorrelated systematic uncertainties, they are added with the statistical errors in quadrature, as shown Table I. in Figs. 1 and 2, i.e., the fit errors have covered uncorrelated systematic uncertainties in the cross sections. The correlated uncertainty from the cross section measurement in each mode is common for all data points [16,17,20,21], which only affects the B × Γ e + e − measurement and is 13.3%, 14.8%, 5.8%, and 4.6% for ωχ c0 , π + π − h c , π + π − J/ψ, and D 0 D * − π + + c.c., respectively.
Instead of using a constant total width, we assume a mass dependent width to estimate the uncertainty due to signal parametrization. To model the π + π − J/ψ cross section near 4 GeV, an exponential function as used in Ref. [20] is taken instead of using the Y (4008) resonance. We consider the systematic bias introduced by possible additional resonances in the processes under study. The fit scenarios include adding an additional phase space term for ωχ c0 ; using three resonances, the Y (4220), Y (4320) and Y (4390), to fit π + π − h c , D 0 D * − π + +c.c., or both of them. The shifts of the masses and widths are taken as systematic uncertainties.
The overall systematic uncertainties are obtained by adding all the sources of systematic uncertainties in quadrature assuming they are independent, which are 16.7 MeV/c 2 and 31.6 MeV for the mass and width of the Y (4008), respectively; 5.1 MeV/c 2 and 6.9 MeV for the mass and width of the Y (4220), respectively; 20.9 MeV/c 2 and 23.1 MeV for the mass I: The resonant parameters from the combined fit to e + e − → ωχ c0 , π + π − h c , π + π − J/ψ, and D 0 D * − π + + c.c.. Here M , Γ, and (B i × Γ e + e − ) j are the mass (in MeV/c 2 ), total width (in MeV), and the product of the branching fraction to specific final state and the e + e − partial width (in eV), respectively, where i presents a final state and j indicates a resonance added in the fit in different processes. The fitted mass and width for each resonance are shown in the upper table separately. All the errors are statistical from fit only. φ is the relative phase (in rad).
The leptonic decay width for a vector state is an important quantity for discriminating various theoretical interpretations of its nature. The magnitude of the leptonic decay width determines how the strong decay widths sum up to the total width. Smaller leptonic decay width means that the strong decay widths will be relatively enhanced and vice versa. As the Y (4220) is the dominant component in e + e − → π + π − J/ψ, we assume the theoretical interpretations of Y (4260) may apply also for the Y (4220).
The recent estimate of Lattice QCD (LQCD) for the leptonic decay width of the Y (4220) is about 40 eV [31] as a feature of the hybrid scenario; the predicted upper limit of the Y (4220) leptonic decay width is about 500 eV if the Y (4220) is a hadronic molecule dominated by DD 1 (2420) [32]; the leptonic decay width is only about 23 eV for the ωχ c0 molecule interpretation [33] where no contributions from the open charm decay channel are included in the analysis.
By considering the isospin symmetric modes of the measured channels, we can estimate the lower limit on the leptonic partial width of the Y (4220) decays. For an isospin-zero charmoniumlike state, we expect Table I, considering the solutions with the smallest B × Γ e + e − , we obtain
By inserting the numbers from
3 ± 0.6 ± 1.5) + ... = (29.1 ± 2.5 ± 7.0) + ... eV > (29.1 ± 2.5 ± 7.0) eV, where the first errors are from fit and the second errors are the systematic errors with the uncertainties from different fit scenarios discussed above included. That is, the lowest value of the Γ e + e − of the Y (4220) is around 30 eV. This lower limit on the leptonic partial width of the Y (4220) is close to the prediction from LQCD for a hybrid vector charmonium state [31].
On the other hand, if we take the Solutions with the largest B × Γ e + e − in Table I, we obtain Γ e + e − = (202 ± 13 ± 23) + ... eV. This means that the leptonic partial width of the Y (4220) can be as large as 200 eV or even higher based on current information, to be compared with the predicted upper limit of 500 eV from the molecular scenario [32]. The other combinations of the Solutions result in Γ e + e − values between (30+...) and (200+...) eV.
However, the assumption that the ωχ c0 , π + π − h c , π + π − J/ψ, and D 0 D * − π + + c.c. modes saturate the Y (4220) decays may not be true. Being well above the thresholds of many final states with η c , such as πρη c , ωη c , and φη c , and final states like ηh c , ππψ(2S), and KKJ/ψ, Y (4220) may decay into such final states with substantial rates. In addition, the decays into open charm final states other than DD * π such as DD, DD * + c.c., D * D * , D + s D − s , D + s D * − s + c.c. are also possible, although the charmed mesons are in relative P-wave. The Y (4220) is very close to the D * + s D * − s threshold, the possible coupling to this mode should also be investigated. Further information on these final states will be important for a deeper understanding of the nature of the Y (4220). | 2017-05-31T15:23:48.000Z | 2017-03-30T00:00:00.000 | {
"year": 2017,
"sha1": "c00e663b0bf770f05be89eb8b96ea5294142086e",
"oa_license": null,
"oa_url": "http://arxiv.org/pdf/1703.10351",
"oa_status": "GREEN",
"pdf_src": "Arxiv",
"pdf_hash": "c00e663b0bf770f05be89eb8b96ea5294142086e",
"s2fieldsofstudy": [
"Physics"
],
"extfieldsofstudy": [
"Physics"
]
} |
79495098 | pes2o/s2orc | v3-fos-license | Bayesian mixture modeling for blood sugar levels of diabetes mellitus patients (case study in RSUD Saiful Anwar Malang Indonesia)
Bayesian statistics proposes an approach that is very flexible in the number of samples and distribution of data. Bayesian Mixture Model (BMM) is a Bayesian approach for multimodal models. Diabetes Mellitus (DM) is more commonly known in the Indonesian community as sweet pee. This disease is one type of chronic non-communicable diseases but it is very dangerous to humans because of the effects of other diseases complications caused. WHO reports in 2013 showed DM disease was ranked 6th in the world as the leading causes of human death. In Indonesia, DM disease continues to increase over time. These research would be studied patterns and would be built the BMM models of the DM data through simulation studies where the simulation data built on cases of blood sugar levels of DM patients in RSUD Saiful Anwar Malang. The results have been successfully demonstrated pattern of distribution of the DM data which has a normal mixture distribution. The BMM models have succeed to accommodate the real condition of the DM data based on the data driven concept.
Introduction
When a set of real data observed have a very small amount and a multimodal distribution then it is necessary need specially handling. The Bayesian statistics proposes an approach that is very flexible in the number of samples and distribution of data. These approach is based directly on the posterior distribution of the data. Therefore Bayesian approach can accommodate the real condition of the data based on the data driven concept ( [1], [2], [3], [4], and [5]). Bayesian Mixture Model (BMM) is one model that uses a Bayesian approach for multimodal models and its parameter models is seen as random variables in the model parameter space [6].
Various studies have previously been carried out by BMM modeling. The modeling of BMM for the DM data in Surabaya is the research that has been done by [7] and the research of [8] has been conducted for the DM data in Malang by BMMA modeling. Some of the BMM research in the other field, among others [9] and [10]. Modeling of cases is very important in order to know the exact model and can be used for decision making in accordance with the cases observed [11]. One disease that is not contagious but it is very dangerous and deadly to humans is Diabetes Mellitus (DM). These disease can cause complications of other very dangerous diseases so the handling costs are very expensive. According to the WHO report in 2013, DM was ranked as the world's sixth largest as the leading causes of death [12]. The impact of the DM disease is very dangerous because it can lead to death of the patient and the number of patients continues to increase sharply over time. A person is said to have the disease if the blood sugar levels had reached more than 200 mg/dL for the condition is not fasting, more than 126 mg/dL for fasting conditions and the average blood sugar levels exceeding 150 mg/dL ( [7], [13], and [14]).
A study of the blood sugar levels in DM patients data modeling is very important because it can be known the exact model for policy making in the management of disease control and prevention of DM diseases. Challenge of the DM data is to have a mixture distribution. The studies of [8] and [15] indicates that the DM data have a mixture normal distribution. These paper would be studied patterns of DM data and would be built the BMM models of the DM data through simulation studies where the simulation data built on cases of blood sugar levels of DM patients in RSUD Saiful Anwar Malang.
Materials and Methods
The data used in this study is simulation data which is constructed based on the cases of blood sugar levels of DM patients in RSUD Saiful Anwar Malang for Lowokwaru District in 2015. In this study, there is 7 villages of Lowokwaru District analyzed. To construct simulation data, we need information on real data distribution and value ranges of data from each village in the Lowokwaru District. Based on the information that has been obtained, the data generation is done as much as the number of real data and is repeated 100 times. The detection of the number of mixture components using RJMCMC algorithms and to build BMM models using MCMC with Gibbs Sampler approach by Winbugs program. Finally, to detect feasibility of this model with the Kolmogorov-Smirnov criterion. In this study hypothesized that normal mixture models (BMM-Normal) proposed as a model for the DM cases in Malang. In building a BMM-Normal model using the R software. The following are descriptions of the DM data DM in the Lowokwaru district of Malang.
Bayesian Analysis
The posterior probability distribution model has become the basis of Bayesian statistical analysis method. These distribution is a blend of the prior information and the likelihood of the data. Thomas Bayes had found this concept in 1702-1761, where in model parameter, θ ∈ Ω, is treated as a random variable ( [16], [8], [16], [17], [18] and [19]).
If f (x | θ) is a likelihood function of observational data x and p(θ) is the prior θ then p(θ | x) is posterior probability distribution of θ is, determined by the rules of probability according to Bayes' theorem [1] as in Equation 1.
where f (x) is a normalized constant [6]. So that 1 can be written as: , the posterior probability that will be used for decision making is proportional to the product of the likelihood function and the prior probability of the model parameters ( [1], [20], [21], [22], [23], and [24]).
BMM Analysis 3.2.1. Mixture Models
Mixture model is a special model for the data that have multimodal distribution, where each proportion of sub-group is a constituent component of the mixture models. The mixture model called the particular model because the model is able to combine some different data but still retains the characteristics of the original data ( [6], [8], [24], [25], and [26]).
where f (x | θ, w) is a function of the probability mixture, g j (x | θ j ) is a j th probability function of k number of sub-group, and θ is a mixture model parameters containing of (θ 1 , θ 2 , . . . , θ k ). Parameter θ j , j = 1, 2, . . . , k represents the characteristic distribution of g j on each component in mixture models. While w is the parameter vector of proportions (weighted) mixture model containing of (w 1 , w 2 , . . . , w k ), where 0 < w j < 1, ∀j and k j=1 = 1 for each model parameterθ j . , [24], and [27]).
BMM Models
Given the value of z i then the observation data x i can be derived from the sub-group as in 5.
Thus the resulting joint posterior distribution of all parameter in the mixture model can be expressed as in Equation 6.
Furthermore, to estimate each parameter in Equation 6 by employing the full conditional distribution of each parameter [27].
Suppose p(θ) is a stationary distribution of parameter θ. The full conditional distribution of parameter θ is constructed by making a partition of θ as shown in Equation 7 ([8], [10], and [24]).
where θ s denote the-s th parameter to be estimated and θ −s denote the complement of θ s that is parameter θ without the-s th component. Therefore the full conditional distribution can be established based on the joint distribution of all the parameters as in Equation 8 ([8], [10], and [28]).
RJMCMC Algorithm Reversible Jump Markov Chain Monte Carlo (RJMCMC) concept
can be used to identify the number of mixture components model in which the number of mixture components is unknown. In its application, RJMCMC algorithm uses the concept of birth/death and split/merge with 6 types of movement, that is w updating, θ updating, z updating, hyperparameter β updating, split/merge components, and birth/death of an empty component. The movement split/merge is a random choice between split (k → k + 1) or merge (k + 1 → k) ( [8], [10], and [27]). This method is more flexible to use than the Bayes Factor method.
MCMC Alogarithm
The posterior distribution in the Bayesian analysis is often very complicate and requires a difficult integration process in determining the marginal posterior of a model parameter. Therefore it takes a numerical approximation with Markov Chain Monte Carlo (MCMC) algorithm ( [3], [8], [23], and [29]). MCMC algorithms with Gibbs Sampler approach can be described as follows: MCMC Algorithm with Gibbs Sampler Approach Step 1. We will provide initial value.
Step 2. We will do the sampling of the parameter. Generate the value of θ j , j = 1, . . . , r from their conditional distribution as follows: Step 2.1. Sampling θ Step 3. We will do iteration.
Execute step 2 as K times with K → ∞ order X (1) , X (2) , . . . , X (n) is the independent random variable with hypothesis distribution,F (x) and F n (x i ) is the empirical distribution as in Equation 9, F n (x i ) = the number of data X i ≤ x i n for i = 1, 2, 3, . . . , k ¡ n, where F n (x i ) is right continuous step function then the formula of test statistic D n can be written as in Equation 10.
In this case, D n is the greatest distance between F n (x) −F (x). If the value of D n smaller than the tested models is better ( [8], [23] and [30]).
Description of the Data Analysis
Description of blood sugar levels of DM patients data for Lowokwaru District in Malang 2015 can be seen that in the Fig. 2. According to Fig. 2 can be indicated that the data have a mixture distribution (multimodal distribution). These can be seen by the existence of different groupings of the data. The next step will be identified distribution data from each village. In this step, the proposed hypothesis is as follows: H 0 : data has a normal distribution versus H 1 : data don't have a normal distribution More identification test results are shown in TABLE I.
Derived from TABLE I can be known that the data have a normal distribution for all 7 villages in Lowokwaru District Malang because P-value for each village is larger than 0.05, so that the null hypothesis is accepted. Therefore based on Fig. 2 and TABLE I can be indicated that the data have a normal mixture distribution. The identification results for the number of components mixture in the normal mixture distribution by RJMCMC algorithm can be seen that in the TABLE I and TABLE II. Based on Table I and Table II can be seen that the mixture normal distribution with 2 components for all villages in Lowokwaru District is the best model because P-value of k2 (with k-Maks= 2) from RJMCMC algorithm is the biggest. This means that the exact model to be built for the DM cases in the Lowokwaru District of Malang is a normal mixture model with 2 components. There are two groups of DM patients in the Lowokwaru district of Malang that each group has a normal distribution. In TABLE III are Based on the weighting values shown in TABLE III it can be seen that the villages of Sumbersari, Lowokwaru, Tulusrejo, and Mojolangu have the second weighting value of mixture components (w2) is greater than the first weighting value of mixture components. Its means that in the four villages have had the proportion of DM patients with higher blood sugar levels more than the number of DM patients with lower blood sugar levels. As for the three other villages namely Tlogomas, Jatimulyo, and Dinoyo have w2 lower than w1. However, the average weighted values, Lowokwaru District has a weighted value of the second mixture components (w2) higher than a weighted value of the first mixture components (w1).
BMM Models and Goodness of Fits BMM Models by KS BMM models and Goodness of Fits BMM models by KS for all villages in Lowokwaru District
Malang can be shown in the Fig. 3
Conslusion
The BMM models for cases of the blood sugar levels of DM patients in RSUD Saiful Anwar Malang in the Lowokwaru District have normal mixture distribution with 2 components, namely DM patients with average blood sugar levels less than 200 mg/dL and greater than or equal to 200 mg/dL with the proportion of the second group is greater than the first group, respectively. Villages that need attention from Local Government in Malang are Lowokwaru, Tulusrejo, Mojolangu, Sumbersari, Dinoyo, Tlogomas, and Jatimulyo, respectively. The BMM-Normal model for Lowokwaru District has an average KS value of 0.35739667 so that these models have high accuracy and able to accommodate the real condition of the DM data with driven data concept.
1234567890
The | 2019-03-17T13:08:10.842Z | 2017-10-01T00:00:00.000 | {
"year": 2017,
"sha1": "31c69b1ebe74e205d0346cb943001df596c93cca",
"oa_license": null,
"oa_url": "https://doi.org/10.1088/1742-6596/893/1/012036",
"oa_status": "GOLD",
"pdf_src": "IOP",
"pdf_hash": "15631cdf3f90b074a872394810fc4b3ddc8ef17f",
"s2fieldsofstudy": [
"Medicine"
],
"extfieldsofstudy": [
"Medicine"
]
} |
259376662 | pes2o/s2orc | v3-fos-license | HITSZQ at SemEval-2023 Task 10: Category-aware Sexism Detection Model with Self-training Strategy
This paper describes our system used in the SemEval-2023 \textit{Task 10 Explainable Detection of Online Sexism (EDOS)}. Specifically, we participated in subtask B: a 4-class sexism classification task, and subtask C: a more fine-grained (11-class) sexism classification task, where it is necessary to predict the category of sexism. We treat these two subtasks as one multi-label hierarchical text classification problem, and propose an integrated sexism detection model for improving the performance of the sexism detection task. More concretely, we use the pre-trained BERT model to encode the text and class label and a hierarchy-relevant structure encoder is employed to model the relationship between classes of subtasks B and C. Additionally, a self-training strategy is designed to alleviate the imbalanced problem of distribution classes. Extensive experiments on subtasks B and C demonstrate the effectiveness of our proposed approach.
Introduction
Sexism is especially rampant online due to the anonymity of the Internet, making the online community unfriendly towards women (Mosca et al., 2021;Abburi et al., 2020;García-Baena et al., 2022). Detecting sexism on social media is meaningful in building a more harmonious Internet world. Sexism detection (Ahuir et al., 2022;del Arco et al., 2022) can be considered as a particular sentiment classification task, applied to predict the categories (the detailed categories are shown in Figure 1) of prejudice or discrimination based on a person's gender in the whole sentence. For example, the sentence "As Roosh said having discussions with woman is a waste of time anyway." is a sexist comment, which belongs to a kind of descriptive attack expressing derogation against women. * Corresponding Author SemEval-2023 Task 10 (Kirk et al., 2023) (Explainable Detection of Online Sexism, EDOS), is an online sexism detection and classification task. The organizers provide a labeled dataset and two unlabeled datasets for training the three subtasks. The labeled dataset consists of 14,000 annotated samples of which 3398 are sexist. For the sexist entries, as shown in Figure 1, fine-grained labels of the 4 categories (subtask B) and 11 vectors (subtask C) are also provided.
Subtask A is a binary classification task aimed at predicting whether a post is sexist or not. Subtask B and subtask C classify the sexist posts into more fine-grained categories. There are four categories for subtask B, which are threats, derogation, animosity, and prejudiced discussions. For subtask C, there are eleven target vectors. The hierarchical structure of the three tasks is shown in Figure 1. One obvious characteristic of subtasks B and C is that categories in subtask C are the detailed classification of categories in subtask B, which means all the instances from the one category in subtask C are labeled the same in subtask B. Labels in subtask B and C put together form a 15-class taxonomic hierarchy.
In this paper, we try to solve subtasks B and C simultaneously and treat these two tasks as one multi-label hierarchical text classification problem (Chen et al., 2020;Huang et al., 2021;Zhang et al., 2022). We investigate the EDOS dataset and summarize the two observations: Class label text holds important textual information regarding the class, and, the hierarchical structure of the classes preserves richer semantics dependency information between tasks. Based on it, we propose an approach based on the pre-trained BERT model (Devlin et al., 2019), Graph Convolutional Network (GCN) (Deng et al., 2021), and a retrieval-based attention mechanism (Vaswani et al., 2017) to obtain the final prediction. First, we use BERT model to obtain the sentence and label representation. Then, we propose a hierarchy-relevant structure encoder to capture the richer class representation. Moreover, a class-aware attention mechanism is proposed to capture the relationship between text and classes. In addition, a self-training strategy (Niu et al., 2020;Soleimani et al., 2022) is designed to alleviate the imbalanced problem of distribution classes, to get a better performance of the sexism detection model. Our system achieves the Macro F1 scores for subtasks B and C of 0.6030 and 0.3847, respectively.
Task Definition
We define the combination of subtasks B and C of EDOS as a detailed hierarchical text classification problem. The hierarchy is predefined according to the class relationships shown in Figure 1.
Given a sentence consisting of n words S = {w 1 , · · · , w n }, we construct a BERT-based pair sequence "[CLS] S [SEP]" and feed the sequence into pre-trained language model BERT (Devlin et al., 2019) to acquire the sentence representation H. As to class labels, 4 classes labeled Y B = {y 1 , y 2 , y 3 , y 4 } are provided for subtask B and 11 classes with label Y C = {y 5 , y 6 , · · · , y 15 } for subtask C. child(i), i ∈ {1, 2, 3, 4} refers to the set of child nodes of label node i. As we consider the two tasks together, the 15 classes are labeled Y = {Y B , Y C } = {y 1 , y 2 , · · · , y 15 }. The goal of the task is to predict one label in Y B and one label in Y C for each input sentence. Figure 2 shows the overall architecture of our system. Our model mainly consists of four modules: 1) Pre-trained label encoder extracts contextualized class-label features through BERT. 2) Hierarchyrelevant structure encoder utilizes graph convolution neural network to produce class-structure feature. 3) Class-sentence attention module computes the attention score between the class feature, which is the concatenation of class-label and classstructure feature, and the input text feature to produce class-relevant text feature. 4) A self-training strategy is adopted to deal with unbalanced class distribution and an insufficient amount of labeled data.
Pre-trained Label Encoder
BERT (Devlin et al., 2019) encoder is utilized to extract class-label features. We input the class labels in the form , 2, · · · , 15} and use the pooled output h y i from the encoder as the class-label feature H y = {h y 1 , h y 2 , · · · , h y 15 }.
Hierarchy-Relevant Structure Encoder
To capture the structure feature between classes, we construct a graph G = {A, H s }, where vertices are the 15 classes in Y with randomly initialized representation (Glorot and Bengio, 2010). Following the study of HiGAM , the prior probability of label dependencies is considered when building the adjacent matrix A. Suppose N i is the number of entries of class label y i , A is formulated as follows: We then use a graph convolutional neural network (GCN) (Kim et al., 2017) to obtain the relationship between classes. Formally, where l is the layer of GCN. After graph convolution, H s l denotes the class structure feature of each class.
Class-Sentence Attention Module
We concatenate class-structure feature H s l and class-label feature H y as the final class feature representation H c = {H s l ; H y }. We compute the attention score between sentence feature H s and class feature H c as: where H att = h att 1 , h att 2 , · · · , h att 15 is used for the final class-relevant sentence representation.
Classification
We use fully-connected layers to calculate the prediction probability distribution for subtasks B and C: where H att , H att C = h att 5 , · · · , h att 15 . The final objective function of subtask B and C are defined by cross-entropy loss, respectively, The final loss the model is: where both α and β are set as 0.5 during training.
Self-training Strategy
The labeled dataset provided by SemEval-2023 Task 10 is very unbalanced. For example, class "2.1 descriptive attacks" has over 700 instances while class "3.4 condescending explanations or unwelcome advice" only has less than 50 instances. As a consequence, we utilize a self-training strategy to mitigate this problem based on CReST. Specifically, we add certain pseudo-labeled data to the training set from the Reddit unlabeled dataset provided by the organizers. After each epoch, the unlabeled data is first tested on the newly attained model. Then, sentences with the prediction probability on both tasks higher than the corresponding terms in threshold list T = {T 1 , T 2 , · · · , T 15 } are added to the training set. Suppose there are a total of N tot instances in the labeled dataset, the threshold list is calculated as follows: Higher thresholds are assigned to more frequent classes and lower thresholds to rare classes. According to CReST, classification models have a much higher accuracy on minority classes. To be more specific, models are usually reluctant to classify instances into minority classes, but once such a prediction is made, the accuracy is very high. Therefore, we make it easier to add less frequent class samples and harder to add the major classes.
Only sexist entries are needed for subtasks B and C, so we select the sexist entries from the original Reddit unlabeled dataset through a BERT-based binary classification model. We train a binary classification model The sexist samples form the unlabeled dataset used in our model.
Experimental Setup
Dataset. We use the EDOS datasaet provided by SemEval-2023Task 10 (Kirk et al., 2023. Detailed statistics of the labeled dataset are illustrated in Table 1. The labeled dataset is divided into a training dataset (80%) and a validation dataset (20%). We randomly sampled 80% of samples for each class in subtask C and used the rest 20% for validation.
Implementation Details. The pre-trained BERT encoder, which is used as the sentence encoder and class-label encoder, is set with a hidden dim of 1024 and a maximum length of token input of 100. The label embedding for class-structure feature H s 0 is initialized with Kaiming uniform . To avoid overfitting, dropout is employed at a rate of 0.5 for sentence embedding and 0.1 for the GCN and attention. We select Adam as the optimizer with a learning rate of 2 × 10 −4 . The model is trained for 30 epochs with a batch size of 32. We assess the prediction results by calculating the accuracy and macro F1 of each subtask.
Experimental Results
To evaluate our model, we compare its performance on the development test dataset with several baselines , such as BERT, RoBERTa, HiAGM-LA, and HTCInfoMax. The comparison results are reported in Table 2 other methods on both evaluation metrics in both tasks, which demonstrates that our model can solve the two sub-tasks effectively. Compared to bertbased models, our model adds additional hierarchical structure information to model the relationship between the labels of the two subtasks. Moreover, the hierarchical classification models only consider the structural relationship without making use of the label text. Our approach, which fully utilized the contextualized information from the label texts, is proven to be better. Among the variants of our model, using bert-large as the pre-trained encoder has the best performance surprisingly. We argue that the training dataset initially includes only 2718 labeled instances for a 15-class classification problem, making larger and more complicated models easily overfitted. This is the main reason why BERT as the encoder outperforms RoBERTa. The best model in the competition reports a Macro-F1 of 73.26 in subtask B and 56.06 in subtask C.
Ablation Study
To evaluate the effectiveness of components of our proposed model, we report the results of the ablation study in Table 3. Our model w/o H y performs significantly worse on subtask C dropping nearly 10%, indicating that the class-label feature H y is effective. The performance of our model w/o H s l also degrades, thus proving class-structure feature H s l improves the performance. The performance of our model w/o self-training is slightly worse on subtask B but drops substantially in terms of subtask C, which confirms the effectiveness of the self-training strategy.
Effect of task weight α and β
To explore the impact of the task weights on the performance of our proposed model, we vary the
Models
Subtask B Subtask C task weight from 0.1 to 0.9 and show the results in Figure 3. The accuracy and macro-F1 of both tasks tend to increase when the weight of subtask B α increases from 0.1 to 0.5 and drops when α continues to grow from 0.5 to 0.9. The same pattern is observed with the weight of subtask C β. The best performance is achieved when equal weights are assigned to the two losses, or α = β = 0.5.
Conclusion
In this paper, we propose a model to address subtasks B and C of SemEval-2023 Task 10 Explainable Detection of Online Sexism. Our goal is to identify sexist statements and explain why they are sexist. The model treats the two subtasks as one multi-label hierarchical classification problem to capture class features from two perspectives: label texts and class hierarchy, utilizing a pre-trained BERT model and a graph convolution neural network respectively. Our model is proven effective by extensive comparison experiments.
Online sexism detection is getting more and more attention as people begin to realize its importance to the Internet community. More efforts need to be put into this cause to make the Internet world better for everybody. | 2023-07-10T13:07:34.567Z | 2023-01-01T00:00:00.000 | {
"year": 2023,
"sha1": "ed21cba2222507f920516b29e76e5d31322704e8",
"oa_license": null,
"oa_url": null,
"oa_status": null,
"pdf_src": "ACL",
"pdf_hash": "ed21cba2222507f920516b29e76e5d31322704e8",
"s2fieldsofstudy": [
"Computer Science"
],
"extfieldsofstudy": [
"Computer Science"
]
} |
259374087 | pes2o/s2orc | v3-fos-license | Protocol for a Wnt reporter assay to measure its activity in human neural stem cells derived from induced pluripotent stem cells
The canonical Wnt signaling is an essential pathway that regulates cellular proliferation, maturation, and differentiation during neurodevelopment and maintenance of adult tissue homeostasis. This pathway has been implicated with the pathophysiology of neuropsychiatric disorders and was associated with cognitive processes, such as learning and memory. However, the molecular investigation of the Wnt signaling in functional human neural cell lines might be challenging since brain biopsies are not possible and animal models may not represent the polygenic profile of some neurological and neurodevelopmental disorders. In this context, using induced pluripotent stem cells (iPSCs) has become a powerful tool to model disorders that affect the Central Nervous System (CNS) in vitro, by maintaining patients’ genetic backgrounds. In this method paper, we report the development of a virus-free Wnt reporter assay in neural stem cells (NSCs) derived from human iPSCs from two healthy individuals, by using a vector containing a reporter gene (luc2P) under the control of a TCF/LEF (T-cell factor/lymphoid enhancer factor) responsive element. Dose-response curve analysis from this luciferase-based method might be useful when testing the activity of the Wnt signaling pathway after agonists (e.g. Wnt3a) or antagonists (e.g. DKK1) administration, comparing activity between cases and controls in distinct disorders. Using such a reporter assay method may help to elucidate whether neurological or neurodevelopmental mental disorders show alterations in this pathway, and testing whether targeted treatment may reverse these. Therefore, our established assay aims to help researchers on the functional and molecular investigation of the Wnt pathway in patient-specific cell types comprising several neuropsychiatric disorders.
Introduction
Wnt is a growth stimulatory factor, inducing cellular proliferation, polarization, and differentiation, which generally implies its relevance during developmental processes (Steinhart and Angers, 2018). Moreover, Wnt distinguishes itself from other growth factors by its exceptional ability to giving shape of expanding tissues (Nusse and Clevers, 2017). Especially during neural development, Wnt has proven to be a driving factor, from defining the anterior-posterior axis of the neural plate (Kiecker and Niehrs, 2001), regulating morphogenesis of the neural tube (van de Ven et al., 2011), modulating stem cell proliferation and differentiation in the developing and mature brain (Bengoa--Vergniory and Kypta, 2015; Gonçalves et al., 2016;Megason and McMahon, 2002), up to participating in the regulation of synaptic plasticity (Mulligan and Cheyette, 2012;Oliva et al., 2013). This outcome of Wnt-signaling is mediated by a downstream signaling cascade, activated by Wnt, which elicits changes in gene expression (Nusse and Clevers, 2017). To date, the most studied Wnt pathways are the canonical pathway, involving β-catenin as a crucial switch in gene expression, and the non-canonical pathway, independent of β-catenin (Ng et al., 2019;Zhan et al., 2017). At a molecular level, the canonical Wnt cascade can be grouped into: a) the heterodimeric receptor complex, consisting of the Frizzled receptor (FZD) and lipoprotein receptor-related protein 5/6 (LRP5/6), b) the multiprotein destruction complex, assembling axin, adenomatous polyposis coli (APC), glycogen synthase kinase 3 (GSK3), casein kinase 1α (CK1α), Beta-Transducin repeats-containing Protein (β-TrCP) and dishevelled (Dvl) proteins, and c) β-catenin (Peifer and Polakis, 2000;Taciak et al., 2018). In absence of the intercellular Wnt signal, the destruction complex binds, phosphorylates, and ubiquitinates β-catenin to be degraded by proteasomes, keeping the intracellular levels of β-catenin low (MacDonald et al., 2009). In an activated state, Wnt binds to its receptor complex, causing phosphorylation of the LRP5/6 protein. The phosphorylation evokes a destabilization of the destruction complex, resulting in an inhibition of β-catenin degradation, permitting an accumulation of free β-catenin in the cytoplasm (Okerlund and Cheyette, 2011). Consequently, free β-catenin transiently converts T-cell factor/lymphoid enhancer factor (TCF/LEF) from a transcriptional repressor to an activator, mediating gene expression (Clevers and Nusse, 2012).
The Wnt pathway is commonly active during healthy development; however, its activity also plays an important role in disease. Alterations in Wnt-signaling has been linked to central nervous system (CNS) disorders such as attention-deficit/hyperactivity disorder (ADHD), autism spectrum disorder (ASD), depression, schizophrenia, bipolar disorder, Alzheimer's disease and intellectual disability (Bem et al., 2019;Folke et al., 2019;Grünblatt et al., 2019;Hussaini et al., 2014;Kwan et al., 2016;Snijders Blok et al., 2015;Voleti and Duman, 2012;Yde Ohki et al., 2020). Available evidence presents genetic disruption in Wnt-pathway associated genes across mental disorders, affecting complex behavior, such as repetitive behavior relevant in ASD (Mulligan and Cheyette, 2017;Zhang et al., 2012), and hyperactivity/impulsivity found in ADHD (Grünblatt et al., 2018). Moreover, a variety of cancers were identified with an abnormal activation of the Wnt-pathway, encouraging cellular proliferation, migration and invasion (Taciak et al., 2018;Zhan et al., 2017). Thus, the understanding of the Wnt-pathway is essential to advance in fundamental developmental processes as well as in neurological and cancerous disease research.
Induced pluripotent stem cells (iPSCs) create a tissue relevant model for the investigation of signaling pathways in a personalized manner. iPSCs retain the same properties as embryonic stem cells (ESCs), however as iPSCs derive from patient-specific somatic cells, which are obtained non-invasively, ethical concerns as found in ESC fall away (Gurwitz, 2016). Additionally, iPSCs show their advantage in recapitulating the genetic background of patients without the need of genetic manipulation (Halevy and Urbach, 2014). This is especially crucial in mental disorders, in which a wide range of genetic variations are linked to one disease, making disease modelling with iPSCs more representative of individuals (Hunsberger et al., 2015).
Reporter gene systems are a valuable tool for investigating molecular pathway activities, such as the Wnt-pathway, by monitoring the expression of the involved promotor or transcriptional response element (TRE) sequence joined to a reporter gene, that are inserted in an expression vector (Badr and Tannous, 2011). In response to the activation of the pathway, the synthesized reporter gene transmits colorimetric, fluorescent, or luminescent signals evoked by enzymatic activities (Cheng et al., 2010). The transient delivery of the expression vector is conducted through either viral transduction or non-viral transfection. Many studies successfully performed lentiviral-mediated reporter gene expression to trace molecular pathway activities (Eggenschwiler et al., 2013;Fuerer and Nusse, 2010), as lentiviral transduction provides an efficient and stable gene expression in cell culture and in vivo (Shuen et al., 2015). However, a recent publication sheds awareness of potential risks that lentiviral vector exposure may carry (Schlimgen et al., 2016). The publication demonstrates studies indicating oncogenic, infectious, and other transformative alterations in the infected cells, implying a biosafety hazard. To prevent such risk, non-viral plasmid transfections provide a safe alternative of reporter gene delivery. In the current method paper, we present an alternative transient plasmid-mediated reporter gene expression method for the observation of the Wnt activity in human iPSC-derived neural stem cells (NSCs) through chemical transfection. The induction of the cells was conducted with a vector (pGL4.49[luc2P/TCF-LEF RE/Hygro]), provided as a gift by Promega, carrying the reporter gene luc2P under the control of TCF/ LEF transcriptional response element, eliciting a bioluminescent signal when the Wnt-pathway is activated. For signal normalization, NanoLuc Luciferase under the control of a TK (thymidine kinase) promoter was used. The successful chemical transfection of a Wnt reporter into human iPSC-derived NSCs is a novel procedure, that has not been published to date. Therefore, the performed non-viral transfection method in human iPSC-derived NSCs can be applied for detecting the Wnt signaling pathway activity across various CNS disorders.
Methods
An overview of the procedure is illustrated in Fig. 1A and a list of reagents is provided in Appendix 1.
Recruitment of individuals
The recruitment of the healthy control male children and adolescents is conducted by the Psychiatric University Hospital, Zürich, University of Zurich, as previously described Yde Ohki et al., 2021, 2023) (Appendix 2).
All individuals and their families voluntarily consented to participate in this project, which was properly approved by the Cantonal Ethics Committee (BASEC-Nr. For this study, 4 NSC lines (from two non-related healthy control male adolescents; 2 clones each) were chosen to establish and test the method (Supplementary Table 1, Supplementary Fig. 1). Additional information about the generation and culture of iPSCs and NSCs may be found in Appendix 3.
Plasmid amplification
In order to guarantee sufficient amount of plasmids for further transfections, the pGL4.49[luc2P/TCF-LEF RE/Hygro] Vector (kindly provided by Promega AG, E4611) and an internal control vector under the control of a TK promoter (pNL1.1.TK [Nluc/TK] Vector; Promega AG, N1501) ( Fig. 1B) were separately amplified in TOP10 E. coli (Life Technologies, C4040-03) using heat-shock bacterial transformation. The whole 5-day process from culture to plasmid isolation and purification was performed as described in Appendix 4.
Transient chemical transfection of NSCs
4) The solution was incubated for 15 min at RT without harsh pipetting. 5) After incubation, the bottom of the tube was gently flipped and 4 μL of the solution was added to each 96-well. Important: reservoirs and multichannel pipettes were not and should not be used! 6) Transfected plates were incubated overnight at 37 • C under 5% CO 2 . was added 10 min prior to DKK-1 treatment, in 40 μL/well, while additional 10 μL of DKK1 in different concentrations (0,4,10,20,30,40 and 50 ng/mL) were added into each well. Therefore, the total volume after both treatments remained 50 μL/well. Cells were incubated overnight (for at least 17 h) at 37 • C under 5% CO 2 conditions.
Luminescence assay
➢ DAY 2 -On the next day of overnight treatment, transfected NSCs were submitted to the luminescence assay in the dark, according to the instructions of the Nano-Glo® Dual-Luciferase® Reporter Assay System (Promega AG, N1610).
2) The plate was incubated for 3 min at RT, at a constant agitation of 150 RPM. 3) Cell lysates were transferred to a white clear-bottom 96 well plate (Berthold Technologies, 24910). 4) Firefly luminescence activity was immediately measured. For our experiments, we used the luminometer function of a Mithras 2 LB 943 Multimode Reader (Berthold Technologies) and the Mikro-Win 2010 (version 5.18) software. The luminometer's parameters were set up as: HiSens mode, with 0.1 s of integration time with a 60-s delay applied prior to the measurement to ensure no excitation due to light exposure before entrance to the machine is measured.
5) Next, 50 μL of the NanoDLR™ Stop & Glo® was added to each well. 6) A thorough pipetting up and down was performed. 7) The plate was incubated for 10 min at RT. 8) Nanoluc luminescence signal was immediately measured using the same settings mentioned on step 4. 9) The raw data was exported as an Excel file for further analysis.
Single x double transfection
To ensure that the transfection efficiency for both plasmids were the same, we performed single transfections (either with the pGL4.49 [luc2P/TCF-LEF RE/Hygro] vector or the pNL1.1.TK [Nluc/TK] vector) and compared the Relative Luminescence Unit (RLU) ratios with the ones obtained from double transfections. Singly and doubly transfected NSCs were then treated with three concentrations of each agonist: Wnt3a (0, 40 and 600 ng/mL) and CHIR-99021 (0, 2.5 and 10 μM).
Data analysis
RLU values from the Firefly Luciferase activity were normalized by the Nanoluc Luciferase signal, well by well. Normalized RLUs were cleaned using the Interquartile Range (IQR) method. For each technical replicate within a cell line, the highest RLU value was set as having Wnt activity as 100% and the other individual values were calculated relatively to it. For DKK1, the mean of RLU values from wells that were treated with Wnt3a EC26 + DKK1's vehicle were set as 26%.
From each cell line, the highest mean of RLUs (considered two technical replicates per cell line) were constrained to 100% of Wnt activity in the case of agonists. As for DKK1, the lowest mean of RLU values were constrained as bottom values. For each condition, the Wnt activity in percentages were plotted in graphs in function of the agonist/antagonist concentration. EC50 and IC50 values (defined as the half-maximal effective and inhibitory concentrations, respectively) were obtained by performing a non-linear regression for each Wnt agonist on GraphPad Prism 9.4.1. EC50 and IC50 values were calculated using GraphPad Prism 9 (version 9.4.1).
Results and discussion
In this study, a reproducible viral-free Wnt reporter assay in human iPSC-derived NSCs was developed (Fig. 1A). Non-viral methods of delivery are less immunogenic and less mutagenic, which offer a great advantage to the use of lentiviruses in reporter systems (Hardee et al., 2017;Loring et al., 2016). For this, we used the Firefly luciferase gene (luc2P) present in the vector pGL4.49[luc2P/TCF-LEF RE/Hygro], as a Wnt reporter under the control of a TCF-LEF responsive gene, allowing the assessment of luminescence signals after various drug treatments that can be translated into Wnt-signaling activity regulation ( Fig. 1A and B).
While this method paper describes the successful transient transfection of NSCs, it should be noted that the stable transfection of iPSCs has initially been assessed. Our initial goal was to further differentiate stably transfected iPSCs (selected through a Hygromycin treatment at 70 μg/mL for one week) into NSCs, which would be submitted to treatment with Wnt agonists and luminescence assays. However, the stable transfection process might be considered more laborious, since the constant selection of clones within the cell population is necessary (Fus-Kujawa et al., 2021). The choice of using iPSCs was based on their indefinite self-renewal potential compared to those of a multipotent lineage (Liang and Zhang, 2013).
However, in our experience, the stable transfection of iPSCs through electroporation based on a protocol from Chatterjee et al. (2011) (Chatterjee et al., 2011 have yielded inconsistent results, indicating low success rate in our cells. Due to these inconsistent results of different experiments conducted with NSCs post-transfection (i.e., different concentration ranges for agonists, integration time, luminescence settings), we further tested whether the plasmid was truly functionally present in the transfected iPSCs. Interestingly, when we performed luminescence assays with transfected Hygromycin-selected iPSCs treated overnight with Wnt3a, we observed that the luminescence signals were very low and inconsistent as similarly observed in the NSCs post-transfection. Supplementary Table 3 shows weak RLU values obtained from the exemplary transfected iPSC K005 z13, while Supplementary Table 4 provides the RLU values from NSCs post-transfection derived from the same cell line under different conditions following treatment with agonists.This observation could be related to a possible partial integration of the vector into the host genome, which would confer Hygromycin resistance to the cells and the formation of different subclones that might not have acquired the luciferase gene. These results have led us to pursue a new alternative that we describe in the current paper: transiently transfecting NSCs, which is a non-integrative method (e.g., not interfering with the host genome, which might be advantageous to studying disorders with genetic etiology) and, unlike required for stable transfection, does not involve the use of antibiotics for selection of successfully transfected cells (Fus-Kujawa et al., 2021).
Following, an efficient transfection method has been chosen. Although electroporation is a common delivery method for stem cells, which are usually described as more resilient to transfection, the method includes high chances of increased cell death and permanent damage (Chong et al., 2021). This led us to choose a chemical transfection using FuGENE® HD (a non-liposomal reagent), which has been previously reported in human embryonic stem cells, being the reagent with the lowest toxicity in this cell type (Suzuki et al., 2008).
To eliminate well-to-well variances (i.e., transfection efficiency and variability in cell number) and obtain trustworthy results, a normalizing vector containing a Nanoluc Luciferase gene under the control of a constitutive promoter (TK) was co-transfected with our vector of interest (Fig. 1B).
Since the transient transfection is a continuous process to be repeatedly performed throughout experiments, it might be necessary to expand the amount of available material. To do so, we transformed competent E. coli with our plasmids, and extracted the amplified DNA after bacterial culture. However, since the use of selective antibiotics might mildly affect the stability of our plasmid following amplification (Wein et al., 2020), we performed a diagnostic enzymatic reaction by restriction enzymes. We found that the integrity of the plasmid was preserved and its original format was intact. Given that HindIII and EcoRI cleave the pGL4.49 vector at positions 437 bp and 2162 bp, respectively, a 6124 bp band for uncut samples and two bands for the digested ones (1724 bp and 3961 bp bands) were to be expected.
On the other hand, by cleaving the Nanoluc vector with HindIII and BamHI at positions 781 and 1595 respectively, the expected products from the double digestion would have 814 bp and 3003 bp. The agarose gels further confirmed that the structure of both vectors has not been affected during the amplification process in E. coli (Fig. 1C and D).
Before starting a transient transfection protocol in the target cells of interest, researchers must consider some important factors. For instance, it should not only be considered how the reporter gene will be delivered, but also how transfection efficiencies may vary according to the nature of the cell lines. In this context, the cell type to be targeted , size (Wang et al., 2021), cell cycle stage (Brunner et al., 2000) and differentiation stage (Stein and Schindler, 2011) could be determinant to a successful transfection. Due to that, the different methodological origins of NSC generation could reflect in a wide range of NSC phenotypes (Galiakberova and Dashinimaev, 2020), which may result in different experimental outcomes than those described in our paper. For our case, the process does not seem to morphologically affect the transfected and treated NSCs, as representatively depicted in Fig. 1E.
Although it is not possible to determine whether each cell was transfected with both of our vectors, the molecular biology community well accepts that a correlated packaging of both plasmids occurs when a transfection reagent is added to the mixture (Gam et al., 2019). Nevertheless, to prove that the transfection was efficient for our vectors, we compared RLU ratios when the NSC line K015 c1 was submitted to single and double transfections. Non-significant differences between single and double transfected wells suggest that the transfection process in both conditions are similar ( Supplementary Fig. 2).
The success of our transient transfection process was functionally observed by the RLU values (Supplementary Table 5) and the ascending dose-curve responses following NSC treatment with the Wnt-agonists, CHIR-99021 and Wnt3a in all four cell lines (two clones from two different individuals) ( Fig. 2A and B, Supplementary Figs. 3 and 4). An additional approach to verify the functionality was assessed by inhibiting Wnt-signals through the treatment of the cells with DKK1, a Wntantagonist (Fig. 2C, Supplementary Fig. 5). Individual EC50 and IC50 values of each NSC line can be found in Table 1, while their regression can be reviewed in Supplementary Figures 2, 3 and 4.
Wnt3a-conditioned media has previously been used in reporter assays as an alternative to the use of purified Wnt3a in human NSCs (Zhao et al., 2012). However, Wnt3a-conditioned media consists of an unknown environment containing undefined factors that might promote differentiation of stem cells (Tüysüz et al., 2017). On the other hand, the high hydrophobicity of purified Wnt3a may be an issue for serum-free media or serum-free diluents. In our case, the presence of HSA (a maximum negligible concentration of 0.008%) was used as a stabilizer that guaranteed the promotion of Wnt activity, as recommended by the manufacturer.
When our NSC lines were analyzed together as one single group, the EC50 values for Wnt3a and CHIR-99021 were 196.4 ng/mL and 5.272 μM, respectively. Our results show that our NSCs are responsive to different Wnt signaling agonists acting on distinct points of the cascade: Wnt3a, an extracellular ligand that binds to LRP5/6 and Frizzled Fig. 2. Dose-curve response after treatment of NSCs with different concentrations of the Wnt-agonists Wnt3a (A) and CHIR-99021 (B), as well as the antagonist DKK1 (C). In the latter, the activity is normalized to 26%, as the cells were treated with Wnt3a′s EC26 (111 ng/mL) prior to the addition of DKK1. The mean and standard deviation (SD) of the four cell lines were taken together to generate a non-linear regression curve (details on non-linear regression statistics can be found in Supplementary Table 6). In the graphs, the highest Wnt activity (100%) for Wnt3a and CHIR-99021 was set as the maximum mean of individual percentages obtained when the four cell lines are plotted together. Conversely, for DKK1, 26% was determined as the mean of the individual percentages from wells treated with 0 ng/mL DKK1 (containing Wnt3a′s EC26). (MacDonald and He, 2012); and CHIR-99021, antagonizes the downstream GSK3β as reviewed by Law and Zheng (2022) (Law and Zheng, 2022). We also observed that increasing concentrations of the Wntinhibitor, DKK1, gradually reduced Wntactivity in our NSCs (Fig. 2C, Supplementary Fig. 5). The IC50 value obtained by the non-linear regression curve was 19.31 ng/mL. Statistical details of the non-linear regression after analyzing the four cell lines together can be found in Supplementary Table 6. In neuropsychiatry, the Wnt signaling has been previously investigated in human NSCs from idiopathic autism (Marchetto et al., 2017) and Pitt-Hopkins Syndrome patients (Papes et al., 2022), which might suggest the association of some of their phenotypes with an imbalance in Wnt activity. Not only neurodevelopmental disorders, but also neurodegenerative diseases (such as Alzheimer's and Parkinson's Disease) have been linked to the Wnt dysregulation as reviewed by Gao and colleagues (Gao et al., 2021). Therefore, elucidating the functionality of the Wnt signaling in human neural cell lines, while investigating CNS disorders at the molecular level is crucial for a deeper understanding of their pathophysiology. Furthermore, since the Wnt pathway coordinates essential cellular processes during embryonic development, neurodevelopment, neurogenesis, and sustainability of cells, understanding its activity alterations may explain the fundamental etiology of certain disorders (Mulligan and Cheyette, 2012). Subsequently, these results might facilitate the search for potential novel therapeutic targets involving the Wnt pathway.
Funding
We would like to acknowledge the Fonds für wissenschaftliche Zwecke im Interesse der Heilung von Geisteskrankheiten (Nr. 8702) and the Waterloo Foundation (reference number 2462/4548) for funding.
Declaration of competing interest
The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Edna Grünblatt reports a relationship with Waterloo Foundation that includes: funding grant. Edna Grünblatt reports a relationship with MEDICE Arzneimittel Pütter GmbH & Co. KG that includes: funding grant.
Susanne Walitza reports a relationship with Swiss National Science Foundation that includes: funding grants. Susanne Walitza reports a relationship with European Commission Seventh Framework Programme for Research that includes: funding grants. Susanne Walitza reports a relationship with Gesamtstrategie Hochspezialisierte Medizin of the Canton of Zurich that includes: funding grants. Susanne Walitza reports a relationship with Federal Institute for Drugs and Medical Devices that includes: funding grants. Susanne Walitza reports a relationship with Zürcher Impulsprogramm zur nachhaltigen Entwicklung der Psychiatrie that includes: funding grants. Susanne Walitza reports a relationship with Hartmann Müller Stiftung that includes: funding grants. Susanne Walitza reports a relationship with Olga Mayenfisch Foundation that includes: funding grants. Susanne Walitza reports a relationship with Gertrud Thalmann Fonds of the UPK Basel that includes: funding grants. Susanne Walitza reports a relationship with Vontobel Foundation that includes: funding grants. Susanne Walitza reports a relationship with Uniscientia Foundation that includes: funding grants. Susanne Walitza reports a relationship with Erika Schwarz Foundation that includes: funding grants. Susanne Walitza reports a relationship with Gesundheitsförderung Schweiz that includes: funding grants. Susanne Walitza reports a relationship with Georg Thieme Verlag KG that includes: consulting or advisory. Susanne Walitza reports a relationship with Hogrefe Publishing Group that includes: consulting or advisory. Susanne Walitza reports a relationship with W Kohlhammer GmbH that includes: consulting or advisory. Susanne Walitza reports a relationship with Springer Nature Deutschland GmbH Heidelberg that includes: consulting or advisory. Susanne Walitza reports a relationship with Beltz Publishing Group that includes: consulting or advisory.
All other authors have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
Data availability
Data will be made available on request. | 2023-07-10T05:04:03.659Z | 2023-06-17T00:00:00.000 | {
"year": 2023,
"sha1": "04276486442ba187d36856f1dbaa888f31928922",
"oa_license": "CCBY",
"oa_url": "https://doi.org/10.1016/j.crneur.2023.100095",
"oa_status": "GOLD",
"pdf_src": "PubMedCentral",
"pdf_hash": "04276486442ba187d36856f1dbaa888f31928922",
"s2fieldsofstudy": [
"Biology",
"Medicine"
],
"extfieldsofstudy": [
"Medicine"
]
} |
85396615 | pes2o/s2orc | v3-fos-license | The Incorporation of Chromium in Rice Straw Fermented with Ganoderma Lucidum
The research was designed to evaluate chromium incorporation by Ganoderma lucidum in rice straw substrate supplemented with chromium chloride. Treatments were combination of Cr level (0, 500, 1000, 1500, 2000, 25000, and 3000 ppm) and fermentation time (0, 2, 3, and 4 wk). The treatments were arranged in a factorial 7 x 4 and allocated in completely randomized design with three replications. G. lucidum was grown in potato dextrose agar (PDA) medium for 10 days and was inoculated to the substrate which have been sterilized and mixed with CrCl3.6H2O. The moisture of substrate was maintained at 70%. Fiber and protein components of growth media of G. lucidum was determined and analyzed for their Cr content. The finding result showed that addition of Cr up to 3000 ppm into the substrate stimulated the G. lucidum growth. Chromium was incorporated into the fiber and protein components of the growth substrate of G. lucidum during fermentation. Incorporation of Cr into the protein of substrate containing3000 ppm Cr was highest when fermented for 4 wk. Protein component of substrate contained 9.29% Cr while in NDF and ADF was 27.20% and 10.55% Cr, respectively. It is concluded that Cr was incorporated into the G. lucidum cells during fermentation.
INTRODUCTION
Chromium (Cr) is one of the micro mineral and its essentiality has been proved in many researches. Chromium is important for animals (NRC, 1997) because of their role in the metabolism of carbohydrates, fats, proteins and nucleic acids (Pechova & Pavlata, 2007). Chromium from inorganic sources is poorly absorbed. The conversion of inorganic Cr to an organic complex improves its bioavailability (NRC 1997;Pechova & Pavlata, 2007). Supplementation of mixed Cr in fermented feed and G. lucidum improved immune response of lactating cows (Agustin et al., 2011). Therefore, efforts should be made to convert inorganic Cr to organic form. Effective Cr level for G. lucidum growth in solid state fermentation of oil palm by-product was 3000 ppm with incorporation value of 68.23% (Agustin et al., 2010). Metal ions including Cr can accumulate intracellularly in fungal cells (Sen & Dastidar, 2010). The ability of microorganism cell to bind metal ions is associated with its cell wall (Shugaba et al., 2012).
G. lucidum is able to use sawdust as a substrate for its growth (Chang & Miles, 2004) and it can also use oil palm by product for its growth (Agustin et al., 2010). A source of fiber such as rice straw is available and its availability is abundant. Rice straw may meet the criteria as a source of fiber for G. lucidum growth. Misra et al., (2007) reported that mustard straw fermented by G. lucidum at a temperature of 35 o C for 21 d can increase in vitro digestibility and delignification. G. lucidum can bind and can accumulate metal ions intracellularly. However, information about the use of rice straw as a growth medium of G. lucidum for the purpose of producing organic Cr have not available yet. The experiment was designed to determine the incorporation of Cr by G. lucidum in rice straw as a main component of substrate. The result obtained is an effort to synthesize organic Cr to be used as a mineral supplement for livestock. In this case the whole fermented substrate is used as the product.
Preparation Starter of Ganoderma lucidum
Inoculum stock of G. lucidum was obtained from the collection of Indonesian Biotechnology Research Institute for Estate Crops (IBRI), Bogor. Starter was obtained by rejuvenating inoculums stock at IBRI Microbial Laboratory and Laboratory of Microbiology, Department of Nutrition and Feed Technology, Faculty of Animal Science, Bogor Agricultural University. The rejuvenation of inoculums was carried out by culturing isolates of stock culture G. lucidum into petridish containing PDA medium (potato-dextrose-agar) (Wagner et al., 2003), and was incubated for 10 d or until the mycelium of G. lucidum met petridish. The production of the inoculums was a crucial step affecting significantly the performance of the subsequent fermentation.
Substrate Preparation and Inoculation
The main material used in this research was derived from rice straw obtained from rice fields. Rice straw was chopped to the size of 2-3 cm and dried in the sun to the moisture content of about 10%. Rice straw was mixed with a solution containing CrCl 3 .6H 2 O with varying Cr concentration: 0, 500, 1000, 1500, 2000, 2500, and 3000 mg Cr per kg of dry matter and conditioned at 70% moisture content. The mixed substrate was inserted into the 300 ml bottle, covered with aluminum foil, and sterilized for 30 min at pressure of 1.2 and temperature of 121 o C. After cooling, sterilized bottle were inoculated with starter aseptically with a plug diameter of 0.5 cm in the laminar flow cabinet. The inoculated substrate was incubated at 27-28 o C for 0, 2, 3, or 4 wk. Fermentation products were harvested according to the time of incubation.
Experimental Design
The experiment was carried out in a 7 x 4 factorial design with 3 replications for each treatment. There were seven levels of Cr supplemented into the rice straw, i.e. 0, 500, 1000, 15000, 1500, 2000, 2500, and 3000 ppm, at the time factor. There were four fermentation times as the second factor, i.e. 0, 2, 3, and 4 wk.
Sample and Data Analysis
Percentage of dry matter (DM) and organic matter (OM) loss were calculated. Total Cr in the fermented biomass was measured by using Atomic Absorption Spectrophotometer (AAS) Shimadzu AA680 models (Carry & Allaway, 1971). The amount of Cr bound fiber and protein components of biomass was measured separately. Measurement of total Cr in solid substrate was conducted by using a sample of 5 g. Samples were crushed in a mortal porcelain and put into a test tube, add 5 mL of 10% TCA and was homogenized with a vortex, and then allowed to stand for one hour. The solution was then centrifuged at 3000 rpm for 10 min. Chromium concentration in supernatant was measured. The content of the neutral detergent fiber fraction of fiber (NDF) and acid detergent fiber (ADF) were determined by analysis of Van Soest (Van Soest & Wine 1967). Levels of Cr contained in the NDF and ADF are determined from the sample after the determination of NDF and ADF. Biomass protein content was analyzed (Bradford 1976) and its Cr levels were measured.
The data were analyzed statistically according to two-way Analysis of Variance (ANOVA) and the difference between treatment means was determined using Duncan's Multiple Range Test (DMRT).
RESULTS AND DISCUSSION
G. lucidum grew rapidly on PDA on the fifth until 10 d of incubation. The mycelium reached maximum growth on stable condition when the level of water was 70%. The water content of the rice straw substrate in this study was higher than recommended by Chang & Miles (2004) which was 60%-65%. This is because the substrate for cultivation of G. lucidum was different. Chang & Miles (2004) used saw dust as a substrate, while in this study we used rice straw as a main component of substrate. The growth of G. lucidum varied widely, depending on the kind of substrate (Erkel, 2009). Fermentation with G. lucidum was carried out at temperatures ranged from 26-28 o C and pH of substrate 5.5. The optimum fermentation condition appeared to depend on temperature and pH, and this substrate conditions was suitable for the growth of G. lucidum according to the recommendation of Chang & Miles (2004). The growth of fungi G.
lucidum on rice straw substrate containing up to 3000 ppm Cr began to appear after the age of 1 wk. After 2 wk of fermentation, its white mycelium had covered half of the substrate surface of the media in the bottle. Mycelium growth in 3000 ppm Cr levels up to 4 wk of incubation was excellent but has not formed fruiting bodies.
Fermented Rice Straw
Dry matter content of fermented rice straw was influenced by the fermentation time and Cr levels (P<0.01) but there was no interaction between the two factors (Table 1). Dry matter content declined sharply in the second week of fermentation, from 29.24% to 26.5%, suggesting that the nutrients contained in the substrate has been used by fungi.
Dry matter content of substrate continued to decrease up to 4 wk of incubation. It was suggested that the nutrients in rice straw was used by G. lucidum to increase the number of cells as indicated by the growth of mycelium. There was a linear relationship between the dry matter content of rice straw media with fermentation time according to the equation, Y= -1.11 X + 29.70 (R 2 = 0.8053, P <0.01 where Y= change in the levels of dry matter, %; X= length of fermentation, days). This means that as the fermentation time increased by one unit there was a reduction of dry matter by 1.11%. Ligninolytic enzyme produced by G. lucidum degraded lignin from the rice straw, and this was associated with fermentation time and the reduce of dry matter. Product of degraded lignin was used by G. lucidum to its mycelium growth.
Chromium supplementation appeared to decrease the rate of decline in dry matter content of rice straw substrate. Slowing decline in dry matter content of rice straw occurred at 1500 ppm Cr levels up to 3000 ppm. It is expected that high levels of Cr inhibits the growth of G. lucidum so that decomposition of organic material into H 2 O and CO 2 slowed and ultimately decreased the proportion of dry matter of fermented rice straw. However, mycelium growth was not impaired as it was observed that mycelium growth covered the media even at Cr levels of 3000 ppm.
The rate of change in the content of organic matter of fermented rice straw was similar to that of dry matter, which was somewhat influenced by fermentation time and Cr levels (P<0.01) (Figure 1). A decline in organic matter content was observed with increasing duration of fermentation from 0 to 4 wk. The decline of organic matter content was from 23.95% to 19:55% in fermentation time of 4 wk. Oxidases such as laccase from G. lucidum are enzymes which were involved in oxidative conversions of organic compounds and materials (Mtui, 2012).
This result showed that the proportion of inorganic materials with organic media remains the same for all the different levels of Cr. It is thought that the formation of organic matter occurred in the form of mycelium and was comparable to the decreasing levels of organic matter or dry matter content.
Fermented Rice Straw
The loss of dry matter and organic matter in fer-ry matter and organic matter in fermented rice straw was significantly increased (P<0.01) significantly increased (P<0.01) with increasing length of fermentation time. There was Note: Means in the same row or column with different superscript differ significantly (P<0.01). no interaction between the fermentation periods with Cr levels ( Table 2). Dry matter loss in rice straw increased with increasing duration of fermentation, followed by growth of the mycelium which filled the surface of the substrate and covered the growing medium. Dry matter loss begins in 2 wk (12.49%) and continued to increase up to 4 wk (17.19%). This condition suggests that with increasing dry matter loss, more nutrients were utilized by G. lucidum for the metabolism and growth of its mycelium. There was a linear relationship between the dry matter loss to the fermentation time of fermented rice straw by following equation: Y= 5.356X-2.35 (R 2 = 0.83, P<0.01 where Y= dry matter loss, %, X= length of fermentation, week), but rate of dry matter loss decreases with increasing Cr at a level of more than 2000 ppm Cr. This suggests that the levels of more than 2000 ppm Cr decreased the growth rate of G. lucidum. In our research, the dry matter loss in fermented rice straw have the different profile with the dry matter loss in oil palm byproduct fermented with G. lucidum. In contrast, in case of oil palm by-product, the increase of Cr level in substrate up to 3000 ppm Cr did not affect the growth rate of G.
Cr level
lucidum (Agustin et al., 2010). The loss of dry matter on cultivation of oyster mushroom in wheat straw substrate was 9.4% (Pant et al., 2006), while in this study, the loss of dry matter was 17.19%. This difference in result could be due to G. lucidum being a white rot fungus which is capable of producing ligninolytic enzyme (laccase, manganese peroxidase, lignin peroxidase) (Ke et al., 2011) and able to degrade fiber components of rice straw. The pattern of organic matter loss was the same with the dry matter loss in fermented rice straw in relation to fermentation time and levels of Cr in media. Means of organic matter loss in fermented rice straw was influenced by the fermentation time (P<0.01) and Cr levels (P<0.05). There was no interaction between Cr levels with long fermentation (Figure 2). Organic matter loss begins at week 2 (14.97%) and continued to increase until at week 4 fermentation period (21.88%). Increasing the rate of organic matter loss in rice straw showed an increase in the amount of nutrients utilized by G. lucidum for its metabolic activity and its cell growth.
At the early stage of fermentation, G. lucidum utilized the digestible nutrients contained in rice straw, after which the fungus utilized structural carbohydrates, especially complex compounds that are bound together with lignin. G. lucidum produces laccase enzyme which is capable of hydrolyzing lignin binding with other compounds (Baldrian, 2003;Ke et al., 2011). Increased organic matter loss decreased with increasing fermentation time. These results indicated that G. lucidum was able to hydrolyze and utilize structural carbohydrates and lignin.
Cr Incorporation in Fermented Products
The element Cr bound in media was probability bound with media components and was converted into mycelial cell component of G. lucidum. Chromium incorporation into the cell of G. lucidum (Figure 3) is thought to occur because it grew well in the rice straw substrate. The binding and incorporation of Cr was significantly affected (P<0.01) by the Cr levels of substrate. There was no interaction between Cr levels with fermentation time.
G. lucidum using Cr together with the use of organic matter of rice straw fiber components. This was indicated by the amount of Cr bound and incorporated in fermentation product. Incorporated Cr in fermentation product of G. lucidum was increase along with the higher Cr levels in substrate. The binding and incorporation of Cr in Cr levels of 1000 ppm was only 120 ppm Cr. The amount of incorporated Cr increase continuously until its value 1410.82 ppm Cr at the Cr levels of 3000 ppm based on dry matter. There was a linear relationship between the binding and incorporation of Cr with a Cr content of the substrate following the equation: Y= 296.72X -471.86 (R 2= 0.98, P<0.01 where Y= Cr bound, %; X= Cr content of the media, 500 ppm). This result showed that G. lucidum was more resistance to supplementation of Cr in high concentration because mycelia growth gave the organism a larger surface area, which provides greater protection to sensitive organelles of the fungi. In addition, fungi are eukaryotic cells, which contain more genes that can inhibit toxic metals (Sen & Dastidar, 2010;Shugaba et al., 2012).
The highest Cr level supplemented into rice straw substrate was 3000 ppm. The highest Cr incorporation value was also found on substrate with Cr level of 3000 ppm (Figure 3). The element Cr was needed in the nutrient metabolism of carbohydrates and protein of G.
lucidum cell for 4 weeks of fermentation. This indicates that G. lucidum was able to adapt to the medium with high levels of Cr. G. lucidum was also able to use the media components that are difficult to degrade.
G. lucidum was able to incorporate Cr into its cell proteins with the value of 9.29% (Table 3) when cultured in substrates containing 3000 ppm Cr. Some Cr was deposited in the neutral detergent fibre (NDF) (27.20%) and acid detergent fibre (ADF) (10.55%). These results showed that there has been a conversion of inorganic to organic forms of Cr by G. lucidum. Cr founded in the protein of G. lucidum cell is an organic compound. The highest efficiency of Cr incorporation was 53.58% showed in treatment with 3000 ppm Cr and fermentation time of 4 wk ( Figure 3). It has been suggested that chromium ions can be incorporated into G. lucidum cells by two step, the first step involves the surface binding of Cr (III) ions to the cell wall and extracellular material, which is not depend on metabolism process. The second step is depending on the metabolic activity because uptake Cr (III) into the cell across the cell membrane and at that time is occurred intracellular accumulation (Sen & Dastidar, 2010).
CONCLUSION
The loss of dry matter and organic matter in fermented rice straw was related to the growth of mycelium of G lucidum. The binding of Cr occurs by fiber matrix of substrate. Inorganic Cr conversion into organic forms by fungi G. lucidum on rice straw substrate was evidenced by the presence of Cr in protein of fermented product. Protein component of fermented product contained 9.29% Cr while in NDF and ADF was 27.20% and 10.55% Cr, respectively at level of 3000 ppm Cr. Chromium was incorporated into the G. lucidum cells during fermentation and G. lucidum is tolerant of high Cr levels up to 3000 ppm. | 2018-12-14T20:38:43.577Z | 2013-05-29T00:00:00.000 | {
"year": 2013,
"sha1": "12390f67760d82aa0b383af62884ed2c264ecb16",
"oa_license": "CCBYSA",
"oa_url": "http://medpet.journal.ipb.ac.id/index.php/mediapeternakan/article/download/6848/5259",
"oa_status": "GOLD",
"pdf_src": "Anansi",
"pdf_hash": "8b1394bd7462478e7368576fed232b96c18616ee",
"s2fieldsofstudy": [
"Agricultural And Food Sciences"
],
"extfieldsofstudy": [
"Biology"
]
} |
252597485 | pes2o/s2orc | v3-fos-license | Acute COVID-19 Presenting as Neurological Disease in a Pediatric Patient
A 14-year-old previously healthy male presented to our facility with a 5-day history of lower extremity myalgias and new onset paresthesias, weakness, and blurry vision. The patient’s symptoms began 5 days earlier with calf pain that progressed to involve his thighs and hips. The patient also started to complain of eye pain as well as blurry vision. He had 1 day of diarrhea and 2 days of non-bloody, non-bilious emesis that had resolved prior to presentation. The patient’s myalgias increased in severity, so his parents took him to an outside emergency room 1 day before presenting to our facility. There he was found to be positive for COVID-19 with all other labs largely unremarkable. The next day, the patient experienced new onset numbness progressing from his toes to his knees as well as his face. His father reported that he had developed a “knock-kneed” gait as well as nystagmus. This prompted the patient to be evaluated at our facility. During this period of symptoms, the patient was never febrile and did not develop other symptoms of cough, rhinorrhea, respiratory distress, chest pain, or headaches. The patient’s father had tested positive for COVID-19 1 or 2 weeks earlier with resolution of symptoms without complications. There were no known tick bites, though the patient and his father had gone on a hunting trip recently. Physical exam revealed a drowsy, ill-appearing male in no acute distress with unlabored breathing, clear lung sounds, and heart auscultation with regular rate and rhythm. Conjunctivae were injected bilaterally, and shotty non-tender anterior cervical lymphadenopathy was palpated. Significant neurological findings on exam were as follows: mild cranial nerve VI palsy bilaterally, nystagmus in all directions, 4+/5 weakness of bilateral hip flexion and knee flexion, dysmetria with finger-to-nose testing bilaterally and abnormal heel-shin, ataxic gait, and 0+ ankle reflexes bilaterally with asymmetric patellar reflexes (0-1+ on left, 2+ on right).
Hospital Course
At our facility, complete blood count, C-reactive protein, basic metabolic panel, hepatic function panel, urine drug screen, and urinalysis were largely unremarkable. Blood cultures showed no growth. Pediatric neurology was consulted and recommended magnetic resonance imaging (MRI) of the brain and lumbosacral spine as well as a lumbar puncture. Initial differential diagnoses included Guillain-Barre Syndrome (GBS) or Miller-Fisher syndrome variant, central brainstem inflammation or infection, and ADEM. Lumbar puncture revealed lymphocytic pleocytosis (cell count 72, 90% lymphocyte) with elevated protein (51) and normal glucose (57). Cerebral spinal fluid (CSF) gram stain was negative with no growth of CSF cultures. Cerebral spinal fluid cryptococcus, herpes simplex virus (HSV), varicella-zoster virus (VZV), west nile virus (WNV), and neuromyelitis optica (NMO) studies were all negative. Cerebral spinal fluid polymerase chain reaction (PCR) for COVID-19 was also negative. MRI brain revealed areas of increased signal on T2-weighted imaging (see Figure 1), and MRI lumbosacral showed increased signal in the conus medullaris without swelling or enhancement (see Figure 2), all suggestive of an acute disseminated encephalomyelitis process. After discussion with our pediatric neurologists, the patient's presentation was deemed most likely secondary to ADEM in the setting of a COVID-19 infection. The patient was started on IVIG and high dose solumedrol during admission and completed 5 days of inpatient treatment with full resolution of weakness, sensory deficits, and ophthalmoplegia. Only mild gait and balance deficits remained after treatment. He was discharged on a prednisone taper with scheduled Pediatric Neurology follow-up.
Discussion
Acute disseminated encephalomyelitis is an immunemediated, demyelinating disease that often occurs after a viral infection and is more common in children than adults. 1 Many different viruses have been associated with ADEM. 2 As the name implies, the disease causes encephalopathy and multifocal neurological deficits. 3 While the typical symptoms of COVID-19 involve the respiratory system, there are cases being reported of neurological symptoms being the primary complaint. 4 While there have been several cases of ADEM associated with COVID-19 reported in adult patients, [3][4][5][6][7] there are fewer cases reported in children. Souza et al presented the case of a 12-year-old girl with history of rash, headaches, and fever that progressed to bilateral motor weakness and eventual respiratory failure requiring intubation and mechanical ventilation. MRI of the brain and cervical spinal cord revealed acute and diffuse encephalomyelitis involving mostly the cerebral white matter, which in conjunction with a recent COVID-19 infection, led to the diagnosis of ADEM. Zoghi et al presented the case of a 21-year-old male with emesis and malaise progressing to weakness and paresthesias of the lower limbs as well as urinary retention and paraparesis of the upper extremities. Nasopharyngeal swabs were negative for COVID-19, but serum IgG levels for COVID-19 were elevated. MRI brain revealed bilateral hyperintensities of the internal capsule, and Axial T2-weighted imaging showed longitudinally extensive transverse myelitis as well as hyperintensities of the dorsal upper pons.
The International Pediatric MS Study Group proposed the following clinical diagnostic criteria when considering ADEM in children: "A first clinical event with a presumed inflammatory or demyelinating cause, with acute or subacute onset that affects multifocal areas of the CNS; the clinical presentation must be polysymptomatic and must include encephalopathy." 2 The required encephalopathy may be in the form of behavioral changes or altered mental status that cannot be explained by other etiologies, has not occurred before, and is followed by clinical and/or MRI improvement. 2 No new clinical or MRI findings emerged 3 months or more following the onset of the event. 2 Brain MRI with FLAIR or T2-weighted images is the preferred imaging modality and usually shows multiple bilateral, asymmetric hyperintensities in central and subcortical white matter that may or may not involve cortical gray-white matter junctions. 8 The most common form of ADEM is a monophasic disease that remits with no recurrence; however, if relapses occur with new lesions, it is defined as multiphasic ADEM. 2 Cerebrospinal studies are often normal or reveal lymphocytic pleocytosis. 2 First-line treatment is with high-dose intravenous corticosteroids, with IVIG and less commonly plasma exchange as second-line options. 2,8 Although COVID-19 is mainly thought to cause primary respiratory disease, there are increasing reports of neurological sequelae or primary neurological disease associated with COVID-19 positivity. Neurological symptoms in adults have ranged from benign anosmia, ageusia, and headache to more significant conditions including encephalopathy, acute hemorrhagic necrotizing encephalopathy, ADEM, GBS, and stroke. 1,9 Krueger et al presented a case series of pediatric patients exhibiting both central and peripheral neurological symptoms deemed secondary to COVID-19 infection. These different manifestations included sensory and motor polyradiculopathy in a 16-year-old female who was simultaneously positive for COVID-19 and dengue in the CSF; GBS in a 15-year-old male with improvement in symptoms after IVIG treatment; acute intracranial hypertension in a 5-year-old female with improvement in symptoms following lumbar puncture and acetazolamide; and focal epileptic seizures in a 2-month-old male who responded well to antiepileptic medication. 10 Stroke and rhabdomyolysis have also been described in pediatric patients in the setting of COVID-19 infection. 11 It is important to note that while multisystem inflammatory syndrome in children (MIS-C) secondary to COVID-19 can present with varying symptomatology, neurologic symptoms such as headache, altered mental status, dysphagia, ataxia, and encephalopathy have been reported. 12 While anosmia and ageusia have become almost synonymous with COVID-19 infection recently, those neurological symptoms have been reported with other viruses as well. 13,14 Though the coronavirus family of viruses is not known as a common cause of neurological disease, they have been shown to cause direct CNS infections and para-infectious disorders, with para-infectious disorders such as GBS, ADEM, and transverse myelitis likely more common than direct viral invasion. 14,15 Mechanisms of COVID-19 neuroinvasion are thought to include direct viral injury, effects of the host immune response after an acute infection such as in GBS, indirect injury due to systemic disease, and/or overactivation of the immune response via cytokine release. 6 Studies have found that sustained CNS inflammation is associated with increased CNS demyelination in animal models. 6 ACE2 receptors expressed on neurons and glial cells have been identified as a potential target of interaction between the virus and host cell, 9,16 and the olfactory bulb has been considered as another potential target of entry for COVID-19 to gain access to the CNS. 6,16 As research continues on the novel coronavirus, a better understanding of the impact of COVID-19 on the central nervous system will help guide diagnoses and therapies in all age groups affected by the virus.
Conclusion
While research on the neurological effects of COVID-19 is ongoing, neurological manifestations of COVID-19 are not uncommon. Acute disseminated encephalomyelitis should be a considered a diagnosis in patients presenting with encephalopathy and/or multifocal neurological symptoms in the setting of a recent COVID-19 infection, even without respiratory symptoms. 3 Although there have been few cases of ADEM secondary to COVID-19 reported in children, the clinician should still consider this on the differential along with others such as transverse myelitis, GBS or Miller-Fischer Variant, meningitis, and encephalitis. The patient presented here displays that the novel COVID-19 viral infection can cause ADEM.
Author Contributions
All authors contributed to the acquisition and/or interpretation of data; the design, drafting, and/or revising of the work; and the final approval of the work to be published.
Declaration of Conflicting Interests
The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.
Funding
The author(s) received no financial support for the research, authorship, and/or publication of this article.
Ethical Approval
Verbal informed consent for publication of their clinical presentation and course was obtained from the patient and his father. | 2022-09-30T06:17:38.447Z | 2022-09-28T00:00:00.000 | {
"year": 2022,
"sha1": "8bd55ba6a8edc52a89079a899aca21a04c31467e",
"oa_license": null,
"oa_url": null,
"oa_status": null,
"pdf_src": "Sage",
"pdf_hash": "053d3510b87e7f27e1331f64a2d0aa3139a27470",
"s2fieldsofstudy": [
"Medicine"
],
"extfieldsofstudy": [
"Medicine"
]
} |
552713 | pes2o/s2orc | v3-fos-license | Increased dietary protein in the second trimester of gestation increases live weight gain and carcass composition in weaner calves to 6 months of age
Genetically similar nulliparous Polled Hereford heifers from a closed pedigree herd were used to evaluate the effects of dietary protein during the first and second trimester of gestation upon foetal, placental and postnatal growth. Heifers were randomly allocated into two groups at 35 days after artificial insemination (35 days post conception (dpc)) to a single bull and fed high (15.7% CP) or low (5.9% CP) protein in the first trimester (T1). At 90 dpc, half of each nutritional treatment group changed to a high- or low-protein diet for the second trimester until 180 dpc (T2). High protein intake in the second trimester increased birth weight in females (P=0.05), but there was no effect of treatment upon birth weight when taken over both sexes. Biparietal diameter was significantly increased by high protein in the second trimester with the effect being greater in the female (P=0.02), but also significant overall (P=0.05). Placental weight was positively correlated with birth weight, fibroblast volume and relative blood vessel volume (P<0.05). Placental fibroblast density was increased and trophoblast volume decreased in the high-protein first trimester treatment group (P<0.05). There was a trend for placental weight to be increased by high protein in the second trimester (P=0.06). Calves from heifers fed the high-protein treatment in the second trimester weighed significantly more on all occasions preweaning (at 1 month (P=0.0004), 2 months (P=0.006), 3 months (P=0.002), 4 months (P=0.01), 5 months (P=0.03), 6 months (P=0.001)), and grew at a faster rate over the 6-month period. By 6 months of age, the calves from heifers fed high nutrition in the second trimester weighed 33 kg heavier than those fed the low diet in the second trimester. These results suggest that dietary protein in early pregnancy alters the development of the bovine placenta and calf growth to weaning.
Introduction
It is well established from epidemiological studies in human populations and experimental studies in a range of animal models that varying maternal nutrition during critical periods of foetal development can alter or 'program' body mass and body composition in later life (Symonds et al., 2004;Micke et al., 2010a;Micke et al., 2011a;Micke et al., 2011b). Range cattle managed under extensive conditions experience such variations in maternal nutrition sufficient to affect foetal programming and thereby the postnatal growth and carcass characteristics of their progeny Greenwood et al., 2006;Martin et al., 2007;Micke et al., 2010a;Summers et al., 2015). Maternal nutrient supply to the foetus regulates the foetal IGF axis (Oliver et al., 1996;Sullivan et al., 2009a) and can programme the postnatal IGF axis (Micke et al., 2010a;Symonds et al., 2012). Calf plasma IGF-1 at birth is positively associated with birth weight (Breier et al., 1988;Micke et al., 2010a), as is postnatal IGF concentration with average daily gain (ADG) and linear growth (Lund-Larsen et al., 1977;Micke et al., 2010a;Micke et al., 2010b). The reported effects of foetal programming upon ADG and carcass development in cattle progeny is however inconsistent (Greenwood et al., 2005;Micke et al., 2011b;Summers et al., 2015). The disparity recorded between these studies may be influenced by the genetic heterogeneity, and age of the dams observed as well as timing of intervention: the Greenwood et al. (2005) study used both pluriparous and nulliparous dams, whereas the Micke et al. (2011b) and Summers et al. (2015) only nulliparous, with each study using different relatively diverse genotypes and intervention periods.
In this study, nulliparous Polled Hereford heifers from a closed stud herd mated at 15 months of age were used, ensuring a reduction in genetic variation within the dams and enabling focus upon the adolescent yearling heifer which has been shown to be more susceptible to foetal growth restriction following gestational nutritional perturbation (Copping et al., 2014;Hernandez-Medrano et al., 2015). Based on our former studies (Perry et al., 1999;Sullivan et al., 2009b;Micke et al., 2010a;Micke et al., 2015), we hypothesise that low protein in the first trimester will enhance placental development and in the second trimester will result in reduced growth and carcass muscling in the offspring.
Materials and methods
Project animals management and treatments All procedures were performed with the approval of the University of Queensland Animal Ethics Committee, approval number SVS/748/08. Genetically similar Polled Hereford heifers (n = 80),~15 months old from the same closed pedigree herd, were selected for inclusion in this trial. Preceding artificial insemination, the heifers were weighed and their reproductive tract palpated for normality. Oestrus was synchronised by insertion of intravaginal progesterone implants ('EAZI-BREED CIDR-B'; Genetics Australia, Bacchus Marsh, VIC, Australia) for 11 days, with an injection of 12.5 mg of dinoprost tromethamine (Lutalyse; Upjohn Pty Limited, Rydalmere, NSW, Australia) and 500 IU of equine chorionic gonadotrophin (ECG) on the day of CIDR removal. Approximately 48 h after the CIDR implants were removed, the heifers were all artificially inseminated on the same day without detection of oestrus. The sire used was a Polled Hereford bull with an estimated breeding value for birth weight of +2.1 kg at 86% reliability.
The study design was a 2 × 2 factorial design. The heifers were stratified by body weight and randomly allocated to two equal first trimester (T1) dietary treatment groups, high (15.7% CP) and low (5.9% CP) protein at 14 days post conception (dpc). Pregnancy diagnosis was completed by manual palpation at 35 days following insemination (taken as 35 dpc). As T1 treatment did not start at conception, the periconception period is, therefore, not addressed. After pregnancy diagnosis, all non-pregnant animals were removed from trial (n = 21). Following an outbreak of Bovine Ephemeral Fever during trimester 1, 17 heifers were recorded to have aborted and were excluded from the study. The remaining numbers of calves in each treatment period were: T1 high protein, n = 10 consisting of six male and four female calves; and T1 Low protein, n = 11 consisting of seven male and four female. At 90 dpc half of each nutritional group was changed to a high (15.6% CP) or low (6.1% CP) protein treatment until 180 dpc (T2). Numbers of animals in T2 high protein: n = 13 consisting of 10 male and three female; and in T2 low protein: n = 8 consisting of three male and five female. This factorial design gave rise to four treatment groups: high/high (HH; four males and two female), high/low (HL; two males and two females), low/high (LH; six males and one female), low/low (LL; one male and three females). After 180 dpc heifers were run together on the same native pasture during the final trimester until term. The diet for this final trimester consisted of native pasture, containing grasses and medicago polymorpha with a CP value ranging between 10% and 13% over the trimester. Both dietary groups of heifers received a grain ration and either ad libitum pasture hay (low) or pasture grazing (high). Details on dry matter intake (DMI) calculations are given in (Table 1).
Before calving, heifers were individually placed in a small yard. Measurements were taken of the newborn calf before suckling (birth weight (BIRW) and biparietal diameter (BPD)), and the foetal portion of the placenta was collected Estimates of DMI from pasture and hay were calculated based upon the National Research Council (NRC) energy requirements for replacement Bos taurus pregnant heifers with a mature weight of 500 kg and a calf birth weight of 32 kg. As the sorghum and copra meal energy content were known (12 and 11.2 MJ/kg DM, respectively) and the heifers on the low-protein diet averaged 340 kg with a rate of gain of 0.8 kg during the first trimester, the DMI of pasture and hay was calculated based upon the energy requirement sufficient to sustain this rate of gain. Similar DMI estimations were completed in the second and third trimesters based on the rate of gain in each treatment group.
2 NRC comparison to ration to NRC (1996) recommended nutrient requirements for pregnant yearling Bos taurus replacement heifers with calf weight of 32 kg.
Miguel-Pacheco, Curtain, Rutland, Knott, Norman, Phillips and Perry immediately upon expulsion. Placental measures included wet weight, cotyledon number and wet weight of cotyledons (CWW) post separation from adjoining membranes. The dry cotyledon weight (CDW) measure was obtained by drying cotyledons overnight at 100°C and then weighing at 1 h intervals, until the same weight was recorded on three consecutive weightings.
After calving, all the heifers were grazed together with their calves on native pasture. Weights from the heifers and calves were recorded monthly. At 6 months of age, ultrasound (model Aloka-500 ® ; Aloka Inc., Tokyo, Japan with 3 MHz linear probe) was used to assess fat depth at the P8 (rump) and 12th rib (FT12) sites (Hopkins, 1989). Anal Fat Fold (AFFT), the thickness of skin and subcutaneous fat situated between the point of the ischium and the base of the tail, was assessed using techniques described by Johnson (1994) by a single experienced technician using calibrated calipers. Heifers were fasted for 6 h and weighed to obtain their empty live weight (ELW). ELW and AFFT were used to calculate the percentage carcass components (muscle, fat and bone) following the published regression equations in Bos taurus Hereford cattle by Johnson (1994).
Quantitative analysis of placental tissue The placenta was immediately collected after expulsion (Stage 3 of labour varying between 0.5 and 6 h) at term and weighed whole before excision of all cotyledons from the surrounding membranes (Perry et al., 1999). Those not completely expelled by 6 h post calving were considered retained foetal membranes and not used. One small (average 4.4 g), one medium (average 24.2 g) and one large cotyledon (average 55 g) were removed from the gravid horn, fixed in neutral buffered formalin (10%) for 48 h, processed and embedded in paraffin wax. Two-micron sections were cut from each cotyledon and stained with haematoxylin-eosin (H&E) with a further section stained with Masson's Trichrome.
Quantitative analysis was used to calculate volume densities and relative total component volumes of major cellular components, surface density and barrier thickness of trophectoderm. An L-36 Merz grid was used in conjunction with a video image analysis system as detailed by Perry et al. (1999) for morphometric analysis, at a final magnification of 250 × (Nikon 80i microscope with Nikon DS camera system software; Nikon, Tokyo, Japan). The points falling on each structure or component were manually assigned using morphological features appropriate to H&E or Masson's Trichrome as appropriate, blood vessels were not differentiated between arteries, veins or capillaries. Ten systematic random fields of each cotyledon, of each size, were analysed (three cotyledons per cow), thus 30 fields were analysed per animal to calculate structural quantities (Weibel et al., 1966). The cellular components that were measured and counted by point and intersection counting were: trophectoderm, connective tissue matrix, connective tissue fibroblasts and connective tissue blood vessels.
Equations detailed by Weibel et al. (1966), were used to calculate relative volume densities of each component, surface density and mean barrier thickness of the foetal trophectoderm. The relative volume densities (calculated at 1 g of placenta occupying 1 cm 3 ) in conjunction with the CWW (collected as detailed above) of each placenta were used to calculate relative total component volumes of cellular components. The surface area of the foetal trophectoderm was also calculated from the number of times the lines on the Merz grid intersected the surface of the trophectoderm. Volume density = number of points falling on structure/total number of test points (V d = P a /P T ). Relative volume of each component = volume density × placental weight in grams (V c = V d × weight (g)). Surface density = 2 × number of intercepts/total length of lines (S v = 2 × L a /L T ). Barrier thickness = trophoblast volume density/surface density of the trophoblast (B T = D d /S v ) (Weibel et al., 1966;Roberts et al., 2001). Every photomicrograph was high quality and could be counted, resulting in 1080 point counts per animal.
Statistical analyses
The study used a 2 × 2 factorial design, with factors being nutrition in the first trimester (T1) and the second trimester (T2). Statistical analyses were performed using StataCorp. 2015 (Stata Statistical Software: Release 14; StataCorp LP, College Station, TX, USA), and significance was set at P < 0.05 for all the results. Normality and equality of variance was checked before any analysis and transformation of the data was applied if it was necessary.
Birth calf weights were analysed using ANOVA with T1 × T2, with sex used as covariate. Birth weight and the monthly calf weights were all subjected to ANOVA with repeated measures, and sex and calf age were added as covariates. For each calf the ADG (kg/day) between birth and the final weighing was computed. This calculation involved just the first and last set of weights. The linear growth rate (g/day), which involved fitting a straight line to the weight data and estimating the slope, was also calculated yielding the linear growth rate. Both calculations gave very similar results with live weights and growth rates showing the same response to the treatments.
Muscle, fat and bone in the live animal were considered as three separate dependent variables, against the fixed effects of nutrition in T1 × T2 and sex. The effect of group was also analysed. ANOVA was used to examine treatment effects upon placental tissues adding calf sex as covariate. Correlation coefficients between treatment effects and other parameters were also calculated using Pearson's correlation coefficient and were also considered statistically significant if P < 0.05.
Placental gross morphometry
There was a trend for placental weight to be increased in the high-protein second trimester treatment group (P = 0.06), Heifer gestational diet increases calf growth but there was no significant effect upon CWW and CDW. In addition, no significant differences were observed between treatments in relation to cotyledon number (Table 2).
Placental cellular components Fibroblast density was increased by the high-protein treatment in the first trimester (P = 0.01; Table 3), whereas low protein intake in the first trimester increased trophectoderm volume (P < 0.05; Table 3). There were, however, no significant effects of treatment upon other density measures of cellular components, matrix density and relative blood vessel volume density (BVVD). There were no statistically significant interactions between treatments for any of the cellular components measured (Table 3).
Calf measures at birth High protein in the second trimester significantly increased BPD (P = 0.05; Table 2), with a greater effect in female progeny (P = 0.02). There was a similar effect of second trimester high-protein diet increasing birth weight in female progeny (P = 0.05), however no effect of treatment on overall birth weight. There was no interaction between treatment and sex. There was increased variability in the birth weight of male calves.
Relationships between birth weight and placental parameters BIRW was positively correlated with placental weight, CWW, and cotyledon number, with a tendency for a positively The observed increase in placental weight by second trimester high-protein treatment was below this level at P = 0.06. Values are unadjusted mean and ± SEM of the cellular composition of bovine placenta by treatment group. Volume densities are a proportion, dimensionless numbers as they are a ratio of two numbers = cm 0 ; Surface density measures surface area per volume or weight of tissue (g of cotyledon) (cm 2 /cm 3 ) = cm − 1 ; Barrier thickness of trophectodermlinear measurement = cm; relative volume of each component assumes 1 g of placenta occupies 1 cm 3 = cm 3 . a,b Different alphabetic superscripts denote mean values that are significantly different (P < 0.05) from each other.
Miguel-Pacheco, Curtain, Rutland, Knott, Norman, Phillips and Perry correlation with CDW. The strongest correlation being with CWW (r = 0.65, P = 0.003; Table 4). Relative matrix volume (P = 0.008) and relative blood vessel volume (BVV) (P = 0.03) were the only cellular parameters correlated with BIRW (Table 4) Preweaning growth of calves Calf live weights and growth rates showed a similar response to the treatments, whereby there was no effect of nutrition in trimester one on ADG, and there was no sex effect observed on ADG and no interaction between protein levels fed in trimesters one and two (P > 0.05). There was a significant effect of nutrition in trimester 2 on ADG (P = 0.01) (Figure 1). Calves from heifers fed the high-protein diet in the second trimester weighed significantly more on all occasions (at 1 month (P = 0.004), 2 months (P = 0.006), 3 months (P = 0.002), 4 months (P = 0.01), 5 months (P = 0.03), 6 months (P = 0.01), and grew at a faster rate over the 6-month period (Figure 1). By 6 months of age, the calves from the heifers fed the high-protein diet in the second trimester weighed 33 kg heavier than those fed the low diet in the second trimester (16% heavier).
In the live carcass measures, there was a sex effect on the amount of muscle (P = 0.0001) and fat (P = 0.0007), but not bone (P = 0.91); males had increased muscle (59.67 ± 0.26) and less fat (23.15 ± 0.38) compared with females (muscle: 57.04 ± 0.51, and fat: 26.71 ± 0.76). For all three variables, there was a significant effect of nutrition in trimester 1 and a significant interaction between nutrition fed in the two trimesters. Decreased protein during trimester 1 led to increased muscle (P = 0.002) and decreased fat (P = 0.002). Animals in the HL group had significantly lower muscle and bone percentages and significantly higher fat percentages than animals in the LL group. No other group was different from another in either muscle bone or fat (Table 5).
Discussion
This study provides clear evidence that nutrition during the first two trimesters of pregnancy alters the growth and body phenotype of the calf. Importantly, decreasing genetic variability in the heifer dams enabled this clear effect of gestational protein manipulation upon progeny growth to be revealed. The analysis of weight gain of progeny supports the hypothesis that providing heifers with a high-protein diet in the second trimester resulted in a faster growth rate and a significant increase in body weight (~33 kg) in their calves at 6 months of age when compared with calves born to heifers fed low protein in the second trimester. Further, this study also shows early gestational dietary protein affects the developing placenta and supports the hypothesis that reduced protein in the first trimester increases physiologically important components of placental development. These findings are of significant interest to the cattle industry in Figure 1 Mean and ± SEM of calf body weight from birth (0 months) until 6 months of age by treatment during the second trimester. Levels of significance indicated by *P < 0.05, **P < 0.01 and ***P < 0.001, respectively.
Heifer gestational diet increases calf growth
Australia as protein is the most deficient nutrient in the Australian rangelands (Norman, 1963;Sullivan et al., 2009a). This downside to utilising these high-value pedigree animals and the farm site of this study was a reduction in the level of intervention permitted. This included prevention of dietary intervention before 35 dpc, which is known to affect foetal development (Copping et al., 2014;Hernandez-Medrano et al., 2015).
Poor maternal dietary protein in early gestation has been shown to impair bovine foetal growth (Long et al., 2009;Micke et al., 2010b;Copping et al., 2014), which was confirmed in this experiment. Low dietary protein intake in the second trimester decreased BPD similar to previous experiments although birth weight was not affected as comprehensively as previously reported Micke et al., 2010c), being only significant in the female. The relatively small numbers in this experiment compared with the Micke paper (Micke et al., 2010c) may have influenced this result as variability was greater in the male calves. Furthermore, birth weight at term may not be indicative of intrauterine growth restriction (IUGR) in earlier gestation (Long et al., 2009;Hernandez-Medrano et al., 2015) in the bovine. BPD and BIRW had a strong positive relationship with placental weight and the placental parameters of BVV, CWW and CDW, concomitant with previous studies (Perry et al., 1999;Sullivan et al., 2009b). There was a trend (P = 0.06) for placental weight to be increased by increased dietary protein in the second trimester, whereas trophectoderm volume was increased by high-protein first trimester as previously described (Perry et al., 1999). The initial rapid growth of the placenta has been linked to the rapid and vigorous development of the foetal trophoblast through proliferation and branching of the foetal villi into the maternal stromal tissue (Bell et al., 1999). Interdigitation between the foetal trophectoderm and maternal microvilli is complete by 28 dpc in the bovine (Wooding and Beckers, 1987), which is before the initiation of our dietary treatments. Placentome weight and surface area in the bovine however, continually increase until term unlike the ovine in which placentome weight is constant or decreasing from 65 dpc (Baur, 1977;Reynolds and Redmer, 1995). The observed increase in trophectoderm volume in the lowprotein heifers during the first trimester may enhance the functional potential of the placenta during this period of maximal villi development and thereby enable an increased nutrient supply to the foetus during later gestation if protein availability increases. Supporting evidence suggesting that the protein restricted bovine foetus signals to the placenta an increased requirement for nutrients is found in its ability to increase blood supply via the uterine artery and increased cotyledonary vasculature at 125 dpc.
The relative vasculature proportions in the foetal cotyledons were not altered in this study, although there was a positive correlation between calf birth weight and vasculature. The literature shows that vascular components Table 5 Anal fat fold (AFFT) measurement and body composition of calves at 6 months of age according to treatment groupEquations used to calculate the body composition were taken from Johnson (1994). Different alphabetic superscripts denote group mean values that are statistically significantly different from each other, a, b and c for P Miguel-Pacheco, Curtain, Rutland, Knott, Norman, Phillips and Perry of the placenta vary greatly depending upon environmental perturbations, dam age, the timing of perturbation and timing of cotyledon excision. Gestational undernutrition found decreased bovine cotyledonary vasculature in the later, but not early stages of placental development, whereas Zhu et al. (2007) showed nutrient restriction increased cotyledonary vascularity at day 125 with no affect at day 250. Furthermore, cows in their third or greater pregnancy did not show effects upon placental composition observed in younger heifers (Long et al., 2009). This may illustrate a window and age-specific effect of nutrition upon placental vascularisation. Connective tissue fibroblast density was increased (P < 0.05) in the placenta of heifers receiving the highprotein dietary treatment in the first trimester (HH, HL) a similar finding to that of a previous study by Roberts et al. (2001) in sheep, where maternal dietary restriction (70% of recommended intake) reduced the total placental surface area for exchange, and the surface density of trophoblast. However, the arithmetic mean barrier thickness for diffusion in this study was increased by this maternal food restriction (Roberts et al., 2001). Similarly, in the current study barrier thickness of the trophectoderm was increased by protein restriction. It has been suggested by Perry et al. (1999) that a restriction in protein in the first trimester in heifers may lead to a larger placenta at term due to the greater development of the microvilli, particularly if this early period of restriction is followed up by a phase of improved nutrition. Indeed the LH group did have the largest CWW (Table 2), but this was only significantly greater than CWW in the HH group (P = 0.03). It has been suggested (Talbot et al., 2000;Shimada et al., 2001;Dunlap et al., 2006) that trophoblasts from ungulates (including cattle), proliferate in the absence of fibroblast growth factors (FGFs), suggesting that the role of FGFs is more limited in maintaining the trophoblast lineage in cattle in comparison with rodents. It has also been shown that fibroblasts and FGFs play a role in angiogenesis, including within the placenta (Klagsbrun and D'Amore, 1991), therefore a concomitant increase in vasculature within these placentae may be expected.
These results suggest that maternal diet restriction affects the structure and function of the placenta as previously reported (Perry et al., 1999;Long et al., 2009;Sullivan et al., 2009b;Sullivan et al., 2009c). Such perturbations to placental development may reduce foetal growth due to decreased nutrient transport via the placenta (Sullivan et al., 2009c) generating a decrease in birth weight (Perry et al., 1999;Micke et al., 2010c), or alternatively dichotomous placental development (as in the LH group) may be attendant to increased birth weight and associated dystocia.
Interpretation of the carcass composition results requires consideration of the stages of foetal bovine muscle development, which are predominantly controlled by IGF-2 expression that peaks between 150 and 160 dpc (Gerrard and Grant, 1994;Florini et al., 1996). The first wave of differentiation however occurs much earlier when primary skeletal muscle fibres (type I) differentiate from primary myotubes at 39 dpc (Robelin et al., 1993), the second at 90 dpc results in secondary fibres; and a third at 110 dpc gives rise to tertiary fibres (Gagniere et al., 1999). These critical events occur during the dietary treatments imposed in this study.
As myocytes are formed from a pool of pluripotent stem cells (Oksbjerg et al., 2004) extrauterine signals such as those regulated by maternal nutrient intake, may affect the number of cells committed to myoblast formation whilst also affecting the rate of myoblast proliferation and thus final myofibre number. It has previously been reported that gestational dietary regimens effectively alter placental signalling hormones (Sullivan et al., 2009c;Summers et al., 2015). These may act to signal foetal skeletal muscle IGF messenger RNA (mRNA) expression and fibre development during the first two trimesters of gestation as previously shown in foetal skeletal sheep muscle where nutritional restriction increased IGF-2 mRNA expression (Brameld et al., 2000) and increased IGF receptor activity (Symonds et al., 2012). Myofibre density is also reduced by maternal nutrient restriction (Costello et al., 2008). As primary fibres provide the scaffolding for secondary fibre formation, a reduction of primary fibre density resulting from decreased maternal nutrient intake during the first trimester may not be fully compensated for by the provision of increased maternal nutrient intake during the latter stages of gestation. In support of this (Micke et al., 2011b), found that the majority of effects of maternal protein intake upon IGF mRNA expression in adult progeny skeletal muscle occurred during the first trimester of gestation suggesting these early stages of foetal development to be the most sensitive to altered nutritional environment. This is attributed to the high rate of cellular differentiation and proliferation during the early stages of gestation compared with the high rate of cellular hypertrophy during the latter stages (Brameld et al., 2000;Tong et al., 2009). This study is a corollary of previous findings (Micke et al., 2011b) as decreased protein during trimester 1 (LL and LH combined) increased muscle (P = 0.002) and decreased fat (P = 0.002) in progeny compared with those animals whose dams received high protein during this period. Though, the predicted values for carcass components presented in this paper are representative of the data presented in Johnson (1994), it is necessary to acknowledge that these predicted measurements may be limited in their inference and accuracy when compared with 'real' carcass measures taken at slaughter.
The treatment effects upon carcass muscle and fat in this study reflect the reciprocal relationship between the development of muscle and adipose tissue, previously reported (Micke et al., 2010a) whereby the in utero diet alters differentiation of the mutual precursor cells of adipocytes and myocytes (Symonds et al., 2012). Furthermore, Micke et al. (2011a) also indicated a possible causal mechanism via the effect of maternal diet upon adipogenic gene expression. The exposure of the developing calf foetus to a high maternal protein intake during the first trimester may result in a relative increase in myostatin gene expression in skeletal muscle as in other species (Liu et al., 2011) and Heifer gestational diet increases calf growth this may result in a decrease in muscle fibre number and an increase in the commitment of stem cells within the muscle to form preadipocytes. This effect may, however, be offset in part by an upregulation of the expression of IGF-1 receptor (IGF-1R) as discussed above which occurs in the progeny of such heifers (Micke et al., 2011b).
In this study the lowest muscling occurred in the HL group (P = 0.05). As the secondary muscle fibres are the largest contributor to postnatal muscle mass of cattle and begin to form at 90 dpc (Robelin et al., 1993) nutrient restriction during the second trimester may have decreased formation of secondary muscle fibres during myoblast proliferation as reported in sheep (Quigley et al., 2005). Importantly, however, both circulating maternal and progeny IGF-1 has been shown to increase following gestational high dietary protein (Perry et al., 2002;Sullivan et al., 2009a;Micke et al., 2010a). This combined effect in the progeny that experienced increased protein in utero during the second trimester may have produced the increased growth rates observed. | 2017-04-27T08:35:35.871Z | 2016-11-08T00:00:00.000 | {
"year": 2016,
"sha1": "9f7e64b35b3c1e8ddd9b8f117712a30e1d784e53",
"oa_license": "CCBY",
"oa_url": "https://www.cambridge.org/core/services/aop-cambridge-core/content/view/F074C0E360136AD81D19002AC29D86F7/S1751731116002330a.pdf/div-class-title-increased-dietary-protein-in-the-second-trimester-of-gestation-increases-live-weight-gain-and-carcass-composition-in-weaner-calves-to-6-months-of-age-div.pdf",
"oa_status": "HYBRID",
"pdf_src": "PubMedCentral",
"pdf_hash": "f70dd4b23a7f9b32808c1ba9a997f8367a119bf3",
"s2fieldsofstudy": [
"Agricultural And Food Sciences"
],
"extfieldsofstudy": [
"Biology",
"Medicine"
]
} |
90436562 | pes2o/s2orc | v3-fos-license | A deep learning approach to pattern recognition for short DNA sequences
Motivation Inferring properties of biological sequences--such as determining the species-of-origin of a DNA sequence or the function of an amino-acid sequence--is a core task in many bioinformatics applications. These tasks are often solved using string-matching to map query sequences to labeled database sequences or via Hidden Markov Model-like pattern matching. In the current work we describe and assess a deep learning approach which trains a deep neural network (DNN) to predict database-derived labels directly from query sequences. Results We demonstrate this DNN performs at state-of-the-art or above levels on a difficult, practically important problem: predicting species-of-origin from short reads of 16S ribosomal DNA. When trained on 16S sequences of over 13,000 distinct species, our DNN achieves read-level species classification accuracy within 2.0% of perfect memorization of training data, and produces more accurate genus-level assignments for reads from held-out species than k-mer, alignment, and taxonomic binning baselines. Moreover, our models exhibit greater robustness than these existing approaches to increasing noise in the query sequences. Finally, we show that these DNNs perform well on experimental 16S mock community dataset. Overall, our results constitute a first step towards our long-term goal of developing a general-purpose deep learning approach to predicting meaningful labels from short biological sequences. Availability TensorFlow training code is available through GitHub (https://github.com/tensorflow/models/tree/master/research). Data in TensorFlow TFRecord format is available on Google Cloud Storage (gs://brain-genomics-public/research/seq2species/). Contact seq2species-interest@google.com Supplementary information Supplementary data are available in a separate document.
Introduction
Many important problems in bioinformatics--including taxonomic profiling, protein family assignment, and detection of regulatory elements--require a method of accurately inferring meaningful labels from short sequences, which we call the sequence-labeling problem. A common approach to labeling these query sequences is to build a database of reference sequences for the property of interest, map the unknown queries to the database using k -mer matching or pairwise-sequence alignment, and finally use the mappings and their scores to assign labels. For example, many top-performing taxonomic binning and profiling tools use an initial k-mer mapping or local alignment of each read and then integrate these per-read mappings into higher-level taxonomic mixture estimates. 5,[32][33][34][35] Hidden Markov Models (HMMs), which are widely used for annotating genes and regulatory elements, represent an alternative approach wherein one builds a statistical model from the relevant database and then uses this model to assign labels to queries. [38][39][40]49 Here, we aim to assess how valuable deep learning might be in approaching these types of sequence-labeling problems using taxonomic classification of short reads of 16S ribosomal DNA as an initial benchmarking problem due to its appropriate level of difficulty and significant practical applications.
The flexibility of deep neural networks (DNNs) and their ability to learn complex patterns make them a promising choice for solving the general sequence-labeling problem. String-matching approaches work well, but are often unable to accurately capture the full complexity of the sequence/label relationship--for example due to loss of positional information in k -mer methods or oversimplified affine gap penalties 41 used in alignment tools like the Basic Local Alignment Search Tool (BLAST) 27 or Burrows-Wheeler Aligner (BWA). 25,26 Recent work using DNNs to learn regulatory elements patterns 42 provide further evidence that neural networks are better at modeling complex dependencies between sequence positions than methods like HMMs. [38][39][40]49 Moreover, using DNNs for sequence-labeling could provide significant performance advantages by leveraging hardware accelerators. We, therefore, design a new DNN architecture to solve the sequence-labeling problem, and explore its behavior on an important example problem to gain initial insights into its behavior and potential utility to the bioinformatics community.
Many sequence-labeling problems in bioinformatics could be used to benchmark our proposed DNN; here we explore that of assigning species-level taxonomy to short reads of 16S ribosomal DNA. This task is inherently challenging due to the presence of closely related organisms and conserved regions, and its difficulty can be easily modulated by holding out information based on phylogeny. Moreover, this problem is both well-characterized--having been used in microbial phylogeny for over forty years 1,2 --and practically important--evidenced by growing public repositories of 16S sequencing data 3,4 and the recent Critical Assessment of Metagenomic Interpretation (CAMI). 7 CAMI's top-performing taxonomic binning and profiling tools use initial k -mer matching (PhyloPythiaS+ 32 and Kraken 33 ) and local alignment (taxator-tk, 34 MEGAN, 5 MetaPhlAn, 6 and MetaPhyler 35 ) steps. Other existing approaches to read-level taxonomic labeling use k -mers via naive Bayes, probabilistic topic models, and Markov models, but tend not to outperform BLAST for typical sequence lengths. 8,9,31,36 Early studies using neural networks for taxonomic classification from DNA barcodes provide initial support for a deep learning approach, but lack sufficient data and experimental validation to provide meaningful guidance on whether a modern DNN solution has any practical advantages over existing tools. 10,11 Despite the recent increase in successful applications of deep learning to genomics, [46][47][48] to our knowledge no previous work demonstrates that DNNs can classify genomic sequencing data of realistic scales at fine taxonomic resolution, or provides an in-depth comparison to existing sequence-matching and read-level taxonomic binning techniques. We show that our proposed neural networks architecture scales successfully to more than 13 thousand distinct species, and moreover that it can accurately analyze both synthetic and experimental read data. We find that these learned, discriminative classifiers outperform k -mer based classifiers and achieve performance which is comparable to that of traditional alignment tools, while also being more robust to both increasing noise and decreasing read length. We show our DNNs are also more robust than Kraken, a popular taxonomic binning tool. We use these results to understand the conditions under which this deep learning solution is most advantageous, and conclude by discussing the generality of our approach.
To evaluate our trained classifiers, we used 16S mock community sequencing data from Nelson et al., 22 Schirmer et al., 29 and D'Amore et al. 23 (PRJEB4688 and PRJEB6244 in the European Nucleotide Archive 24 ). We used six Illumina MiSeq replicates (ERR348713-5 and ERR619081-3) of the 20-organism mock community (PRJEB4688) and all 51 runs from the 59-organism community (PRJEB6244). Appendix 7 details the contents of each replicate.
Model Architecture
Each canonical base (A, C, T, G) or IUPAC ambiguity code in a given input was encoded as an appropriate four-dimensional probability distribution over the four canonical bases (Extended Data Figure 1). Similarly, we one-hot encoded species labels as a 13,838-dimensional vector. We used a model architecture comprised of three layers of depthwise separable convolutions (sequential spatial and pointwise convolution operations), two to three fully-connected layers, a pooling layer, and a softmax output layer that produces a probability distribution over possible species labels. We apply dropout regularization 30 and leaky rectified-linear activation 17,18 to each convolutional and fully-connected layer (Appendix 2). When inputs are longer than the width of the fully-connected layers, these layers are tiled as needed and the pooling layer combines the intermediate outputs before the softmax output layer. We found that average pooling worked best for this application (Appendix 4). Appendix 3 describes model selection and gives the best configuration we found for each minimum read length. To compute probability distributions over higher taxonomic labels, we marginalized the predicted species-level distributions as follows: Given an input read x , fixed model DNN , and higher-order taxon of interest: 1. Let DNN ( x ) i denote the predicted probability assigned by the model to the i th species label, and take I j = { i : i th species is contained in j th higher-order taxon}
Training and Implementation
We implemented our neural networks using the open-source software library TensorFlow. 16 Starting with randomly initialized parameters (Appendix 2), we used a random mini-batch of 500 read and species label pairs on each iteration to update model parameters to minimize cross entropy loss between the true and predicted species labels (using TensorFlow's implementation of the ADAM optimizer 19 ). 3 We also explored classification performance as a function of noise rate. When we injected random base-flipping noise into input sequences, we mutated each base b with fixed probability r according to the following rule: If b is a canonical base: Flip b to one of the other three canonical bases with equal probability. Otherwise: Flip b to one of the four canonical bases with equal probability. We trained models with five different noise rates r = {0%, 2%, 4%, 8%, 16%} and evaluated on data with injected noise rates of r = {0%, 0.5%, 1%, 2%, 5%, 10%}.
Baselines
We computed the Bayes optimal accuracy for a fixed set T of training examples as follows: 1. Partition T into subgroups of (read, species label) pairs, where N is the , T , ... , T T 1 2 We repeat this process for each higher taxonomic rank.
For our alignment baselines, we used BLAST 27,28 and BWA 25,26 to align queries against the original NCBI reference sequences and computed the accuracy of randomly guessing a label for each read from the set of labels for all references with equally good mappings. For BWA, we consider all references with edit distances as least as good as the primary mapping, and for BLAST we use the bit score (Appendix 6).
Our naive Bayes classifier (based on the RDP Classifier 8 ) uses 8-mer matches of queries reads to the original references sequences. We transformed input sequences into vectors indicating the presence or absence of each possible 8-mer subsequence. For each such 8-mer, the prior probability used for its presence and genus-specific conditional probability were computed as in Wang et al. 8 We handled IUPAC ambiguity codes in the 8-mer vector representation by assigning each possible DNA 8-mer a fractional presence (Appendix 6).
We also compared the sensitivity, positive predictive value (PPV), and F1 scores of our DNNs to Kraken2 33 ( https://github.com/DerrickWood/kraken2 , downloaded 2018-09-28) a popular taxonomic binning tool that emits read-level predictions. We generated a kraken2 database in exact correspondence with the taxonomy of organisms in our NCBI training data (Appendix 6), and ran kraken2 on FASTQ files of query sequences using the following command: $ kraken2 --db kraken2_database --threads 48 kraken.fastq > output.kraken kraken2 emits whether each read was classified (C) or unclassified (U), and gives a predicted taxonomic rank and entity for each classified read. All ranks above the emitted rank are marked with the entity implied by traversing up the taxonomy from the predicted rank, while all ranks 4 below the predicted rank are marked as no-call. We calculated counts of true positives and false positives, along with derived sensitivity, specificity, PPV, false discovery rate, and F1 values at each rank by comparing to the true taxonomic rank and entity while respecting no-calls (i.e. sensitivity includes no-calls as false negatives while specificity ignores no-calls).
Mock Community Evaluation
Base-flipping noise does not capture all the interesting aspects of real sequencing errors; 37 we thus also applied our DNNs to experimental reads with real error profiles. We used the following iterative method to obtain mixture estimates from read-level predictions: 1. Using a fixed model, DNN , initialize a matrix of read-level probabilities P so that for each row row i , where is the i th read in the set of raw sequencing reads.
Initialize a vector of mixture estimates E as the uniform distribution over possible species labels. 3. For each iteration: a. Compute a matrix where where is element-wise We used 10,000 reads and 75 iterations for the smaller mock community, and 20,000 reads and 150 iterations for the larger. We took the list of species with estimated mixture fractions over 0.001 in the final E as the predicted set of community members. We repeated this procedure to estimate genus-level composition by marginalizing the model's predictions to the genus level, and initializing E as a uniform distribution of the appropriate size.
Training Performance
For each of five minimum read lengths L = {25, 50, 100, 150, 200}, we explored whether a neural network, DNN L , of the structure in Figure 1 can learn to predict accurate read-level labels. While our architecture's pooling layer allows the models to tolerate variation in read length, we found it informative to train multiple models optimized for different lengths (Figure 2b). We trained each DNN L independently on the synthetic reads in NCBI L . We compare to the Bayes optimal classifier, which gives the maximum achievable accuracy on the training set for a perfect read mapping. Overall, classification accuracy increased with read length, and each DNN achieved species classification accuracy within 2.0% of the Bayes optimal rate (Figure 2a). Species-level classification accuracies using BWA fell, on average, 4.6% below Bayes optimal. Higher-order predictions obtained by marginalizing the models' predicted probability distributions over species (Methods) were also highly accurate, coming within 1.0%, 0.6%, and 0.5% of Bayes optimal rates at the genus, family, and order levels, respectively. DNN 25 attained 84.4%, 90.0%, and 99.9% read-level accuracies on class, phylum, and superkingdom classification; the remaining models surpassed 95.7% accuracy on all these tasks. On NCBI 250 all the models performed well at higher-order classification tasks, attaining upwards of 98.3% read-level accuracy on order prediction. At the species-level, models trained on longer reads achieved noticeably better performance on NCBI 250 , falling within 4.1% of the Bayes optimal rate, compared to 6.1% and 13.6% for DNN 25 and DNN 50 , respectively.
Figure 3: Accuracy and consistency of our deep learning approach on reads from held-out species relative to existing tools. Heatmap
showing the read-level accuracy of our deep learning approach (using 8% noise injection during training for 200 base pairs and 4% for all others) relative to naive Bayes and alignment approaches on the task of assigning reads from held-out species to correct genera for several combinations of read length and noise rate. Here, alignment represents the better of our BLAST and BWA baselines, and each cell is labeled with the proportion of reads placed into the correct genus when the corresponding method ( y -axis) is used to assess held-out reads corrupted by the given noise rate ( x -axis).
We retrained DNN 25 , DNN 50 , DNN 100 , and DNN 200 on synthetic reads from a subset of 12,598 of the 13,838 species in the NCBI data, with base-flipping noise randomly injected into inputs at train time. We compared the efficacy of these models at making genus-level predictions for novel species to alignment and naive Bayes baselines (Methods). Figure 4 shows genus-level accuracy for each method on synthetic reads in the validation set, comprised of synthetic reads from 16S references of the 1,240 species held-out from the training set. The first column of Figure 4 shows that genus-level accuracy on errorless validation reads varies significantly across the different methods, with our approach significantly outperforming for every read length except 200 base pairs where the results are roughly comparable to alignment.
Across the 24 read length and noise rate pairs we tried, our DNNs outperformed the naive Bayes baseline by between 1.9% and 51.9% (Figure 3). In 20 of these 24 settings, our DNNs outperformed alignment baselines; these improvements ranged from 0.1% to 32.5%. For the remaining 4 low-noise experiments on 200 base pair reads, BLAST attained no more than a 1.3% advantage over DNN 200 . Our DNNs performed well even when BWA failed to align a significant portion of the reads; for example, BWA achieved less than 3% read-level genus accuracy on 200 base pair validation reads with 5% or 10% injected base-flipping noise, whereas DNN 200 achieved 81.1% and 76.1% accuracy, respectively. Thus, in general we found that, compared to k -mer and alignment baselines, our proposed deep learning approach tends to assign more accurate genus labels to reads from novel species and is more robust to sequencing errors and the high mutation rate involved in bacteria genomes. Figure 4 compares the sensitivity, PPV, and F1 scores of our DNNs to Kraken2 33 on the validation reads from held-out species. Because our method predicts a genus label for every read, its scores are equivalent across the three performance metrics. In contrast, Kraken emits labels for only a subset of the reads, in general allowing it to attain high PPV at the cost of lower sensitivity. Kraken2's PPV was highest on validation sets with low noise rates; once the noise rate increased upwards of 1%, the PPV attained by our DNNs was comparable in most cases, and higher for 100 and 200 base pair reads. Sensitivity achieved using Kraken2 was much lower than our DNNs, even when no noise was added to the validation reads. Indeed, our proposed approach achieved high sensitivity and good PPV in general, resulting in much higher F1 scores on genus-level prediction for the held-out species compared to Kraken for every combination of read length and noise rate evaluated in Figure 4. The performance of our deep learning approach was thus more consistent and degraded less with increasing noise. Using DNN 100 to analyze 57 sets of empirical mock community sequencing data, 22,23,29 we demonstrated that our DNNs achieve reasonable performance on real reads generated using a variety of typical next-generation sequencing protocols. Due to the lack of read-level ground-truth labels for the mock community sequencing reads, we introduce a method of integrating over our model's read-level predictions to produce model-based community reconstructions (Methods). We chose to keep this model-based estimation method as simple as possible so results directly reflect the model's sequence-labeling performance, but note that integrating the model's outputs into a more sophisticated analysis pipeline (for instance, inspired by MetaPhlAn 6 or MetaPhyler 35 ) would likely improve profiling results.
Kraken Comparison
For five of six 20-organism mock community replicates, our model-based estimation perfectly reconstructed the list of 17 genera (Figure 5b). At the species level, our method identified, on average, 21.8 distinct species (16.3 truly present and 5.5 false discoveries). For the remaining 51 mock community sequencing datasets, we correctly identified on average 36.0 of 45 genera and 32.6 of 56 species based on the model's probability assignments. We consistently failed to recover six Archaeal genera: Nanoarchaeum (which is not contained in our NCBI dataset), Methanocaldococcus , Pyrococcus , Sulfolobus , Archaeoglobus , and Ignicoccus . This is likely due, in part, to the "failure of the V1-V9 primers in amplifying the Archaea" 23
Discussion
We proposed a deep learning method that performs at or above state-of-the-art levels on a specific sequence-labeling problem: predicting species of origin for individual reads of 16S ribosomal DNA. We demonstrated this DNN is capable of near-optimal species classification, and moreover that these species-level predictions marginalize accurately to higher taxonomic ranks ( Figure 2). When we examined the DNN's individual predictions along a fixed reference sequence, the majority of confident, correct species-level predictions were achieved for queries covering parts of the 16S hypervariable regions (Appendix 5). As these hypervariable regions are well-known and exploited features of 16S sequences, 44,45 the ability to learn the importance of these regions from training data is an important demonstration that 1) the DNN learned important biological structure of 16S, and 2) we might be able to discover unknown structure by inspecting the DNN's behavior. We also validated that our approach works on experimental mock community data produced using typical Illumina sequencing protocols ( Figure 5).
Our DNNs perform well compared to a diverse set of existing programs. They achieved read-level order classification accuracy comparable to Khawaldeh et al. 11 despite modeling 22-fold more distinct orders (Figure 2b), and assigned genera to reads from novel species more accurately than naive Bayes approach based on the RDP Classifier 8 (Figure 3). Though alignment baselines were slightly more competitive, our DNNs classified reads from held-out species more accurately for almost every read length we tried and were more robust to noise ( Figure 3). This suggests our approach of injecting random base-flipping noise at train time allows the DNNs to learn a more robust noise model than the one implicitly incorporated by aligners like BLAST and BWA by allowing gaps and mismatches to be tolerated. Similarly, compared to Kraken2, our DNNs achieve PPVs which degrade less with increasing error rates and strike a better balance between sensitivity and precision overall ( Figure 4). Therefore, we demonstrated that our deep learning approach is robust to high error rates where other popular approach struggle, an improvement which is particularly important considering newer technologies which produce more errorful reads, such as those being developed by Pacific Biosciences and Oxford Nanopore Technologies. 43 Runtime performance is also a critical element of sequence-labelers; tools like Kraken2 are heavily optimized for speed. Our model architecture uses depthwise separable convolutions, which have been shown empirically to use parameters more efficiently than regular convolutions. [12][13][14] The DNNs used in the current study consists of roughly 1.2 million floating-point operations per query and thus, though actual performance likely vary by application, common hardware accelerators for machine learning could allow them to process roughly 13 million (on a V100 GPU) to 156 million (on a Cloud TPU) reads per second. Moreover, our modeling choices make our DNNs well-suited for adaptation to mobile hardware, in contrast to approaches like alignment requiring access to large databases.
While our initial results are highly suggestive that downstream metagenomic analyses could benefit from complementing or replacing existing sequence-labeling substeps with our deep learning approach, more work is needed to fully understand its behavior. The current work does not explore performance on paired data, though it would be simple to extend our model to leverage this data by concatenating the reads. Additional adjustments for factors like unequal coverage in training data may be needed to properly calibrate the models' predicted probability estimates (Appendix 5). More generally, it is possible that other deep learning architectures could further improve performance. When applying our approach to full metagenomic data, it might be necessary to incorporate some mechanism for detecting queries outside the training data's domain, similar to the way Kraken2 attains higher PPV by leaving some queries unclassified. Finally, while our study on the effect of base-flips implies robustness of the method against sequencing errors and mutation rates, some of the higher error rates where our DNNs make the largest improvement may not be realistic for widely-used sequencing technologies like Illumina; follow-up analyses should specifically explore the behavior of our approach on reads produced by more errorful sequencers.
We emphasize that the model and methods proposed here are highly general, making this new approach an excellent candidate for complementing or replacing solutions to many large-scale bioinformatics problems which currently rely on string-matching or less flexible statistical modeling. Due to the improved robustness of our deep learning approach, impact may highest for applications where data is inherently noisy including analysis of bisulfite sequencing, viral DNA, immune-mapping, or ancient DNA. Overall, the current study acts as a first step towards our long-term goal of developing a general-purpose deep learning model that can successfully perform any task framed as the assignment of labels to short biological sequences. | 2019-04-02T13:14:47.924Z | 2018-06-22T00:00:00.000 | {
"year": 2019,
"sha1": "1d2d381fd1d2fe3d2b65e3b1e371717c5536ded1",
"oa_license": "CCBY",
"oa_url": "https://www.biorxiv.org/content/biorxiv/early/2019/08/10/353474.full.pdf",
"oa_status": "GREEN",
"pdf_src": "BioRxiv",
"pdf_hash": "6ede8c5e7d68a1580b634724799c5d831f243de5",
"s2fieldsofstudy": [
"Computer Science",
"Biology"
],
"extfieldsofstudy": [
"Biology",
"Computer Science"
]
} |
198927777 | pes2o/s2orc | v3-fos-license | Standard State Space Models of Unawareness (Extended Abstract)
The impossibility theorem of Dekel, Lipman and Rustichini has been thought to demonstrate that standard state-space models cannot be used to represent unawareness. We first show that Dekel, Lipman and Rustichini do not establish this claim. We then distinguish three notions of awareness, and argue that although one of them may not be adequately modeled using standard state spaces, there is no reason to think that standard state spaces cannot provide models of the other two notions. In fact, standard space models of these forms of awareness are attractively simple. They allow us to prove completeness and decidability results with ease, to carry over standard techniques from decision theory, and to add propositional quantifiers straightforwardly.
Introduction
Dekel, Lipman and Rustichini [8], hereafter, "DLR", claim to show that "standard state space models preclude unawareness".Their claim has achieved the status of orthodoxy. 1The first task of this paper is to clear the way for standard state space models of unawareness by showing that the formal result DLR present does not establish their headline conclusion.DLR informally motivate certain axioms concerning unawareness, but in their formal impossibility result, they rely on the claim that these axioms hold at all states in the model.As section 2 argues, the assumption that axioms hold at all states of the model is unwarranted; in fact, DLR themselves reject it.While DLR's formal results are valid, they are not sufficiently general to rule out standard state space models of unawareness.As we show, the impossibility results do not hold if one assumes only that DLR's explicit assumptions about unawareness hold at some "real" states, as opposed to at all states.Even strengthening those explicit assumptions considerably does not reinstate the results.
But this does not yet vindicate standard state space models of unawareness.Section 3 presents a novel impossibility result which uses widely shared assumptions about awareness.The new impossibility theorem relies on the assumption that an agent who is aware of a conjunction is aware of its conjuncts.If awareness satisfies this assumption, then standard state space models do in fact preclude unawareness.We then distinguish three notions of awareness, and suggest that two important ones do not satisfy this assumption, leaving open the possibility that they could be adequately modeled by standard state space models.
The remainder of the paper continues in a more positive vein.We describe a simple class of standard state space models which represent key features of awareness.In section 4, we establish completeness and decidability for the logic of these models.We also show that adding propositional quantifiers, a topic which has presented major difficulties for existing approaches to awareness, is straightforward in our standard state space models.In section 5, we present one way of implementing a choice-based approach to decision theory within these models, and show how non-trivial unawareness is consistent with speculative trade.Section 6 concludes.
Standard State Space Models
Standard state space models for the knowledge and awareness of a single agent can be understood as certain tuples Ω, k, a .Ω is required to be a set, called the set of states, from which a set of events is derived by taking an event to be a set of states.k and a are functions on events, which represent the agent's knowledge and awareness, respectively: k maps each event E to the event k(E) of the agent knowing E; a similarly takes each E to the event a(E) of the agent being aware of E.
Such models are straightforwardly used to interpret a formal language in which one can talk about knowledge and awareness.Let L be such a language built up from proposition letters p, q, . . ., using a unary negation operator ¬, a binary conjunction connective ∧ and two unary operators K and A, respectively ascribing knowledge and awareness to the agent.Formulas of this language are interpreted relative to a model M = Ω, k, a and a valuation function v which maps each proposition letter p to the event v(p).The interpretation uses a function • M,v which maps each formula ϕ of L to the event expressed by ϕ in M, which can be understood as the set of states in which ϕ is true in M. To state the constraints on such a function let −E = Ω\E.
The agent being unaware of something can of course be understood as it not being the case that she is aware of it.We therefore syntactically use U ϕ as an abbreviation for ¬Aϕ.Similarly, we introduce the other connectives of classical propositional logic as abbreviations, using ∨ for disjunction, → for material implication, ↔ for bi-implication, and ⊤ and ⊥ for an arbitrary tautology and contradiction, respectively.On the semantic side, we adopt the convention of writing f g for the composition of functions f and g, which allows us two write, e.g., k − a(E) instead of k(−(a(E))).
In order to express general constraints on these models, we say that a formula ϕ is valid on M if ϕ M,v = Ω for each valuation function v; this can be understood as requiring ϕ to be true in every state of M according to every valuation function.In order to limit this constraint to a particular state ω ∈ Ω, we say that ϕ is valid in ω if ω ∈ ϕ M,v for each valuation function v.
These models count as "standard" in the sense of DLR.First, the events expressed by Aϕ and Kϕ are each a function of the event expressed by ϕ. (DLR call this "event-sufficiency".)Second, negation is interpreted as set-complement and conjunction as intersection, so that all tautologies of classical propositional logic, such as p ∨ ¬p, are interpreted as the set of all states in every model.(DLR call this assumption "real states".)
DLR on Standard State Space Models
DLR introduce three constraints on awareness, which can be stated using the following three axioms: Their constraints on knowledge can be stated using the following three axioms: Their main results are then: Theorem 1 (DLR).Let M = Ω, k, a be a model on which Plausibility, KU-Introspection and AU-Introspection are valid.
2 If Weak Necessitation is valid on M, then K p → Aq and Ap ↔ Aq are valid on M.
Our presentation of DLR's result differs in superficial respects from their original presentation.DLR do not present their constraints in terms of the validity of certain axioms.Thus, for example, instead of requiring KU-Introspection to be valid on M, they require k − a(E) = / 0 for all events E. However, it is a routine exercise to show that this condition is equivalent to the validity of our corresponding axiom.The same point holds for the other axioms.In short, our later models will not be escaping their triviality result by a sleight of hand which depends on this presentation.
One reason for the variant presentation is that it will facilitate the later exposition.It also serves to demonstrate that standard state space models as discussed here are equivalent to what are now commonly known as neighborhood or Scott-Montague frames (see [56] and [44]).It is well known that given certain restrictions on the function interpreting knowledge, this function can be turned into a binary relation among states along the lines of those used by [37] and [29].This representation as a binary relation is, in turn, formally interchangeable with the "possibility correspondences" introduced by [2] (see also [3]) and used throughout economic theory (see, e.g.[11]).
Two Kinds of States
In response to their triviality results, DLR suggest distinguishing informally between "real" states and "subjective" states.As we understand it, this distinction can be explained as follows.An epistemic model makes predictions about how an agent or group of agents will or would behave in particular situations.The model makes predictions about these situations by including states which represent them.The real states in a model are the states which represent situations the model is intended to describe.The model predicts an agent will behave a certain way in a particular situation just in case the agent behaves that way in the real state which represents that situation.The predictions of a single model are given by what holds at all its real states; the behavioral theory of a class of models is given by what holds at all real states in all its models.
A state in an epistemic model is subjective if it figures in the specification of what the agent knows or is aware of at some real state.According to this way of understanding real and subjective states, states may be both real and subjective.Suppose we wish to represent an agent who knows that a particular coin will be flipped, but who will not learn the outcome of this coin flip.If our model is intended to make predictions no matter how the coin lands, the subjective states needed to specify the agent's knowledge (heads and ignorant, tails and ignorant) will be exactly the real states; every state will be both real and subjective.But as DLR recognize, there is no reason to require all states to play both roles.In the earlier example, if we only wanted to make predictions about the situation in which the coin comes up heads, we would not count one of the states (tails and ignorant) as a real state; even so, to represent the agent's ignorance given heads, a state where the coin comes up tails would still have to be included as a subjective state.The point can also be illustrated with a less artificial example.Consider an analyst who wishes to model the interactions of agents who are rational, but who do not believe each other to be rational.To represent the beliefs of these agents, the analyst must include subjective states in which the agents are irrational.But although she includes these subjective states, the analyst has no intention of eliminating the claim that the agents are rational from the predictions of her theory.Rather, it is understood informally that these subjective states are not real; they do not represent situations the analyst aims to describe.
Put in the terms of our presentation, DLR's proposal is to allow subjective states in which the law of excluded middle, p ∨ ¬p, may not be true.DLR have no intention of eliminating the law of excluded middle from the predictions of their theory.Rather, they introduce these subjective states to specify the agent's knowledge and unawareness at real states where classical logic holds.The theory of DLR's models is given by what holds at these classical real states, not by what holds at all states whatsoever.Still, since classical tautologies may fail in DLR's subjective states, their models violate the "real states" assumption, and so are not standard state space models.
But once we acknowledge that some states may not be intended to represent situations the analyst wishes to describe, a question arises: Why should one require DLR's three axioms on unawareness to be valid in all states?DLR do not argue for this assumption.At best, their arguments in favor of their axioms only motivate imposing these axioms at real states.These arguments provide no motivation for imposing the axioms on subjective states that are not real, since these states are merely included in the model to specify the agent's knowledge and unawareness at real states.
There is a general methodological principle in epistemic modeling that axioms are to be imposed at all states.But in the literature on awareness, following DLR, this methodological principle has long been abandoned.DLR's own non-standard models violate this requirement, as do the current leading proposals for representing awareness, for example that of [24].The subjective states in DLR's models include states in which logical axioms, including the law of excluded middle, do not hold.In DLR's models, as in the ones we will propose, even logical axioms are allowed to fail at some states.The only difference between our proposal and theirs concerns which axioms are allowed to fail.DLR preserve their own axioms at all states, and move to non-standard models in which classical propositional logic may fail at subjective states.We will preserve classical propositional logic at all states, and work with standard models in which DLR's axioms may fail at subjective states.
Still, one might ask: How should we understand a state where DLR's axioms are false?DLR interpret their own subjective states as "descriptions of possibilities as perceived by the agent" (p.171).This interpretation does not seem appropriate for our models, in which DLR's axioms may fail at subjective states.But such metaphorical interpretations of these states are unnecessary.Subjective states where the axioms of awareness are invalid are simply to be understood in terms of the agent's knowledge and awareness at real states where the axioms are valid.
In fact, we can give a direct argument for not imposing DLR's axioms at all states, and in particular, for including states where KU-Introspection is invalid.First, by AU-Introspection, if an agent is unaware of p, then she must be unaware of being unaware of p.But then, by Plausibility, the agent does not know that she does not know that she is unaware of p. (Essentially, DLR already give this argument on p. 169.) In epistemic models, we generally represent an agent's not knowing q by including a state in which q is false.So, to allow for real states in which the agent does not know that she does not know that she is unaware of p, we must include subjective states in which the agent knows that she is unaware of p, and so violates KU-Introspection. 2o sum up: after rejecting standard state space models, DLR propose that we should use models in which the laws of logic fail at subjective states.They implement this proposal by countenancing states where propositional logic fails, so that their models are non-standard.But if we allow models in which the law of excluded middle may fail at subjective states, we must also consider models in which other axioms, including DLR's, may fail at subjective states.DLR's formal results only apply to standard state space models in which their axioms are imposed at all states; the results do not concern standard state space models in which the axioms are imposed only at real states.As a consequence, these formal results cannot provide a basis for the conclusion DLR draw: that standard state space models preclude unawareness.
Non-Triviality
We have not argued against the validity of DLR's three axioms in real states -states representing the situations to be modeled.In our setting, we can formalize the distinction between real states and subjective states which are not real, by only assuming the validity of the axioms in some subset of the states in a model.We can then ask: is assuming the validity of the three axioms in such distinguished real states enough to lead to triviality?The following example shows that it is not: Theorem 2. There is a model M = Ω, k, a , state α ∈ Ω and event E ⊆ Ω such that Necessitation is valid on M, Plausibility, AU-Introspection and KU-Introspection are valid in α, and α / ∈ a(E).
Proof.Let Ω = {α, ω 1 , ω 2 }.Define a binary (accessibility) relation R on Ω as follows: R induces a possibility correspondence P such that P(σ ) = {τ : Rσ τ}.With P, define k and a such that for all F ⊆ Ω: It is routine to check that M = Ω, k, a , α and E = {α, ω 1 } witness the claim to be proven. 3his shows that DLR's Theorem 1(i) cannot be extended to standard state space models in which DLR's three axioms are only required to be valid in real states.In fact, the model used in the above proof of Theorem 2 can also be used to show that DLR's other two results cannot be extended either.For 1(ii), note that Monotonicity is valid on M, and that α ∈ k(Ω) although α / ∈ a(E).For 2, note first that Weak Necessitation is valid on M, and that α ∈ a(Ω) but as before α ∈ k(Ω) and α / ∈ a(E).More generally, any state in any model which satisfies both Necessitation and Monotonicity, in addition to DLR's three axioms, will be a counterexample not just to extensions of DLR's Theorem 1(i), but also to extensions of their Theorems 1(ii) and 2.
We conclude that none of DLR's three triviality results show that standard state space models preclude unawareness.One might wonder whether plausible strengthenings of the axioms on knowledge and unawareness allow us to reinstate the triviality results.In the full paper, we argue first that this cannot be achieved by strengthening their axioms governing knowledge, and, second, that it cannot be achieved by a particular strengthening of the axioms governing unawareness.The theorems and proofs may be found in Appendix A.
Three Kinds of Awareness 3.1 A New Triviality Result
DLR's result had limited implications for state space models because it depended on the validity of their axioms at all states.Is there a triviality result which only uses the validity of axioms on awareness in real states, rather than their validity in all states?In fact, as we now show, widely accepted axioms on awareness do lead to triviality even if they are imposed only at real states.The result uses the following two axioms: Awareness is widely assumed to satisfy both of these axioms; see, e.g., [43, pp. 274-275], [18, p. 331] (axioms A1 and A2) and [25, p. 309] (axioms 1 and 2).
As the next theorem shows, these axioms lead straightforwardly to triviality.
Theorem 3. Let M = Ω, k, a be a model and α ∈ Ω such that AS and AC are valid in α.Then Ap → Aq is valid in α.
The crucial difference between this and DLR's triviality result is that AS and AC are only assumed to be valid in a distinguished state, for which it is shown that non-trivial unawareness in it is ruled out.
But does awareness really satisfy both AS and AC?In the following, we will focus in particular on AC, arguing that for some important notions of unawareness, being aware of a conjunction does not entail being aware of its conjuncts.
Attending vs. Conceiving vs. Processing
In the literature on awareness, it is uncontentious that there is no single attitude of awareness; what is expressed by "aware" is a loose cluster of notions.This was noted at the very start of the literature, as witnessed by the lengthy discussions in [10]; another detailed discussion can be found in [55].We will argue that that at least some important notions of awareness do not satisfy AC (for others, as we will see, the situation is more complex)..In order to do so, we roughly distinguish the following three ways of understanding a claim of the form "The agent is aware of . . .": (i) The agent is attending to . . .
(ii) The agent has the conceptual resources required to conceive of . . .
(iii)
The agent is able to process . . .We will introduce these notions -and distinguish between them -using various examples found in the literature.Consider first attention.An influential example, which first appeared in [16], and which is discussed at length by DLR and numerous places in the subsequent literature, is based on the following quote from one of Arthur Conan Doyle's Sherlock Holmes stories [9]: "'Is there any other point to which you would wish to draw my attention?' 'To the incident of the dog in the night-time.' 'The dog did nothing in the night-time.' 'That was the curious incident' remarked Sherlock Holmes." Holmes's interlocutor is Inspector Gregory, a Scotland Yard detective.Before Holmes pointed out to Gregory that the dog did nothing in the night-time, Gregory was unaware of the dog doing nothing in the night-time.Gregory's state of unawareness is naturally understood as one of inattention -Holmes makes Gregory aware of the dog doing nothing in the night time in the sense of bringing this fact to his attention.
Gregory's failing to attend to the dog doing nothing in the night-time must be sharply distinguished from Gregory's not being able to conceive of the the dog doing nothing in the night-time.Before Holmes alerted Gregory to the dog doing nothing in the night-time, Gregory possessed the concepts required to entertain thoughts about the dog doing nothing in the night time.Contrast this with the following example for unawareness from [10, p. 40]: "How can someone say that he knows or doesn't know about p if p is a concept he is completely unaware of?One can imagine the puzzled frown of a Bantu tribesman's face when asked if he knows that personal computer prices are going down!"The relevant state of unawareness in this example is not merely a matter of the agent failing to attend to the relevant event or subject matter.For example, if one is unaware in the sense of being unable to conceive of an event, it must be that one does not understand the words for those notions in any language.Contrast this with the case of Inspector Gregory.Gregory understands what Holmes says: he can conceive of the dog's doing nothing.But the purported example of inconceivability does not have this structure: the tribesman is supposed to be unable to think about computers using any of his conceptual resources, no matter what he attends to.The two notions of awareness -attending to versus being able to conceive of -are therefore clearly distinct.
The third notion of unawareness we want to single out is one which [10] (see also [19] and [20]) focus on; it can be understood as an attempt to deal with what is known as the "problem of logical omniscience" in epistemic logic.In standard state spaces, if two sentences ϕ and ψ are equivalent in classical propositional logic, then Kϕ and Kψ will be true in the same states.In particular, if K(p ∨ ¬p) is true in a given state, then so is Kτ for any propositional tautology τ.One of Fagin and Halpern's reasons for developing a logic of awareness is to obtain logics which do not have this property.They write: "The notion of awareness we use in this approach is open to a number of interpretations.One of them is that an agent is aware of a formula if he can compute whether or not it is true in a given situation within a certain time or space bound.This interpretation of awareness gives us a way of capturing resource-bounded reasoning in our model." Being unaware of ϕ in the sense of not being able to process ϕ is clearly distinct from failing to attend to ϕ: although Gregory did not attend to the dog doing nothing in the night-time, he had no difficulties processing the claim that the dog did nothing in the night-time.Not being able to process ϕ is also clearly distinct from not being able to conceive of ϕ: Gregory might not have been able to process an extremely complicated propositional tautology using only negation, conjunction and the sentence "the dog did nothing in the night-time", but he clearly possessed all the concepts required to entertain it.
Awareness of Conjunctions
Let's return to the new triviality result introduced at the start of this section.As already advertised, we believe that the principle AC, which says that an agent who is aware of a conjunction is aware of its conjuncts, may be plausible for one notion of awareness, but it is not for the other two.
Consider first awareness as the ability to process.This is plausibly a relation of agents to sentences, as part of what it takes to process . . . is to be able to find out what the sentence ". . ." means.AC may hold if awareness is understood as the ability to process: It is natural to assume that an agent who is able to process a conjunction "... and -" is also able to process "..." and "-".As noted already in [10, p. 54], even this may fail: an agent might be able to recognize that a very long sentence has the form ϕ ∧ ¬ϕ, and so be able to process it, although she is unable to process the complex ϕ on its own.Resolving this controversy may require distinguishing further among different notions of processing, and the appropriate resolution may depend on the intended application.
The relations of attention and conceivability are different from the ability to process.In particular, they are plausibly relations agents have not to sentences but to what sentences express.We might call these entities contents or propositions, but in keeping with the terminology employed above, we call them events.In the full paper, we discuss the difference between sentences and events in more detail, and motivate the assumption of a coarse-grained theory of events, according to which events form a complete atomic Boolean algebra.A consequence of this assumption -which is implicit in all standard state-space based modeling techniques -is that sentences which are equivalent in propositional logic have identical contents.
With this understanding of attention and conceivability as relations of agents to coarse-grained events, consider first attention.Assume that after the conversation with Holmes quoted above, Gregory is alone thinking about the case, and attending to the event of the dog barking in the night (p).He is not, however, attending to event of Holmes at that moment smoking a pipe (q).It is then natural to say that Gregory is also not attending to the conditional event that if Holmes is currently smoking a pipe then the dog barked in the night (q → p).But notice that according to the coarse-grained Boolean theory of events, the event that the dog barked in the night (p) is identical to the event that if Holmes is smoking a pipe then the dog barked in the night, and the dog barked in the night ((q → p) ∧ p).So if AC were valid, then since Gregory is attending to the event of the dog barking in the night, he would be attending to the event that if Holmes is smoking a pipe then the dog barked in the night.But by assumption Gregory is not attending to this last event.Thus it follows from the coarse-grained theory of events that AC must be rejected.
A similar example can be given if we understand awareness as conceivability.Assume our agent does not have the conceptual resources to entertain the event of there being a black hole.According to the assumed coarse-grained theory of content, the event of there being a black hole and there being no black hole is identical to any event expressed by a propositional contradiction, such as the event of there being a sheep and there being no sheep.The agent might well have the conceptual resources to entertain the event of there being a sheep and there being no sheep, without having the conceptual resources to entertain the event of there being a black hole.
If we adopt, as usual, a theory of events which identifies the event expressed by sentences which are equivalent in propositional logic, AC appears to be inappropriate.Thus the new triviality result with which we started this section also does not establish that standard state space models preclude unawareness understood as inattention or inability to conceive.
Partitional Models
So far, we have shown that standard state-space models escape certain putative impossibility results for models of attention and conceivability.But this does not establish that standard space models can provide fertile models of these notions.In the remainder of the paper, we define, motivate, and examine a class of standard state space models for representing attention and the ability to conceive.
To show that our models generalize smoothly to the multi-agent case, from now on we use a language L I parametrized to an arbitrary set of agent-indices I which is defined as the language L above, except that the operators A i and K i are indexed to i ∈ I. Models are consequently tuples of the form Ω, k i , a i i∈I .The models we will be working with are defined as follows: i∈I is a partitional model if Ω is a set and for each i ∈ I, R i is a binary relation on Ω which is reflexive and transitive, and ≈ i a function which maps each ω ∈ Ω to an equivalence relation Here and in what follows, we make use of the fact that each equivalence relation corresponds to a unique partition, and vice versa; accordingly, we treat them as interchangeable.
Partitional models can be used to generate standard models in the following way: R i specifies states of knowledge just as in Theorem 2. The idea behind ≈ i is that the events the agent is aware of at ω are the events which are unions of sets of equivalence classes of ≈ i ω (equivalently: unions of sets of cells of the induced partition).So for each i ∈ I, let R i and ≈ i determine functions k i and a i on events on Ω as follows: k i (E) = {σ ∈ Ω : P i (σ ) ⊆ E}, where P i (σ ) = {τ : R i σ τ} a i (E) = {σ ∈ Ω : for all ρ and τ such that ρ ≈ i σ τ, ρ ∈ E iff τ ∈ E} Let the standard model determined by a partitional model Ω, R i , ≈ i i∈I be Ω, k i , a i i∈I , with k i and a i as just defined.On such a standard model, L I can be interpreted as above; obviously, this induces a way of interpreting L I directly on partitional models.
The Attitude of Attention
In order to motivate partitional models as models of limited attention, we suggest that attention in the sense we have been using the term should primarily be understood as an attitude towards questions.There are many available formal approaches to modeling questions (for an overview, see [35]).For concreteness, we'll adopt a standard approach, representing questions as partitions of the state space (see [17], building on [23] and [32]).Although we think the attitude to questions is primary, we will follow the literature on awareness, in axiomatizing a notion of attention which has events as its objects.The relationship between this attitude to events and the attitude toward questions will be as follows: an agent attends to the question Q if and only if the agent attends to every partial answer to Q.Using partitions to model questions, partial answers are unions of sets of cells, corresponding to how standard models are derived from partitional models. 4
Partitions for Conceivability
To motivate the use of partitional models of conceivability, assume that the agents to be modeled have the concept of negation and (infinitary) conjunction, so that the set of events they can conceive of are closed under complement and arbitrary intersection.This is mathematically equivalent to requiring that this set is derived from an equivalence relation as above.
An Example
It will be useful to have a concrete partitional model before us, as a running example.The following model shows that there are non-trivial partitional models; for simplicity, a single-agent case is specified.Let M = Ω, R, ≈ , with ωRν iff ω = 1 or ω = ν, and ≈ given by the following equivalence classes: Thus, at 1, the strongest event known by the agent is Ω, and at each other state n, it is {n}.At each state n, the events the agent is aware of are the events which don't distinguish between any states in Ω\{1, n}.
Drawing the four states in a circle, starting with 1 at the top and going clockwise, we can draw each equivalence relation in a similar smaller circle, connecting two states by a sequence of lines if they are related by the relevant equivalence relation: This is a partitional model in which there is non-trivial unawareness at each state.We will appeal to it below in order to show the consistency of various constraints.
Axioms
Given a class of models C, a set of sentences Σ ⊆ L I is the logic of C if and only if Σ contains exactly those sentences which are valid on C. Characterizing the logic of a class of models gives us a formal perspective from which to assess what assumptions our models encode about agents knowledge and awareness.
Thus we may ask: What is the logic of partitional models?Standard techniques on completeness results in modal logic are easily adapted to obtain the following result.
Theorem 4. A formula is valid on all partitional models if and only if it is derivable in the calculus with the following axiom schemas and rules:
PL: Any substitution instance of a theorem of propositional logic.
A proof is given in Appendix B.
DLR Once More
Consider again DLR's three axioms.Given our discussion above, it is natural to consider partitional models where DLR's axioms are required to be valid in some distinguished state: i∈I is a partitional model and Plausibility, KU-Introspection and AU-Introspection (for each i ∈ I) are valid in α.
We now show that DLR's triviality result cannot be revived in partitional models: Theorem 5.There is a partitional DLR model Ω, α, R i , ≈ i i∈I and an event E ⊆ Ω such that α ∈ U (E).
Proof.Simply distinguish state 1 in the model presented in section 4.3.
We conjecture that the logic of partitional DLR models can be axiomatized as follows: Conjecture 1. Add the following axioms to the theorems of the axiom system in Theorem 4 and close under modus ponens:
is derivable in this calculus if and only if it is valid in every distinguished state of every partitional DLR model.
Note that ¬KU ϕ can be derived using P, AU and K-T.The present result shows that we can impose the DLR axioms without trivializing partitional models.But we confess to doubts about whether these axioms are appropriate.Just as with AC, once we understand more clearly the character of attention and conceivability, as well as the distinction between sentences and what they express, DLR's axioms become much less compelling.The clearest case concerns Plausibility and attention.A consequence of the contrapositive of Plausibility is Kϕ → Aϕ.But this principle is false for attention.You know that there are more than four stars in the universe, but we doubt that you were attending to the question of how many stars there are prior to reading the previous clause.As we discuss in more detail in the full paper, the coarse-grained conception of content together with clarity about the notion of awareness to be modeled cast doubt on DLR's axioms.
Propositional Quantification
A challenge to some approaches to unawareness is to represent propositionally quantified statements.E.g., earlier models by Halpern made the claim that the agent knew she was unaware of something unsatisfiable (cf.[21] and [22]).In standard state space models such as ours, it is trivial to add propositional quantifiers without any such consequences.To do so, we write v[E/p] for the valuation function which maps p to E and every other proposition letter q to v(q): To illustrate that these quantifiers behave just as one would expect, note that in state 1 of the example described in section 4.3, the agent knows that she is unaware of something without there being something that she knows to be unaware of: K∃pU p ∧ ¬∃pKU p is true in this state.
Closure and Automorphisms
In partitional models, what agents are aware of (attend to/can conceive) is closed under negation and conjunction.One might wonder whether we can also impose the constraints that what agents are aware of must be closed under awareness and knowledge.In other words, whether there are models on which the following axioms are valid: To provide models which validate these principles we adapt the coherence constraint of [13]. 6The idea behind it is most easily described for awareness as conceivability, taking the equivalence relations of partitional models to represent a relation of indistinguishability using conceptual resources available to the relevant agent at the relevant state.Coherence requires that if two states are indistinguishable in this way, then there must be a way of permuting the state space in a way which preserves all structural facts about knowledge and awareness, as well as all the events which the relevant agent is aware of at the relevant state.
Let M = Ω, R i , ≈ i i∈I be a partitional model.A permutation f of Ω is an automorphism of M if for all i ∈ I, A state x ∈ Ω coheres if for all i ∈ I and y, z ∈ Ω such that y ≈ i x z there is an automorphism f of M such that f (y) = z and f ⊆ ≈ i x (i.e., ω ≈ i x ω for all ω ∈ Ω).It's routine to verify that A-4ij and AK-4 are valid in any coherent state of a partitional model.
Once again, the model presented in section 4.3 demonstrates the satisfiability of this constraint: every state in this model is coherent.Since the model also satisfies the DLR axioms at state 1, it shows that even if we were to uphold the DLR axioms, imposing them together with coherence would not trivialize state space models of awareness.
Decision Theory
In section 4.5 the example of the number of stars illustrated how one may believe and know things to which one is not attending; clearly this kind of inattention may also affect choice-behavior.One advantage of standard state spaces is that we can use the usual decision-theoretic framework to represent the effects of inattention on choice-behavior. 7he usual decision theoretic representation of an agent's beliefs uses a measure-space S, B, µ . 8To generate a partitional model, we enrich this description of the agent by selecting B C , a complete atomic subalgebra of B, to generate a representation of what the agent attends to in the context of choice: S, B, µ, B C .The atoms of B C are a partition of S, so this structure gives rise to a partitional model of unawareness.The distribution the agent "uses" in a choice context is given by letting µ C (E) = µ(E) for all E ∈ B C and undefined otherwise.The events the agent "explicitly believes" in the context can then be defined as the events of which the agent is certain in µ C .The algebra B C can also be used to parametrize "expanding" and more generally "changing" awareness, represented as transitions between different complete atomic sub-algebras of B. 9 Since different algebras will determine different explicit beliefs in different contexts, this changing awareness can also represent effects of limited attention such as framing effects or failures of recall.
An approach along these lines has already proven fruitful in epistemic game theory.[33] and [34] develop Harsanyi type spaces in which players' beliefs may be defined on different σ -algebras.If the algebras are taken as the events the agent is attending to, one may interpret these models as examples of agents who fail to attend to questions about the higher-order beliefs of others, and thus do not have explicit beliefs over events which can be defined only by the level-n beliefs of others for large enough n.
Speculative Trade
An important test of approaches to unawareness has been how they fare with speculative trade ( [26]).Building on the work of [2], [41] proved their famous "no-trade" theorem, illustrating the extreme strength of S5 knowledge together with a common prior.One aim of representing "bounded" agents such as those with limited attention is to escape such paradoxes (for this perspective, see [45], [38]).Accordingly, we now provide a partitional DLR model with a common prior in which speculative trade is possible.
As is well known (see [16], [50], [49]), the "no-trade" theorem does not hold in general if agents' accessibility relations R i are merely transitive and reflexive, but are not required to form an equivalence relation.Plausibility is incompatible with the R i forming an equivalence relation ( [42]).Still, the DLR axioms together with partitional awareness models impose substantial further constraints, which might be thought to rule out speculative trade.We now construct a partitional DLR model to show that speculative trade can still occur in the presence of DLR's axioms and nontrivial unawareness.
Conclusion
Standard state space models of attention and conceivability are at least as successful as current nonstandard state space models.The non-standard models are, however, more complicated, and it is unclear that this complexity affords any advantages in predictive strength or accuracy.Standard state space models of these phenomena promise to lead to a rich and rewarding theory, posing technical and conceptual challenges, and offering connections to related work by linguists, philosophers and logicians -as well as work on bounded reasoning elsewhere in economic theory. | 2016-06-24T00:31:42.000Z | 2016-06-24T00:00:00.000 | {
"year": 2016,
"sha1": "e00687bbe8b500c1c10cf522fba67f2ff532fc56",
"oa_license": "CCBYNCND",
"oa_url": "https://arxiv.org/pdf/1606.07520",
"oa_status": "GOLD",
"pdf_src": "Arxiv",
"pdf_hash": "af977d7fef63a70313d601aedefaa78e99d1b50e",
"s2fieldsofstudy": [
"Philosophy"
],
"extfieldsofstudy": [
"Mathematics",
"Computer Science"
]
} |
148751830 | pes2o/s2orc | v3-fos-license | The Challenges of Higher Education in Growing Dialogue Culture and Understanding Cultural Pluralism
The contemporary world is increasingly multicultural and the identity crisis resulting from this sometimes threatens sustainable human development. Nigeria is a plural society in terms of its multi-ethnic and multi-religious nature. Of all the federal democracies in the world, only India can match Nigeria’s cultural complexity. If well managed, this factor of unity in diversity would have been a major asset to the Nigerian state, but the contrary is the case. Nigeria’s cultural diversity is politicized and exploited by the elite in such a way that retards the nation’s growth and progress. This makes the promotion of understanding and dialogue to be a prime issue in the management of multiculturalism, global peace and security. This paper attempts to answer these questions by taking a critical look at the situations in Nigeria – one of the most culturally-complex countries in the world. The paper is divided into three parts. In the last part, which is actually the fulcrum of the presentation, the point is made that these challenges notwithstanding; the Nigerian universities still manage to make some outstanding contributions in the direction of promoting dialogue among the contending forces in the country. The Nigerian case study is internationally instructive.
A. Introduction
Nigeria is a plural society in terms of its multi-ethnic and multireligious nature. The country has more than 400 ethnic groups (Suberu 1998:277) and two major religions (Islam and Christianity). Of all the federal democracies in the world, only India can match Nigeria's cultural complexity (Joseph 2006:15). If well managed, this factor of unity in diversity would have been a major asset to the Nigerian state, but the contrary is the case.
Nigeria's cultural diversity is politicized and exploited by the elite in such a way that retards the nation's growth and progress. The problem affects all aspects of Nigeria's national life -federal and even local resource allocation, management of public institutions (Dudley 1973;Egwu 1993;Ake 1996: Anber 1967, and youth development (Babawale 2003;Akinyele 2001).
The most threatening of the problems faced by the country is ethnicity. Osaghae defined the concept as "the employment or mobilization of ethnic identity and difference to gain advantage in situations of competition, conflict or cooperation" (Osaghae 1995:11). This definition challenges the position of the primordialists who argue that ethnicity is so much a matter of "shared traits or cultural commonalities".
It is rather the result of the interplay between external categorization and self-identification (Brubaker, Loveman and Stamatov 2004:31-32).
In other words, the problem of ethnicity in Nigeria is easier understood in terms of the competition between the ethnic groups in the country for the scarce resources available to the federation. The second point that seemed to have been underscored by Osaghae's definition is that ethnicity could affect any aspect of a society: most especially the ownership and management of national resources. In a related work, van den Berghe (1973) argued that public institutions in multi-ethnic and At national level, the ethnic conflicts in Nigeria are largely among the three dominant groups in the country: the Hausa/Fulani, Yoruba and Igbo. At local level, the conflicts are between ethnic neighbours and these vary from one State to the other. At each of the locations, groups compete for the available economic, material and political resources and institutions and these are often done in a manner that threatens national peace and stability.
B. Effect on Higher Education
The national crisis negatively affects higher education in terms of how ethnic groups compete, sometimes acrimoniously, for the location and management of Federal Universities, Polytechnics, Colleges of Education and Colleges of Agriculture. The university system is one of the most contested by the contending groups in the country.
The aggressive competition between the diverse groups in Nigeria for the control of the universities derives from the assumption that these institutions have significant roles to play in elite formation and recruitment in addition to the fact that the institutions generate local employment and economic regeneration. Within this framework, ethnic and sub-ethnic groups in the country are sensitive to the location of universities, appointment of their Vice Chancellors, staff recruitment as well as admission of students. In most cases, the people of the States and communities where the Federal and State universities are located see the institutions as their personal property and would want them managed as such.
The most controversial is the appointment of Vice Chancellors. Ethnic groups in Nigeria come together to "fight" one another once a new Vice Chancellor is to be appointed. There are several cases of this type of problem in the country. Vice Chancellors are also under pressure when staff and students of the universities are to be recruited and admitted respectively.
It is interesting to note that many of these conflicts over vacant Vice Chancellorship positions started to occur in the late 1990s when the issue of
C. Culture of Dialogue and Tolerance
The foregoing makes the need for fostering the culture of dialogue and understanding a major national project for Nigeria. What is "dialogue" and how do we want it applied to the issue of Nigeria's multiculturalism? Dialogue, as a social science concept, derives from two Greek words "dia" and "logos". "Dia" means "through' or "with each other" while "logos" means "the word". To this end, the word dialogue is etymologically understood to mean a free flow of information or meaning between people. In a multicultural society, it refers to an organic exchange information between and amongst peoples of diverse ethnic or religious orientations in such a way that helps to break down stereotypes and poor understanding of how others think or perceive the world around them (Weimann 2014: 19).
The significance of dialogue in this respect is embedded in the fact that poor communication is a major cause of identity conflict around the world. Explaining how this type of conflict crystallizes, Weimann argued that: Our interpretation of the message we have received from another person, as well as the decoding of the message, depends on our knowledge of this person. But, if the reality in which the message was formulated or encoded is too different from the reality it is interpreted in, or decoded, then the message received will not resemble the message emitted (Weimann 2014:23).
A Nigerian example suffices to support the above position. It has been clearly established at various meetings of the Nigeria Inter-religious Council (NIREC) which I coordinate that the major cause of religious crisis in Nigeria is that many adherents of the two major religions in the country -Islam and Christianity -do not have sufficient information on what each other's religion preaches. The Muslims are poorly educated about Christianity and the Christians are poorly educated about Islam. The reason is that there is limited opportunity for exchange of information between the adherents of the two religions. NIREC was established to deal with this problem.
Dialogue is a collaborative exercise; it requires the readiness of the interacting social actors. It is also voluntary; it cannot be forced on anybody. It requires trust, sincerity, and the willingness to accept diversity in human nature. It entails collective reflections, learning and communication between groups and a tolerance of paradox (or opposing views), the suspension of judgment and empathic listening. Its main goal is to promote societal cohesion by making complex issues to be collectively explored (Isaacs 1993;McGinn 2004).
D. Dialogue and the Nigerian University System
Dialogue in the context of higher education in Nigeria refers to two main situations: (i) the extent to which Nigerian universities are able to facilitate a healthy interaction among Nigerians most especially through capacity building (ii) the extent to which the university campus could be said to be a locus of intercultural exchange. The achievability of these two objectives is much dependent on the extent to which the government (most especially the Federal Government) is able to deal with the problem of identity crisis at national level. This is because, as said earlier, the problems come from the top.
It is thus compelling for us at this point to examine a number of federal policies that promote the culture of dialogue, understanding and tolerance to the extent of having powerful impact on the functioning of the university system in Nigeria. The first of such policies is contained in Section 14, sub-secti0n 3 of the 1979 Constitution which provides as follows: The composition of the Government of the Federation or any of its agencies and the conduct of its affairs shall be carried out in such a manner as to reflect the federal character of Nigeria and the need to promote national unity, and also to command national loyalty, thereby ensuring that there shall be no predominance of persons from a few states or combination of a few ethnic or other sectional groups in that Government or any of its agencies.
Section 277, sub-section 1 of the 1979 Constitution defined "federal character of Nigeria" as "the distinctive desire of the people of Nigeria to promote national unity, foster national unity and give every Nigerian a sense of belonging to the nation as expressed in Section 14(3) and 4 of this Constitution".
Since 1979, the federal character principle and others deriving from it (e.g. Section 157, sub-section 5; Section 197, sub-section 2; Section 199) have provided the basis for location of universities, polytechnics, Colleges of Education and even Federal Secondary Schools (High Schools) in Nigeria as well as the personnel to man these institutions. This policy is aimed at promoting equity in the Nigerian society and making all Nigerians to have a sense of belonging. A body known as the Federal Character Commission has been established by the Federal Government to promote, enforce and monitor compliance with provisions of the Federal Character Clauses of the Nigerian Constitution.
It is necessary to note however that unlike what obtains in Lebanon, Belgium, Cyprus, India and Malaysia where comparable constitutional provisions and public policy exist, the Nigerian Constitution does not reserve or earmark any quotas for any designated ethnic groups. Thus, each group in the federation adopts its own peculiar self-help strategies to get what it considers to be its own fair share of the "Nigerian national cake". The end product is widespread suspicion among groups and sub-groups in the country and this makes dialogue and understanding to be highly expedient at the national, state and communal levels but difficult to attain in the country. Higher education has significant roles to play in dealing with this problem.
Additionally, the Revised National Policy on Education which came into effect in 1981 specified that the growth and development of the university system in the country should ensure (a) " a more even geographical distribution (of universities) to provide a fairer spread of higher education facilities" in the country and that (b) "admission of students and recruitment of staff into universities and other institutions of higher learning should be on a broad national basis" (FGN cited in Yoloye 1989:75). This policy, in my consideration, is merely calling attention once again to the need to reflect "federal character". Ethnic and religious politics have made this policy a source of conflicts in many Nigerian universities today.
The Nigerian state also has a system for promoting dialogue and understanding on issues relating to students admission into federal universities. The prevailing regulation for the admission of students into the federal universities in the country was set out in a circular (Ref. No. FME/S/518/Vol. 1/99 of Sept. 2, 1983) in which all universities in the countries are enjoined to promote diversity in their admission policies. Similarly, the Association of American Universities (AAU), consisting of 62 leading North American research universities, adopted a statement on April 14, 1997 that expresses strong support for continued attention to diversity in university admissions. The US diversity scheme takes into account a wide range of considerations -including ethnicity, race, and gender (AAU 1997). The AAU statement, which is significant for putting the Nigerian policy in global perspective, provided the following as the rationale for the diversity policy: We believe that our students benefit significantly from education that takes place within a diverse setting. In the course of their university education, our students encounter and learn from others who have backgrounds and characteristics very different from their own. As we seek to prepare students for life in the twenty-first century, the educational value of such encounters will become very important, not less, than in the past.
A very substantial portion of our curriculum is enhanced by the discourse made possible by the heterogeneous backgrounds of our students. Equally, a significant part of education in our institutions takes place outside the classroom, in extracurricular activities where students learn how to work together, as well as to compete; how to exercise leadership, as well as to build their consensus. If our institutional capacity to bring together a genuinely diverse group of students is removed -or severely reduced -then the quality and texture of the education we provide will be significantly reduced… In this respect, we speak not only as educators, but also as concerned citizens. As presidents and chancellors of universities that have historically produced many of America's leaders in business, government, the professions, and the arts, we are conscious of our obligation to educate exceptional people who will serve all of the nation's different communities. The US statement on diversity admission contained a statement that requires that we shed more light on the equivalent policy in Nigeria. The AAU statement noted: "We do not advocate admitting students who cannot meet the criteria for admission to our universities. We do not endorse quota or "set asides" in admissions. But we do insist that we must be able, as educators, to select those students -from among many qualified applicants -who will best enable our institutions to fulfill their broad educational purposes".
The Nigerian admission policy favours what the AAU refers to pejoratively as "quota" or "set asides". However, the policy is not to compromise meritocracy but rather provide opportunities for the best candidates from all regions of the country to be provided access to university education. The first regulation is that all students to be admitted must have met the minimum standards of the affected universities.
The following admission criteria were provided in the Nigerian circular mentioned above: (a) Merit -40 percent (b) Catchment/Locality area -30 percent (c) Educationally Less Developed States -20 percent (d) others -10 percent. In other words if 100 students are to be admitted, the first best 40 are admitted first, the next 30 best are admitted from the locality and catchment areas, the next best 30 are admitted from the States of the federation considered to be educationally less developed, and the university bases the rest 10 admissions on any criteria considered best. In all, the 100 students admitted are among the best qualified candidates. No admission is given to any unqualified students.
There is the need to throw more light on the admission criteria. "Merit" as used above is determined by each candidate's score in the competitive examination conducted by the Joint Admissions Matriculation Board (JAMB) or the Advanced Level Certificate Examination conducted by the West African Examinations Council, the University of London and other related examination bodies. Under this criterion, the higher the score of a candidate, the higher the chances of his/her being admitted. "Catchment Area or Locality" is determined on the basis of states contiguous to the states in which each federal university is located. The "Educationally Less Developed States" are the later starters in western education. Candidates from these states are given special concession in the admission policy to enable them catch up with their counterparts from the more advanced states. Most of these states are from northern Nigeria. "Discretion" is used to admit students who could not be admitted based on the three earlier criteria but who in the opinion of the administrators of the concerned university deserve to be given consideration usually on humanitarian grounds (Yoloye 1989:65-68).
E. The Role of Higher Education in Promoting Dialogue and Understanding
The federal character policy in the appointment of Vice Chancellors and staff of the universities as well as in the admission of students has a positive impact on the promotion of a culture of dialogue, understanding and tolerance in Nigeria. It makes every Nigerian university to be a microsm of the Nigerian society in terms of generous mixture of all the ethnic and religious groups. Students from different parts of the country are thus forced to live side by side in the hostels; they do class assignments together and discuss the problems of Nigeria together. By the time many of these students return to their communities, they are different species of Nigerians altogether having towered above those ethnic and religious stereotypes that bedevil the Nigerian society and retard the growth of the country. Staff members go through the same experience. Their neighbors in the office, as a result of the Federal Character policy, are from other parts of Nigeria. Through the gossips and discussions of national issues in the office, they too are forced to understand the other groups in the country and to learn to work harmoniously with them.
The committee system in the university, Senate traditions and the wide ethnic and religious distribution of the teaching staff and principal officers of the university also constrain any Vice Chancellor from being sectional in his or her admission. He or she has to consult very widely and get the approval of appropriate bodies for all the steps that he or she takes. A good Vice Chancellor, under the present system in Nigeria, cannot afford not to be a good social mixer. He is daily invited by his staff and even students to naming ceremonies, funeral rites and other social functions that could in some cases offend his beliefs. He must demonstrate tolerance and understanding by attending many of these social functions.
The Higher Education in Nigeria, like their counterparts elsewhere, have three important mandates: to produce high-quality manpower for promoting national and international development; to carry out cutting-edge research; and to engage in community service. Issues pertaining to "fostering dialogue and understanding" point in the direction of the "community service" responsibility of the university. The fact remains, however, that in the process of teaching and doing research, universities equally promote dialogue and understanding. Some intervention projects of Nigerian universities can be used to shed more light on this.
F. Conclusion
The point made in this paper is that the Nigerian society is divided by the factors of ethnicity and religion and this works against the smooth running of the universities in the country most especially in terms of staff appointment, management of the institutions and recruitment of students into them. All these limit the extent to which the university system in the country could effectively foster the culture of dialogue and understanding. The paper defined the nature of this problem but attempted to underscore some modest efforts that some universities are making to still reach out to the people most especially in terms of making significant contributions to peacebuilding in the country.
The point was made in the presentation that the universities are merely a microsm of the larger Nigerian society and, within this framework, it is argued that the problems of ethnicity and religiosity in them would not go away until these two problems are played down at national level through the institutionalization of the policy of true federalism and a culture of tolerance most especially by the elite who exploit ethnicity and religiosity for personal gains. This does not mean that the universities themselves cannot solve the problems besetting them through in-house activities. These include the need for VCs to begin to see themselves as not representing any particular ethnic or religious groups as it often happens but as being the "fathers of all".
The management of the universities must place emphasis on meritocracy rather than mediocrity. This would make both staff and students to begin to see their future as being a function of how hardworking they are rather than a matter of sycophancy tied to ethnicity and religiosity. Universities must also organize regular seminars, workshops and conferences that build bridges across ethnic and religious divides. The lesson of the peace studies programmes at the Universities of Ibadan and Ilorin is that universities can silently contribute to the fostering of dialogue and understanding by training quality manpower for these activities at the grassroots level. | 2019-05-11T13:05:57.752Z | 2017-09-28T00:00:00.000 | {
"year": 2017,
"sha1": "afb0bcf7b91294eb1bf2cc53639a245a674bb1f6",
"oa_license": "CCBYSA",
"oa_url": "https://journal.scadindependent.org/index.php/jipeuradeun/article/download/183/261",
"oa_status": "HYBRID",
"pdf_src": "Adhoc",
"pdf_hash": "60ee6a7b367636ea84b62a4610a9006b2c4b795e",
"s2fieldsofstudy": [
"Education",
"Sociology",
"Political Science"
],
"extfieldsofstudy": [
"Political Science"
]
} |
256116483 | pes2o/s2orc | v3-fos-license | A note on isoperimetric inequalities of Gromov hyperbolic manifolds and graphs
We study in this paper the relationship of isoperimetric inequality and hyperbolicity for graphs and Riemannian manifolds. We obtain a characterization of graphs and Riemannian manifolds (with bounded local geometry) satisfying the (Cheeger) isoperimetric inequality, in terms of their Gromov boundary, improving similar results from a previous work. In particular, we prove that having a pole is a necessary condition to have isoperimetric inequality and, therefore, it can be removed as hypothesis.
Introduction
It is well-known that isoperimetric inequalities are of interest in applied and pure mathematics [22,48]. The Cheeger isoperimetric inequality is related with many conformal invariants in First author supported in part by a Grant from Ministerio de Ciencia, Innovación y Universidades (PGC2018-098321-B-I00), Spain. Second author supported in part by two Grants from Ministerio de Economía y Competitividad, Agencia Estatal de Investigación (AEI) and Fondo Europeo de Desarrollo Regional (FEDER) (MTM2016-78227-C2-1-P and MTM2017-90584-REDT), Spain. Also, the research of the second author was supported by the Madrid Government (Comunidad de Madrid-Spain) under the Multiannual Agreement with UC3M in the line of Excellence of University Professors (EPUC3M23), and in the context of the V PRICIT (Regional Programme of Research and Technological Innovation). We would like to thank to Paloma Martín for her support during this research.
B Álvaro Martínez-Pérez alvaro.martinezperez@uclm.es José M. Rodríguez jomaro@math.uc3m.es 1 Riemannian manifolds and graphs, namely the exponent of convergence, the bottom of the spectrum of the Laplace-Beltrami operator, Poincaré-Sobolev inequalities, and the Hausdorff dimensions of the sets of both escaping and bounded geodesics in negatively curved surfaces [4,13], [18, p. 228], [23,[27][28][29][30][31]43,46], [52, p. 333]. Isoperimetric inequality is also closely related to Ancona's project on the space of positive harmonic functions of Gromov-hyperbolic graphs and manifolds [7][8][9]. In fact, in the study of the Laplace operator on a hyperbolic manifold or graph X , Ancona obtained in those three last papers several interesting results, under the hypothesis that the bottom of the spectrum of the Laplace spectrum λ 1 (X ) is positive. If we denote by h(X ) the isoperimetric constant of X , the well-known Cheeger's inequality λ 1 (X ) ≥ 1 4 h(X ) 2 guarantees that λ 1 (X ) > 0 when h(X ) > 0 (see [17] for a converse inequality). Therefore, the results in this paper are useful in order to apply these Ancona's results: We provide the minimal hypotheses in order to guarantee h(X ) > 0 (in fact, we have a characterization of this fact), then λ 1 (X ) > 0 and we can apply Ancona's results to ensure the existence of solutions for elliptic partial differential equations in graphs and manifolds, among many other results (also, recall that in fact h(X ) > 0 and λ 1 (X ) > 0 are equivalent conditions for a large kind of graphs and manifolds).
There is a natural connection between isoperimetric inequality and hyperbolicity. Recall that one of the definitions of Gromov hyperbolicity involves some kind of isoperimetric inequality (see [3,34]).
The Cayley graph of the group Z is an example of hyperbolic graph without isoperimetric inequality. Cao proved in [21] that hyperbolicity implies isoperimetric inequality (with an extra hypothesis on the Gromov boundary).
In [41] we studied the relationship of hyperbolicity and Cheeger isoperimetric inequality in the context of graphs and Riemannian manifolds with bounded local geometry.
A celebrated theorem of Kanai [37] gives that quasi-isometries preserve several interesting properties (including isoperimetric inequalities) between Riemannian manifolds and graphs with bounded local geometry. The isoperimetric inequality is also preserved with weaker hypotheses in the context of Riemann surfaces [20,33].
In [41] we characterized, in terms of their Gromov boundary, the uniform hyperbolic graphs and a large class of hyperbolic manifolds satisfying isoperimetric inequality (see, respectively, Theorems 2 and 4). In Theorems 2, 4 and [21, Theorem 1.1] it is used the hypothesis of the existence of a pole (in fact, although [21, Theorem 1.1] apparently uses a different hypothesis, actually, in hyperbolic spaces, it is equivalent to the existence of a pole). The hypothesis on Gromov hyperbolicity is natural (we need it in order to deal with the Gromov boundary). However, although the hypothesis on the pole is technically needed in the proofs, it does not look natural. The goal of this paper is to remove this hypothesis in the statements of Theorems 2 and 4. Thus, in Theorems 7 and 8 we prove that having a pole is not needed as an hypothesis but it is also a necessary condition of the characterization; this characterization appears in Theorems 5 and 6.
Background
We include in this section the definitions and results that we will need in Sect. 3.
Definition 1 Given any Riemannian n-manifold M, the Cheeger isoperimetric constant of M is defined as
where U ranges over all non-empty bounded open subsets of M, and Vol m (S) denotes the m-dimensional Riemannian volume of S.
Along this paper we consider any graph G = (V , E) = (V (G), E(G)) endowed with its natural metric d G obtained by considering shortest paths (we always assume that every edge has length 1). Given any vertex v ∈ V (G) and m ∈ N we denote, as usual, by the open ball, the closed ball and the sphere in G with center v and radius m, respectively.
Definition 2 The combinatorial Cheeger isoperimetric constant of G is defined to be
where U ranges over all non-empty finite subsets of vertices in G, and |U | denotes the cardinality of the set U .
A graph or Riemannian manifold X satisfies the (Cheeger) isoperimetric inequality if h(X ) > 0, since this means that for every finite set of vertices U if X is a graph, and for every bounded open set U in the Riemannian n-manifold X .
We just consider graphs and manifolds X which are connected (recall that if X has connected components {X m }, then h(X ) = inf m h(X m )).
Let (X , d X ) and (Y , d Y ) be two metric spaces, and a, b constants satisfying It is easy to see that to be quasi-isometric is an equivalence relation.
Definition 3 Given a graph G and
We say that G is uniform or that G has bounded local geometry if it is μ-uniform for some constant μ.
We say that a Riemannian n-manifold M has bounded local geometry if there exist positive constants r , a, satisfying the following property: for each p ∈ M there exists a diffeomor- If M is a complete Riemannian manifold, the injectivity radius inj(x) of x ∈ M is the largest radius for which the exponential map at x is a diffeomorphism. If M has non-positive sectional curvatures, then the injectivity radius can be defined, also, as the supremum of those r > 0 such that B M (x, r ) is simply connected or, equivalently, as half the infimum of the lengths of the (homotopically non-trivial) loops based at x. The injectivity radius inj(M) of M is defined as One can check that M has bounded local geometry if it has a lower bound on its Ricci curvature and positive injectivity radius [10].
Given a metric space X and x, y, w ∈ X , we define the Gromov product of x, y with respect to w as Definition 4 Given δ ≥ 0 and a metric space X , we say that X is δ- (Gromov) hyperbolic if (x|y) w ≥ min{(x|z) w , (z|y) w } − δ for every x, y, z, w ∈ X . We say that X is hyperbolic if it is δ-hyperbolic for some δ ≥ 0.
If X is hyperbolic, we denote by δ(X ) the sharp hyperbolicity constant of X : If X is not hyperbolic, then we define δ(X ) = ∞.
Recall that a geodesic metric space X is a metric space such that for every x 1 , x 2 ∈ X there exists a geodesic joining them. A geodesic ray γ emanating from v ∈ X is a geodesic γ : [0, ∞) → X with γ (0) = v.
Definition 6
If X is a geodesic metric space and x 1 , x 2 , x 3 ∈ X , the union of three geodesics [x 1 x 2 ], [x 2 x 3 ] and [x 3 x 1 ] is a geodesic triangle that will be denoted by T = {x 1 , x 2 , x 3 }. We say that x 1 , x 2 , x 3 are the vertices of T . The geodesic triangle T is δ-thin if any side of T is contained in the δ-neighborhood of the union of the two other sides. We denote by δ th (T ) the sharp thin constant of T , i.e., δ th (T ) := inf{δ ≥ 0 : T is δ − thin }. We say that the space X satisfies the Rips condition with constant δ (or is δ-thin) if every geodesic triangle in X is δ-thin. We denote by δ th (X ) the sharp thin constant of X , i.e., δ th (X ) := sup{δ th (T ) : T is a geodesic triangle in X }.
It is well-known that a geodesic metric space is hyperbolic if and only if it satisfies the Rips condition for some constant [3,32]. Although there are many different definitions of Gromov hyperbolicity (see, e.g., [3,32]), they are equivalent in the following sense: if X is δ-hyperbolic with respect to one definition, then it is δ -hyperbolic with respect to another definition (for some δ related to δ).
Since we consider any graph G as a geodesic metric space, we need to identify any edge uv ∈ E(G) with the interval [0, 1] in R. Thus, the points in G are the vertices and, also, the points in the interior of any edge of G. Therefore, any connected graph G has a natural distance defined on its points (vertices and interior points), induced by taking shortest paths in G, and we can see G as a metric graph. (X , d) and a constant A > 1, we say that (X , d) is Auniformly perfect if there exists some ε 0 > 0 such that for every 0 < ε ≤ ε 0 and every x ∈ X there exist a point y ∈ X such that ε/A < d(x, y) ≤ ε. We say that (X , d) is uniformly perfect if there exists some A such that (X , d) is A-uniformly perfect.
Definition 7 Given a metric space
Let us recall the concept of boundary of a hyperbolic space. For further information we refer the reader to [14,19,32,34].
Let X be a hyperbolic space and fix w ∈ X . A sequence of points {x i } ⊂ X converges to infinity if One can check that this property is independent from the choice of w. Two sequences One can check that this defines an equivalence relation for sequences in X converging to infinity. The sequential boundary at infinity ∂ ∞ X of X is the set of equivalence classes of sequences converging to infinity. For every ξ, ξ ∈ ∂ ∞ X , its Gromov product with respect to w ∈ X is defined as where the infimum is taken over all sequences {x i } ∈ ξ , {x i } ∈ ξ . A metric d on ∂ ∞ X is visual if there exist w ∈ X , A > 1 and positive constants k 1 , k 2 , such that for all ξ, ξ ∈ ∂ ∞ X . In this case, we say that d is a visual metric with respect to w and A. Note that ∂ ∞ X is a bounded and complete metric space for any visual metric.
Theorem 2 [41, Theorem 4.15] If G is a hyperbolic uniform infinite graph with a pole, then h(G) > 0 if and only if ∂ ∞ G is uniformly perfect for some visual metric.
Let (X , d) be a metric space, Y ⊂ X and t > 0. The subset Y is t-separated in X if d(y 1 , y 2 ) ≥ t for any y 1 , y 2 ∈ Y with y 1 = y 2 . A t-approximation of X is a maximal (with respect to the inclusion) t-separated set in X .
Let X be a complete Riemannian manifold with induced metric d. If A ε is an εapproximation of X , the graph Γ A ε = (A ε , E) with E := {x y : x, y ∈ A ε , 0 < d(x, y) ≤ 2ε} is called an ε-net of X .
Proposition 1 [37, Lemmas 2.5 and 4.5]
If X is a complete Riemannian manifold with bounded local geometry and Γ is an ε-net in X , then Γ and X are quasi-isometric, and h(X ) > 0 if and only if h(Γ ) > 0. In [21] appear several sufficient conditions that guarantee that a Riemannian n-manifold is hyperbolic. Besides, see [53] for the case n = 2 of Riemannian surfaces.
Main results
In this section we prove that having a pole is a necessary condition for a hyperbolic uniform graph or a complete Riemannian manifold with bounded local geometry satisfying isoperimetric inequality. Therefore, it can be removed as hypothesis in the statements of Theorems 2 and 4.
In fact, we are going to prove the following results.
Theorem 5 Given a hyperbolic uniform graph Γ , then h(Γ ) > 0 if and only if Γ is an infinite
graph with a pole and ∂ ∞ Γ is uniformly perfect for some visual metric.
Theorem 6 Let X be a hyperbolic complete Riemannian manifold with bounded local geometry. Then, h(X ) > 0 if and only if X is non-compact and has a pole and ∂ ∞ X is uniformly perfect.
These results are a consequence of Theorems 2, 4, and the two following results.
Theorem 7
Let Γ be a uniform graph. If Γ is hyperbolic and h(Γ ) > 0, then Γ is an infinite graph with a pole.
Theorem 8 Let X be a complete Riemannian manifold with bounded local geometry. If X is hyperbolic and h(X ) > 0, then X is non-compact and has a pole.
Let us now proceed with the proof of Theorem 7.
Proof Seeking for a contradiction assume that Γ is a finite graph. Since h(Γ ) > 0, if we consider the set U = V (Γ ), then a contradiction. Therefore, Γ is an infinite graph. Seeking for a contradiction assume that Γ does not have a pole. Let μ be a constant such that Γ is μ-uniform.
Fix v ∈ V (Γ ) and denote by K the union of the geodesic rays starting from v. Note that there exists at least a geodesic ray, since Γ is an infinite graph, and so K = ∅. Since Γ does not have a pole, for each n there exists v n ∈ V (Γ ) with d Γ (v n , K ) ≥ 4n. Let η n : [0, n ] → Γ be a geodesic joining v n with K and such that η n (0) = v n and d Γ (v n , K ) = L(η n ) = n ≥ 4n.
Given s ∈ R, denote by s the lower integer part of s, i.e., the largest integer not greater than s. Assume that the ball B Γ η n (t), δ th (Γ ) + 1 intersects every geodesic joining v n with some point of K , for some t ∈ Z + with δ th (Γ ) < t ≤ 4n ≤ n . Thus, the connected component A n of Γ \B Γ η n (t), δ th (Γ ) + 1 containing v n satisfies that its boundary ∂ A n is contained in the sphere S Γ η n (t), δ th (Γ ) since Γ is a graph.
Consider now the geodesic triangle T n = {v, x n , y n } in Γ , and z n ∈ [x n y n ]. Since [vx n ] ∪ [vy n ] ⊂ K , we have and so, δ th (Γ ) ≥ n for every n. This is the contradiction we were looking for, since Γ is hyperbolic, and we conclude that Γ has a pole.
Finally, let us now proceed with the proof of Theorem 8.
Proof Seeking for a contradiction assume that X is a compact manifold. Since h(X ) > 0, if we consider the set U = X , then 0 < h(X ) ≤ Vol n−1 (∂ X ) Vol n (X ) = 0, v n v x n y n z n η n (2n) δ' Fig. 1 If there is a geodesic σ n = [v n y n ] from v n to K which does not intersect the ball B Γ η n (2n), δ , where δ := δ th (Γ ) + 1, then there is a point z n ∈ [x n y n ] which is far from K a contradiction. Hence, X is non-compact.
Note that X is a proper geodesic metric space since it is a complete Riemannian manifold. Also, Γ is a proper geodesic metric space since it is a uniform graph by Lemma 1.
By Proposition 1, X and Γ are quasi-isometric and so, Γ is hyperbolic by Theorem 3. Theorem 7 gives that Γ has a pole, and by Proposition 2, X has a pole.
Remark 1 Theorem 5 also allows to improve Theorem 4.12 in [42] which is based in the same results. In the same way, having a pole is a necessary condition and can be removed as hypothesis.
Remark 2 Theorem 5.11 in [44] states that for geodesic, visual, Gromov hyperbolic spaces, having uniformly perfect boundary at infinity is equivalent to being uniformly equilateral. Thus, Theorems 5 and 6 can also be written using this property and without using the boundary at infinity. | 2023-01-24T15:35:03.735Z | 2021-06-26T00:00:00.000 | {
"year": 2021,
"sha1": "f9f03f9c15773d7c3373b095de7551f711a95658",
"oa_license": "CCBY",
"oa_url": "https://link.springer.com/content/pdf/10.1007/s13398-021-01096-2.pdf",
"oa_status": "HYBRID",
"pdf_src": "SpringerNature",
"pdf_hash": "f9f03f9c15773d7c3373b095de7551f711a95658",
"s2fieldsofstudy": [
"Mathematics"
],
"extfieldsofstudy": []
} |
118873599 | pes2o/s2orc | v3-fos-license | Properties of polycrystalline nanoparticles with uniaxial and cubic types of magnetic anisotropy of individual grains
The influence of the crystal structure inhomogeneities on the magnetic properties of cobalt nanoparticles with different aspect ratio and spherical nanoparticles of chromium dioxide, cobalt ferrite and magnetite has been studied by means of numerical simulation. The polycrystalline nanoparticles are modeled by means of subdivision of the nanoparticle volume into tightly bound single-crystal granules with randomly distributed directions of the easy anisotropy axes. The probability of appearance of quasi uniform and vortex states in sufficiently large assemblies of polycrystalline nanoparticles of various types have been calculated depending on the nanoparticle diameter. It is shown that the subdivision of a nanoparticle into single-crystal granules with different orientations of the easy anisotropy axes substantially reduces the effective single-domain diameters for particles with uniaxial type of anisotropy of individual granules. However, for particles with cubic type of magnetic anisotropy the influence of the crystal structure inhomogeneities on the equilibrium properties of the particles is not so important even for magnetically hard cobalt ferrite nanoparticles. It is practically absent for magnetically soft magnetite nanoparticles.
I. INTRODUCTION
Magnetic nanoparticle assemblies are promising for various technological and biomedical applications, in particular for magnetic resonance imaging contrast enhancement, targeted drug delivery and magnetic hyperthermia [1][2][3]. However, in order to fully understand the magnetic properties of nanoparticles, detailed experimental information on the particle crystal structure is important. Are the magnetic particles singlecrystal or do they consist of a set of several monocrystalline grains? In the latter case what the average size of the monocrystalline granules is? What are the type of magnetic anisotropy and the direction of the easy anisotropy axes of individual granules in this nanoparticle? Such detailed information is available in some cases for very small nanoparticles with dimensions on the order of several nanometers [4,5]. But for magnetic nanoparticles of larger sizes experimental information on the particle crystal structure is scarce. It is usually tacitly assumed that magnetic nanoparticles with dimensions on the order of several tens of nanometers, which are used in particular in biomedical applications [2,3], are monocrystalline. However, it does remain unclear why the properties of these nanoparticles, for example, the saturation magnetization, the magnetic anisotropy constants, etc. differ substantially from the corresponding bulk values.
Meanwhile, the formation of polycrystalline particles in the course of chemical reactions is quite likely [6], if the growth of large nanoparticles occurs as a result of the coalescence of single-crystal nuclei. It has been found recently, that monodisperse iron oxide nanoparticles of sufficiently large sizes prepared by thermal decomposition of organometallic precursors [7] or other methods [8][9][10][11] may have complicated crystalline structure. Multi-core nanoparticles [12,13] consisting of monocrystalline magnetic grains can also be considered as polycrystalline nanoparticles if there is exchange interaction of appreciable value between the constituting grains.
It should be noted that now, due to a significant increase in computer performance, numerical modeling makes it possible to study the equilibrium and kinetic properties of magnetic nanoparticles in all details. Single-domain diameters of different types of magnetic particles can be obtained both with the help of analytical estimates [14][15][16][17][18] and using numerical modeling [18][19][20][21][22][23][24]. It was shown in particular [23] that effective singledomain diameters of non ellipsoidal nanoparticles, such as cube or cylinder, do not differ considerably from single-domain diameter of an ellipsoid of a proper shape. At the same time, the experimental values for singledomain diameters of various magnetic nanoparticles, their dependence on the particle shape, possible composition variations, crystal structure, etc., are practically unknown.
Investigation of the effect of the crystal structure heterogeneities on the equilibrium magnetic properties of nanoparticles is important also for understanding the behavior of nanoparticles in a quasistatic and alternating magnetic field. Recently [25], the magnetic properties of polycrystalline cobalt nanoparticles of spherical shape have been studied theoretically. In this paper, single-domain diameters for oblate and elongated single-crystal cobalt particles, as well as for spherical nanoparticles of chromium dioxide, cobalt ferrite, and magnetite are determined using numerical simulation. Particles of cobalt and chromium dioxide have uniaxial type of magnetic anisotropy, whereas particles of cobalt ferrite and magnetite belong to the cubic type of anisotropy, with a different number of equivalent directions of the easy anisotropy axes. In addition, the nanoparticles studied have different magnetic hardness. The latter is characterized by a dimensionless parameter 14,17,18], where N z is the demagnetizing factor of the spheroidal particle along the symmetry axis, M s is the saturation magnetization, and K is the absolute value of the particle anisotropy constant.
Analogous calculations have been also carried out for assemblies of polycrystalline nanoparticles of the same compositions, with different amounts of singlecrystal granules in the particle volume. In the absence of more specific information, it is assumed that the easy anisotropy axes in various single-crystal granules of a polycrystalline nanoparticle are randomly oriented. It is shown that for polycrystalline nanoparticles with uniaxial anisotropy the effective single-domain diameter D c,ef is significantly reduced in comparison with that of monocrystalline nanoparticle, D c0 . The difference between these diameters increases with increasing magnetic hardness of the nanoparticle, that is, with a decrease in the parameter p. On the other hand, for nanoparticles with a cubic type of magnetic anisotropy, a decrease in the effective single-domain diameter was obtained only for magnetically hard nanoparticles of cobalt ferrite. It has been found that the diameters D c,ef and D c0 coincide for magnetically soft magnetite nanoparticles.
II. Numerical simulation
Dynamics of the unit magnetization vector ( ) r r r α of a polycrystalline nanoparticle is described by the Landau -Lifshitz -Gilbert (LLG) equation [14,17] ( ) where γ is the gyromagnetic ratio and κ is the ( Here V is the nanoparticle volume, M s is the saturation magnetization, C = 2A is the exchange constant, Typically, several thousands of numerical cells, N ~ 10 3 -10 4 , are necessary to approximate with sufficient accuracy stationary magnetization distributions in nanoparticle volume. The equilibrium micromagnetic configurations in the nanoparticles studied were calculated starting from arbitrary initial micromagnetic state, the magnetic damping parameter being κ = 0.5. In accordance with the Eq. (1), the final magnetization state is assumed to be stable under the condition where i α r and To simulate a distribution of the easy anisotropy axes in a polycrystalline magnetic nanoparticle, we first select randomly within a particle volume several seed cells, N g = 4 -16, which serve as the nuclei of the monocrystalline grains to be constructed. Then we consistently attach the closest numerical cells to every embryo until all available numerical cells are connected to one of the growing grains. Using such an algorithm one can generate within the nanoparticle volume the disjoint continuous areas representing the monocrystalline grains of a polycrystalline nanoparticle. Interestingly, this algorithm leads to the partition of the particle volume into N g grains of similar volume. Other partitioning algorithms that have been tried lead to similar results.
The directions of the easy anisotropy axes in the crystallites created in such a manner were selected randomly. For a nanoparticle with uniaxial magnetic anisotropy the magneto-crystalline anisotropy energy density of the j-th monocrystalline grain is given by where K is the uniaxial anisotropy constant and n j is the unit vector parallel to the easy anisotropy axis of the given grain. For a nanoparticle with cubic anisotropy the corresponding expression reads Here K c is the cubic magnetic anisotropy constant, and (e 1j , e 2j , e 3j ) is a set of orthogonal unit vectors that determine an orientation of j-th monocrystalline grain of the nanoparticle. One may hope that this numerical model is capable to describe the distribution of the easy anisotropy axes in real polycrystalline nanoparticles created as a result of the coalescence of monocrystalline embryos originally formed by a chemical reaction in a solution [6]. Fig. 1 shows typical examples of a random partitioning of a quasi-spherical nanoparticle into different numbers N g = 4, 8, 16 monocrystalline granules of approximately equal volume obtained by the algorithm described above. It is assumed that the type of the magnetic anisotropy in all granules of the given magnetic nanoparticle is the same. However, as we mentioned above the easy anisotropy axes are randomly oriented in different granules of a polycrystalline nanoparticle.
When a subdivision of a particle of a given diameter into monocrystalline granules is carried out, and the directions of the easy anisotropy axes are assigned to each numerical cell, the stationary magnetization distributions existing in a given nanoparticle can be obtained by solving the dynamic LLG Eq. (1). The calculations show that for a given polycrystalline nanoparticle in the range of diameters studied only one or two stable stationary micromagnetic states usually exist. They are a quasi-uniform state with average magnetization close to the saturation magnetization, and a vortex with relatively small remanent magnetization. In this paper, for each nanoparticle of the given type and geometry, a sufficiently large number of independent random particle partitions with the fixed number N g of monocrystalline granules was generated. Then, for this assembly of polycrystalline nanoparticles the average energy and average magnetization of low-lying micromagnetic states were calculated. The material parameters of various types of the nanoparticles studied are given in Table 1. In order to investigate the effect of complicated crystal structure on the particle magnetic properties, the nanoparticles of both uniaxial (Co, CrO 2 ) and cubic (CoFe 2 O 4 , Fe 3 O 4 ) types of magnetic anisotropy were considered.
III. Results and discussion
Let us consider the results of numerical simulation obtained for polycrystalline magnetic nanoparticles listed in Table 1, in comparison with the properties of monocrystalline nanoparticles of the same composition.
Cobalt nanoparticles
The magnetic properties of spherical polycrystalline cobalt nanoparticles have been studied in Ref. 25. Meanwhile, in the experiment cobalt nanoparticles of various shapes can be found. Therefore, in this paper oblate and elongated spheroidal cobalt nanoparticles with aspect ratios D z /D = 2/3 and 3/2, respectively, were also studied for completeness. Here D z and D are the longitudinal and transverse diameters of a spheroid, respectively. It is assumed that monocrystalline cobalt granules have a hexagonal crystal structure, so that the energy density of magnetic anisotropy of individual cobalt granules is given by Eq. (4a) with the corresponding uniaxial anisotropy constant, K = 4.3×10 6 erg/cm 3 . Fig. 3a correspond to the case of a monocrystalline cobalt nanoparticle with easy anisotropy axis parallel to the short diameter of the spheroid, D z . In this nanoparticle for all investigated diameters there is a stable uniform state with a magnetization parallel to the easy anisotropy axis (curve 4). The vortex state with the vortex axis oriented perpendicular to the easy anisotropy axis competes in energy with the uniform magnetization, (curve 5 in Fig. 3a). Curve 6 in Fig. 3a shows the energy of the vortex whose axis is oriented parallel to the easy anisotropy axis. The intersection of the curves 4 and 5 in Fig. 3a determines the single-domain diameter, D c0 = 41 nm, of the oblate monocrystalline cobalt nanoparticle with aspect ratio D z /D = 2/3. For the oblate particle the transverse vortex remains stable in the range of diameters D ≥ 40 nm. Curves 1-3 in Fig. 3a show the average energies of stationary micromagnetic states in assemblies of polycrystalline oblate nanoparticles with different amounts of crystalline granules, namely, N g = 4 for curve 1, N g = 8 for curve 2, and N g = 16 for curve 3, respectively. To obtain statistically reliable results, curves 1-3 were obtained by averaging over 200 -250 independent realizations of polycrystalline oblate nanoparticles of fixed diameter in the range of sizes 24 nm ≤ D ≤ 60 nm. The dependence of the average reduced magnetic moment of oblate cobalt nanoparticles on the transverse particle diameter is shown in Fig. 3b. Curves 4 and 5 in Fig. 3b give the magnetization of a monocrystalline cobalt nanoparticle in uniform and transverse vortex states, respectively, Curves 1-3 correspond to polycrystalline nanoparticles with various number of granules N g = 4, 8 and 16, correspondingly.
Comparing the curves 1-3 in Fig. 3a and 3b, one can conclude that the stationary states in polycrystalline particles with the smallest number of granules, N g = 4, (curves 1), have the smallest average energy and, at the same time, the largest average magnetic moment. With an increase in the number of granules in the polycrystalline nanoparticle, the average particle energy increases, whereas average magnetic moment decreases. This effect is a direct consequence of a subdivision of the nanoparticle volume into the domains with different directions of the easy anisotropy axes.
The calculations show that only quasi-uniform states with a reduced magnetic moment M e /M s > 0.9 are realized for oblate polycrystalline cobalt nanoparticles with diameters D ≤ 28 nm, and only vortex states are realized in the range of diameters D ≥ 52 nm. In the intermediate range of sizes, 28 nm < D < 52 nm, both vortex and quasi-uniform micromagnetic states can be found in oblate polycrystalline nanoparticles. Based on the results obtained, one can conclude that in oblate polycrystalline cobalt nanoparticles quasi-uniform micromagnetic states arise in the interval of transverse diameters D ≤ 28 nm, regardless of the number of monocrystalline granules in the particle. Accordingly, the value D c,ef = 28 nm can be taken as the effective single-domain diameter of the polycrystalline cobalt nanoparticle with aspect ratio D z /D = 2/3. The latter is thus much smaller than the single-domain diameter, D c0 = 41 nm, of the oblate monocrystalline cobalt nanoparticle. a monocrystalline cobalt nanoparticle with an easy anisotropy axis parallel to particle symmetry axis. Curve 5 corresponds to the vortex with the vortex axis parallel to the easy anisotropy axis. As can be seen in Fig. 5a, the single-domain diameter of an elongated monocrystalline cobalt nanoparticle with aspect ratio D z /D = 1.5 equals D c0 = 56 nm. The vortex state in this nanoparticle is stable at D ≥ 36 nm. Detailed calculations carried out for polycrystalline cobalt nanoparticles with aspect ratio D z /D = 1.5 in the range of transverse diameters 20 nm ≤ D ≤ 64 nm showed that only quasi-uniform states with reduced magnetic moment close to unity exist in the interval D ≤ 24 nm, whereas in the range of transverse diameters D ≥ 44 nm only vortex states are realized. In the intermediate range of diameters, 24 nm < D < 44 nm, in elongated polycrystalline cobalt nanoparticles both vortex and quasi uniform micromagnetic states can be found. Thus, the value D c,ef = 24 nm can be taken as the effective single-domain diameter of polycrystalline cobalt nanoparticle with aspect ratio D z /D = 1.5.
Chromium dioxide
Similar calculations have been made for spherical chromium dioxide nanoparticles with uniaxial type of magnetic anisotropy, Fig. 6. a) Energy diagram of stationary micromagnetic states in chromium dioxide nanoparticles; b) the total reduced magnetic moment as a function of particle diameter. Curves 1-3 correspond to polycrystalline nanoparticles, and curves 4, 5 to monocrystalline ones, respectively. but much lower saturation magnetization than that of cobalt.
As can be seen in Fig. 6a, for a monocrystalline particle of chromium dioxide the single-domain diameter is given by D c0 = 60 nm. The calculations of stationary magnetization distributions for polycrystalline chromium dioxide nanoparticles were carried out in the interval of diameters 40 nm ≤ D ≤ 120 nm. It was found that only quasi-uniform states are realized in the range of diameters D ≤ 44 nm, and only vortices exist for D ≥ 60 nm. In the transition region, 44 nm < D < 60 nm, both vortices and quasi uniform magnetization distributions are realized with various probabilities. Based on these calculations, the effective single-domain diameter of polycrystalline chromium dioxide nanoparticles was determined to be D c,ef = 44 nm.
Cobalt ferrite
To investigate the effect of the type of magnetic anisotropy on the magnetic properties of polycrystalline nanoparticles, spherical nanoparticles of cobalt ferrite, with positive cubic anisotropy constant, K c = 2.0×10 6 erg/cm 3 , were considered. Consequently, monocrystalline cobalt ferrite granules have 6 equivalent directions of the easy anisotropy axis. The energy density of magnetic anisotropy of individual cobalt ferrite grain is given by Eq. (4b).
As Fig. 7a shows, for a monocrystalline cobalt ferrite nanoparticle the single-domain diameter is given by D c0 = 140 nm. The calculations of stationary magnetization distributions for polycrystalline cobalt ferrite nanoparticles were carried out in the interval of diameters 60 nm ≤ D ≤ 150 nm. In Figs. 7b -7e the probabilities of the appearance of stationary micromagnetic states with different mean magnetization are shown for polycrystalline cobalt ferrite nanoparticles of various diameters consisting of N g = 4 monocrystalline grains. One can see that for polycrystalline cobalt ferrite nanoparticles with diameter D = 110 nm most of the stationary micromagnetic states have large average magnetization, M e /M s > 0.9. The same is true for nanoparticles with diameters D < 110 nm. On the other hand, for nanoparticles with diameter D ≥ 120 the probability of appearance of non-uniform micromagnetic state with average magnetization, M e /M s ~ 0.5, increases gradually as a function of the particle diameter. One can conclude safely, that the lower bound for the effective single-domain diameter of polycrystalline cobalt ferrite nanoparticles is given by D c,ef = 110 nm.
Magnetite
The magnetic properties of polycrystalline magnetite nanoparticles are particularly interesting, since magnetite nanoparticles are widely used in biomedicine [1][2][3]. Magnetite nanoparticles also have cubic type of magnetic anisotropy. But unlike cobalt ferrite, the cubic anisotropy constant for magnetite is negative, K c = -1.0×10 5 erg/cm 3 . As a result, monocrystalline magnetite granules have 8 equivalent directions of the easy anisotropy axis. Fig. 8 shows the energy of stationary micromagnetic states calculated for spherical monocrystalline and polycrystalline magnetite nanoparticles with different amounts of crystalline granules in the range of diameters 32 nm ≤ D ≤ 96 nm. As can be seen in this figure, the effect of complicated crystal structure on the properties of magnetite nanoparticles is insignificant. In particular, singledomain diameters for monocrystalline and polycrystalline magnetite nanoparticles coincide, D c0 = D c,ef = 64 nm.
In Table 2 we present the calculated single-domain diameters, D c0 , of the monocrystalline nanoparticles studied in comparison with the effective single-domain diameters of the polycrystalline ones, D c,ef . In addition, the values of the parameter , which characterizes the magnetic hardness of the nanoparticles, are also given in Table 2. Note, that one has p >> 1 and p < 1 for particles of soft and hard magnetic types, respectively. First of all, one can see that a noticeable difference between the D c0 and D c,ef values exists for magnetic nanoparticles with uniaxial type of magnetic anisotropy. Furthermore, this difference increases with increasing of magnetic hardness of the nanoparticle. The greatest difference between the D c0 and D c,ef diameters was obtained for elongated cobalt nanoparticles with p = 0.667. On the other hand, for nanoparticles with cubic anisotropy, relatively small difference in D c0 and D c,ef diameters exists only for cobalt ferrite nanoparticles with a small parameter p = 0.185. For magnetically soft magnetite nanoparticles the single-domain diameters D c0 and D c,ef coincide. Obviously, the individual monocrystalline grains with cubic anisotropy have a large number of equivalent directions of the easy anisotropy axis. Therefore, for polycrystalline nanoparticle with cubic anisotorpy the probability to get an inhomogeneous distribution of easy anisotropy axes over the particle volume is much smaller than for particles with uniaxial type of magnetic anisotropy. As a result, some difference between the D c0 and D c,ef values for polycrystalline nanoparticle with cubic anisotropy appears only for small values of parameter p. This gives a qualitative physical explanation for the results of the numerical simulation presented in Table 2.
IV. Conclusions
In this paper the influence of the crystal structure heterogeneities on the equilibrium magnetic properties of cobalt nanoparticles with different aspect ratio and spherical nanoparticles of chromium dioxide, cobalt ferrite and magnetite has been studied using numerical simulation. The nanoparticles studied possess both uniaxial and cubic types of magnetic anisotropy and have different magnetic hardness. For monocrystalline nanoparticles of these types the single-domain diameters D c0 have been determined comparing the total energies of quasi-uniform and vortex micromagnetic states. The polycrystalline nanoparticles are modeled by means of subdivision of the nanoparticle volume into tightly bound single-crystal granules with randomly distributed directions of the easy anisotropy axes. The probability of appearance of quasi uniform and vortex states in sufficiently large assemblies of polycrystalline nanoparticles of various types have been calculated depending on the nanoparticle diameter. For polycrystalline nanoparticles the effective single-domain diameters, D c,ef , have been determined under the condition that only quasi-uniform states with reduced magnetic moment M e /M s > 0.9 exist in the polycrystalline nanoparticle of a given type in the range of diameters D < D c,ef .
It is shown that the subdivision of a nanoparticle into single-crystal granules with different orientations of the easy anisotropy axes substantially reduces the effective single-domain diameters for particles with uniaxial type of anisotropy. Moreover, for these nanoparticles the difference between the diameters D c0 and D c,ef increases with increasing of the particle magnetic hardness. At the same time, for particles with cubic type of magnetic anisotropy, the influence of the crystal structure inhomogeneities on the equilibrium properties of the particles is not so important even for magnetically hard cobalt ferrite nanoparticles. It is practically absent for magnetically soft magnetite nanoparticles. This is a consequence of the fact that in particles with cubic type of magnetic anisotropy the probability to create an inhomogeneous easy axis distribution over the nanoparticle volume is small due to the presence of a large number of equivalent directions of the easy anisotropy axes in the individual granules. | 2019-04-13T20:17:29.795Z | 2018-01-04T00:00:00.000 | {
"year": 2018,
"sha1": "56a8e5ec57646d490274db289ccac2d754d788d8",
"oa_license": null,
"oa_url": "http://arxiv.org/pdf/1801.01266",
"oa_status": "GREEN",
"pdf_src": "Arxiv",
"pdf_hash": "56a8e5ec57646d490274db289ccac2d754d788d8",
"s2fieldsofstudy": [
"Materials Science"
],
"extfieldsofstudy": [
"Materials Science",
"Physics"
]
} |
214720036 | pes2o/s2orc | v3-fos-license | 14 Years’ experience of esophageal replacement surgeries
Background Esophageal replacement is a challenge to the therapeutic skills of surgeons and a technically demanding operation in the pediatric age group. Various conduits and routes have been described in the literature, each with their specific advantages and disadvantages. We carried out this retrospective study to share our experience of esophageal replacement. Methodology This study was conducted at the department of pediatric surgery The Children’s Hospital and The Institute of Child Health, Lahore. The records of patients treated for esophageal replacement were reviewed. The patients under follow-up were called for clinical evaluation and assessed of long terms complications if any. Results A total of 93 patients with esophageal replacement were included in the study. Esophageal replacement was done with gastric transposition in 84 cases (90%), colon interposition in 7 cases (7.5%) including one case of redo colonic interposition, and jejunal interposition in 2 cases (2%). Routes of esophageal replacement were trans-hiatal in 71 (76%), retrosternal in 13 (14%), and trans-hiatal with thoracotomy in 9 (10%) patients. Postoperatively, all of the conduits maintained viability. Wound infection was seen in 10 (11%), wound dehiscence in 5 (5%), anastomotic leak in 9 (10%), anastomotic stenosis in 12 (13%), fistula formation in 4 (4%), aortic injury 1 (1%), dumping syndrome 8 (9%), reflux 18 (19%), dysphagia 15 (16%) and death occurred in 12 patients (13%). Conclusion There are problems with esophageal replacement in developing countries. In this context, gastric conduit appeared as the best conduit for esophageal replacement, using the trans-hiatal route for replacement, in the authors’ experience.
Introduction
In children, indications for esophageal replacement include esophageal corrosive strictures not amenable to dilatations, long gap esophageal atresia, long-segment congenital esophageal stenosis (CES), esophageal malignancies and some rare esophageal disorders [1,2]. The various conduits used for replacement are gastric pull-up, gastric tube formation, colonic interposition, and jejunal interposition. The option selected depends upon anatomic factors, previous surgery, the extent of corrosive injury to esophagus and stomach and surgical expertise of the surgeon [3]. Every conduit and route of esophageal replacement has its own merits and demerits [1]. Additional procedures may be required in case of various short term and long term complications. Meticulous surgical technique and postoperative care are required for optimal outcome [4].
Herein, we share our 14-years experience of esophageal replacement, with various conduits especially with gastric pull-up, in terms of etiology, surgical details, complications encountered and their management, outcome, and follow-up.
Methodology
This retrospective descriptive case series was conducted at the department of Pediatric Surgery, The Children's Hospital and The Institute of The Child's Health Lahore from July 2005 to June 2019. Permission was taken from the Institutional Ethical Review Board. The charts of all patients treated for esophageal replacement were reviewed in depth.
3
A self-structured Pro-forma was used for data collection. The data includes the demographic details (age, gender) presenting complaints, indications of esophageal replacement, investigations, operative procedure, complications, outcome, and follow-up. The patients were called for clinical evaluation during the data collection and assessed for any late complications. The collected data were analyzed using SPSS version 24. The descriptive frequencies were analyzed for quantitative data and percentages were documented for qualitative data.
Results
A total of 93 patients with esophageal replacement were included. Males were 61 (65.59%) and females 32 (34.41%) with male to female ration of 1.9:1. Seven of these were referred from other provinces and Afghanistan. The mean age was 63.87 months ± 50.16 with a range of one day to 180 months.
The majority of the patients were booked through outpatient clinics for esophageal replacement who were on our regular follow-up and declared non-dilatable in case of corrosive injury or with esophagostomy and gastrostomy for long gap esophageal atresia. The patients of congenital esophageal stenosis and esophageal tumor presented the complaints of dysphagia, weight loss, regurgitation, and repeated chest infections. Neonates were booked from nursery ICU for esophageal replacement. The etiology of esophageal replacement included caustic ingestion in 68 (73.11%) patients, isolated long gap esophageal atresia in 13 (13.97%), long gap esophageal atresia with tracheaesophageal fistula in 5 (5.37%), congenital esophageal stenosis in 4 (4.30%), esophageal tumor in 2 (2.15%), and one patient with repeated failed surgeries for long segment cardiac achalasia.
These patients underwent various assessments such as nutritional status, blood labs, chest X-ray and contrast esophagogram, and esophagoscopy before surgery. In 44 (47.31%) patients feeding jejunostomy and in six patients feeding gastrostomy was already done for feeding purpose (these were corrosive patients); whereas, in 15 (16.12%) patients feeding gastrostomy and esophagostomy was done for long gap esophageal atresia. Twelve patients (12.90%) with corrosive injury, who had relatively good health, required total parenteral nutrition through a central venous line for weight optimization for a short period before surgery. The remaining 13 (13.97%) patients,(4 CES, 2 tumor, 6 corrosive, 1 Cardiac achalasia) were on oral nutrition as they were in relatively better health and was still able to take nutrition orally. They were directly subjected to replacement surgery. Feeding jejunostomy was preferred method for nutritional support in corrosive stricture patients if needed, as we wanted to spare the stomach for pull up. These are the patients who had weight less then 70th percentile for age and had esophageal strictures that were non-negotiable on dilatation and unable to take orally. Feeding gastrostomy was preferred in esophageal atresia patients, as jejunostomy was considered unsafe in the neonatal age group. All these patients were discussed with nutritionist and case-specific diet plan and response was monitored during fallow up visits.
After optimization, these patients underwent esophageal replacement. Three patients underwent primary gastric pullup. These are patients with isolated long gap esophageal atresia. The most common conduit was stomach as gastric pull-up based on right gastric and right gastro-epiploic vessels and next common conduit was colonic interposition, based on middle colic artery (right colon-isoperistaltic). The commonly used route was trans-hiatal route (80 patients) either isolated this route (71 patients) or combined with thoracotomy (9 patients) in difficult cases. Table 1 describes the details of conduits utilized and routes of replacement in our series. Our preferred conduit was gastric pull-up (84 patients). Next preference was colon (7 patients) if not available then we used jejunum in two patients. Additionally, in one patient pyloroplasty was done, whereas in 45 (48.39%) patients of gastric pull-up, pyloromyotomy was done to avoid hold up in the chest. Moreover, we used a GIA linear cutter/stapler for gastroesophageal junction excision in all cases of gastric pull-up. Whenever the transhiatal route was used native esophagus was removed, however, where retrosternal route was used esophagus was left in situ ( Fig. 1). The fundus was then anastomosed with the upper esophageal pouch. In one patient with jejunal interposition, the lower esophagus and stomach both were very scarred due to corrosive fibrosis; therefore, a roux-en-y loop of jejunum was pulled-up to the chest and anastomosed with the upper esophagus in the chest because the vascular arcade of jejunum posed difficulty in the procedure. In one case of colonic interposition using the retrosternal route, the lower end of colon had to be anastomosed with jejunum due to complete scarring of the stomach. In another patient with a corrosive long esophageal stricture along with gastric antral/ pyloric stricture (who developed complete re-stenosis after pyloroplasty), colonic interposition through the retrosternal route was performed with anterior wall of stomach along with gastroduodenostomy to bypass the pylorus (Fig. 2). In three patients with retrosternal route, the manubrium sterni was also removed per operatively due to compression on the conduit. This is based on the finding that two patients with dysphagia after esophageal replacement improved after resection of the manubrium sterni. Accordingly, in the following cases using the retrosternal route we made this standard that when the thoracic inlet is too tight that even two fingers can not be negotiated (subject to author's assessment) manubrial resection should be done. In nine cases, the chest had to be opened in patients with CES, tumor, cardiac achalasia and one for technical difficulty due to either fibrosis secondary to corrosive ingestion and one due to injury to the aorta. One patient was referred from another hospital with dehisced colonic interposition. This patient was operated by median sternotomy and re-anastomosis with both upper esophagus and colon was performed. In eight patients, anastomosis was performed in the chest (four CES and two tumors done using a partial gastric pull-up, and two jejujnal replacement for caustic stricture). In 85 patients, the anastomosis was performed in the neck. Table 2 describes early and late per/postoperative complications in our series. Per operatively, we have encountered one aortic arch injury, which was dealt with help of cardiac surgeon after open thoracotomy. Postoperatively, various complications were encountered. Common early complications were pneumothorax in 10 (10.75%) (3 on table and 7 in early postoperative period), anastomotic leak in 9 (9.68%) [3 in chest (one with jejunal transposition, 2 with partial gastric pull-up) and 6 in neck], and wound infection in 10 (10.75%) patients. The pneumothorax was dealt with tube thoracostomy. Anastomotic leaks (nine patients) were managed conservatively; out of those, seven healed ultimately. Wound infection was dealt with daily dressings and wound management. One patient who presented with anastomotic stricture needed median sternotomy and revision of anastomosis 5 years after the previous gastric pull-up through retrosternal route. Three more patients with anastomotic stricture with gastric pull up needed revision of anastomosis in neck, years after initial replacement surgery. One of these was operated 7 years after initial replacement, succumbed to death due to aspiration. Rest of patients with anastomotic strictures, were dealt with anastomotic dilatation with metallic/gum elastic bougies. In two patients, the manubrium was contributing to the postoperative dysphagia so manubrium sterni was removed afterward. One of patient with colonic transplant had signs of obstruction in the early postoperative period and on exploration revealed internal herniation in leftover defect of mesentery. We encountered one case of colonic dilatation post-operatively where we performed a re-do surgery. However, no case of gastric dilatation was seen in our series. So our four patients needed revision of anastomosis, one through median sternotomy. Dumping syndrome was observed in eight patients (8.6%) of gastric pull-up and all of these patients had pyloromyotomy as additional procedure. In our experience, there was no difference of gastric content hold up with or without pyloromyotomy; it was associated with dumping syndrome in eight patients. Patients with reflux were managed medically.
In this series, 12 patients succumbed to death. One patient of isolated long gap esophageal with gastric pullup through trans-hiatal route could not be weaned off ventilator due to high intrathoracic pressure and ultimately acquired sepsis and died after 15 days. Two patients died due to mechanical ventilator failure, which remained undetected. Two patients with isolated long gap atresia having primary gastric pull died due to sepsis in the early postoperative period. Two patients (one with carcinoma of the lower esophagus and other one CES with partial gastric pull up) died in the early postoperative period due to a leak in the chest. One with esophageal dilatation for corrosive stricture suffered from a leak and developed empyema. Despite decortication, esophagectomy and replacement carried out in the same setting, the patient succumbed to death postoperatively. One patient died due to aspiration after revision of anastomosis for anastomotic stricture. The remaining three patients died due to sepsis. After the initial follow-up of 2 years, the majority of patients were lost to follow-up. Currently, only 20 patients are on follow-up and doing fine. Out of these, 8 patients required esophageal dilatations (Table 3).
Discussion
Historically, in nineteenth-century first attempt for the reconstruction of esophagus was made using a subcutaneous skin tube. A host of conditions and diseases may require esophageal replacement such as long gap esophageal atresia, non-dilatable long esophageal corrosive strictures, more than 3-5 cm long congenital esophageal stenosis, and esophageal tumors [5]. In our series, the main indications of esophageal replacements were corrosive intake in 76.34% patients followed by esophageal a systemic review of the literature on pediatric esophageal replacement and found the most common reason for replacement was esophageal atresia followed by corrosive injury [6]. Also Soccorso and Dilshad et al. reported esophageal atresia and peptic disease as common indication for esophageal replacement [1,7]. These reports are contrary to our findings where majority of the patients belonged to corrosive ingestion and esophageal atresia was the second common etiology. This is due to lack of awareness among local community for safety measures regarding handling of corrosive material as compared to the developed countries where public awareness and industry collaborations have markedly reduced the incidence of corrosive injuries [1]. The commonly practiced conduits for esophageal replacement are colonic interposition, gastric pull-up, gastric tube interposition, jejunal interposition and jejunal free graft. The replacement can be performed through the esophageal hiatus, transthoracic, retrosternal, and occasionally through subcutaneous route. All the above mentioned conduit and routes used for replacement in literature have their own advantages and disadvantage. Choice is mainly depends upon patient's anatomy, available structures and surgical expertise. We preferred gastric-pull up (> 90% of the patients) in our series, because of its technical advantages over other techniques in addition to our extensive experience for this procedure. It is because of the straight forward mobilization of stomach, with the blood supply based on the right gastric and right gastroepiploic vessels, and does not involve multiple anastomoses, thus the risk of anastomotic leakage is low. These factors have a direct impact on patient's morbidity and mortality. Although 6 patients developed anastomotic leakage in the neck, all were managed conservatively with free salivary drainage. We have a limited experience with colonic interposition (seven cases) and jejunal interposition (two cases) in patients with scarred stomach. In one patient we faced colonic dilatation and in cases of jejunal interposition, we encountered difficulty in bringing up the jejunum owing to its limited vascular arcade as endorsed by Giampiero and Dakeshesh [1]. These were the other reasons that we preferred gastric pull-up procedure. According to Spitz et al. [8] and Delshad et al. [7], esophageal replacement with complete stomach is the ideal procedure because it has low complication rate, however, Delshad et al. and others have noted that colon interposition is still the commonest practiced method for esophageal replacement in the world literature [6,7,[9][10][11][12]. Hirschl et al. [13] reported that a colon interposition is a more complex endeavor than a gastric transposition. Other studies conclude that gastric pullup is the procedure of choice for centers in Europe and the United States [14]. Similarly, in our series, we had gastric pull-up for the majority of our patients [6,8,[14][15][16][17][18].
Trans-hiatal route is the most commonly used route for esophageal replacement, as after esophagectomy through the hiatus, this route is readily available for any pull-up [1]. We used trans-hiatal route without thoracotomy in 76.34% cases and combined with thoracotomy was done in 9.7% of cases. In our series, the retrosternal route was used in 14% of cases. This is similar to the approach described by others, including Gupta et al. [15][16][17].
Complications are divided into intraoperative, early and late. In one case we had aortic tear at the level of arch of aorta during surgery, which was managed by open thoracotomy with the help of our cardiac surgeon. Other intraoperative complication encountered were related to operative difficulty in esophagectomy owing to massive adhesion with the surrounding structures and bleeding, which were dealt with by opening the chest. The most common early postoperative complications in this series were pneumothorax in 10.75% of cases, leak in 9.68% of patients, wound infection and dehiscence in 16.12% of patients, and one patient had internal herniation. Pneumothorax was commonly seen with trans-hiatal route and identified either on table or after shifting to surgical ICU and managed with immediate chest intubation. In several patients, we deliberately passed chest tube fearing a pneumothorax especially in cases with difficult esophagectomy owing to adhesions. Almost same incidence of pneumothorax (10-14%), was reported in the literature. [18][19][20] In our series second common early complication was anastomotic leak (9.68%). Our results were comparable with Bradshaw et al. [19] who encountered anastomotic leak in 14% of patients. Awad et al. noted it in 20% of his patients [21]. Choudhury et al. [18] found much higher leak rate 14/19 after gastric pull up. According to a systemic review, anastomotic leak with gastric pull up was seen in 22.8% while with colonic transposition in 19.7% of cases [20].
The most common late complication encountered in our series was dysphagia (16.13%). This has not been well documented; in his systemic review, Garritano et al. [6] reported that only in two among 14 studies was this Table 3 Route vs conduit Route Stomach Colon Jejunum Total Transhiatal 70 1 0 71 Transhiatal combined with thoracotomy 6 1 2 9 Retrosternal 8 5 0 13 84 7 2 93 complication discussed. Nakahara et al. [22] reported that 42.9% of his cases suffered from dysphagia. The current study showed anastomotic stenosis/stricture in 12.90% and reflex in 19.35%. Our results of stenosis/stricture were comparable with a systemic review conducted by Liu et al. [20] who reported that early anastomosis stricture were 10.2% (13/127) with gastric replacement, 11.9% (40/335) with colonic interposition, and 17.8% (8/45) with jejunal interposition. Awad et al. [21] in a review of 22 studies reported that 22% of children developed anastomotic stricture after esophageal replacement with stomach in early follow up which was later increased to 70% of the cases on long-tern follow-up. Another important complication is reflux; our results for postoperative reflex were similar to Jain et al. [23] (10%). Lee et al. [24] found a very high rate of reflex ( ≥ 90%) after esophageal replacement, using gastric conduit. Gupta et al. [15] mentioned that after gastric pull up the incidence of reflux was 60% at 3 months, 50% at 6 months and 40% at 1 year post-procedure. In our series current dumping syndrome was seen in 8 patients (8.60%). Dumping syndrome and delayed gastric emptying was also under reported in many studies, however, Jain et al. [23] evaluated 10 patients at 90.5 months of follow-up evidencing a mean gastric emptying was 39.1 min.
Overall mortality associated with esophageal replacement is variable in different series depending upon available resources, disease severity and surgical expertize [6]. In our series, we had 12 mortalities (12.90%), which were mainly due to postoperative complications such as failure to wean off from ventilator, pneumonia, sepsis, anastomotic leak, esophageal tumor and two mechanical ventilatory failures. Awad et al [18] in a systemic review of esophageal replacement with stomach found 4.8% mortality rate (27/558). In another systemic review, Garritano et al. [6] reported that the overall mortality rate varies between 2-4% in various studies. Our mortality rate was higher, likely due to disease severity, and compromised intensive care setting.
Conclusion
There are problems with esophageal replacement especially in developing countries, with low resources and suboptimal intensive care services. In our experience, the gastric conduit as a pull-up is the best conduit as esophageal replacement, which has the advantage of single anastomosis and trans-hiatal route is best shortest route for replacement. Esophageal replacement has morbidity as well as mortality and needs long-term follow-up in many patients for the management of various late complications. The surgeons should be well versed for using various conduits and routes as the patients may have variable extent of corrosive injury to esophagus and stomach and limitation due to previous surgery. The treatment plan must be individualized as seen in our series. | 2020-03-31T17:10:30.316Z | 2020-03-31T00:00:00.000 | {
"year": 2020,
"sha1": "9039ab2f41fcf6f00e5a96c39caacfe8ca784a3a",
"oa_license": null,
"oa_url": "https://link.springer.com/content/pdf/10.1007/s00383-020-04649-5.pdf",
"oa_status": "BRONZE",
"pdf_src": "PubMedCentral",
"pdf_hash": "9039ab2f41fcf6f00e5a96c39caacfe8ca784a3a",
"s2fieldsofstudy": [
"Medicine"
],
"extfieldsofstudy": [
"Medicine"
]
} |
75137042 | pes2o/s2orc | v3-fos-license | Narrowband IoT: An Appropriate Solution for Developing Countries
Internet of things (IoT) is very much attractive for several sensor based applications. It provides large coverage of the services with small amount of resources. Its applications span from the ordinary scenarios such as sensing in the common digital ecosystem to the far more complicated processes of modern manufacturing, agriculture, security provisioning, location tracking and health care. Several types of IoTs have been proposed for the recent applications. Narrowband IoT (NBIoT) is one of the economical versions of the IoTs. It is a low power wide area network technology and thus suitable for resource limited scenarios. In the developing countries, the resources are scarce and economical solutions are always preferable. Therefore, NBIoT is an attractive solution for the developing countries. In this article, we present its features and functions which make it suitable for developing countries. We also provide several sector based analysis which are suitable for the NBIoT deployment.
I. INTRODUCTION
The Internet of Things (IoT) is now an integral part of the modern digital ecosystem. It has several ramifications and can be applied to a number of sensor based application scenarios. It was initially started as a value added system for cellular communication networks. However, now they appear in several cellular and non-cellular forms including sensor networks, control networks and independent networks. IoTs can be of different types depending on their features. One of the standardized forms of IoT is narrowband IoT (NBIoT). As the name suggests, this IoT needs narrow band frequencies for its operations. Due to the narrow band requirement, it has several advantages over other forms of IoTs. Green and sustainable technologies are in high demand due to their energy efficiency and environment friendly features. These low energy technologies are very popular in different forms of IoTs and sensor based applications. These days, green IoTs are well researched areas in which optimization of resources takes the central position [1] - [5]. In the developing countries, they have even more important roles due to the shortage of resources [1]. In the recent years, several works have been done on different types of IoTs to make them more energy efficient. In [1], Green IoTs have been studies from several technological aspects. In [1], NBIoT has been proposed as the inherent green technology for several applications. In [2], energy efficient and sustainable initiatives for IoT based smart world have been addressed. It provides several recent progressive trends and technologies which are able to make the entire IoT networks energy efficient. It also gives insight for the segment-wise greening processes in the framework of existing information and communication technologies (ICTs) across different application domains. In [3], a detailed survey of the IoT marketplace of last few years has been presented. It explains the recent industrial demands and applications of IoT and its associated technologies in different processes. It also elaborates through justified logic that manufacturing and automation will have a large share of industrial IoT. In [3], a similar survey is provided which focuses on the technological prospective of IoT. It emphasizes that the wide deployments need to be energy efficient for the practical reasons. In [4], a comprehensive survey on the practical aspects of IoT has been provided. It first deals with the enabling technologies that make IoT a reality and then show the main protocols which are instrumental in the IoT operations. Then it provides a long list of applications in which IoT can play a significant role. The authors pointed that the overall success of IoT is very much dependent on the efficiency of the entire IoT networks, their components and end devices. In [5] and [6], the energy aspects of 4.5G and 5G are dealt with detailed information. Green initiatives of 5G are discusses in depth in [5].
In [7], the recent trends of consumer electronics driven data communication networks are provided. It shows the volume of the resources needed for the global communication networks. The data storage and communication infrastructure required for the current ICT sectors too have been presented in this article. In [8], sustainability aspects of communication networks have been presented. It shows the carbon emission contributions of global communication sector. Finally, it provides outlooks for overall sustainability. In [9], research and development initiatives of 5G and IoT are presented in which energy efficiency and overall network optimizations are analyzed. In [10], the green radio communication technologies and emerging methods are highlighted with proper background information. It also provides insights for emerging networking technologies for energy efficiency. In [11], 5G energy efficiency related aspects are presented which discusses the general system characteristics and their overall effect on the system. In [12] and [13], energy efficiency related aspects of the Internet and wireless Internet such as mobile Internet and IoT related aspects in specific have been discussed. It also covers the energy aspects related to IoT. In [14], green technologies for clouds are analyzed. Cloud energy efficiency is essential for green IoTs as clouds are going to be their integral parts in the long term. IoT data management needs clouds for the efficient ICT activities.
In this article, we provide the main initiatives being taken to make the IoTs efficient in resource utilization. We present the main features of NBIoT, its deployment issues and the commonly expected application domains. We show that narrowband IoT (NBIoT) is certainly the right choice for the developing countries now. We present its main motivating features, deployment options and applications in different sectors.
The reminder of this paper is organized in three sections. In Section II, we present the energy efficient and greener version of the current IoTs. Then we show that these features are found in NBIoT. We also presented its deployment options and applications in different sectors. In Section III, we present the overall initiatives being taken in the recent times to make NBIoT energy and resource efficient. We showed its suitability for the developing countries. In Section IV, we conclude with the main points of this article.
II. GENERAL FEATURES OF NBIOT
NBIoT is the low energy and low bandwidth version of IoT which is designed for the massive machine-to-machine communications. As the name suggests, it uses narrow bands for its different functions and operations. It needs a bandwidth of just 180 kHz to 200 kHz for its designated processes. In LTE Release 13 and 14, 180 kHz has been proposed as the operating bandwidth for NBIoT. It is a low power wide area (LPWA) technology which can save a lot of power when compared with other forms of IoTs. It is good for large scale economical deployment of IoT for different applications. In true sense, it is leaner, thinner and greener than other IoTs proposed in the recent years. It can be deployed in both the cellular and non-cellular forms. However, cellular forms are popular as they can use the existing cellular architectures for its operations. In LTE Release 13 and 14, it has been standardized according to the compatible LTE provisions and also proposed for connected living environments [15].
A. Basic LPWA Features of NBIoT
NBIoT is an energy efficient version of IoT. Main attraction of NBIoT is its LPWA nature. It can save a large amount of energy and bandwidth of a network. Its architecture and protocols have been standardized in the recent releases of LTE for different environments. Deployment bandwidths of NBIoT have been agreed by the standardization committee in Release 13. Depending on the situation, different spectral bands can be used by different operators. According to Release 13 of 3GPP LTE, the maximum usable bandwidth of an end device is 200 kHz. In fact, for the communication purpose 180 kHz is used [16] - [20]. At this bandwidth, the upper limits of the uplink and downlink data rates are set at 150 kbps. Half duplex mode has been recommended for NBIoT communications [17]. In Release 14, these specifications have been further enhanced for advanced applications. The power from the transmitter is kept quite small so that a single battery can supply power to the NBIoT devices or nodes for more than 10 years. In fact, in the recent NBIoT standards, two power levels have been specified: 20 dBm and 23 dBm [1]. The sensitivities of the NBIoT sensors are really good. They can receive the signal at a power level as low as 64 dBm. These are the exclusive features of NBIoT which is not found in other versions of IoTs proposed so far.
B. NBIoT Architecture
A systematic NBIoT architecture is required for its planning, dimensioning, cost estimation, design and final deployment. It does not have a legacy to follow as it is one of the earliest IoTs of its type. However, it is similar to the WSN and the WSNs are there for several years. The existing WSN architecture and topologies can be helpful in its further advancement. It is noteworthy that WSNs did not have a structured and well defined architecture like the cellular systems such as the LTE networks which will form the backbone of NBIoT. Therefore, an LTE cellular framework for NBIoT is the right choice at the moment [17]. The layered structure of NBIoT is helpful in its planning and deployment. In Release 13, several specifications for different layers have been mentioned. NBIoT can be separated into 6 layers as shown in Fig. 1. The physical layer is at the bottom and it is normally the air interface. Physical layer does the similar functions as other WSNs and some added functions as defined in Release 13. Above it is the medium access control (MAC) layer. This has the similar functions like the MAC layer of other networks. It incorporates the protocols for medium access and multiple access techniques. There is a radio link control layer in between the MAC and the upper layers. This layer makes the adaptation of the MAC layer information for radio links. Above it is the packet data convergence protocol layer which provides routing, traffic scheduling, networking and other related tasks. Then above it is the radio resource control layer which takes care of the radio resources of the packets in the channels. NBIoT uses user datagram protocol (UDP) and other cellular mechanism to carry this function [18]. UDP is effective in the wireless networks and thus suitable for NBIoT as well. The topmost layer is the Non- Access Stratum (NAS) which establishes the communication between the user equipment (UE) and the main server of the NBIoT also known as NBIoT central node.
C. Deployment Options for NBIoT
NBIoT deployment options have been agreed in Release 13. According to it, NBIoT can be deployed in three different forms. The first being the standalone deployment in which an independent microwave band is provided in the 700MHz or 800 MHz range for its deployment. The second one is the guard band deployment in which the spectrum for IoT is the unused GSM and LTE guard bands. These guard bands are normally not used for communication purposes in GSM and LTE cellular systems. With the arrival of NBIoT, these guard bands find a new application. The third one is the in-band deployment. In this case, some part of the GSM or LTE spectrum is allocated to the NBIoT. In Fig. 2, we show these 3 deployment scenarios. In practice, either of them can be used depending on the availability of the bands and its suitability. If required, even two or all three options can also be used in specific cases.
D. Applications of NBIoT
NBIoT is attractive in the LPWA regime. It has tremendous potential to operate in the resource limited scenarios. Here, we shed the light on some of the typical sectors where it is currently considered for deployment. Several applications of NBIoT are possible and it can be deployed for any LPWA applications in which the six elements of IoT are available. Both local and nonlocal applications of NBIoT are equally possible. Emerging applications are found in several technology and social sectors with every passing month. There are several new sectors in which NBIoT has potential applications. Some of these applications are indoor and the others are outdoor. NBIoT can be instrumental in designing smart homes and smart cities [16]. Similarly, it can help in healthcare as well as the security monitoring of public places. In Fig. 3, we present some typical sectors in which NBIoT can have significant role. These sectors are: smart city management and monitoring; smart home applications; large scale manufacturing; pet tracking; kids' tracking; healthcare monitoring; safety and security related applications; smart agriculture and farming; energy and utility management; automobile and vehicular management; retail management; policing and law enforcement assistance; detection of environmental degradation; waste management; and several other LPWA applications. In fact, for rural areas NBIoT is considered as the only choice due to its economical features.
III. NBIOT FOR DEVELOPIONG COUNTRIES
In Section II, we have seen the attractive LPWA features of NBIoT. It is very much energy efficient and provides significantly large coverage which is not possible by other versions of IoTs. The LPWA features are suitable for resource limited scenarios. In developing countries resources are scarce and economical solutions are always an advantage. Furthermore, the legacy systems such as GSM are widely found in the developing countries. NBIoT is compatible with GSM as well as the recent versions of the LTE such as 4G and 4.5G. However, other forms of IoTs are compatible with only the recent versions of LTE. Therefore, NBIoT is a natural choice for the developing countries.
A. NBIoT for Agriculture in Developing Countries
NBIoT can be deployed in the agricultural sector for several performance enhancement related aspects. In agriculture, several resources are used to increase the harvest. Many of those resources are underutilized due to the lack of facilities to monitor their utilities. For optimal use of the resources, NBIoT can be used which can sense the level of utilization and improve the utilization efficiency. For instance, water is an essential resource for agriculture. It is not available in sufficient amount in every part of the world. In the dry areas, water has to be utilized very efficiently. This utilization can be monitored in the agricultural farms through the water sensors deployed under the soil. Similarly, fertilizer concentration, pesticide intensity and humidity levels too can be monitored using different types of sensors as a part of NBIoT in agriculture. These resources can also be remotely controlled through the NBIoT. As the energy levels for these sensing operations are very low, the cost of this IoT based monitoring is very much economical for the farmers. On the other hand, the harvest can be maximized due to the efficient nutrition distribution to the plants. These initiatives are already being deployed in several countries and found to be very effective. Of course, it will be made even more efficient in the future.
B. NBIoT for Manufacturing in Developing Countries
Manufacturing is an important economic sector for developing countries. Recently, manufacturing companies in the developing countries are experiencing extreme competition, mainly due to the increasing pressures from technological changes and global business challenges. These pressures result in the globalization of manufacturing, characterized by faster transfers of materials, complex payment systems and the compression of products' life cycles, which drives the integration of technologies with increasingly sophisticated customers' needs [21]. To be successful companies try to anticipate future trends by developing ideas, products or services to rapidly and effectively meet future demands. In addition to that they are responding to their current customers' or organizational needs to sustain competitive advantage. Among all the sets of pressures of a technological nature, the advent of the Internet has deeply affected companies' approach to production and has strongly reshaped organizational and operational structures. However, the role of the Internet in manufacturing is still understudied as it is for the IoT phenomenon. Advanced manufacturing technologies strongly rely on various ICT technologies to achieve higher productivity, higher quality and lower production costs. Such an effect is especially focused on processes of manufacturing automation, and of information systems. Indeed, the advent of Internet-based technologies has led to the emergence of new manufacturing philosophies and new forms of organization, such as virtual organizations, remote manufacturing, computer-integrated manufacturing systems, and Internetbased manufacturing, i.e. wireless milling machines, coordinated measuring machines, networked sensor arrays and surveillance systems. For example, "design anywhere, manufacture anywhere" is a new approach to production which shares design and manufacturing data across multiple platforms and infrastructures [22]. Recent studies have confirmed such trends, indicating that the future of manufacturing firms will be mostly information-oriented and knowledge-driven, leading to a much more flexible and an abundance of automated operations systems. In all these changing scenarios, overall efficiency and automation can be provided by NBIoT [23]. Thus, it is a hot choice for the industrial automation in the developing countries.
C. NBIoT for Healthcare in Developing Countries
Healthcare provisioning in any country is one of the basic services. It is also a large sector due to the very nature of the services provided by this system. Healthcare system is multitiered and need proper collaboration among each of the tiers. In several healthcare processes, NBIoT can play a role of game changer. For instance, in emergency proceedings it can help the patients to get appropriate treatments while coming to the hospital from their residence. Using the sensors, the vital information of the patients can be sent to the doctor who in return can provide the appropriate support through the sensors till the patient reaches the hospital. Similarly, the telemedicine applications too can be improved through the NBIoT based sensor assistance.
D. NBIoT forResource Management in Developing Countries
Basic resources such as water, electricity, gas and other essential commodities are not properly managed in the developing countries. That leads to the poor resource utilization efficiency and loss of revenue for the government and associated public sectors. NBIoT based solutions already exist to improve the resource utilization efficiency in these areas. Smart metering and efficient resource distribution can be a game changer in these sectors. NBIoT based automation in the metering and resource distribution is quite cost effective and efficient. It can provide a real-time resource monitoring framework to the resource distributors. This is how the operators can directly monitor the efficiency of resource utilization. Thus NBIoT is being deployed in several countries for the public resource management.
IV. CONCLUSIONS
Narrowband IoT is unique in the low power regime of the IoTs. It is also special in the wide coverage and low cost of its deployment. In this article, we have presented the main features of NBIoT, its architecture and an OSI model for its layer wise architecture which is needed in its design and implementation. NBIoT is very much suitable for the developing countries. Its low cost and wide coverage makes it the first choice for the common applications in the developing countries. NBIoT is very much effective in agriculture, manufacturing, healthcare, resource management and social applications such as policing. We presented the overall effectiveness of NBIoT in the developing countries. In the future there will be many new applications of NBIoT in the developing countries. | 2019-03-12T11:30:52.000Z | 2019-03-12T00:00:00.000 | {
"year": 2019,
"sha1": "3e4dcf730cd4a67527be852a8cd751aff0be4e93",
"oa_license": null,
"oa_url": null,
"oa_status": null,
"pdf_src": "Arxiv",
"pdf_hash": "3e4dcf730cd4a67527be852a8cd751aff0be4e93",
"s2fieldsofstudy": [
"Engineering",
"Environmental Science",
"Computer Science"
],
"extfieldsofstudy": [
"Computer Science",
"Engineering"
]
} |
236470111 | pes2o/s2orc | v3-fos-license | U-Shaped Association of Body Mass Index with the Risk of Peripheral Arterial Disease in Chinese Hypertensive Population
Background High body mass index (BMI) is a well-recognized risk factor for cardiovascular diseases. But its role in peripheral artery disease (PAD) remains perplexing. Our study aims to evaluate the association of BMI with PAD in the Chinese hypertensive population. Methods This is a cross-sectional study with enrollment data from the Chinese H-type Hypertension Registry.10896 hypertensive patients aged ≥18 years were included in the final analysis. Results The prevalence of PAD diagnosed by ABI in this study was 3.2% (n=351). A U-shaped association between BMI and PAD was found. Per SD increment (3.6 kg/m2) on the left side of the BMI threshold (BMI < 25.7 kg/m2) was associated with a 27% decrease in the adjusted risk of PAD [OR, 0.73; 95% confidence interval (CI) 0.60, 0.89; P=0.002]; BMI was significantly positively associated with the risk of PAD (OR, 1.52; 95% CI 1.52, 1.93; P=0.001) in those with BMI ≥25.7 kg/m2. Conclusion In summary, a U-shaped association between BMI and the risk of PAD in the Chinese hypertensive population was found. BMI with the lowest risk of PAD was estimated to be 25.7 kg/m2.
Introduction
Peripheral arterial disease (PAD) is the third leading atherosclerotic disease after coronary heart disease and stroke, 1 mainly caused by the accumulation of lipid and fibrous material between the intima and media of lower limb arteries, resulting in luminal stenosis (focal or diffuse). It is well known for a sharp increase in the prevalence of PAD with advanced age. 2,3 With the aging of the Chinese population, PAD has become an increasingly severe clinical and social problem. Allison et al also showed ethnic differences were independent factors in the prevalence of PAD. 4 Compared to Whites, Blacks seem to be more vulnerable to PAD, while Asians seem to have a lower prevalence of PAD. 5 The prevalence of PAD was higher in people with underweight, but the association between BMI and PAD was uncertain due to a variety of potential covariates. 6,7 A small prospective cohort study showed that obesity independently predicts severe PAD. 8 However, the recent observational study with more than 3 million sample size has found J-shaped relationship between BMI and PAD only in females. 9 Epidemiology of Dementia in Central Africa (EPIDEMCA) study recruited the elderly in the Central African Republic and the Republic of Congo, showed underweight and obesity were all associated with the risk of PAD. 10 Due to the inconsistent and the evidence of association between BMI and prevalence of PAD in the Chinese was still lacked. Our study aims to explore the association between BMI and the risk of PAD in Chinese hypertensive patients.
Study Design and Participants
The study population was drawn from the China Hypertension Registry (Registration number: ChiCTR1800017274), a real-world observational registry of hypertension designed to investigate the prevalence and treatment of hypertension in China and to assess prognostic risk factors. Details of the inclusion and exclusion criteria for the study have been published. 11 From March 2018 to August 2018, we recruited a total of 14,268 study participants in Wuyuan, Jiangxi Province, China as our study population, and finally analyzed the data of 10,802.
Ethics Statement
The study was conducted in accordance with the Declaration of Helsinki and approved by the Ethics Committee of the Institute of Biomedicine, Anhui Medical University (No. CH1059). All patients signed informed written consent before enrollment in this study.
Laboratory Biochemical Examination
All subjects were asked to do an overnight fast Venous blood samples were obtained from all study participants and analyzed by Biaojia Biotechnology Laboratory in Shenzhen, China. Lipids (including total cholesterol (TC, mmol/L), triglycerides (TG, mmol/L), highdensity lipoprotein-cholesterol (HDL-C, mmol/L), lowdensity lipoprotein cholesterol (LDL-C, mmol/L)), 12,13 estimated glomerular filtration rate (eGFR, mL/min/ 1.73 m 2 ), fasting blood glucose (FBG, mmol/L) and homocysteine (Hcy, μmol/L) were measured using automatic clinical analyzers (Beckman Coulter, USA) and the laboratory staff were blind to the research protocol.
Measurement of BMI
The height and weight of the subjects were measured by trained staff using standardized equipment in accordance with standard operation procedure. BMI = Weight (kg)/Height (m) 2 .
Measurement of ABI and Definition of PAD
The ABI of each lower limb was calculated by dividing the systolic pressure of the ankle artery of the corresponding lower limb by the systolic pressure of the brachial artery. Subjects rested quietly in a warm room for more than 10 minutes and fully exposed their upper limbs and ankles. Trained technicians used the Omron Colin BP-203RPE III device (Omron Health Care, Kyoto, Japan) to simultaneously measure bilateral brachial and ankle arterial systolic pressures in supine subjects. And the software automatically calculates the bilateral ABI data according to the above calculation formula. All measurements were conducted in accordance with strict standard protocols. PAD was defined as an ABI ≤ 0.9 in either lower limb. 14 Subjects with ABI >1.4 were excluded because of abnormal elevation of ABI may due to calcification of the arterial wall. 15
Other Variables
Variables included age (years), sex, systolic blood pressure (SBP, mmHg) and diastolic blood pressure (DBP, mmHg) measured by electronic sphygmomanometers after the subjects had rested for 10 minutes. Qualified researchers were trained to collect information by using standardized questionnaires, including smoking status (never, former, current), alcohol consumption (never, former, current), antihypertensive drugs (yes or no), the history of comorbid diseases including diabetes mellitus (yes or no), stroke (yes or no), and coronary heart disease (yes or no).
Statistical Analysis
Normally distributed variables were presented as mean ± standard deviation (SD); for non-normally distributed data the median and inter-quartile range (IQR) are given, and categorical variables as percentage (%). Population characteristics were described according to BMI classify. To reduce redundancy, variance inflation factors (VIF) were used to assess collinearity between independent variables before our data analysis, with a variable having VIF > 5 considered collinear with other variables. In comparison, LDL-C (VIF=5.9) had to be excluded from the next analysis because of its collinearity to other variables. The dose-response relationship between BMI and the risk of PAD was estimated using generalized additive regression model and smoothing curve (penalized spline method) with adjustment for age, sex, systolic and diastolic blood pressure, pulse rate, smoking status, alcohol consumption, TC, TG, HDL-C, eGFR, Hcy, antihypertensive drugs, diabetes mellitus, stroke, coronary heart disease. If nonlinear was detected, threshold effect analysis was used for inflection points of BMI by using segmented regression model, LRT test and bootstrap resampling method. Multivariate logistic regression was used to analyze the relationship between BMI and the risk of PAD around threshold value. P value for interaction was used to compare whether there was a significant difference in the correlation between BMI and the risk of PAD before and after inflection point. In addition, possible modifications of the association between BMI and PAD were assessed for variables including sex, age, blood pressure controlled, pulse rate, Hcy, lipids profile, smoking status, history of diabetes mellitus and stroke.
Baseline Characteristics of Participants
As shown in Table 1, a total of 10,896 hypertensive patients with a mean age of 63.9 ± 9.3 years were included in this study. The prevalence of PAD was 3.2%, the mean BMI was 23.6 ± 3.6 kg/m 2 , and 47.1% were male. BMI was stratified to four groups: underweight (BMI<18.5 kg/ m 2 ), normal (BMI≥18.5, <25 kg/m 2 ), overweight (BMI≥25, >30 kg/m 2 ) and obesity (BMI≥30 kg/m 2 ) to describe demographic characteristics. The underweight of participants accounted for 6.3% of the total population, and obesity was only 4.2%. The prevalence of PAD in underweight was the highest (6.7%) and followed by obesity (4.4%), while overweight was only 2.3%. Compared with the other three groups, underweight participants were older, with higher tHcy, HDL-C, current smoking rate, and lower TC, TG, eGFR, the prevalence of diabetes mellitus and the use of the antihypertensive drug.
Association Between BMI and PAD
As shown in Figure 1, the relationship between BMI and the prevalence of PAD showed a U-shaped curve, and threshold saturation effect analysis showed that BMI value with the lowest risk of PAD was estimated to be 25.7 kg/m 2 . We stratified BMI by 25.7 kg/m 2 and used logistic regression analysis models (Table 2). Per SD increment (3.6 kg/m 2 ) on the left side of the threshold (BMI< 25.7 kg/m 2 ), BMI was associated with a 27% decrease in the risk of PAD [adjusted odds ratio (OR), 0.73; 95% confidence interval (CI)0.60, 0.89; P= 0.002]; however, BMI was significantly positively associated with the risk of PAD (adjusted OR, 1.52; 95% CI 1.52, 1.93; P=0.001) in those with BMI ≥ 25.7 kg/m 2 . Further adjusted lipid-lowering drugs as a sensitivity analysis, no change to the result suggested that the result was stable (Supplemental Table 1).
Discussion
In our analysis of this community-based hypertension registry study in China, we noted a U-shaped relationship between BMI and risk of PAD. The BMI value with lowest risk of PAD was estimated to be 25.7 kg/m 2 .
A number of studies have reported the relationship between BMI and the risk of PAD. However, the association between BMI and PAD risk was not consistent. Epidemiological studies more than two decades ago reported a positive association between BMI and intermittent claudication in middle-aged males in Israel. 16 However, many population studies after adjusting for the relevant covariates fail to support the significant association between BMI and the prevalence of PAD. 4,17 In addition, the San Diego study reported an independent and significantly inverse association between BMI and prevalence of PAD (OR: 0.88) in multi-ethnic population. 18
3629
Taiwan showed that compared with diabetic patients without PAD, the BMI of patients with PAD was lower (23.5 ±3.2 vs.24.8±3.5 kg/m 2 , P < 0.005). Heffron et al who gathered data from more than 20,000 sites (n= 3,250,350) in the United States from 2003 to 2008, recently reported BMI and the prevalence of PAD in females showed a J-shaped nonlinear relationship; a significant positive correlation between obesity and PAD in females, while only a slight positive correlation between obesity (BMI ≥ 40kg/m 2 ) and PAD in males (OR=2.98 vs 1.37). 9 Stepwise logistic regression analysis showed that the association between BMI and PAD was inverse. 19
3630
To our knowledge, the U-shaped relationship between BMI and the risk of PAD shown in our study was the first reported in Chinese hypertensive population. Different from the very large sample population studies 9 in the United States, where participants were nearly 30% obese and 3.4% underweight, as well as study of the prevalence of PAD in African, 10 where obesity was only 4.5%, 34.1% underweight, we were 6.3% (691) underweight and only 4.2% (454) obesity, nearly 90% of the population was normal BMI and overweight. Over a third of the study population was underweight. A U-shaped relationship between BMI and the risk of PAD was observed. Compare to the subjects with normal BMI, underweight and obesity were statistically significant association with Figure 1 Smoothing curve of association between BMI and the risk of PAD. Adjusted for: age, sex, systolic and diastolic blood pressure, pulse rate, smoking status, alcohol consumption, total cholesterol, triglyceride, high density lipoprotein cholesterol, fasting blood glucose, estimated glomerular filtration rate, total homocysteine, antihypertensive drugs, diabetes mellitus, stroke, coronary heart disease. 10 However, Heffron et al found a "J-shaped" relationship between BMI and PAD only in females, not in males, which may be due to the height and weight data used in this study for self-reporting of participants. Self-reported data may lead to personal BMI classification appear serious mistakes, 20 difficult to correct the mistakes, 21 especially in the stratified analysis according to gender. 22 Thus, self-report bias may have contributed to the fact that this study found a "J-shaped" relationship between BMI and PAD risk only in females, and not in males.
At present, few studies have elaborated on the possible mechanism of the correlation between BMI and PAD. A cross-sectional study of hemodialysis patients reported a lower prevalence of atherosclerosis and lower levels of inflammation (CRP) in patients with normal BMI and overweight compared with those with underweight and obesity. 23 Lower levels of inflammation and atherosclerosis may be associated with the lowest risk of PAD in this population (normal BMI and overweight).
Not only that, there have been also many reports on the U-shaped relationship between BMI and cardiovascular disease and death. A meta-analysis of 97 studies showed that obesity (all grades) and grades 2 and 3 obesity were significantly associated with all-cause mortality relative to normal BMI. However, overweight was associated with a significant reduction in all-cause mortality. 22 Among more than 1 million East Asian populations in the Asia Cohort Consortium BMI Project, including Chinese, Japanese, and Korean, the Cox proportional hazard regression model was used to analyze the relationship between BMI and mortality risk, which showed that the population with BMI between 22.6 and 27.5 had the lowest mortality risk. 24 Based on this, we speculate that the "U-shaped" relationship between BMI and peripheral atherosclerosis may, on one hand, explain the causes of the lowest cardiovascular disease risk and all-cause mortality in normal BMI/overweight.
Limitations and Future Directions
Nonetheless, these results must be interpreted with caution, and a number of limitations should be borne in mind. First, subjects in our analysis were middle-aged and elderly patients with hypertension. The U-shaped relationship between BMI and the risk of PAD was not necessarily applicable to the general population, but as an independent risk factor for PAD, exploring the relationship between BMI and the risk of PAD in the hypertensive population can serve the high-risk population more precisely. In addition, the association between BMI and the risk of PAD was still controversial. By design, our study was a crosssectional study and cannot study the chronology of BMI and PAD. There might be a reverse causal relationship. The weight change caused by the disease may distort the Figure 2 Subgroup analyses on the association between BMI and the risk of PAD. Adjusted for: age, sex, systolic and diastolic blood pressure, pulse rate, smoking status, alcohol consumption, total cholesterol, triglyceride, high density lipoprotein cholesterol, fasting blood glucose, estimated glomerular filtration rate, total homocysteine, antihypertensive drugs, diabetes mellitus, stroke, coronary heart disease, except for the stratifying variable. relationship between BMI and PAD. In the future, large prospective cohort studies on PAD were urgently needed. Final, the obesity rate in our study was low. It has no enough power to assess the relationship between different degrees of obesity or morbid obesity and the risk of PAD. However, our study reflects the real situation of hypertension population in Chinese hypertension, and the results obtained were more suitable for the application of hypertension in middle-aged and elderly people in China.
Conclusions
Our study reported the prevalence of PAD was 3.2%. The U-shaped association between BMI and the risk of PAD was found in Chinese middle-aged and elderly patients with hypertension. BMI with the lowest risk of PAD was estimated to be 25.7 kg/m 2 in our study.
Data Sharing Statement
The raw data used to support the findings of this study are available from the corresponding author upon request. | 2021-07-29T05:27:11.087Z | 2021-07-01T00:00:00.000 | {
"year": 2021,
"sha1": "2959ddea2fe274e0d228cf2a2b32d738d4434287",
"oa_license": "CCBYNC",
"oa_url": "https://www.dovepress.com/getfile.php?fileID=71846",
"oa_status": "GOLD",
"pdf_src": "PubMedCentral",
"pdf_hash": "2959ddea2fe274e0d228cf2a2b32d738d4434287",
"s2fieldsofstudy": [
"Medicine"
],
"extfieldsofstudy": [
"Medicine"
]
} |
14079918 | pes2o/s2orc | v3-fos-license | Putting “sticky notes” on the electronic medical record to promote intra-hospital referral of hepatitis B and C virus-positive patients to hepatology specialists: an exploratory study
Background Currently, no system for appropriate intra-hospital collaboration regarding hepatitis virus positive individuals exists, even in medical institutions with hepatologists among their staff. The main objective of this study was to explore a simple alert system to promote the referral of patients with hepatitis B surface antigen (HBsAg)- or anti-hepatitis C virus (HCV) antibodies positivity to hepatologists through electronic medical records. Methods Since April 2014 at Osaka City Juso Hospital, “sticky notes” have been put on the electronic medical records of patients newly diagnosed with HBsAg- or anti-HCV- antibodies positivity to recommend intra-hospital referral of those patients to specialists. In this study, we investigated the number of referrals to hepatologists before vs. after the introduction of this system (that is, in fiscal years 2013 [Period 1] and 2014 [Period 2], respectively), and the subsequent clinical courses of the patients. Results The proportions of patients with HBsAg and anti-HCV antibody positivity did not show statistically significant differences between Period 1 and Period 2 (1.6 % [43/2,757] vs. 1.3 % [39/2,891], p = 0.58; and 5.8 % [156/2,674] vs. 5.3 % [147/2,790], p = 0.39, respectively). However, the referral proportions for patients with HBsAg- and anti-HCV antibody positivity were significantly higher in Period 2 (73 % [11/15] and 65 % [41/63], respectively) than in Period 1 (28 % [5/18] and 17 % [9/54]) (p = 0.009 and p < 0.001, respectively). Among patients who were referred to hepatologists, 2 HBsAg-positive and 4 anti-HCV antibody positive patients initiated antiviral treatment. Conclusion Our simple electronic medical record based alert system effectively promoted intra-hospital referral of hepatitis virus-positive patients, who have been detected by screening tests, to hepatologists. Electronic supplementary material The online version of this article (doi:10.1186/s12879-016-1765-y) contains supplementary material, which is available to authorized users.
Background
Chronic infection with hepatitis B virus (HBV) and hepatitis C virus (HCV) is the major cause of end-stage liver diseases, including cirrhosis and hepatocellular carcinoma [1,2]. However, disease progression and cancer development can be inhibited by antiviral treatment [3][4][5][6], especially with the recent advances in these antiviral treatments. Thus, referral of hepatitis virus-positive individuals to a hepatologist at least once in their lifetime is recommended [7]. Unfortunately, in many Western countries, even though patients are successfully screened, many do not receive confirmatory testing for HBV or HCV [8][9][10][11].
In Japan, although medical check-ups for HBV and HCV infection are conducted nationwide, not all those who are found to be positive for these viruses seek specialist medical attention or receive proper treatment from a hepatologist. Thus, the government is collaborating with two in-country civil society groups-the Japan Hepatitis Council and the Viral Hepatitis Research Foundation of Japan-to develop and implement its viral hepatitis prevention and control program [12]. In hospitals, testing for HBV and HCV is performed as part of daily practice, as a preoperative or pretransfusion screening test. However, the efficiency of intra-hospital collaboration with hepatologists in relation to the follow-up treatment of HBV-or HCV-positive individuals detected by screening tests remains unclear. Recently, Furukawa et al. reported on the current management practices for patients with hepatitis B surface antigen-(HBsAg-) and anti-HCV-antibody positivity in nonhepatology departments at Saga University Hospital [13]. The prevalence proportions of HBsAg-and anti-HCV antibody positive individuals were 1.9 and 5.6 %, respectively. However, 79 % of HBV-and 82 % of probable HCV-positive patients were not referred to hepatologists. To address this issue, a system to manage hepatitis virus-positive patients and to ensure that they receive specialist care should be established as soon as possible in medical institutions that have hepatologists among their staff.
We developed a simple alert system to promote the referral of HBsAg-or anti-HCV antibody positive patients to hepatology specialists through their electronic medical records. In this exploratory study, we investigated the changes in the number of referrals to hepatologists and in the distribution of patients referred after the introduction of the system, as well as the subsequent clinical courses of the patients following these referrals. Herein, we present our results. Further improvements needed for intrahospital collaboration are discussed based on our findings.
Design
This was a single-center study of both inpatients and outpatients conducted in a secondary-care hospital. The investigation consisted of two parts, a retrospective investigation of the pre-intervention period (from April 1, 2013, to March 31, 2014; defined as "Period 1") and a prospective investigation during the post-intervention period (from April 1, 2014, to March 31, 2015; defined as "Period 2").
Development of an alert system for promoting referrals of HBsAg-or anti-HCV antibody positive patients to a specialist department Osaka City Juso Hospital is a community hospital with 263 beds (5 wards). Outpatient services are provided by 15 clinical departments, and approximately 500 patients visit the hospital daily. The personal identification numbers of newly diagnosed patients with HBsAg-or anti-HCV antibody positivity were sent from the hospital's central laboratory to the principal investigator through the hospital intranet every Monday afternoon during Period 2. After receiving these numbers, the principal investigator entered the following comment, referred to as a "sticky note," in the electronic medical records of any patient who had not been referred to a hepatologist : "This patient was found to be positive for HBsAg (or anti-HCV antibody) in this test. Please consider referring the patient to a hepatologist." This was done every Thursday evening. Once "sticky noted," every user of the medical record was forced to see this "sticky note". The cut-off for the presence or absence of referral for the purposes of the study was set as 1 month after the medical records were "sticky noted." We compared the number of patients screened for either HBV or HCV in Period 2 with that in Period 1. The distribution across the referring departments of patients who were referred to a hepatologist from nonhepatology departments and the patients' clinical courses after the referral was examined. The primary outcome of this study was the referral proportion to a hepatologist. The referral proportion was calculated as follows: referral proportion = number of patients referred to a hepatologist/ number of patients who attended a non-hepatologist appointment to receive a positive test result of HBsAg or anti-HCV antibody × 100.
The study protocol and informed consent procedure were approved by the Institutional Review Board at Osaka City Juso Hospital, and the study was conducted in accordance to the concepts of the Declaration of Helsinki. The Institutional Review Board gave permission to collect data without informed consent.
Hepatitis virus testing
HBsAg was measured using the Architect HBsAg QT assay (Abbott Japan, Tokyo), and anti-HCV antibodies were measured using the Architect HCV assay (Abbott Japan, Tokyo). HBV-DNA was measured using COBAS TaqMan HBV Test v. 2.0 (Roche Diagnostics K.K., Tokyo, Japan), and HCV-RNA was measured using the COBAS TaqMan HCV Test v. 2.0 (Roche Diagnostics K.K.).
Statistical analysis
Statistical analysis was conducted using JMP 11.0 software (SAS Institute, Inc., Cary, NC). Fisher's exact probability test or the chi-square test was used for categorical factors. As no participant appeared in the study periods, these tests were considered valid and appropriate. Differences with p values <0.05 were considered statistically significant.
Status of HBsAg and anti-HCV antibody testing
In Period 1, of the 2,757 patients in whom HBsAg was measured, 43 (1.6 %) were found to be positive, and of the 2,674 patients who underwent anti-HCV antibody measurements, 156 (5.8 %) were found to be positive (Fig. 1a). In Period 2, of the 2,891 patients in whom HBsAg was measured, 39 (1.3 %) were found to be positive, and of the 2,790 patients who underwent anti-HCV antibody measurements, 147 (5.3 %) were found to be positive (Fig. 1b). The proportions of patients with HBsAg and anti-HCV antibody positivity did not show statistically significant differences between Period 1 and Period 2 (p = 0.58 and p = 0.39, respectively). We put "sticky notes" on the records for the 23 HBsAg-positive and 81 anti-HCV antibody positive patients who were not immediately referred to a hepatologist (Fig. 1b). Figure S1).
Outcomes of patients referred to hepatologists
As shown in Fig. 2a, b, of the 23 HBsAg positive patients who were "sticky noted," 11 ultimately consulted a hepatologist; of these, two started nucleoside analogue treatment, while nine were found to be inactive HBVpositive patients who required only periodic follow-up. Of the 81 anti-HCV antibody-positive patients who were "sticky noted," 41 ultimately consulted a hepatologist; of these, four started interferon-based or first-generation interferon-free treatment, ten were listed as being considered for next-generation antiviral treatment, and 27 were negative for HCV-RNA. The clinical backgrounds of the six patients who received antiviral treatment are shown in Additional file 2: Table S1.
Discussion
We developed a simple alert system to refer HBsAg-or anti-HCV antibody-positive patients to hepatologists Fig. 2 HBsAg or anti-HCV antibody positive subjects in non-hepatology departments referred to hepatologists ("sticky noted"). a in Period 1. b in Period 2. HBsAg, hepatitis B surface antigen; HCV, hepatitis C virus through electronic medical records. The main finding of this study was that our system could increase the number of referrals of hepatitis virus-positive patients detected by screening tests to hepatologists. Similarly, Shimomura et al. recently reported that the implementation of a fully automated reporting system relying on electronic medical records at Okayama University Hospital also increased the number of referrals of HBsAg-or anti-HCV antibody-positive patients to the Hepatology department [14], indicating that such alert system may have the potential to promote intra-hospital referrals in other areas as well. For example, HbA1C is widely used as a screening tool for diabetes mellitus [15], and thus may help promote intra-hospital referrals of high-risk diabetic patients to specialists.
Electronic medical records have the potential to provide substantial benefits to physicians, clinical practices, and health care organizations [16], with the system optimization process being defined as the continuing effort to help users maximize their proficiency in the use of the system [17]. Our alert system has certain important advantages. First, this system is easy to operate. Although it is a manual system, it requires little effort to implement. We created approximately 100 "sticky notes" on the patients' electronic medical records over the course of 1 year; this is equivalent to 1 "sticky note" every 3 days, and it takes only a minute or two to place such a "sticky note". This level of additional workload seems to be acceptable even for busy medical staff. Next, this system is free to use, and it is easy to optimize the existing electronic medical record-based systems. Moreover, in relatively small community hospitals like ours, where care decisions are generally made in only a short time, it is easy to familiarize the staff with this system.
In Japan, the numbers of anti-HCV antibody-and HBsAg-positive individuals are estimated to be 1.5-2 million and 1.3-1.5 million, respectively. However, Tanaka et al. estimated that approximately 800,000 anti-HCV antibody-positive and 900,000 HBsAg-positive individuals are not aware that they are infected [18]. Moreover, the national Ministry of Health, Labour, and Welfare Ministerial Notification No. 160 (released on May 16, 2011) requested that medical institutions properly explain the results of hepatitis virus testing to the individuals tested. Although antiviral treatment has advanced substantially, if infected patients screened on various occasions do not consult a hepatologist and receive proper treatment, they will not benefit from the recent advances made in antiviral treatment. Additionally, even when the viruses are eliminated, cancer may sometimes develop, especially in older patients and in those with advanced fibrosis. Thus, unless proper follow-up is continued over the lifetime of the patients, reductions in liver-related deaths cannot ultimately be achieved.
Even with "sticky notes", the referral proportion was still comparably low in some departments in our hospital (<50 % referral proportions in the Orthopedics, Urology, Surgery, and Ophthalmology Departments). The reason for this low proportion is unclear, but it is probable that the referral proportion varies with the varying awareness of the physicians regarding appropriate responses for patients with hepatitis virus infection. It is also necessary for hepatologists to make efforts to inform physicians in non-hepatology departments about the recent advances in antiviral treatments at any opportunity, for example at conferences.
Some limitations of this study should be mentioned. First, the referral proportion did not directly reflect the number of patients who subsequently received care for HBV or HCV infection. One reason is that some patients referred to hepatologists have already received care for HBV or HCV in other hospitals. Second, this was a singlecentre study conducted in a secondary care hospital. A manual "sticky note" system as utilized in this study may be of little relevance or clinical assistance in tertiary care hospitals or public health settings. Third, some confounders, such as staff education, awareness of clinical trials, and availability of antiviral therapy for HBV and HCV may have affected the referral proportions.
Conclusions
In conclusion, the simple, electronic medical recordbased alert system described herein was found to be effective for promoting intra-hospital referrals of hepatitis virus positive patients detected by screening tests to hepatologists. In addition, improved knowledge and awareness of HBV and HCV infection among healthcare service providers appear important in terms of enabling intra-hospital collaborations and specialist referrals. | 2018-04-03T05:20:51.595Z | 2016-08-12T00:00:00.000 | {
"year": 2016,
"sha1": "10366c06e73271ceaef53c209d6f08c5c28830a4",
"oa_license": "CCBY",
"oa_url": "https://doi.org/10.1186/s12879-016-1765-y",
"oa_status": "GOLD",
"pdf_src": "PubMedCentral",
"pdf_hash": "10366c06e73271ceaef53c209d6f08c5c28830a4",
"s2fieldsofstudy": [
"Medicine",
"Biology"
],
"extfieldsofstudy": [
"Medicine"
]
} |
219644659 | pes2o/s2orc | v3-fos-license | On Teachers and Students’ Interaction
—The object of this paper is the interaction between teachers and students in classroom. Starting with the concept of interaction, the final goal is that teachers and students can participate widely in teaching activities and students can acquire knowledge and improve their personality under happy condition. Besides, the interaction action is analysed in three aspects of language interaction, emotion interaction and learning motivation. And then quite a little ways are offered to help to develop a duplex interactive model. At last, the paper will introduce a new way of interaction. That is to say that we can join the classroom and Internet to achieve a better effect of interaction
I. INTRODUCTION
When the view of quality education is gradually filtering into people's minds, the classroom undoubtedly draws people's attention as the basis of carrying out such an education and improving education reform. However, oriented by examination, in classroom existed the unidirectional spread form of knowledge, students were blinded by it so that their language and emotion are lied in gloomy and passive statement, and the classroom atmosphere isn't active and even shows apathy. Such a means of education goes against the quality tenet seriously. And most obviously, it violates the education tendency to emphases the humans as the basis and students as the subject. Compared with this way, the duplex interaction model of teachers and students caters for the developing current of education in Chain. In particular, the rapid development and popularisation of the Internet pours infinite energy into the interaction teaching.
A. The Meaning of Interaction
Going after high score in the examination education, students could study by the single spread form, in other words, they accepted the given knowledge in definite time and lost their active right in schoolroom. An opposite form is the duplex interactive mode, which means the mutual action and influence between persons and persons, persons and groups, groups and groups through language exchange, emotion interchange or other interchange ways. The interaction of teachers and students is meant that teachers are viewed as the main organizers in the course of teaching, they and students who are considered as the host corporate harmoniously by means of language exchange and feeling exchange so as to come out the common development of teaching and learning in schoolroom. This method requires that all learners must give and take, and thus generate a duplex interchange form affectively. As a result, each individual can naturally achieve his or her desirable goals of improving psychological and physical quality.
B. The Essence of Interaction
Teachers' duty is to tell students learning ways, spread knowledge and solve difficult problems in the course of teaching. The point was the traditional view as to teachers and to teaching in China. This idea attaches important to the authority of teachers and the unchangeable teaching direction from teachers to students in the procedure of spreading knowledge, and also shows the decisive status of teachers in ancient times. This view handed down from generation to generation. But with the times changing, the education view also transforms to certain degree. New view about the relationship between teachers and students is concerning the equality, democracy of teachers and students, and emphasising teachers as organizers merely in classroom. New talent view is focusing on People oriented but not the basis traditionally of spreading knowledge and accepting it. New learning view advises that students should study actively but not to accept passively. If to carry out the above views in classroom successfully, the interactive way must be the best-suited way to achieve the aim. As a teacher, he must own the sense of equality, lay aside the authority and really keep equality relationship with students, and thus he successfully views himself as an organizer and let the students become the host of the total course. As a student, in light of the new requirement of creative capacity from social developing, he must be able to discover issues, think about them himself and determine actively and then independently copy whit them. Besides he possesses definite capability and quality of resolving problems, and can control himself and good at cooperating with others.
In a word, the teachers should esteem their particular personality of students and guarantee the personality developing freely in interactive condition. In addition, they foster their spirits of self-support, self-respect and self-determination and help them to get along with others and keep harmony with society. As a result of the common endeavour of teachers and students' interaction, teachers and students both win the feeling of satisfactory, and raise experience and ability.
C. The Ways of Interaction
According to various standards, interaction can be sorted out different classifications in practical course. Here, we-separately discuss two different ways of classification.
1. Considering the teacher's action, interaction is divided into three kinds of interaction between teacher and each student, teacher and class, and teacher and group of students.
a. The interaction of teacher and every student. It means that teacher interchanges with every student in schoolroom. According to the research, the interaction occupies 54.4% of the whole interaction in primary school.
b. The interaction of teacher and the total class. That is to say, the teacher's behaviour is point to the whole class. This means is equal to 54.4% of the total time of teachers and students' interaction.
c. The interaction of teacher and groups of students is that the teachers' behaviour points to certain team of students. The interactive method is applied hardly.
2. Due to the crucial interactive individual, effective interaction way is fallen into teacher-students interaction, student-student interaction and group-group interaction.
a. Teachers and students interaction. Interactive teaching viewpoint considers, in classroom teacher-student interaction practically is such an exchange and communication, which they understand each other in terms of their own experience. In view of carrying out that teaching pattern, discipline is generated and teaching organization and means are the effects of the pattern. Meanwhile, how to arrange detail content and structure of teaching is taken the consequence into account.
b. Students and students' interaction. It means that each individual interacts with each other. It may be assorted into two kinds of cooperation and competition. Cooperation enables the individual gain more dignity, more capacity of society interchange and better mentality accommodation capacity; based on competition, students may promote together, learn the merits each other, and overcome our shortcomings by studying from others strong points.
c. Groups and groups' interaction. From the literal meaning of these words, we understand it -one group and another group work together in classroom. Research indicates when the contemporary groups learning together may help to eliminate much indeterminacy.
Among these kinds interactions, teacher-student interaction takes a primitive and decisive effect on interactive action, and a good teachers and students interaction may propel forward the student-student and group-group interaction.
II. INTERACTIVE PROBLEMS IN SCHOOLROOM
During concrete teaching, teacher-students' interaction represents the overt language interaction and implicit feeling interaction, and other factors which influence directly on the two interactive forms, for instance, learning motivation. It consists of evoking motivation and maintaining it.
A. Language Interaction
The teachers and students' teaching activity mainly takes language as medium to hand over with each other a function.
Interaction action between teachers and students is a kind of mutual effect. Teaching always takes place in the particular circumstance. Linguistic performance is the most primitive teaching form in classroom, and occupies about 80% of the total teaching activity. During the teaching course, language as the most important element is applied to spread knowledge and strengthen interaction exchange as a means, in other words, the goal of using language is to provide more opportunities for students to exchange. This partner stands out the position of students oriented, and encourages them to participate in teaching activity. It also enhances each other's interchange and mostly fosters their learning initiative and interest. Besides, the important point to use language is to create a good atmosphere for teachers and students interaction. Therefore, teachers ought to stress the skills of applying language, and try hard to unify the euphonic, tone, speed, expression etc. To generate a good atmosphere will be in favour with comprehension, students would acquire more knowledge in such enjoyable condition. Because a good atmosphere will enable students easier and thus they are willing to interchange with teachers or with each other.
B. Emotion Interaction
Traditional dignity of teachers enables the classroom atmosphere nervous very much. In modern times, teachers and students almost meet every day for several times, but they are short of virtual exchange really. They talk about the overwhelming majority of issues all that relevant to learning and how to learn. A number of facts show that the interchange of teachers and students is merely confined to spread and acceptance of knowledge but lacks feeling interaction which pays a great role in teaching and learning and creating a harmonic atmosphere in classroom.
Once Karl Jasperes noted, the course of education first is the course of growing up of students, and then is a part of the course of acquiring knowledge scientifically. In the way of teaching, emotion takes great effect on teaching and
THEORY AND PRACTICE IN LANGUAGE STUDIES
learning from various aspects. Because teachers stand for authority, they can influence profoundly students on developing viewpoints by interchanging, estimating and controlling. Their language form, expression, body language and line contact of vision all enable students attain a host of relational information which includes the assessment from teachers and their value in teachers' eyes, and teachers' attitude. Those students can feel teachers' positive assessment and attitude in learning performance and potential, they often develop positive learning attitude, and nurse a fever of anticipation to earn better marks and do their best to achieve their prospective aims. In contrast, those students feel passive estimate and attitude of teachers; they don't hope to attain good marks. What's more, these passive assessment, passive attitude and passive exchange given by teachers will lead to passive or misery which may persist in their minds for a long time, and even influence on developing right view of live and value. From this point, the teachers can't ignore their subtle emotion and expression. Main classification of emotion in classroom. Emotion interaction isn't visible like language exchange, so it's necessary to analyze the main emotion. Spielberg and Starr thought that curiosity and worry are the two most important emotions in schoolroom. Curiosity is defined as a kind of searching tendency of physiology arousal level or cognition conflict aroused by novelty maters, that is to say the individual yearns for discovery. They worry about learning result because they don't have distinct aims at achieving desirable degree. When students think they can't competent learning tasks, meanwhile they understand the performance requests them to finish the tasks, the worry appears naturally. The worry often accompanies with the emotions of anxiety and horror. The relationship of worry and learning efficiency can be shown by n-curve graph. That is to say, the moderate degree worry will contribute to improving learning efficiency; lower worry may prevent individual from disencumbering some distances so that they obtain slow and non-precise results about relevant examination in the course of learning; the higher degree worry makes the range of their attention more sorrow, weaken the transferring capacity of attention and finally don't find out main rules when they learn and accept knowledge.
C. To Stir up and Maintain Learning Motivation
As a factor irrelevant with intelligence, learning motivation doesn't determine directly the individual learning performance. But in fact, it has a great influence on studying interest, studying attitude, and endeavor degree and task maintenance of students. For this reason, it indirectly affects studying consequence of students. It is not difficult to understand whether the learning motivation being efficiently stirred up or not will directly impact upon their learning effects and also influence the results of the language interaction and emotion interaction between teachers and students in classroom.
Achievement goal theory insists, the different persons will develop different cognitions about accomplishment, and the difference would result in diverse achievement goal orientations, which may fall into mastery goal orientation and performance goal orientation. The former aims at learning and mastering knowledge primarily, whereas the latter is meant to pursuing good performance and marks.
Arming at mastery goal orientation, the students attach importance to develop new capability, and they believe that the most efficient way of achieving success is to do their almost in the course of studying. As a result, they can earn the sense of satisfactory by comparing with themselves. And most obvious, they always maintain active attitude to deal with any problems in learning, and love those tasks filled with challenges; besides, they apply deep-seated manufacture tragedy efficiently and regulate their learning method on time. Facing with difficulty and failure, they always maintain positive emotion and continue to plunge into the new learning tasks and strive for the next aim. This continuous learning motivation is a bracing and adaptive method to make sure success at last. In contrast, arming at performance goal orientation, those students focus on earning a high mark and fame which is viewed as a symbol to demonstrate their abilities. Therefore, they can obtain their desirable sense of satisfactory and performance. They tend to consider the endeavor and capability as opposite two sides. In other words, they think-the more you try, the less your ability is. In addition, they apt to make use of superficial learn tragedy. For instance, they get into habit of bearing in mind in the course of studying. When encountering frustration during learning, they will generate these emotions such as losing heart, feeling shame and depressing. And to make matters worse, they judge themselves in error and easily give up effort, when they are defeated. That kind of learning motivation embodies their helpless and unsuited features.
Based on their dissimilar learning goal orientations, teachers are in duty bound to create a favorite atmosphere for mastery goal orientation in classroom. The features of the atmosphere are to concentrate on mastering novel skills and knowledge and developing their ability from disparate aspects. At largely, learning motivation can be evoked and maintain in the following aspects.
1. In the respect of assigning tasks and giving right. The design plan of activities and tasks assigned by teachers should abide by three principles of variety, novelty and participation. Besides, the plan of designing ought to meet almost students' curiosities, challenges and self-controlling. The novelty and varied tasks don't easily stir up their interest and decrease the opportunities of comparing with others in society, but also lower the possibility of taking tasks performance simply as a kind capability symbol. Another advantage helps students to concentrate on completing tasks and mastering technique. What's more, the real, detail and short-time aim may increase their self-confidence to accomplish tasks triumphantly and increase their willingness to try hard for finishing them. When they witness themselves close to their desirable goals, the sense of self-competence will appear.
If given more opportunities and rights to chose actively and take part in determination in classroom, students will go into habit of self-decision that enhances their internal motivation and be apt to develop the mastery goal orientation. The investigation indicates that self-orientation atmosphere is favor of fostering competence feeling of students. Since the right of self-decision can enable students profit a lot from learning, teachers ought to give students more rights to determinate and chose in classroom. And they should conduct students to take part in learning activities so as to increase their learning initiative and responsibility. 2. In the field of affirmation and assessment. Affirmation is the positive estimate to the individual and takes affirmative influence on the developing of psychology and mentality of students. From the point of view, teachers should praise their students according to concrete matters. If necessary, teachers might commend them with certain intention. If not, the external commend may influence and even decrease their learning initiative. Based on the individual progress and enhancement, teachers gift the positive affirmative to students' performance. Their assessment would contribute to increasing the sense of pride and satisfactory of students. Once they know their proud feeling and satisfactory feeling originate from the result by comparing with themselves rather than with others, they will focus on boosting new capacity, mastering new learning tasks and generating mastery goal orientation. If the commend can be given to students in private, the effect will be better. In other words, teachers use proper ways to praise as possible as they could. The estimate content shouldn't only be limited to the marks, but should take other factors into account. If more opportunities can be created to enable students find out mistakes and go after a higher assessment, then they may realize that to make mistakes is a part of learning and doesn't mean failure or bad capability. At last, they understand what they really try to do is to concentrate on accomplish tasks, mastering knowledge and raise capability.
3. Learning teams. A number of investigations indicate that, during the three goal structures of competition, cooperation and individualization, competition emotion easily stirs up the conception of capacity variance and results in performance goal orientation. The goal of cooperation and individualization contributes to fostering mastery goal orientation. Under cooperation conditions, society comparison and assessment both base upon the representation of the whole group so that the subtle difference among each individual isn't distinct very much. Except for bumping up the studying interest and self-confidence of those students who earn a bad performance, team learning is able to deepen their comprehension to knowledge and then master it during the course of playing a part of students. Since the team learning allows students to control the whole course of learning, their competence feeling will increase during the course.
Obeying by the above aspects, students' learning motivation can be stirred up reasonably and maintain all the time in classroom. The most important point is to achieve the aim of making sure the classroom activity and help students really join in the teaching. All this promotes the interaction of teachers and students into a new level.
III. INTERACTION STRATEGIES
In schoolroom, teachers are required to change the traditional interpersonal relationship between teachers and students. The tendency of change is from authority and obedience into the democracy and harmonic friendship. In deal and friendly atmosphere filled with feeling, teachers impart knowledge efficiently and guarantee to carry reality, confidence and comprehension to effect by being on their shoes. If they want to help students to internalize learning motivation, they may conduct students to realize rightly the learning attitude and the learning performance. To adhere on the conception of interaction all the time in teaching classroom, the following strategies should be taken measure.
A. Language Interchange Strategy
In classroom of going on duplex spread, teachers clearly control the whole class though the form of substantive enactment and the exchange during groups is very little. On the condition, teachers play the role as lecturers, and students of course play the role of listeners and accept the given information passively. If teachers want to develop a proper statement of teachers and students' interaction, the teachers must encourage students to express their viewpoints boldly and make sure their Conversation flow freely. In addition, language interchange shouldn't only be confined to take place between teachers and students, but also between teachers and students groups, different groups of students and even each student. Only when the language interchange may go well, then it is possible that the good relationship of teachers and students could be developed. In other words, the language interchange is the premise of developing a good interaction of teachers and students.
B. Reasonable Expectation Strategy
The strategy can compel students to generate the self-efficacy feeling. Then they can participate in the classroom learning, and try their best to generate an adapted circumstance of learning. Being a teacher, he ought to possess optimistic attitude to students and encourage them timely by the direct or indirect ways. Some researches studied by Good and Brophy in 1994 have shown that, the teachers who keep hopeful attitude to students will present more supportive information in classroom, for instance, more contact of line of sight, and to keep smiling and friendly expression. By the contrast, those teachers lacking in positive attitude will express less encouraging information irrelevant with language when they spread knowledge to students. And they usually criticize and condemn students. In campus, teachers' assessment and attitude of praise, criticizing, friendship and so on is a mirror to provide the students' self-assessment and self-experience with basis rule. Because the development and diversification of the students'
THEORY AND PRACTICE IN LANGUAGE STUDIES
opinion reflects the assessment and attitude given by the authority to a certain degree. Their dignity could be maintained by means of the affirmative estimate of the authority.
C. Emotion Regulation Strategy
The teachers are considered as the promoters who are abundant in knowledge, emotion and patience, and also as assistant of cognition. In the process of classroom teaching, teachers should flexibly adopt measures to induce and regulate the emotion of students. Meanwhile, they ought to do their best to create a kind of harmonic and friendly atmosphere of teaching psychology. And the atmosphere guarantees students to take easy and feel "mentality safety" in classroom. In the course of searching and resolving difficult issues, students may produce the proper worry and interest, which will help them to generate positive cognition, lure new knowledge and bring forth new ideas. Teachers can assess them in virtue of means of checking to encourage students. Students can profit from their assessment and attain the sense of success, satisfactory and pride that may contribute them to experience the bitter and the sweet of studying.
Another important point is that teachers can stand on their position and angle to think about matters and observe their inward world. Such a teaching method is bestowed with affection and humanism. On such conditions, the every link of teaching is going to proceed on cordiality, open and freedom atmosphere. Students may express their thoughts safely and freely, and they can share common responsibility with teachers when the learning motivation is stirred up.
D. To Improve Assessment Strategy
The former way of estimating from teachers is a singular judgement standard which takes a point to replace other merits of students. And the evaluation criterion focuses on their marks mainly. Nowadays teachers should change the outdate evaluation way into developing a multiplex way. We name the way as appreciation assessment that embodies the education humanism. Every one is a particular, they all exert their potentiality on learning as long as there is proper circumstance; all people are equal, they can act a part in their position only if teachers treat them equally.
Appreciation assessment means giving up the former critical and negative judgment way and fining out others' merits and praising them modestly. The method will offer them more opportunities of experiencing joy, increasing self-confidence and self-pride, and encouraging them to show themselves. Meanwhile, it will set a good example for students so that they can view others' merits as a new point to improve, to develop and to acquire. They profit so much from the novelty evaluation way in maintaining dignity and interest. Its deeply influence will contribute to establish good relationship of teachers and students and develop efficient interaction of teachers and students. However, the appreciation estimate is given by others. In the assessment of education, on the one hand, the estimate from others is very important, and on the other hand, it is crucial considerably to encourage the students to develop self-assessment. Only when the way of estimate is changed completely, the classroom atmosphere is improved radically, and the real interaction can generate at last.
IV. THE NEW INTERACTION WAY OF WEB-BASED INSTRUCTION
When the internet industry develop quickly, various new techniques based on the internet pop up naturally. Their appearances are changing people's life style, and at the same it also changes traditional teaching style of the face-to-face between teachers and students. The new teaching way will benefit teachers and students a lot from various respects, and it might bring them new challenges.
A. The Essence of Web-based Interactive Teaching
1. The Web-based interactive teaching is a kind online learning way according to the techniques of medium and network. The web-based instruction circumstance distinguishes from the traditional and face-to-face teaching, that is to say the teachers and students aren't affirmative in the interactive classroom of based Web. The network techniques provide the teachers and the students with various communication mechanizes and ways for exchange and interaction. Gilberthe Moore, the distance-teaching expert pointed out in 1998: the new features of web-based instruction enable us to apply the learning theory of interaction and proper learning tragedy to strengthen students' study. Therefore, under the computer -based teaching circumstance, it's necessary that we understand the interactive features, and design appropriate plans. And then the learners and media, teachers and learner can interchange and study each other by applying the Internet.
2. In Internet teaching circumstance, the interaction teaching may fall into two classifications: teaching interaction activity and social interaction activity. The teaching interaction activity means to apply the hypermedia link skill of the computer to organize information. And the learners may utilize the explorer to obtain useful information from the recourses of non-linear organization and also can distribute their information or resources on the web page to share with others. This is the reason that people can catch more information and exchange ideas with each other. These interactive activities mainly include how to obtain teaching resources, to link content, to download information and to distribute message. This kind of interactive activity offers the learner dynastic control ability of information, and the opportunities to control learning circumstances. The non-linear information organization forms can accommodate the learners to take the information into practical usage, at anytime. The web system allows the individual to seek for information according to their ways.
The social interaction activity means to the interchange between people and people. In the network environment, sociality interaction is try hard to exchange with other computers by means of various media such as e-mail, language mailbox, list system, chatting room, call-board, online meeting and others, using these exchanges activities, the learners and teaching, the learners and learners can be online to carry on lifting to ask, answer, argue and consult, and then they can carry out cooperation and exchanges and others availably. These advantages can't be obtained from the classroom teaching and are drawing growing people's attention.
B. The Features of Web-based Interaction
The interactive activities in network are a dynastic course, and exist in the whole learning course of the learners. If the learners want to go in for the learning in the Internet they must carry out mutual interchange and information communication. The main characters in network circumstance present the following aspects.
1. The interactive activities are initiative. The web interaction is to take the learners as a central really. Whether the learners want to take port in the web-based instruction or not? And how do they carry out the learning activity in the course of exchange? They can make decision completely according to their demand and, they are really the subject of learning and control the interactive activities themselves availably. Based on its character, students must master the basis operate of the Internet and have clear goal about the information to be searched.
2. The interactive forms are flexible and various. In traditional teaching environment, the interchange form is sole and finite, whereas the web offers the learners various kinds of interactive ways and interchange means. Since the learners can choose a different step method, such as the BBS, e-mail, message book, etc. they can also make use of a chatting room, electronics whiteboard, on-line meeting, and etc. synchronous way to carry on interactive exchange with each other.
3. The network interaction is full of independence and expansibility. The interactive channels copy with the various the information respectively and independently, and don't disturb with each other. In classroom teaching environment, the students must ask questions in order and they acquire limited information about the questions. But in web circumstance, the students can ask questions and express ideas at the same time by different channels and then they can gain a great deal of related information that they need to understand.
Here, we only discuss the above features of the Internet. In fact, a number of characters or merits may be found out and be used to help us. Of course, everything has two sides; the online teaching has its sides also. The interactive activities in Internet are complex for any user. If the learners don't make good use of it, maybe it will mislead the learners and result in overloading information and feeling of frustration. So we should apply correctly it to serve for my learning.
C. The Staff's Role in the Interaction Teaching of Web-based
Staff development is essential to ensure that staff have the necessary skills and expertise to become effective online tutors. The staff development must offer opportunities for self-reflection. It is essential to develop staff as reflective practitioners to enable them to challenge their own beliefs, attitudes and practices, and to personalize their experiences and set them in context. Many online tutors have to deal with a vast range of new information and experiences beyond their immediate role of online tutor. So they master the skill of handing information. Because of the other side of the Internet, the teachers should try their best to conduct the students to use the Internet with correct means and ways, and also provide them precise and useful information in the course of interaction teaching in classroom. On the other hand, they must help students learn to understand the interaction way of web so that students can acquire good results in the learning. In a word, the teacher plays a role as an assistant, conductor and supervisor.
V. CONCLUSION
However, whether we chose to use the interaction teaching of face-to-face or the interaction way of web-based, our goal is to enable students to learn more initiatively and effectively. Only we can change the traditional teaching aimed at higher examination result, and bring the interaction teaching into full play. This paper has explored these advantages of the two different ways, and it also explains how to apply them and what should we play attention. All that will give us some enlightenment to go in for this interaction teaching. | 2020-05-28T09:12:12.698Z | 2020-06-01T00:00:00.000 | {
"year": 2020,
"sha1": "daec86c68e070a21fd0f62cff84e33d4b70fac19",
"oa_license": null,
"oa_url": "https://doi.org/10.17507/tpls.1006.09",
"oa_status": "GOLD",
"pdf_src": "MergedPDFExtraction",
"pdf_hash": "5581ad23fac818a7507771e5e52f168ac5af542c",
"s2fieldsofstudy": [
"Education"
],
"extfieldsofstudy": [
"Psychology"
]
} |
3657003 | pes2o/s2orc | v3-fos-license | Effects of Foliar Selenite on the Nutrient Components of Turnip (Brassica rapa var. rapa Linn.)
We administered foliar applications of 50, 100, and 200 mg L−1 selenium (Se, selenite) on turnip (Brassica rapa var. rapa Linn.) and detected the changes in the main nutrient components in fleshy roots. Results showed that the foliar application of Se (IV) significantly increased the Se content in turnip, and Se (IV) positively affected the uptake of several mineral elements, including magnesium, phosphorus, iron, zinc, manganese, and copper. Se (IV) treatments also improved the synthesis of protein and multiple amino acids instead of crude fat and total carbohydrate in turnip, indicating that the foliar application of Se (IV) could enhance Se biofortification in turnip and promote its nutritional value. We recommended 50–100 mg L−1 Se treatment for foliar application on turnip based on the daily intake of Se for adults (96–139 μg person−1 day−1) and its favorable effects on the nutrient components of turnip.
INTRODUCTION Selenium (Se) is an essential micronutrient for humans (White and Brown, 2010). Se deficiency can weaken the immune system and increase the risks of several diseases, including hypothyroidism, cardiovascular disease, or various cancers (Fairweather-Tait et al., 2011;Rayman, 2012;White, 2016). Excessive dietary Se intake can also cause selenosis in humans (Fairweather-Tait et al., 2011;Rayman, 2012;Sperotto et al., 2014), and its symptoms are similar to those caused by heavy metals (White, 2016). As such, the World Health Organization (WHO) has recommended the dietary allowance of ∼55-200 µg Se day −1 for adults (Wu et al., 2015), and the Institute of Medicine (USA) has suggested a tolerable upper intake of 400 µg Se day −1 for adults (White, 2016). Se supplementation greatly depends on the production of crops, vegetables, or edible mushrooms on soils with substantial Se content or phytoavailability (Broadley et al., 2006;Chilimba et al., 2011;Joy et al., 2015;Dogan et al., 2016). Se accumulation in tissues can be used as a basis for the classification of angiosperm species into three ecological types: non-accumulator, Se-indicator, and Se-accumulator species (White et al., 2007;White, 2016). Some studies have focused on the potential of plants, such as Se-accumulator species, for the phytoremediation of Se-contaminated soils (Banuelos and Dhillon, 2011;Wu et al., 2015). However, much interest has been directed toward developing Se-biofortified agricultural products because of severe Se deficiency worldwide (Wu et al., 2015). For example, inorganic Se fertilizers have been applied to effectively increase Se contents in diets and thus improve the Se status and health of humans (White and Broadley, 2009;Alfthan et al., 2015).
For plants, Se is a beneficial element because it can stimulate plant growth and enhance plant tolerance or resistance to abiotic or biotic stress (Quinn et al., 2007;Pilon-Smits et al., 2009;Feng et al., 2013). However, Se toxicity in plants has also been reported (Fu et al., 2011;Mao et al., 2011;Longchamp et al., 2015). A low Se concentration is considered advantageous for plant growth and development, whereas a high Se concentration can be toxic to plants (Fu et al., 2011;Mao et al., 2011;Gupta and Gupta, 2017). These effects may be attributed to changes in biochemical and metabolic processes caused by Se. For example, Se can mediate reactive oxygen species metabolism and trace element uptake in plants (Gupta and Gupta, 2017). Appropriate Se concentrations also improve the nutrient components, including mineral elements, polysaccharides, proteins, amino acids, and vitamin C, of some vegetables or edible mushrooms (Shang et al., 1998;Du et al., 2004;Zhao et al., 2004;Wang et al., 2005Wang et al., , 2006. Se enhances the synthesis of glucosinolates, which are important secondary metabolites found mainly in cruciferous plants (Sams et al., 2011;Malagoli et al., 2015;Schiavon et al., 2016). These reports fully support the conclusion that Se assimilation affects sulfur (S) and nitrogen (N) metabolic pathways in plants (Malagoli et al., 2015).
Turnip (Brassica rapa var. rapa Linn.), a cruciferous biennial plant, has been widely cultivated as a long-term vegetable or fodder in Europe, America, and Asia. It is rich in vitamin C, riboflavin, dietary fiber, and various mineral elements but is low in calories (Parveen et al., 2015;Ma et al., 2016). This biennial plant also contains antioxidants and can reduce the risk of high blood pressure, diabetes, and different cancers (Parveen et al., 2015). In China, turnip is mainly cultivated in the Qinghai-Tibet Plateau and its surrounding areas. However, these areas are exposed to severe Se deficiency. In a previous study, we analyzed the absorption and translocation characteristics of selenite and selenate in turnip by soil addition or foliar spraying (Li et al., 2018). We reported that turnip may be a potential Se-indicator species, and Se (IV) should be mainly selected as artificial Se fertilizer for turnips (Li et al., 2018). These results provided preliminary information on Se intake from turnip for local people, but the risk of Se biofortification, such as the assimilation of Se in plants and the effects of Se on turnip quality, is still unknown in turnips. In the present study, we examined the changes in nutrient components in turnips treated with Se (IV) concentration gradient via foliar application. We assumed that suitable Se concentrations can promote the nutrition level of turnip. Our results would further improve the understanding of the potential and value of Se biofortification in turnips.
Plant Cultivation and Treatment
Turnip seeds from Ninglang County of China (Landrace No. KTRG-B54) were germinated and grown in a natural environment. After 25 days, seedlings with consistent growth were neatly transplanted into uniform flowerpots (d = 18.5 cm, h = 17.5 cm) with equal amounts of Se-free mucky soil (one seedling in each pot). The soil condition is shown in Table 1. The
Sample Preparation and Biomass Measurement
The fleshy roots of plants were harvested and washed with distilled water. The fresh weights of the root samples were measured, and the samples were subsequently dried in an oven at 80 • C for 48 h to determine their dry biomasses. The conversion factors necessary to convert the fresh weight to the dry weight (DW) of the experimental plants were calculated for each treatment. The dry samples were then used for the following measurements. Three biological replicates were prepared for each treatment.
Mineral Analysis
Calcium (Ca), potassium (K), magnesium (Mg), and phosphorus (P) contents were determined in accordance with the method described by by using an inductively-coupled plasma spectrometer (ICP-OES Optima 8000, Perkin Elmer, USA). The contents of Se, copper (Cu), iron (Fe), manganese (Mn), and zinc (Zn) were determined in accordance with the method of Li et al. (2017) by using an inductively-coupled plasma mass spectrometer (ICP-MS, Thermo Fisher Scientific, USA). The daily intake of Se for adults was calculated on the basis of the Se concentrations as follows: daily intake of Se = Se concentrations in plants (µg kg −1 DW) × conversion factor × daily intake of vegetables. The conversion factors 0.120, 0.116, 0.104, and 0.093 were used to convert the fresh weight to DW of the samples Se0, Se50, Se100, and Se200 in this study, respectively. The average daily vegetable intake for adults is 0.345 kg person −1 day −1 (Parveen et al., 2015).
Macronutrient Analysis
The crude protein content (N × 4.38) was determined using Kjeldahl method Liu et al., 2016). The crude fat content was determined through Soxhlet extraction with petroleum ether as a solvent Liu et al., 2016). The total carbohydrate content was measured using a phenolsulphuric acid method (Liu et al., 2016). The total energy was calculated on the basis of the following equation: total energy (kJ) = 17 × (g crude protein + g total carbohydrate) + 37 × (g crude fat) (Liu et al., 2016).
Amino Acid Analysis
The hydrolyzed amino acid compositions of the different samples were determined using a Thermo Fisher U3000 highperformance liquid chromatography system. Approximately 2 g of the samples was mixed with 16 mL of 6 M HCl in 20 mL hydrolysis tubes and subsequently vacuum degassed for 30 min. The tubes were then sealed with N 2 and hydrolyzed at 110 • C for 22-24 h. When cooled, the mixtures were transferred to 50 mL volumetric flasks, and their volumes were fixed to the scale. The hydrolysate (1 mL) of each sample was vacuum dried, and the precipitate was dissolved with 1 mL of 0.02 M HCl. Afterwards, 500 µL of the solution was mixed with 250 µL of 1 M triethylamine acetonitrile solution, and 250 µL of 0.1 M phenyl isothiocyanate acetonitrile solution. The obtained mixture was subsequently left at room temperature for 1 h. The mixture was added with 2 mL of n-hexane, intensely shaken and allowed to stand for 10 min. The resulting solution was filtered with a 0.22 µm aqueous filter membrane for analysis.
Statistical Analysis
Statistical analyses were performed using SPSS version 18.0. Oneway ANOVA was conducted to analyse significant differences among multiple samples at a 0.05 level.
Contents of Mineral Elements
The contents of the mineral elements expressed on a DW basis in different samples are shown in Figure 1A and Table 2. The results of Se concentrations indicated that the foliar application of selenite could significantly increase Se accumulation in turnip fleshy roots (0.09 µg g −1 DW in the control samples and 2.39-7.54 µg g −1 DW in the Se-treated samples; P < 0.05; Figure 1A). On the basis of the Se concentrations in the fleshy roots, we estimated the following daily intake of Se for adults by feeding the turnip samples treated with the respective foliar applications of 0, 50, 100, and 200 mg L −1 Se (IV): 3.89, 96.08, 139.23, and 242.77 µg ( Figure 1B).
To assess the effects of Se biofortification on the nutrients of turnip, we detected other essential mineral elements in the samples. In this study, the foliar application of Se (IV) did not elicit significant effects on the uptake of the macroelement Ca (4.36-7.27 mg g −1 DW) and K (8.45-11.48 mg g −1 DW) ( Table 2). The Mg content in turnip significantly increased at 200 mg L −1 Se (IV) treatment (2.12 mg g −1 DW) ( Table 2). The Se (IV) treatment at 100 mg L −1 increased the P content (3.17 mg g −1 DW) in the turnip fleshy roots, but a higher Se (IV) concentration (200 mg L −1 ) did not produce the same stimulus effect ( Table 2). By comparison, the Se (IV) treatment further affected the uptake of microelements (Table 2). Furthermore, 100 mg L −1 Se (IV) treatment significantly improved the contents of Fe, Zn, Mn, and Cu in the turnip fleshy roots (P < 0.05) compared with those of the control samples, whereas 50 mg L −1 Se (IV) treatment had no such effects ( Table 2). The contents of all of the four microelements decreased at 200 mg L −1 Se (IV) compared with those at 100 mg L −1 Se (IV) (P < 0.05). However, the contents of Zn and Cu remained higher than those of the control samples (P < 0.05; Table 2).
Contents of Macronutrients
The macronutrients and the total energy in different samples are presented in Table 3. The determined contents in a descending order were total carbohydrate (59.47-71.63 g/100 g DW), crude protein (5.95-9.35 g/100 g DW), and crude fat (5.90-7.76 g/100 g DW). The total energy (1414-1537 kJ/100 g DW) was also estimated. The crude protein content was significantly higher at 100 and 200 mg L −1 Se (IV) treatment (increased by 31.09 and 57.14%, respectively) than at the control concentration (Table 3). By contrast, Se (IV) treatment did not significantly affect the changes in the contents of total carbohydrate and crude fat and the amount of total energy ( Table 3).
Contents of Hydrolyzed Amino Acids
The changes in the hydrolyzed amino acids (17 amino acids) in the turnip fleshy roots that were treated with different Se concentrations are presented in Table 4. The results showed that the foliar application of 50-200 mg L −1 Se (IV) did not change the contents of glutamate (3.67-5.89 g kg −1 DW), leucine (5.79-7.46 g kg −1 DW), and phenylalanine (4.19-4.60 g kg −1 DW) in turnip ( Table 4). The contents of the 13 amino acids were improved by Se (IV) application at various levels. The contents of cystine, alanine, proline and lysine significantly increased when Se (IV) reached 100 and 200 mg L −1 (P < 0.05). By comparison, the contents of aspartic acid, glycine, histidine, arginine, threonine, tyrosine, valine, methionine, and isoleucine were not markedly induced until 200 mg L −1 Se (IV) was applied (P < 0.05; Table 4). The content of serine began to significantly increase at 50 mg L −1 Se (IV) (P < 0.05; Table 4). Overall, the contents of the total amino acids increased as the Se treatment concentrations increased (Table 4).
Effects of Se (IV) on the Contents of Mineral Elements
Consistent with the results from our previous study (Li et al., 2018), the foliar application of Se (IV) could significantly increase Se accumulation in the turnip fleshy roots. On the basis of the Se concentrations in the fleshy roots, we estimated the daily intake of Se for adults according to the formula used by Parveen et al. (2015). The daily Se intake from the turnip treated with the foliar application of 50 and 100 mg L −1 Se (IV) was in accordance with the dietary allowance of 55-200 µg Se day −1 for adults recommended by WHO (Wu et al., 2015). However, in our previous study, the daily Se intake from turnip treated with similar methods reaches 239 and 340 µg (Li et al., 2018). These values did not exceed the tolerable upper intake of 400 µg Se day −1 for adults as suggested by the Institute of Medicine (USA) (White, 2016), indicating that the foliar application of selenite should be an advisable method to consider turnip as Se-enriched food by biofortification. However, Se concentrations should be further validated according to actual environmental conditions, especially soil properties.
Interactions between Se uptake and other mineral elements have been studied in many plants, but some studies have reported ambiguous conclusions on these interactions. Some reports have supported our results that suitable Se concentrations could improve the uptake of various mineral elements, such as Mg, P, Fe, Zn, Mn, and Cu. Arvy (1992) considered that Se content is positively correlated with Mn, Zn, cobalt (Co), P, and molybdenum (Mo) concentrations in plants. Similarly, Chen and Liu (1996) reported that Se within the physiological concentration range can improve the uptake of P, K, Ca, Mg, S, Fe, Mn, Cu, Zn, and Mo in plants. Hu et al. (2015) also showed that Mn, Zn, Cu, Ni, and Co in the roots of Danshen are high when Se (Na 2 SeO 4 ) is added. However, some of these studies have demonstrated that Se uptake can subsequently decrease the concentrations of other elements. For example, Arvy (1992) reported that Se content is negatively correlated with Fe, aluminum, and arsenic concentrations in plants. Singh and Singh (1978) also found that Se application reduces Fe, Mn, and Zn concentrations. Thus far, the interaction mechanism between Se uptake and other mineral elements is undefined. Se-induced decrease in the concentrations of some other elements may be attributed to ion antagonism. Fu et al. (2011) found that low Se (selenate or selenite) concentrations can improve the uptake of N, P, K, S, Mg, and Zn, whereas high Se concentrations inhibit the uptake of these elements in pak choi. In addition, some environmental factors may regulate the antagonism among different microelements. Singh and Singh (1978) found that increased Se levels reduce Zn and Cu contents in plants with a low P level, whereas Se application significantly increases Zn, Cu, and Mn contents when P fertilizer is applied. Thus, the effects Values are means ± standard deviation. Means within a column with different letters are significantly different at P < 0.05. of Se on the uptake of other mineral elements possibly depend on Se concentrations and soil conditions. However, our results provided evidence that the foliar application of Se (IV) could help improve mineral nutrient contents in turnip.
Effects of Se (IV) on the Contents of Macronutrients
Our results showed that Se could improve protein synthesis in turnip, and similar results have been reported in several mushroom or plant species. Zhao et al. (2004) reported that Se can enhance protein content in Ganoderma lucidum. Yin et al. (2015) found that the crude protein content in potato increases by 4.87-5.44% after Se (0.379 kg hm −2 ) is applied. Nawaz et al. (2016) found that Se foliar spray (40 mg L −1 ) increases crude protein content by 47% in maize plants under drought stress conditions. These results indicated that Se affects N metabolism in mushrooms and plants. In contrast to our results, previous findings showed that Se can improve the contents of total carbohydrate, polysaccharide, or soluble sugar in mushrooms or plants (Shang et al., 1998;Zhao et al., 2004;Wang et al., 2006;Yin et al., 2015). For example, Shang et al. (1998) found that Se application can increase the total carbohydrate content in lettuce, and similar results are observed in the fleshy root of carrot (Wang et al., 2006). Related studies have shown that Se application positively affects the production of some organic nutrients, including carotene, crude fiber, vitamin C, and amino acids (Shang et al., 1998;Zhao et al., 2004;Wang et al., 2006;Yin et al., 2015). However, few studies have reported that Se can change the contents of total carbohydrate or crude fat. Du et al. (2004) revealed that 0.60 and 3.00 mg kg −1 of soil Se fertilizer significantly increase the content of crude fat in eggplant. Overall, our results were consistent with existing knowledge that Se can improve organic nutrients in plants. The Se-induced enhancement of organic nutrients may be closely related to Se assimilation and metabolism in plants, but the exact mechanism by which Se improves organic nutrition remains unclear.
Effects of Se (IV) on the Contents of Hydrolysis Amino Acids
In this study, Se uptake elicited various positive effects on the synthesis of different amino acids, and the results were consistent with the change in crude protein. Previous studies indicated that Se can improve the contents of amino acids in some mushroom and plant species. Zhao et al. (2004) showed that moderate amounts of Se added in a culture medium can increase the contents of total amino acids and most components, except cystine in G. lucidum. Similar results have also been reported in two other studies (Niu et al., 1998;Wang et al., 2001). Du et al. (2004) found that soil Se fertilizer (0.60 and 3.00 mg kg −1 ) significantly improves the contents of total essential amino acids but does not obviously influence total amino acids in eggplant. However, they also observed that 0.15 mg kg −1 of soil Se fertilizer negatively affects the contents of amino acids in eggplant. In addition, Nawaz et al. (2016) demonstrated that 40 mg L −1 Se application enhanced the accumulation of total free amino acids (40%) in water-stressed maize plants. Thus, our results were partly consistent with previous findings, indicating that the effects of Se on amino acid synthesis were related to Se treatment concentrations and environmental conditions for different species. Se-induced changes in S and N metabolism in plants may be an important factor causing an increase in amino acid contents (Malagoli et al., 2015). For example, the contents of methionine and cystine can be improved to synthesize selenomethionine and selenocystine by inorganic Se because these amino acids contain S . This conclusion is supported by the detected Se-methyl-SeCys in foliar selenate-treated radish (Schiavon et al., 2016) because the metabolism of inorganic Se in plants is usually carried out via the S metabolism pathway (Singh and Singh, 1979).
Among the Se-induced amino acids in our study, threonine, valine, lysine, methionine, histidine, and isoleucine are essential amino acids for humans (Galili et al., 2016). In addition, alanine, glycine, serine, and proline are considered as sweeteners, whereas aspartic acid exhibits umami characteristics (Ma et al., 2016). Thus, Se (IV) application for turnip might help improve amino acid nutrition and textures. The promotional effects of Se on amino acids were also observed at 50 and 100 mg L −1 Se (IV) ( Table 4), which are the recommended concentrations for Se biofortification in turnip.
CONCLUSION
This study demonstrated that the foliar application of selenite on turnip could significantly increase the Se content in turnip fleshy roots. Se (IV) application positively affected the uptake of several other mineral elements, such as Mg, P, Fe, Zn, Mn, and Cu. The foliar application of Se (IV) could improve the synthesis of proteins and multiple amino acids, including several essential amino acids, in turnip. Although Se (IV) treatment did not affect the contents of Ca, K, crude fat, and total carbohydrate and the amount of total energy, the results indicated that the foliar application of Se could enhance Se biofortification in turnip and promote its nutritional value. We recommended 50-100 mg L −1 Se (IV) treatment for foliar applications on turnip based on the daily intake of Se for adults. These Se treatment concentrations elicited favorable effects on the nutrient components of turnip. We also considered that the effects of Se on the nutrient components of plants are closely related to the soil environment. This study presented the feasibility and benefits of Se biofortification in Chinese turnip. | 2018-03-02T14:02:34.778Z | 2018-03-02T00:00:00.000 | {
"year": 2018,
"sha1": "b54c9014bd5a5e2466f9781cb005cd7f2fc1b6b1",
"oa_license": "CCBY",
"oa_url": "https://doi.org/10.3389/fchem.2018.00042",
"oa_status": "GOLD",
"pdf_src": "PubMedCentral",
"pdf_hash": "b54c9014bd5a5e2466f9781cb005cd7f2fc1b6b1",
"s2fieldsofstudy": [
"Agricultural and Food Sciences"
],
"extfieldsofstudy": [
"Chemistry",
"Medicine"
]
} |
119161418 | pes2o/s2orc | v3-fos-license | Irreducible Ginzburg-Landau fields in dimension 2
Ginzburg-Landau fields are the solutions of the Ginzburg-Landau equations which depend on two positive parameters, $\alpha$ and $\beta$. We give conditions on $\alpha$ and $\beta$ for the existence of irreducible solutions of these equations. Our results hold for arbitrary compact, oriented, Riemannian 2-manifolds (for example, bounded domains in $\mathbb{R}^2$, spheres, tori, etc.) with de Gennes-Neumann boundary conditions. We also prove that, for each such manifold and all positive $\alpha$ and $\beta$, the Ginzburg-Landau free energy is a Palais-Smale function on the space of gauge equivalence classes, Ginzburg-Landau fields exist for only a finite set of energy values, and the moduli space of Ginzburg-Landau fields is compact.
Introduction
Ginzburg-Landau theory is a phenomenological model for superconductivity which gives variational equations for an Abelian gauge field and a complex scalar field. The gauge field is the electromagnetic vector potential, while the scalar field can be interpreted as the wave function of the so-called Bardeen-Cooper-Schrieffer ground state (a single quantum state occupied by a large number of Cooper pairs); the norm of the scalar field is the order parameter of the superconducting phase. Thus Ginzburg-Landau fields with non-vanishing scalar field describe superconducting phases, while vanishing scalar field corresponds to the normal phases of the material. This paper investigates the existence -and non-existence -of superconducting phases in the 2-dimensional Ginzburg-Landau theory. The topic has a vast literature in the case where the background is a planar (flat) domain in R 2 with various boundary conditions; we recommend [1] for references.
Throughout this paper Σ denotes a compact, oriented, Riemannian 2-manifold, which can have a non-empty, smooth boundary ∂Σ. The Riemannian metric and the orientation together define a orthogonal complex structure j and a symplectic form ω. These structures together make Σ a Kähler manifold. The volume form of Riemannian metric is ω. Let L → Σ be a smooth, complex line bundle with hermitian metric h. Fix a unitary curvature tensor F 0 with finite L 2 -norm. We call F 0 the external magnetic field. Finally fix two positive coupling constants α, β ∈ R + . For each smooth unitary connection ∇ and smooth section φ consider the Ginzburg-Landau free energy: Physically, if ∇ 0 is a unitary connection that satisfies then the energy (1.1) is the energy difference between the states described by (∇, φ) and (∇ 0 , 0). The variational equations of the energy (1.1) -called the Ginzburg-Landau equations -are gauge invariant, non-linear, second order partial differential equations. If a solution (∇, φ) is twice (weakly) differentiable, then it satisfies the de Gennes-Neumann boundary conditions. These boundary conditions describe superconductor-insulator interfaces; see in Section 2 for details.
Each pair (∇ 0 , 0) that satisfies equation (1.2) is a solution of the Ginzburg-Landau equations. We call such a pair a normal phase solution, because the order parameter vanishes identically. The energy (1.1) of any normal phase solution is zero. Note that another pair (∇, 0) is normal phase solution if and only if the 1-form a = ∇ − ∇ 0 is closed. In Abelian gauge theories, a pair (∇, φ) is called reducible if φ is identically zero, and irreducible otherwise. It is easy to see that a solution of the Ginzburg-Landau equations is reducible if and only if it is a normal phase solution.
For the rest of the paper let This non-negative real number depends on the geometric data (Σ, j, ω, L, h) and the curvature 2-form F 0 , but independent of the coupling constants, α and β. The quantity λ 1 plays a key role in the main theorems of this paper, which are listed below: The results of Main Theorem 3 also hold in the borderline β = 1 2 case, which was proved in [2, Section 4] by Bradlow. In fact Bradlow proved that for a closed (that is compact and without boundary) Kähler n-manifold X, in the β = We remark, that on surfaces with boundary there is a similar phase diagram, since 2πd, in inequality (1.7) can be replaced by the magnetic flux of the external magnetic field, Φ 0 ; see equation (2.5).
Our last main theorem uses a recent result of Feehan and Maridakis about the Lojasiewicz-Simon inequality for analytic functions on Banach spaces. We show that solutions for the Ginzburg-Landau equations (2.2a) and (2.2b) with de Gennes-Neumann boundary conditions exist only at finitely many energies, and the moduli space of Ginzburg-Landau fields, that is the quotient of the set of critical points of energy (1.1) by the action of the gauge group G (see definition in Section 2), is compact. Finally, we remark that there are similar results to Main Theorem 1 and Main Theorem 2, in the context of Abrikosov lattices, by Sigal and Tzaneteas; see [9,10]. Abrikosov lattices are Z 2 -(gauge)periodic solutions to the Ginzburg-Landau equations on the euclidean plane, R 2 . Here R 2 is viewed as the universal cover of the flat torus, and Z 2 acts via deck transformations. Furthermore, it came to our attention during the preparation of this paper that Chouchkov et al. has generalized these results to surfaces of higher genus with hyperbolic metrics [3]. This paper is organized as follows. Section 2 is a brief introduction to the Ginzburg-Landau theory on compact surfaces. In Section 3 we establish Main Theorem 1 by proving that Ginzburg-Landau free energy (1.1) is a Palais-Smale function on the space of gauge equivalence classes of fields. In Section 4.1 we prove a technical lemma about the solutions of the Ginzburg-Landau equations; this is used in Section 4.2 to prove Main Theorem 2. In Section 5 we combine results from the previous sections and a theorem of Feehan and Maridakis to prove Main Theorem 4.
Acknowledgment. I wish to thank my advisor, Tom Parker, for his advice during the preparation of this paper. I greatly benefited from the discussions with Paul Feehan and Manos Maridakis about the Lojasiewicz-Simon inequality. I am also grateful for the help of Benoit Charbonneau.
Ginzburg-Landau equations on compact surfaces
As is standard in gauge theory we work with the Sobolev W k,p -completions of connection and fields. For the rest of this paper we fix a connection ∇ 0 that satisfies equation (1.2). The Sobolev norms are defined via the Levi-Civita connection of Σ and the connection ∇ 0 . The norm of any W k,p space are denoted by . k,p . Furthermore . p stands for . 0,p , and .|. stands for the real L 2 inner products. In dimension 2, W 1,2 embeds in L 4 . The weakest Sobolev norm in which the energy (1.1) is C 1 (in fact analytic) is W 1,2 . Thus let C L be the W 1,2 -closure of the affine space of smooth unitary connections on L and Ω 0 L be the W 1,2 -closure of the vector space of smooth sections of L. Similarly, let Ω k and Ω k L be the W 1,2 -closure of k-forms and L-valued k-forms, respectively. Note that C L is now an affine space over iΩ 1 . The configuration space The gauge group is canonically isomorphic to the infinite dimensional Abelian Lie group W 2,2 (Σ, U(1)) whose Lie algebra is W 2,2 (Σ; iR). Elements g ∈ G act on smooth pairs which defines a smooth action of G on C L ⊕ Ω 0 L .
The energy (1.1) extends to a smooth function on C L ⊕ Ω 0 L which now can be written as Each critical point (∇, φ) ∈ C L ⊕ Ω 0 L of the energy (1.1) satisfies the following equations: If the pair (∇, φ) is in W 2,2 , then equations (2.1a) and (2.1b) are equivalent to the Ginzburg-Landau equations: with the boundary conditions (see [6, page 345]): De Gennes showed that conditions (2.3a) and (2.3b) describe an interface with superconducting material on one side and insulator or vacuum on the other (see [4, page 229]). Equation (2.3a) corresponds to the continuity of the magnetic field on the boundary. The 1-form j = Im(h(φ, ∇φ)) represents the supercurrent, the current of superconducting Cooper pairs, thus equation (2.3a) means that no supercurrent is leaving or entering the surface. Since in mathematics equations (2.3b) and (2.4) are also called the Neumann boundary conditions, we refer to them as the de Gennes-Neumann boundary conditions. Chern-Weil theory and equation (2.3a) implies that The interpretation of equation (2.5) is that the magnetic flux through the surface, Σ, is fixed.
Existence
To prove the existence of irreducible solutions of equations (2.2a) and (2.2b) when α > λ 1 we show two things: (i) absolute minimizers of the energy (1.1) exist for all α, β ∈ R + , and (ii) if α > λ 1 , then reducible (normal phase) solutions are not absolute minimizers. It follows that the absolute minimizers are irreducible.
We say that a function E on a Banach space satisfies the Palais-Smale Compactness Property if every sequence, on which E is bounded and DE (the derivative of E) converges to zero in the dual of the Banach space, has a convergent subsequence. The next lemma shows that the energy (1.1) satisfies a gauged version of the Palais-Smale Compactness Property.
Proof. Since smooth pairs in iΩ 1 ⊕ Ω 0 L are dense and the energy (1.1) is a continuous (in fact an analytic) function on C L ⊕ Ω 0 L , it is enough to prove the statement for smooth sequences. One can complete the square in the last two terms of the energy (1.1) to get a lower bound in terms of the coupling constants and the area: The right hand side of (3.1) is greater than or equal to the constant − α 2 2β Area(Σ). Since all terms are positive or constant, they are bounded individually. Equation (3.1), and the hypotheses of the theorem give us the following: (1) Since {F ∇ 0 +a k } k∈N is bounded in L 2 (by equation (3.1) and the hypotheses of the theorem) the sequence {∇ 0 + a k } k∈N is has a subsequence, which is gauge equivalent to a weakly convergent sequence in C L , by [12,Theorem 3.6]. Thus, after replacing the original sequence with a gauge equivalent one, and then taking a subsequence, we can assume that {a k } k∈N is weakly convergent and hence bounded in iΩ 1 . We can still assume smoothness, due to the density of smooth fields and gauge transformations. By the Sobolev inequality, {a k } k∈N is also bounded in L 4 . (2) We can also require the Coulomb gauge fixing condition to hold (for a subsequence), that is by to the following argument: For all k ∈ N let f k ∈ Ω 0 be the unique solution of the equations The sequence {d * a k } k∈N is smooth and bounded in L 2 , so by elliptic regularity {f k } k∈N is smooth and bounded in W 2,2 , and thus it has a weakly convergent subsequence, {f k l } l∈N , in W 2,2 . Setting g l = exp(if k l ) for all l ∈ N and replacing {∇ 0 + a k } k∈N with {g l (∇ 0 + a k l ) = ∇ 0 + a k l + idf k l } l∈N gives us equation (3.2), while not ruining the weak convergence and the smoothness of the sequence.
(3) The integrals Σ α β − |φ k | 2 2 ω are uniformly bounded. Since Σ has finite volume, Jensen's inequality implies that (4) Combining (1), (3), and Hölder's inequality shows that { a k φ k 2 } k∈N is bounded. Let ∇ LC be the Levi-Civita connection acting on elements of iΩ 1 . Since iΩ 1 ⊕ Ω 0 L is Hilbert space, there is a sequence, for all k ∈ N. By hypothesis, the left hand side of equation (3.5) converges to zero, which implies that {(b k , ψ k )} k∈N converges to zero in iΩ 1 ⊕ Ω 0 L as well. On the other hand, by using the definition of the derivative we get (3.6)
Combining equations (3.4) and (3.6) gives us the equation
for all (b, ψ) ∈ iΩ 1 ⊕ Ω 0 L . Equation (3.7) is a linear, elliptic partial differential equation, and the sequence {(a k , φ k )} k∈N is smooth, thus so is {(b k , ψ k )} k∈N , by elliptic regularity. Let ∆ = d * d + dd * be the Laplacian on forms and κ is the Gauss curvature of the Riemannian metric of Σ. By the Weitzenböck identity, ∇ LC * ∇ LC = ∆ − κ on iΩ 1 . Since both {(a k , φ k )} k∈N and {(b k , ψ k )} k∈N are smooth, we can integrate by parts in equation (3.7). Recall that d * a k = 0, and so ∆a k = d * da k for all k ∈ N, to get the following equations: Equations equation (3.8a) and (3.8b) imply the following equations in the interior of Σ: Since Σ is compact, κ is a bounded function. This fact and the observations (1)- (6) imply that the right hand sides of equations (3.9a) and (3.9b) are bounded in L 2 . Since {(a k − b k , φ k − ψ k )} k∈N is also bounded in L 2 , elliptic regularity implies that it is bounded in W 2,2 as well. The embedding W 2,2 ֒→ W 1,2 is completely continuous (see [8,Proposition 2.6]), thus {(a k − b k , φ k − ψ k )} k∈N has a convergent subsequence in iΩ 1 ⊕ Ω 0 L . Since {(b k , ψ k )} k∈N converges to zero by the argument after equation (3.5), we conclude that {(∇ 0 + a k , φ k )} k∈N has a convergent subsequence in C L ⊕ Ω 0 L .
Corollary 3.2. The infimum of energy (1.1) is achieved by some smooth field
Proof. The energy (1.1) bounded below by (3.1), thus its infimum is not −∞. Pick a minimizer sequence {(∇ 0 + a k , φ k )} k∈N . By [5, Proposition 2.6], we can assume that {DE(∇ 0 + a k , φ k )} k∈N converges to zero in the dual of iΩ 1 ⊕Ω 0 L . By Lemma 3.1, there is a subsequence, {(∇ 0 + a k l , φ k l )} l∈N , and a sequence of gauge transformations, {g l } l∈N , such that {g l (∇ 0 + a k l , φ k l )} l∈N converges in C L ⊕ Ω 0 L . Since the energy (1.1) is gauge invariant and analytic, the limit is an absolute minimizer and hence a critical point of the energy (1.1). By [8,Theorem 2.4], all critical points are gauge equivalent to a smooth pair (∇, φ) ∈ C L ⊕ Ω 0 L .
Finally, we show that the infimum cannot be a reducible (normal phase) solution when α > λ 1 . Proof. Since all reducible solutions satisfy E F 0 α,β = 0, it is enough to show that the minimum of the energy (1.1) is negative. Assume that α > λ 1 , and pick a positive ǫ which is less than α − λ 1 . Using the definition of λ 1 , equation (1.3), we get that there exist a normal phase solution, (∇ 0 , 0), and a non-zero section, φ ∈ Ω 0 L , such that Thus the pair (∇ 0 , t φ) ∈ C L ⊕ Ω 0 L satisfies which is negative for small enough t since λ 1 + ǫ − α < 0. Thus the minimum of the energy (1.1) is negative, and the hence absolute minimizer found in Corollary 3.2 is irreducible.
Together Corollary 3.2 and Lemma 3.3 prove Main Theorem 1.
1b)
If equality holds at any point in inequality (4.1a), then it holds everywhere and L is trivial.
Proof. When (∇, φ) is reducible, and hence φ is identically zero, we get F ∇ = F 0 by equations (2.2a) and (2.2b). Thus inequalities (4.1a) and (4.1b) hold. We can therefore assume that (∇, φ) is irreducible. Because the inequalities in the lemma are gauge invariant, and every solution is gauge equivalent to a smooth solution, we assume that (∇, φ) is smooth. Set Using equation (2.2b) straightforward computation (see [11, Section III]) provides Since 2β|φ| 2 and |∇ψ| 2 are both non-negative, the maximum principle (cf. [8,Proposition 3.3]) implies that w is either strictly positive, or vanishes identically, thus proving inequality (4.1a) and the claim about the case of equality at point. Hence when equality holds φ is a nowhere zero section of the line bundle L, thus L is trivial, Let ⋆ be the convolution of functions. Let G φ be the Green's function of the positive, elliptic operator ∆ + |φ| 2 on Ω 0 with Neumann boundary conditions, and similarly, G 0 be a Green's function of the scalar Laplacian, ∆, on Ω 0 with Neumann boundary conditions, which can be chosen to be everywhere positive. Let y ∈ Σ any, then for all x = y Thus by the maximum principle G 0 (x, y) G φ (x, y). Hence Furthermore recall that 0 w α β . Thus by inequalities (4.4) and (4.5) and equation (4.6) we get that Finally, assume that (∇, φ) is irreducible and F 0 solves Maxwell's equation. Thus f 0 is constant, and equations (4.3) and (4.6) and the fact that G φ ⋆ |φ| 2 = 1 give us: which proves inequality (4.2). The proof of Main Theorem 2. First we prove the statements about λ 1 . Let (∇ 0 ) 0,1 be the Cauchy-Riemann operator associated to the connection ∇ 0 . Using the Kähler identities and the de Gennes-Neumann boundary conditions we get that
The
L . The lower bound can be achieved by choosing φ to be holomorphic. Such φ exists, because f 0 is non-negative. Similarly, for a negative f 0 and the inequality is sharp for non-zero, anti-holomorphic sections, which exist due to the sign of f 0 . Hence whenever F 0 solves Maxwell's equations λ 1 equals to B 0 . Now we prove the claim about non-existence when Σ is closed. Assume that (∇, φ) is an irreducible solutions of equations (2.2a) and (2.2b), and ∂Σ = ∅. Chern-Weil theory tells us that in this case |F 0 | has to be 2π|d| Area(Σ) , where d is the degree of the line bundle L. By inequality (4.2) and Chern-Weil theory again, we get that Strict inequality holds in the last step since φ = 0 somewhere. Thus there are no irreducible solutions (by the contrapositive of the previous implication) when When Σ is not closed, that is ∂Σ = ∅, the magnitude of the external magnetic field, B 0 , can be any non-negative number. Equations (2.3a) and (2.5) and inequality (4.2) gives us Strict equality cannot hold in the last step if φ = 0 somewhere, which proves that there are no irreducible solutions when max α, α
Compactness
A real number E is a critical value of the energy (1.1) if there is a critical point, (∇, φ) ∈ C L ⊕ Ω 0 L , such that E F 0 α,β (∇, φ) = E. In this section we prove that there are only finitely many critical values and furthermore the moduli space of all Ginzburg-Landau fields is compact.
Proof of Main Theorem 4. First, we prove that the set of critical values is bounded. By (3.1) we see that the energy (1.1) is bounded below. If (∇, φ) is a critical point, then using equation (2.1b) with ψ = φ, and inequality (4.1b) give us α,β , which proves that the critical values are bounded above.
Next, we show that the set critical values inherits the discrete topology from R, using a result of Feehan and Maridakis about the Lojasiewicz-Simon gradient inequality. First of all, note that the energy (1.1) is constant on gauge equivalence classes, hence we can impose the Coulomb gauge fixing condition for the proof, that is we fix a smooth critical point (∇, φ) and define the Coulomb slice to be S C = (∇ + a, ψ) ∈ C L ⊕ Ω 0 L d * a = 0 & a(n) = 0 ∀n ⊥ ∂Σ . By the argument shown in the proof of Lemma 3.1, every gauge equivalence class intersects S C . The energy (1.1) is still analytic on the affine Hilbert manifold S C , thus its Hessian at (∇, φ) is a symmetric bilinear form on the tangent space of S C , and it is defined as for any (a, ψ), (b, χ) ∈ T (∇,φ) S C . Straightforward computation using equation ( In particular, if (∇ ′ , φ ′ ) is also a critical point, then E F 0 α,β (∇ ′ , φ ′ ) = E F 0 α,β (∇, φ). Now assume that the set of critical values in infinite, and let {(∇ 0 + a k , φ k )} k∈N ⊂ S C be a sequence of critical points with distinct energies. The sequence {(∇ 0 + a k , φ k )} k∈N satisfies the conditions of Lemma 3.1, because {E k = E F 0 α,β (∇ 0 + a k , φ k )} k∈N is bounded and the derivatives, DE(∇ 0 + a k , φ k ), are zero for all k ∈ N. Thus there is a subsequence, {(∇ 0 + a k l , φ k l )} l∈N , and a sequence of gauge transformations, {g l ∈ G} l∈N , such that {g l (∇ 0 + a k l , φ k l )} l∈N converges in C L ⊕Ω 0 L to a pair (∇ 0 + a ∞ , φ ∞ ). Since the energy (1.1) is an analytic function on C L ⊕ Ω 0 L , (∇ 0 + a ∞ , φ ∞ ) also a critical point with energy Let δ, Z > 0 be the constants corresponding to (∇ 0 + a ∞ , φ ∞ ) in inequality (5.2). By the definition of convergence there exist i cr such that if i > i cr , then (∇ 0 + a ∞ , φ ∞ ) − g l (∇ 0 + a k l , φ k l ) 1,2 < δ. The Lojasiewicz-Simon gradient inequality (5.2) then implies that E k l = E for all i > i cr which contradicts our assumption that there are infinitely many critical values.
The compactness of the moduli space of Ginzburg-Landau fields, M F 0 α,β , defined in (1.8), also follows from Lemma 3.1: Since the space C L ⊕ Ω 0 L /G is a metric space, it has the Bolzano-Weierstrass Property, that is a subset M ⊂ C L ⊕ Ω 0 L /G is compact if only if every sequence in M has a convergent subsequence in M. By the previous observation, the energy (1.1) is bounded and has constantly zero derivative on the space of all solutions of the equations (2.1a) and (2.1b). Thus every sequence in this space satisfies the conditions of Lemma 3.1, thus has a subsequence that is gauge equivalent to a convergent one. That means that the projection of the space of all solutions of equations (2.1a) and (2.1b) to C L ⊕ Ω 0 L /G, which is (by definition) M F 0 α,β , has the Bolzano-Weierstrass Property, and thus compact. | 2018-01-31T15:27:58.824Z | 2016-07-01T00:00:00.000 | {
"year": 2016,
"sha1": "7c0bbcf1f1e8ec91f7926ee12471c11127f79811",
"oa_license": null,
"oa_url": "http://arxiv.org/pdf/1607.00232",
"oa_status": "GREEN",
"pdf_src": "Arxiv",
"pdf_hash": "7c0bbcf1f1e8ec91f7926ee12471c11127f79811",
"s2fieldsofstudy": [
"Mathematics"
],
"extfieldsofstudy": [
"Mathematics",
"Physics"
]
} |
218861018 | pes2o/s2orc | v3-fos-license | Fetal magnetic resonance imaging contributes to the diagnosis and treatment of meconium peritonitis
Background Meconium peritonitis (MP) is a rare fetal disease that needs to be urgently identified for surgical intervention. We report a series of 35 patients diagnosed prenatally with MP by magnetic resonance imaging (MRI), illustrate the imaging findings and investigate the predictive value of these findings for postpartum management. Method A consecutive cohort of patients diagnosed with MP who were born at our institution from 2013 to 2018 was enrolled retrospectively. The prenatal ultrasound and MRI findings were analyzed. Fisher’s exact probability test was used to evaluate the predictive value of MRI for surgical intervention between the operative group and the nonoperative group. Results Ascites (30/35) and distended bowel loops (27/35) were two of the most common prenatal MP-related findings on fetal MRI. Of the 35 infants, 26 received surgical intervention. All fetuses with MRI scans showing bowel dilatation (14/26, p = 0.048) and micro-colorectum (13/26, p = 0.013) required surgery. There were no significant differences in the number of fetuses with meconium pseudocysts and peritoneal calcifications between the two groups. Conclusion Fetuses with bowel dilatation and micro-colorectum on MRI may need postpartum surgical intervention. Infants with only a small amount of ascites and slight bowel distention were likely to receive conservative treatment.
Background
Meconium peritonitis (MP) is a type of aseptic chemical peritonitis caused by the leakage of meconium from small bowel perforations in fetuses. MP has a low incidence of 1 in 30,000 live births [1] and was a fatal disease with a mortality rate of 80-90% in the early 1950s [2]. MP is usually caused by the imperforation or/and perforation of the intestinal tract, but the inherent pathogenic mechanism remains unclear [3][4][5]. Because of the compensatory function of the maternal placenta, fetuses show few or even no abnormalities when MP appears. It is difficult to diagnose infants with MP without prenatal imaging. The fatality rate remains over 40% according to the report by Shyu M. K et al in 2003 [6]. Identifying and diagnosing MP with an effective method may provide some clues for postpartum management in an earlier period.
Ultrasound is the most commonly used method for prenatal examinations, but sometimes the results are disturbed by many factors [7]. In recent years, the development of fetal MRI technology has been suggested to be safe and feasible. He et al. reported that fetal MRI could be used to define MP features in 8 cases [8]. He et al summarized the MRI findings of MP, including massive meconium ascites, meconium pseudocysts, dilated bowel loops and micro-colorectum. However, the limited number of reported cases was not convincing, and the value of fetal MRI in predicting the need for surgical intervention among infants with MP has not yet been clarified.
The purpose of this study was to summarize the MRI findings for identifying MP and to investigate the value of these findings for predicting the need for postpartum surgical intervention. We collected all patients diagnosed with MP who underwent fetal MRI at the Guangzhou Women and Children's Medical Center from January 1st, 2013, to January 1st, 2018. Then, the patient characteristics, ultrasound and MRI findings, postnatal management strategies and outcomes were analyzed.
Methods
Infants diagnosed with MP who were born at our institution from January 1st, 2013, to January 1st, 2018 were analyzed retrospectively. A total of 35 infants who received fetal MRI scanning were finally included in this research. The fetal MRI and medical histories of the fetuses and mothers were systematically collected, and a follow-up (1 year at least) was performed. All parents provided written informed consent for fetal MRI before scanning. All of the information involving the private messages of these patients is protected. Written informed consent was waived by our institutional ethics committee review board for this retrospective case series.
Ultrasound imaging
In our hospital, routine prenatal two-dimensional (2D) transabdominal ultrasound scans were performed by an obstetric specialist in prenatal ultrasonography diagnosis using a color Doppler ultrasonography (Volusion E8, General Electric, USA; HD11, Philips, Netherlands). The obstetric specialist who diagnosed the infants based on fetal US had no conflicts of interest in the study.
Magnetic resonance imaging
Noncontrast enhanced fetal MRI was performed on a 1.5-Tesla (Achieva, Philips, the Netherlands) or 3.0-Tesla (Skyra, Siemens, Germany) superconducting MRI scanner with a four-channel phased-array abdominal coil. Sedation was not used for either the fetus or mother. Our fetal MRI protocol included the following principles and sequences to scan the fetus: (1) All pregnant women received an MRI scan in the supine position in the morning and were told not to eat for at least 3 h and not to drink for at least 2 h so that the fetus would be less active, and the MRI examination was performed during this period. (2) The 1.5-T scanning parameters were as follows: axial T1-weighted turbo spin echo (TR: 200 ms, TE: 5 ms, slices: 3 mm) and coronal, axial and sagittal T2-weighted sense-body fetal turbo spin echo (TR: 15000 ms, TE: 120 ms, slices: 6 mm). On occasion, fatsuppressed T2-weighted spectral presaturation attenuated inversion recovery (SPAIR) echo sequences (TR: 598 ms, TE: 80 ms, slices: 3 mm) and balanced fast field echo sequences (TR: 3.5 ms, TE: 1.74 ms, slices: 4 mm) were preferred. (3) The 3.0-T scanning parameters were as follows: axial T1-weighted turbo spin echo (TR: 4.0 ms, TE: 1.8 ms, slices: 3 mm) and coronal, axial and sagittal T2-weighted sense-body fetal turbo spin echo (TR: 2000 ms, TE: 87 ms, slices: 6 mm) were performed. On occasion, T1-weighted imaging vibe fat-suppressed (TR: 4.2 ms, TE: 2.0 ms, slices: 3 mm) sequences were preferred. Sixteen patients underwent imaging with a 1.5-T scanner, and 19 patients underwent imaging with a 3.0-T scanner (since June 2015). Serial ultrasound examinations were performed to monitor disease progression after the MRI diagnosis.
The images were independently reviewed by two experienced fetal MRI radiologists, both with 15 years of experience and who were blinded to the postnatal outcomes. A third radiologist was included when disagreements about the diagnosis existed. The fetal MRI report consisted of abnormal findings, including seroperitoneum and total abdominal tube morphology, and the presence of amniotic fluid. The fetal outcomes were identified by medical records and autopsy.
Follow-up and statistical analysis
All infants were followed up for at least 1 year. Descriptive statistics were used to analyze the number of fetal abnormalities in the study cohort. Fisher's exact probability test was used to evaluate the surgical predictive value of MRI between the operation group and observation group in a 2 × 2 table. The MRI findings were also reported using a descriptive approach.
Results
Thirty-five infants diagnosed with MP who underwent MRI scanning were collected and analyzed in our study. The median gestational age was 31 weeks (range, 24-39 weeks) at the time of MRI scanning. There was no family history of MP in any mothers in our cohort. The median maternal age was 28 years old (19 to 34 years old). Of all mothers, nine mothers had hepatitis B virus (HBV), and nine had mild alpha thalassemia. Nine developed intrahepatic cholestasis of pregnancy, including eight mothers with HBV and one with alpha thalassemia.
The imaging findings on both ultrasound and MRI are summarized in Table 1, and representative imaging findings are shown in Fig. 1 (ascites, gathered bowel loops, bowel dilatation, meconium pseudocysts, peritoneal calcification and hydroceles) and Fig. 2d (micro-colorectum). Ascites and bowel dilatation were two of the most common findings on both ultrasound and MRI. Distended bowel loops (27:4 on MRI or ultrasound), micro-colorectum (13:1), meconium pseudocysts (10:3) and hydroceles (13:4) were more likely to be depicted by MRI than by ultrasound. Of all 35 patients, 26 received surgical management. The intraoperative findings corresponded to the findings on MRI scanning. Atresia and perforation, two of the main causes of MP, were visualized in 22 and 12 infants, respectively.
To investigate the value of fetal MRI findings for predicting the need for surgical intervention, we divided all infants into an operative group and an observation group according to whether they received a postpartum operation, and we performed Fisher's exact probability tests to analyze the correlations ( Table 2). The presence of bowel dilatation (14/26, p = 0.048) and microcolorectum (13/26, p = 0.013) seemed to be associated with the need for postpartum surgical intervention (Fig. 2). Of the 9 infants with nonsurgical interventions after delivery, 8 infants had ascites or/and distended bowel loops. There was only one infant with MRI signs of peritoneal calcifications or meconium pseudocysts who received conservative treatment (Fig. 3). Although the number of infants with meconium pseudocysts or peritoneal calcifications was not significantly different between the two groups, surgical intervention was considered when the MRI also revealed the presence of other findings other than ascites or distended bowel loops. Infants with hydrops in an enlarged scrotum did not need to undergo surgical treatment because of the self-healing capabilities of the body.
In 26/35 (74.3%) cases, the parents elected for exploratory laparotomies at a median age of 1 day old (range, 0-9 days). Intestinal atresia (n = 14), perforation (n = 4) or both (n = 8) that occurred intraoperatively caused the development of MP. Most infants (n = 24) underwent resection and anastomosis, and the remaining infants (n = 2) underwent simple repairs. There were 3 cases of mortality (8.6%), including one intraoperative death and two postoperative deaths. After a one-year follow-up, 9 infants managed with expectant treatment and 23 managed with surgical intervention recovered well. In addition, 13 infants were confirmed to have hydroceles, but all recovered 6 months after birth.
Discussion
MP, initially described in the English literature as early as 1838 [9], is a kind of chemical peritonitis caused by fetal autologous meconium. In our present study, we collected 35 cases of MP at our Women and Children's Center from 2013 to 2018. The characteristics of the pregnancies and fetuses with MP and the ultrasound (14) 15 (14) Micro-colorectum 1 (14) 13 (14) Meconium pseudocyst 2 (6) 7 (6) Peritoneal calcification 3 (10) 12 ( and MRI findings were systemically analyzed. Then, we retraced the treatments, pathological reports and outcomes of the fetuses with MP. In our current study, we found that fetal MRI could visualize more MP findings of intestinal perforation and atresia than other imaging methods, and the findings of bowel dilatation and micro-colorectum on MRI were two predictive factors for the need for surgery. Similar to previous studies [1,5,10], the common characteristics of MP included fetal ascites, distended bowel loops, bowel dilatation, micro-colorectum, hydroceles, meconium pseudocysts and peritoneal calcifications in our current analysis. In 1983, Smith et al first reported that MRI could be used for scanning during pregnancy [11]. In the next decades, the development of rapid MRI technology has solved the disadvantage of long scanning times. Half-Fourier acquisition single-shot turbo spin-echo (HASTE) and true fast imaging with steady-state precession (true FISP) are two common sequences used for fetal MRI scanning and could acquire high-quality fetal images without the need for sedation or breath holding for 1 s. Early in 2002, Ertl-Wagner et al noted that fetal MRI was safe for both mothers during pregnancy and fetuses [12]. The American Food and Drug Administration suggested that fetal MRI could be used for scanning 3 months after conception. In the last 10 years, fetal MRI technology has been implemented and rapidly developed in China. However, systematic research on MP was lacking in Asian populations. Fetal ascites was the most common antenatal finding of MP with both ultrasound (26/35, 74.3%) and MRI scanning (30/35, 85.7%) in our cohort, which is similar to the findings reported by Ping et al [13]. Sometimes, only fetal ascites appeared, and differential diagnoses such as fetal edematous syndrome (also called Bart's syndrome) should be considered. Fetal pleural effusion, pericardial effusion and/or skin hydroncus can also be observed in addition to fetal ascites if Bart's syndrome is present. Distended bowel loops was the second most common finding of MP on MRI. This symptom may be caused by fibrinous exudate and adhesions. A small pseudocyst does not influence the function of the digestive tract. In some cases, a large meconium pseudocyst required postnatal surgical treatment [6]. Infants who only had ascites and mild bowel loop distention were more likely to receive conservative treatment than to undergo surgery. It is speculated that the unexplained bowel perforation had been sealed off. Hydroceles and polyhydramnios were the third most common findings of MP on MRI in our study. When the ascites moved from the abdominal cavity into the scrotum, hydroceles developed. Fetal hydroceles could gradually be absorbed postnatally. Intestinal obstructions result in a decrease in swallowed amniotic fluid, which causes an increase in amniotic fluid in the uterus.
Dilated bowel, micro-colorectum and peritoneal calcifications were some of the other findings of MP. Bowel atresia could cause dilation of the proximal bowel. Nyberg et al suggested that a bowel internal diameter over 7 mm and a length over 15 mm should be diagnosed as intestinal dilation [14]. For the distal colon, the diameter reduces in size because of disuse atrophy. Then, the atretic intestinal tract appears perforated, and the meconium can enter into the abdominal cavity. In some cases, the location of the bowel perforation was difficult to distinguish. A perforation alleviates the pressure of the dilated bowel, which may lead to remission of the dilation of the proximal bowel. In our study, only 4 fetuses had symptoms of bowel dilation. The peritoneal calcifications formed because of the calcium salt particles of the meconium in the abdominal cavity reacted with the peritoneal inflammatory exudate. Shyu et al. reported that peritoneal calcifications on prenatal ultrasound were the most common finding of MP [6]. In our study, fetal ascites and distended bowel loops were the most common findings of MP on both ultrasound and MRI. The existence of ascites and/or distended bowel loops can interfere with the sonographers' observation of other abnormities, while fetal MRI can provide more information to help determine the postpartum management strategy.
Surgery is the main method for the postnatal management for MP. Recent studies have achieved a better survival rate than that reported in the 1960's, [15,16]. In our cohort, 9 infants received conservative treatment, and 26 infants underwent surgical intervention. The overall mortality rate was 8.6%, which was similar to that reported in the literature [1,13]. Patients with only a small amount of ascites and mild distention of the bowel loops may not need surgery, according to our current study.
Conclusions
In conclusion, our current study demonstrated that fetal MRI could help diagnose MP with various findings, including abnormal fetal ascites, distended bowel loops, hydroceles, micro-colorectum, bowel dilatation, peritoneal calcifications and meconium pseudocysts. Particularly, the existence of micro-colorectum and/or bowel dilatation reflected the need for postnatal surgical intervention.
Abbreviations MP: meconium peritonitis; MRI: magnetic resonance imaging; SPAIR: spectral presaturation attenuated inversion recovery echo sequences; TR: time of repetition; TE: time of echo; HBV: hepatitis B virus; HASTE: Half-Fourier acquisition single-shot turbo spin-echo; true FISP: true fast imaging with steady-state precession
Acknowledgments
We sincerely appreciate all infants who participated in this study.
Authors' contributions YT L contributed to the study concept and design, literature research, clinical studies, data analysis and manuscript preparation; B A contributed to the literature research, clinical studies and data analysis; WJ Z contributed to the literature research and clinical studies; HG L contributed to the study concept and design and manuscript editing. All authors have read and approved the manuscript.
Funding
This work was supported by funds from the Guangzhou Institute of Pediatrics/Guangzhou Women and Children Medical Center (grant number: YIP-2019-023), Guangzhou Science and Technology Department (grant number: 201804010142) and Guangdong Science and Technology Department (grant number: 2016ZC0227). The funding body had no role in the design of the study, collection, analysis, and interpretation of data, or in writing the manuscript.
Availability of data and materials
The dataset analyzed during the current study is available from the corresponding author on reasonable request pending the approval of our institution and trial/study investigators who contributed to the dataset.
Ethics approval and consent to participate Written informed consent was waived by the Review Board of Guangzhou Women and Children's Medical Center Ethics Committee for this retrospective case series. | 2020-05-24T13:59:10.852Z | 2020-05-24T00:00:00.000 | {
"year": 2020,
"sha1": "c1f5a84a0e2367dd39a6d0288f6e010b12270f9b",
"oa_license": "CCBY",
"oa_url": "https://bmcmedimaging.biomedcentral.com/track/pdf/10.1186/s12880-020-00453-8",
"oa_status": "GOLD",
"pdf_src": "PubMedCentral",
"pdf_hash": "c1f5a84a0e2367dd39a6d0288f6e010b12270f9b",
"s2fieldsofstudy": [
"Medicine"
],
"extfieldsofstudy": [
"Computer Science",
"Medicine"
]
} |
255675637 | pes2o/s2orc | v3-fos-license | Bilateral Congenital Retinal Macrovessel: A Case Report
Congenital retinal macrovessel (CRM) is a congenital anomaly where an aberrant vessel supplies both the macula on either side of the horizontal raphe. It is usually an incidental finding but may be associated with other retinal findings that may impact vision. We present an unusual case of bilateral congenital retinal macrovessel in a 40-year-old patient. The patient presented to the ophthalmology clinic for diabetic retinopathy screening and had no visual complaints. The patient underwent a complete ophthalmological examination including fundus imaging, fundus fluorescein angiography, and optical coherence tomography (OCT) imaging. Dilated fundus examination showed bilateral hypertensive retinopathy with optic disc collateral vessels and congenital retinal macrovessels. The multimodal imaging techniques that were used to confirm the finding indicated that the vessel is arterial.
Introduction
Congenital retinal macrovessel (CRM), first described by Mauthner in 1869 [1], is a rare finding of an aberrant retinal vessel that crosses the macula and supplies or drains the retina inferior and superior to the horizontal raphe [2]. Later, it was classified as a form of arteriovenous malformation of the retina by Archer et al. [3]. This finding, while commonly incidental, has been seen in association with other pathologies such as branch retinal artery occlusion, macroaneurysm, telangiectasia, macular hemorrhage, and cavernous hemangioma [4][5][6][7][8]. More recently, an association with venous anomalies of the central nervous system has been proposed [9].
Case Presentation
A 40-year-old male who is known to have diabetes mellitus, dyslipidemia, and hypertension for the last eight years presented to the ophthalmology clinic for diabetic retinopathy screening. He had no previous ocular history. On examination, his best corrected visual acuity was 6/6 in both eyes. Slit lamp examination of the anterior segment was normal in both eyes. Dilated fundus examination revealed bilateral grade 2 hypertensive retinopathy with optic disc collateral vessels. In both eyes, an aberrant vessel was found originating from the optic nerve head and traversing the papillomacular bundle inferotemporally. In the right eye, the vessel gives off a branch that crosses superiorly to supply the fovea, whereas in the left eye, the entire vessel crosses the horizontal raphe as shown in the fundus image below ( Figure 1). The caliber of the vessel was similar to that of a retinal artery in both eyes.
FIGURE 1: Color fundus imaging of the right (A) and left (B) eyes.
White arrowheads pointing to the congenital retinal macrovessels and the red arrowhead pointing to the branch crossing the horizontal raphe in the right eye.
Fundus fluorescein angiography showed the filling of the vessel early in the arterial phase, and no leakage was noted ( Figure 2). Optical coherence tomography (OCT) and optical coherence tomography angiography (OCT-A) scans were obtained using Zeiss Cirrus HD-OCT (Zeiss, Oberkochen, Germany). In both eyes, no enlargement of the foveal avascular zone (FAZ) was observed ( Figure 3).
Discussion
Congenital retinal macrovessel is an abnormal macular vessel with distribution above and below the horizontal raphe [1,2]. The term retinal venous malformation by Pichi et al. [9] has been suggested in order to highlight the potential presence of cerebral venous malformations found in association with this condition. In their cross-sectional multicenter study, all cases were unilateral, and the presence of cerebral venous malformation was established in 24% of the patients, with 83% being ipsilateral to the CRM and 75% involving the frontal lobe [9]. In our case, the patient did not consent to have a brain magnetic resonance imaging (MRI) done; therefore, the presence of a cerebral venous malformation could not be determined. Most cases of congenital retinal macrovessel are found incidentally with no impact on visual acuity suggesting a possible underestimation of its prevalence. Rarely, it may be found in conjunction with vitreous hemorrhage, foveal cyst, serous retinal detachment, branch retinal artery occlusion, macroaneurysm, telangiectasia, macular hemorrhage, or cavernous hemangioma [4][5][6][7][8][9][10][11][12]. The occurrence of the aforementioned associations can result in the deterioration of visual function. Fundus fluorescein angiography findings include early filling with delayed emptying of the vessel and the enlargement of FAZ occasionally. Other findings seen on angiography include dilated capillaries, areas of capillary nonperfusion, and dye leakage [9,13,14]. To our knowledge, no case of bilateral involvement has been reported.
Conclusions
In conclusion, we report the case of bilateral congenital retinal macrovessel, which we believe is arterial in origin. This finding was observed incidentally as the patient was asymptomatic. Further investigation with imaging modalities highlighted the presence of this retinal vascular anomaly bilaterally. No macular thickening or FAZ enlargement was observed on OCT and OCT-A imaging, respectively.
Additional Information Disclosures
Human subjects: Consent was obtained or waived by all participants in this study. Conflicts of interest: In compliance with the ICMJE uniform disclosure form, all authors declare the following: Payment/services info: All authors have declared that no financial support was received from any organization for the submitted work. Financial relationships: All authors have declared that they have no financial relationships at present or within the previous three years with any organizations that might have an interest in the submitted work. Other relationships: All authors have declared that there are no other relationships or activities that could appear to have influenced the submitted work. | 2023-01-12T16:26:21.713Z | 2023-01-01T00:00:00.000 | {
"year": 2023,
"sha1": "89b2b75837a6b4ed05df91d9d9cfe8394c1744a6",
"oa_license": "CCBY",
"oa_url": "https://www.cureus.com/articles/131754-bilateral-congenital-retinal-macrovessel-a-case-report.pdf",
"oa_status": "GOLD",
"pdf_src": "PubMedCentral",
"pdf_hash": "4e833110e36f7e40efaf120ad3db224162ffcf6e",
"s2fieldsofstudy": [
"Medicine"
],
"extfieldsofstudy": [
"Medicine"
]
} |
17506952 | pes2o/s2orc | v3-fos-license | The impact of inspiratory pressure on stroke volume variation and the evaluation of indexing stroke volume variation to inspiratory pressure under various preload conditions in experimental animals
Purpose Stroke volume variation (SVV) measures fluid responsiveness, enabling optimal fluid management under positive pressure ventilation. We aimed to investigate the effect of peak inspiratory pressure (PIP) on SVV under various preload conditions in experimental animals and to ascertain whether SVV indexed to PIP decreases the effect. Methods Mild and moderate hemorrhage models were created in nine anesthetized, mechanically ventilated beagle dogs by sequentially removing 10 and then an additional 10 ml/kg of blood, respectively. In all the animals, PIP was incrementally increased by 4 cmH2O, from 5 to 21 cmH2O. SVV was measured by arterial pulse contour analysis. Stroke volume was derived using a thermodilution method, and central venous pressure and mean arterial pressure were also measured. Results SVV increased according to PIP with significant correlation at baseline, with mild hemorrhage and moderate hemorrhage. PIP regression coefficients at baseline and in the mild and moderate hemorrhage models were 0.59, 0.86, and 1.4, respectively. Two-way repeated-measures analysis of variance showed that PIP and the degree of hemorrhage had a significant interaction effect on SVV (p = 0.0016). SVV indexed to PIP reflected the hemorrhage status regardless of PIP changes ≥9 cmH2O. Conclusions PIP is significantly correlated with SVV, even under hypovolemia, and the effect is enhanced with decreasing preload volumes. Compared with SVV, the indexed SVV was less susceptible to higher inspiratory pressures.
Introduction
Stroke volume variation (SVV) is a hemodynamic parameter derived from arterial pulse contour analysis that reflects the respiratory changes in stroke volume (SV) under positive pressure ventilation. This widely used indicator is clinically applied in emergency departments, intensive care units, and operating rooms. The accuracy of SVV-based measurements of fluid responsiveness at tidal volumes (Vt) of 8-10 ml/kg has been evaluated previously [1][2][3][4]. These studies showed that an increased SVV reflected a hypovolemic state and could be used as a sensitive indicator of fluid responsiveness. However, regarding current critical care, particularly in patients with acute respiratory distress syndrome (ARDS), ventilation using such a high Vt is not recommended. Instead, protective lung strategies using low Vt and high positive-end expiratory pressures (PEEP) are generally adopted [5,6]. In contrast, high Vt ventilation (15-20 ml/kg) with no PEEP is used in patients with cervical spinal cord injury [7,8]. Thus, it is important to understand how SVV varies with different ventilatory settings under different preload conditions and to make appropriate adjustments for the prediction accuracy of SVV.
Abstract
Purpose Stroke volume variation (SVV) measures fluid responsiveness, enabling optimal fluid management under positive pressure ventilation. We aimed to investigate the effect of peak inspiratory pressure (PIP) on SVV under various preload conditions in experimental animals and to ascertain whether SVV indexed to PIP decreases the effect. Methods Mild and moderate hemorrhage models were created in nine anesthetized, mechanically ventilated beagle dogs by sequentially removing 10 and then an additional 10 ml/kg of blood, respectively. In all the animals, PIP was incrementally increased by 4 cmH 2 O, from 5 to 21 cmH 2 O. SVV was measured by arterial pulse contour analysis. Stroke volume was derived using a thermodilution method, and central venous pressure and mean arterial pressure were also measured. Results SVV increased according to PIP with significant correlation at baseline, with mild hemorrhage and moderate hemorrhage. PIP regression coefficients at baseline and in the mild and moderate hemorrhage models were 0.59, 0.86, and 1.4, respectively. Two-way repeated-measures analysis of variance showed that PIP and the degree of hemorrhage had a significant interaction effect on SVV (p = 0.0016). SVV indexed to PIP reflected the hemorrhage status regardless of PIP changes ≥9 cmH 2 O. by Vt, improve the accuracy for prediction of fluid responsiveness at lower Vt. We aimed to investigate the effects of peak inspiratory pressure (PIP) on SVV under various preload conditions, and to verify whether the SVV values indexed to PIP reduce the confounding effects caused by variation of the inspiratory pressure.
Methods
The present study was performed following The Science Council of Japan guidelines for animal experimentation after approval from the ethics committee for Animal Experimentation of Osaka Prefecture University, Japan.
Nine beagle dogs weighing approximately 10-12 kg were evaluated. A cannula was inserted into a peripheral vein, and butorphanol tartrate was continuously administered at a rate of 0.1 mg/kg/h. Following the subcutaneous injection of 0.025 mg/kg atropine, 0.5 mg/kg diazepam was injected intravenously during preoxygenation. Propofol was continuously injected at a rate of 8-16 mg/kg/h, and the animals were intubated with a cuffed endotracheal tube, with an internal diameter of 6.0-7.0 mm, once anesthesia was established. To enable controlled mechanical ventilation, spontaneous respiration was suspended by administration of a 1.0 mg/kg bolus of rocuronium bromide with train-of-four monitoring. Mechanical ventilation was performed using the pressure control mode with 50 % oxygen, a PIP of 5-7 cmH 2 O, an inspiration to expiration ratio of 1:2, and a respiratory rate of 20 breaths/ min (Evita 4, Dräger Medical, Lübeck, Germany). PEEP was not applied to avoid its confounding effects on hemodynamics. During preparation, end-tidal CO 2 was adjusted to within 35-45 mmHg by changing the respiratory rate. Arterial pressure was measured continuously using a cannula inserted into the tarsal artery. SVV was measured using the Vigileo-FloTrac™ system (Edwards Lifesciences, Irvine, CA, USA) based on an arterial pulse contour analysis. The animals' age, converted to human terms, and body surface area according to the conversion table by Nelson et al. [12] were inputted during the system set up. A thermodilution catheter (132F5, Edwards Lifesciences, Irvine, CA, USA) was inserted through an introducer (RR-A60G10S, TERUMO, Tokyo, Japan) into the right internal jugular vein to obtain continuous data of central venous pressure (CVP) and intermittent data of cardiac output derived by the thermodilution method (COtd). Following anesthesia induction, 10 ml/kg hydroxyethyl starch was administered, as needed, in order to maintain the mean arterial pressure (MAP) >60 mmHg and pulse rate within 100 beats/ min, with supplementation for dehydration at baseline.
Next, PIP was incrementally increased by 4 cmH 2 O, from 5 to 21 cmH 2 O, with observation for at least 2 min between steps. Baseline measurements of SVV, CVP, MAP, COtd, heart rate (HR), and Vt were recorded at each PIP, following a stabilization period of at least 2 min. The CVP, MAP, and HR were obtained from a patient monitor (BP-608 Evolution II, Omron Colin, Tokyo, Japan). We used the thermodilution method for COtd measurements, injecting 5 ml of saline at a temperature of <8 °C through the central vein. This procedure was performed three times at PIPs of 5, 13, and 21 cmH 2 O, to avoid fluid loading.
The hemorrhage models were prepared by withdrawing blood via an introducer catheter in two steps, first removing 10 ml/kg of blood (mild hemorrhage model), followed by removal of an additional 10 ml/kg (moderate hemorrhage model). Measurements were repeated at the five PIPs under the two hemorrhage conditions using the same methods as those used during PIP changes at baseline. The withdrawn blood, which was temporarily stored in a blood bag during these protocols, was carefully readministered at the end of the experiment. SV derived from a thermodilution method (SVtd) was calculated using the formula: SVtd = COtd/ HR × 1,000 (ml). SVV was also indexed to PIP and Vt.
The data were analyzed using the JMP9 software program for Windows (SAS Institute Inc., Cary, NC, USA). Correlations between two variables were analyzed using a linear regression model based on the least-squares method. Dunnett's test was used to compare the values under hemorrhage with those at baseline, two-way repeated measures analysis of variance (ANOVA) was used to analyze the effect of the relationship between PIP and hemorrhage on SVV, and the Tukey-Kramer test was used to analyze the indexed SVV disparity at other PIPs. Differences were considered significant for p values <0.05. In the tables, all data are presented as the means and 95 % confidence intervals.
Results
The dogs' body weight ranged 10.3-12.7 kg. All hemodynamic data recorded during the study are presented in Table 1.
Effect of PIP on hemodynamic parameters at each preload condition
Vt and PIP were significantly correlated at all time (p < 0.01, R 2 = 0.84). In addition, SVV was significantly correlated with PIP and Vt at each preload condition ( Table 1). The regression coefficients between SVV and PIP at baseline, with mild hemorrhage, and moderate hemorrhage were 0.59, 0.86, and 1.4, respectively (Fig. 1).
Although at baseline there was no correlation between CVP and PIP (p = 0.31), CVP increased according to PIP in both hemorrhage models (p < 0.01, each) ( Table 1). MAP and SVtd, calculated based on the COtd, and HR were not affected by PIP.
Changes in hemodynamic parameters in association with changes of preload
All of the parameters, except for COtd, were significantly affected by moderate hemorrhage at each PIP. However, COtd was significantly decreased at a 13 cmH 2 O PIP, but not at the other PIPs. Only CVP decreased with mild hemorrhage (Table 1). Two-way repeated-measures ANOVA demonstrated that increases in PIP and hemorrhage level significantly increased SVV (p < 0.01, each). Moreover, there was a significantly positive interaction between the effects of hemorrhage and PIP on SVV (p = 0.016) (Fig. 2).
Discussion
This is the first published study statistically demonstrating the relationship between PIP and SVV under hypovolemia and elucidating the mechanism of the interaction between PIP and preload on SVV. We made three main observations, as follows. First, SVV correlates with PIP, a determinant of Vt, even under hypovolemic conditions without any intravascular volume change. Second, there is a significant interactive effect between the relationship of PIP and SVV and that of preload and SVV, i.e., the effect of PIP on SVV is enhanced by hypovolemia. Third, SVV indexed to PIP reduces the effect of a variation of inspiratory pressure on SVV when the PIP is high enough. Fig. 2 Effects of hemorrhage on stroke volume variation (SVV) at each peak inspiratory pressure (PIP). According to two-way repeated-measures analysis of variance, high PIP and hemorrhage significantly increased SVV (p < 0.01, each); moreover, there was a significant interaction between the effects of PIP and hemorrhage on SVV (p = 0.016) Reuter et al. [13] and Szold et al. [14] demonstrated that SVV was significantly correlated with the magnitude of Vt. Moreover, Kang et al. [15] reported that SVV was elevated by increased PIP in pediatric patients. Therefore, as Lansdorp et al. [16] indicated, the predictive value of SVV is reduced in routine clinical practice because of the lower Vt used. However, to our knowledge, there are no reports regarding how the relationship between Vt and SVV changes with hypovolemia. Protective lung strategies using low Vt ventilation of 6 ml/kg and high PEEP have been recommended for the treatment of ARDS [17,18]. The effects of this strategy on SVV have yet to be determined in detail, and we are planning a future study on the effects of inspiratory time, mean airway pressure, and PEEP on SVV. Alveolar compliance is low in patients with ARDS [19]; therefore, they require a higher ventilatory pressure to maintain the prefixed Vt, suggesting the need for a study protocol under a higher PIP.
Here, increasing the PIP resulted in SVV elevation. In addition, the positive correlation between hemorrhage and SVV had significant interactive effects with increasing PIP. This indicates that a hypovolemic state enhanced the effects of PIP on SVV.
These observations can be explained by the mechanisms of SVV. A maximal SV (SVmax) is a measure of the inspiratory elevation of the left ventricle (LV) SV [20][21][22].
Mesquida et al. [23] demonstrated that intrathoracic blood volume, calculated as the difference between the right ventricle and LV SV, gradually increases in the expiratory phase, and decreases in the inspiratory phase of mechanical ventilation. This filling of the intrathoracic blood volume in the expiratory phase temporarily reduces LV preload and results in a minimal SV (SVmin) at the beginning of expiration. Hence, SVmin indicates the expiratory fall of the SV. Preisman et al. [24] clearly indicated that increasing the Vt reduces the pulse pressure at the beginning of expiration. In the late expiratory phase, however, SV remains stable [25], i.e., the reference SV (SVref) is maintained. Figure 3 shows how SVV is affected by PIP and preload, and substantiates the evidence provided by Renner et al. [26] that SVV at a lower Vt is not sensitive to loss of preload, and that use of SVV as a guide to fluid management at a higher Vt can lead to volume overloading.
Based on our investigation of the correlation between SVV and PIP, SVV can be indexed to PIP. Indeed, this substantiates the reports by Vistisen et al. [9][10][11] that indexed dynamic parameters improve the accuracy for prediction of fluid responsiveness at lower Vt. We also investigated the correlation between indexed SVV and PIP under various preload conditions. Elevation in SVV with increasing PIP was eliminated by indexation with a PIP of ≥9 cmH 2 O. However, the indexed SVV was significantly larger with a Fig. 3 The concept of stroke volume variation (SVV) explained on the cardiac function curve. This figure illustrates that the determinants of SVV are cardiac function, preload, and changes in the preload of the left ventricle with the respiratory cycle. When preload is at normal levels (circle), delta stroke volume up (ΔSVup) and delta stroke volume down (ΔSVdown), generated by respiration-induced changes in the preload of the left ventricle, are small. When preload reduces and shifts to the triangle, however, a large difference between the maximal stroke volume (SVmax) and minimal stroke volume (SVmin) is observed, which indicates increased SVV in comparison with a normal preload state. In addition, a higher inspiratory pressure can increase both the ΔSVup and ΔSVdown, resulting in an increased SVV, the effect being larger in the lower preload state than the normal preload state 1 3 PIP of 5 cmH 2 O than with a higher PIP. This may indicate that there is a limit to indexing SVV to PIP under the conditions of low PIP, which means low Vt ventilation.
There are several limitations to the current study. First, it was performed in dogs; however, previous studies on SVV extracted using the pulse contour technique were also conducted in dogs [27][28][29]. Vascular responsiveness, lung compliance, relative volume of pulmonary blood flow, responses to the effects of respiratory changes, and pulse contour changes during tachycardia may differ between humans and dogs. The lung compliance of dogs is so high that their Vt reached >40 ml/kg for the maximum PIP of 21 cmH 2 O. However, we adopted the current study protocol as a higher PIP is needed for the prefixed Vt when lung compliance is low, e.g., in the case of ARDS and for the high Vt ventilation in cervical injury patients. Second, we only investigated up to the level of moderate hemorrhage of 20 ml/kg of blood, which is a safe volume of blood donation for the animals. Therefore, because we planned a non-lethal animal experiment, we could not evaluate these parameters in a model of severe blood loss. Third, we measured SV using the thermodilution method. Since the observed SVtd and MAP express the averaged values for preload rather than the beat-to-beat values for LV output, SVtd and MAP did not decrease with increasing PIP. This contrasts with the results of Morgan et al. [30], who demonstrated that increasing airway pressure and the inspiratory/expiratory ratio, decreases SV in expiration. Finally, changing PIP without PEEP surely affects end-tidal CO 2 , which can affect the SVV by changing pulmonary and systemic vascular tonus, although Kubitz et al. [31] verified that SVV is not affected by changes in cardiac afterload. To improve the clinical utility of SVV, further studies are required to reveal the precise relationship between respiratory change of the LV preload and effects of other ventilator settings.
In conclusion, we found that SVV correlates with PIP, a determinant of Vt, even under hypovolemic conditions, and that the increase in SVV induced by PIP is greater in cases of decreased preload. The indexed SVV was less susceptible to higher inspiratory pressures and should be a better parameter to evaluate fluid responsiveness under such conditions.
Open Access This article is distributed under the terms of the Crea- | 2016-05-12T22:15:10.714Z | 2015-03-15T00:00:00.000 | {
"year": 2015,
"sha1": "7a512505400406fa287b806ba0ca7633ea300f84",
"oa_license": "CCBY",
"oa_url": "https://link.springer.com/content/pdf/10.1007/s00540-015-1995-y.pdf",
"oa_status": "HYBRID",
"pdf_src": "PubMedCentral",
"pdf_hash": "7a512505400406fa287b806ba0ca7633ea300f84",
"s2fieldsofstudy": [
"Medicine"
],
"extfieldsofstudy": [
"Medicine"
]
} |
237942743 | pes2o/s2orc | v3-fos-license | Potential Environmental Risk Characteristics of PCB Transformation Products in the Environmental Medium
The complementary construction of polychlorinated biphenyl (PCB) phytotoxicity and the biotoxicity 3D-QSAR model, combined with the constructed PCB environmental risk characterization model, was carried out to evaluate the persistent organic pollutant (POP) properties (toxicity (phytotoxicity and biotoxicity), bioconcentration, migration, and persistence) of PCBs and their corresponding transformation products (phytodegradation, microbial degradation, biometabolism, and photodegradation). The transformation path with a significant increase in environmental risks was analyzed. Some environmentally friendly PCB derivatives, exhibiting a good modification effect, and their parent molecules were selected as precursor molecules. Their transformation processes were simulated and evaluated for assessing the environmental risks. Some transformation products displayed increased environmental risks. The environmental risks of plant degradation products of the PCBs in the environmental media showed the maximum risk, indicating that the potential risks of the transformation products of the PCBs and their environmentally friendly derivatives could not be neglected. It is essential to further improve the ability of plants to degrade their transformation products. The improvement of some degradation products for environmentally friendly PCB derivatives indicates that the theoretical modification of a single environmental feature cannot completely control the potential environmental risks of molecules. In addition, this method can be used to analyze and evaluate environmentally friendly PCB derivatives to avoid and reduce the potential environmental and human health risks caused by environmentally friendly PCB derivatives.
Introduction
Polychlorinated biphenyls (PCBs) are considered persistent organic pollutants (POPs) that spread into the environment in large quantities. The global production of PCBs is estimated to be approximately 1 to 2 million tons, out of which 0.2-0.4 million tons have produced environmental hazards [1]. The degradation or metabolism of PCBs in the environment can occur by using a variety of pathways. For instance, PCBs can be degraded to benzoic acid products by using the expression of dioxygenase degradation genes in tobacco and Arabidopsis plants [2][3][4]. Microorganisms can reduce the dechlorination of PCBs and degrade highly chlorinated PCBs to less-chlorinated ones [5]. In addition, microorganisms can also degrade PCBs by using cytochrome P450 enzymes (CYP450) in vivo to produce hydroxy PCB products with hydroxyl groups (OH-PCBs) [6]. The metabolism of PCBs by organisms can produce polychlorinated biphenyl methane sulfonate (MeSO 2 -PCB) through reactions such as oxidative substitution [7]. Under natural light radiation conditions, PCBs in the environment can absorb ultraviolet light and undergo
Data Sources
(1) The data sources of the PCB environmental characteristics The Stockholm Convention determines whether a chemical substance can be classified as a POP by using the following four characteristics: toxicity (phytotoxicity and biotoxicity), bioconcentration, migration, and persistence. Each characteristic requires its own characteristic parameters for evaluating the degree of each characteristic. Bioconcentration factors (BCFs) are used to represent biological enrichment. Bioconcentration as a component of risk assessment determines a meaningful BCF value for hazardous substances. It indicates the potential hazardous capacity of a substance and is the basis for assessing environmental and human risks [16]. As the evaluation criterion for the retention time of PCBs in environmental media, the half-life (t 1/2 ) was used. The larger the t 1/2 of a PCB is, the longer the retention time in the environmental media will be [17]. The octanol air partition coefficient (K OA ) can respond to the migration ability of PCBs to some extent. As the K OA decreases, PCBs easily volatilize into the air [18]. When PCBs enter the plant body, they can cause the oxidation of the cell membrane and several organelles, inhibit peroxidase activity, and damage the health of the plant body [19][20][21]. Therefore, this paper selected the total score of the PCB interactions with peroxidase for characterizing the phytotoxicity of the PCBs and with estrogen receptors for representing the biotoxicity (estrogen toxicity) of the PCBs. The 3D-QSAR models of the PCB bioconcentration [16], migration [17], and persistence [18] refer to the existing models. The 3D-QSAR models of PCB phytotoxicity and biotoxicity were constructed in this paper. The structures of the receptor enzyme for phytotoxicity (1CCK) and estrogen receptor enzyme (3MDJ) for estrogen toxicity were derived from the Protein Data Bank (http://www1.rcsb.org; accessed on 15 February 2021).
(2) The data sources of transformation pathways and transformation products of PCBs in the environmental media Toxics 2021, 9,213 3 of 20 The literature review summarized the following four transformation pathways of the PCBs [7,[22][23][24][25]: plant degradation pathway, microbial degradation pathway, biometabolism pathway, and photodegradation pathway ( Figure 1). The pathway for the degradation of PCBs by plants is usually a preferential attack on the non-chlorine-substituted benzene ring by dioxygenases. However, the oxidation reaction can also occur on the chlorinesubstituted ring if there is no barrier at the 2 and 3 carbon positions. For instance, first, PCBs are oxidized by dioxygenases at the 2 and 3 carbon positions in order to produce 2,3-dihydro dihydroxy PCB products; then, 2,3-dihydro dihydroxy PCBs undergo dehydrogenation reactions in order to produce 2,3-dihydroxy PCB products; thereafter, 2,3-dihydroxy PCBs undergo the meta-ring opening reaction by oxidation; finally, the resulting meta-ring opening mixture is hydrolyzed in order to produce polychlorinated benzoic acid [26]. In the microbial degradation pathway, PCBs can be oxidized to epoxides through oxidation, primarily by attacking the meta-and para-substitution sites of PCBs. The epoxidation products after meta-and para-oxidation can be directly metabolized into hydroxy PCBs by adding hydroxyl groups. In addition, PCBs can also undergo a reductive dechlorination reaction, in which the main reduction sites are meta and para, and the ortho-reaction is relatively less [27]. First, the biometabolism of PCBs is catalyzed by enzymes to produce epoxidation intermediates and carry out the methylation of PCBs by the nucleophilic reaction, dehydration, and methylation. Finally, PCBs with methyl benzenesulfonic acid were synthesized by catalytic oxidation [7]. The essence of the photodegradation of PCBs is the change of molecular energy under the action of light radiation from a low-energy state to a high-energy state, chemical bond breaking, and chemical reaction. PCBs can absorb ultraviolet light directly [28]. Fifty PCB degradation or metabolism products are summarized in Table 1. In this paper, we used SYBYL-X 2.0 software for molecular structure mapping. The PCBs were studied by using the Minimize module of SYBYL-X 2.0 software. The energy convergence was limited to 0.005 kJ/mol by using the Powell conjugate gradient method with the Gasteiger-Huckel charge, and the Tripos force field was selected for 10,000 iterations. The optimized molecules were stored in the database, and the PCBs with the highest environmental risk values in the PCB samples were used as the common skeleton for superposition.
StockholOpen was used as the molecular library of the training set. The environmental risk values of some PCBs were input into the database in turn, and the model parameters were automatically calculated using the calculate properties function. In order to establish the relationship between the structure and biological activity of the target compounds, the partial least-squares (PLS) analysis was used. By using the leave-one-out (L-O-O) method, the training set compounds were cross-validated, and the cross-validation coefficient q 2 and the best principal component n were calculated. Then, by using the non-crossvalidation function (No Validation), the regression analysis was performed. Finally, to ensure a reliable 3D-QSAR estimation model for the PCB risk characteristics, the non-crossvalidation coefficient r 2 , standard deviation SEE, and the test value F were calculated [16].
3D-QSAR Model Construction and Evaluation of PCB Toxicity (Phytotoxicity and Estrogen Toxicity)
Based on the CoMFA method using the total score of 70 PCBs docked with the 1CCK enzyme as the dependent variable and their molecular structures as the independent variables, the 3D-QSAR model for the phytotoxicity of the PCBs was constructed. In this process, 60 PCBs were randomly selected as the training set, and the remaining 10 molecules were selected as the test set. Based on the CoMFA method, the results showed that the best principal component n and the cross-validation coefficient q 2 of the constructed 3D-QSAR model were 8 and 0.695 (q 2 > 0.5), respectively, which indicates that the model exhibited good estimation ability. The non-cross-validation coefficient R 2 , the standard deviation SEE, and the test value F were estimated as 0.914 (R 2 > 0.9), 2.854, and 67.952, respectively, which indicates that the constructed model fulfilled the stability requirements and exhibited good fitting and estimation abilities [32]. Based on the CoMFA method, Figure 2 shows the linear fit plots of the experimental and estimated values of the 3D-QSAR model for the phytotoxicity of PCBs. The results showed that all the data were concentrated around the trend line, and the R-value was 0.956, which indicates that the linear fit between the experimental and estimated values was good. The model exhibited a high internal estimative power [32]. This model can be used for estimating the phytotoxicity values of PCBs and their derivatives.
were selected as the test set. Based on the CoMFA method, the results showed that the best principal component n and the cross-validation coefficient q 2 of the constructed 3D-QSAR model were 8 and 0.695 (q 2 > 0.5), respectively, which indicates that the model exhibited good estimation ability. The non-cross-validation coefficient R 2 , the standard deviation SEE, and the test value F were estimated as 0.914 (R 2 > 0.9), 2.854, and 67.952, respectively, which indicates that the constructed model fulfilled the stability requirements and exhibited good fitting and estimation abilities [32]. Based on the CoMFA method, Figure 2 shows the linear fit plots of the experimental and estimated values of the 3D-QSAR model for the phytotoxicity of PCBs. The results showed that all the data were concentrated around the trend line, and the R-value was 0.956, which indicates that the linear fit between the experimental and estimated values was good. The model exhibited a high internal estimative power [32]. This model can be used for estimating the phytotoxicity values of PCBs and their derivatives. Based on the CoMFA method, the constructed 3D-QSAR model of phytotoxicity in the PCBs estimated 70 PCBs with known experimental values. The results showed that the relative error between the experimental and estimated values of the phytotoxicity in 70 PCBs was less than 10% [16]. The estimated values of the phytotoxicity in 209 PCBs are shown in Table 2. In addition, the total-score of 48 PCBs docked with the 3GZX enzyme were selected for representing the estrogen toxicity, and 38 PCBs and 10 PCBs were randomly selected in the training and test sets of the model for constructing the 3D-QSAR model of estrogen toxicity. Based on the CoMFA method, the results showed that the 3D-QSAR model of estrogen toxicity in PCBs showed a good estimation ability by using the best principal component n with a value of 7 and the cross-validation coefficient q 2 with a value of 0.671 (q 2 > 0.5). The constructed model fulfilled the stability requirements and exhibited a good fitting ability (the non-cross-validation coefficient R 2 of 0.90 (R 2 > 0.9), the standard deviation SEE, and test values F of 2.509 and 38.578, respectively) [32]. The experimental and estimated values of the test and training sets of the estrogen toxicity model in PCBs were linearly fitted ( Figure 3). As shown in Figure 3, all the data were concentrated near the trend line with an R-value of 0.949, which indicated a high correlation coefficient and estimate capability for the linear fit of the relationship between the experimental and estimated values [14]. This model can be used for estimating the estrogen toxicity values of PCBs and their derivatives ( Figure 3). The 3D-QSAR model of estrogen toxicity in PCBs was used for estimating the estrogen toxicity values of 209 PCBs. The relative errors between the experimental and estimated values of the estrogen toxicity in 48 PCBs were less than 10% [16]. In addition, the total-score of 48 PCBs docked with the 3GZX enzyme were selected for representing the estrogen toxicity, and 38 PCBs and 10 PCBs were randomly selected in the training and test sets of the model for constructing the 3D-QSAR model of estrogen toxicity. Based on the CoMFA method, the results showed that the 3D-QSAR model of estrogen toxicity in PCBs showed a good estimation ability by using the best principal component n with a value of 7 and the cross-validation coefficient q 2 with a value of 0.671 (q 2 > 0.5). The constructed model fulfilled the stability requirements and exhibited a good fitting ability (the non-cross-validation coefficient R 2 of 0.90 (R 2 > 0.9), the standard deviation SEE, and test values F of 2.509 and 38.578, respectively) [32]. The experimental and estimated values of the test and training sets of the estrogen toxicity model in PCBs were linearly fitted ( Figure 3). As shown in Figure 3, all the data were concentrated near the trend line with an R-value of 0.949, which indicated a high correlation coefficient and estimate capability for the linear fit of the relationship between the experimental and estimated values [14]. This model can be used for estimating the estrogen toxicity values of PCBs and their derivatives (Figure 3). The 3D-QSAR model of estrogen toxicity in PCBs was used for estimating the estrogen toxicity values of 209 PCBs. The relative errors between the experimental and estimated values of the estrogen toxicity in 48 PCBs were less than 10% [16].
The Estimation of the Environmental Risk Characteristics of PCB Transformation Products in Environmental Media
To determine the degradation path of PCB degradation and transformation products with the greatest environmental risk, a total of 50 PCB transformation products were estimated by using the phytodegradation pathway, microbial degradation pathway, biometabolism pathway, and photodegradation pathway of the PCBs. Five kinds of environmental risk characteristics (phytotoxicity, estrogen toxicity, bioconcentration, persistence, and migration) of the PCB transformation products were evaluated. As shown in Table S1, the ranges of the phytotoxicity, estrogen toxicity, bioconcentration, persistence, and migration for the different PCB transformation products were as follows: for PCB phytodegradation products: −1.36% to 22.92%, −11.01% to 8.32%, 22.08% to 61.26%, 56.87% to
The Estimation of the Environmental Risk Characteristics of PCB Transformation Products in Environmental Media
To determine the degradation path of PCB degradation and transformation products with the greatest environmental risk, a total of 50 PCB transformation products were estimated by using the phytodegradation pathway, microbial degradation pathway, biometabolism pathway, and photodegradation pathway of the PCBs. Five kinds of environmental risk characteristics (phytotoxicity, estrogen toxicity, bioconcentration, persistence, and migration) of the PCB transformation products were evaluated. As shown in Table S1, the ranges of the phytotoxicity, estrogen toxicity, bioconcentration, persistence, and migration for the different PCB transformation products were as follows: for PCB phytodegradation products: −1.36% to 22.92%, −11.01% to 8.32%, 22 Figure 4 is a heat map of the environmental risk characteristics (phytotoxicity, estrogen toxicity, bioconcentration, persistence, and migration) of PCB transformation products under different degradation pathways (the phytodegradation pathway, the microbial degradation pathway, the biometabolism pathway, and the photodegradation pathway). The color of the heat map is divided into 10 levels. The higher the color level, the greater the variation range of environmental risk characteristics of PCBs degradation products. As shown in Figure 4, the region of PCBs plant degradation products has the darkest color. Combined with the data of Table S1, the environmental risk characteristics of the PCB degradation products showed a maximum increase of 421.71%. Therefore, the environmental risk of the PCB plant degradation products was the highest. Improving the degradation of PCBs by plants is of great significance for environmental health. In addition, the change of environmental risk of the microbial products in all the PCB degradation products is relatively small, indicating that microbial degradation methods have little impact on secondary environmental pollution. The microbial anaerobic degradation method has certain advantages over the microbial aerobic degradation method. The phytotoxicity of anaerobic degradation products was higher than that of aerobic degradation products, and other properties were improved than that of the aerobic degradation products. −19.68% to 18.51%, respectively; for PCB microbial anaerobic degradation products: −8.82% to 32.53%, −5.76% to 12.92%, −13.09% to 4.05%, −25.45% to 9.64%, and −19.10% to −3.99%, respectively; for PCB biometabolism products: −8.97% to 19.40%, −8.74% to 1.89%, −4.45% to 19.39%, −5.69% to 42.10%, and −2.92% to 20.42%, respectively; for PCB photodegradation products: 13.35% to 36.06%, 12.63% to 40.28%, −22.93% to −11.85%, −60.70% to −15.36%, and −16.20% to −1.89%, respectively. Figure 4 is a heat map of the environmental risk characteristics (phytotoxicity, estrogen toxicity, bioconcentration, persistence, and migration) of PCB transformation products under different degradation pathways (the phytodegradation pathway, the microbial degradation pathway, the biometabolism pathway, and the photodegradation pathway). The color of the heat map is divided into 10 levels. The higher the color level, the greater the variation range of environmental risk characteristics of PCBs degradation products. As shown in Figure 4, the region of PCBs plant degradation products has the darkest color. Combined with the data of Table S1, the environmental risk characteristics of the PCB degradation products showed a maximum increase of 421.71%. Therefore, the environmental risk of the PCB plant degradation products was the highest. Improving the degradation of PCBs by plants is of great significance for environmental health. In addition, the change of environmental risk of the microbial products in all the PCB degradation products is relatively small, indicating that microbial degradation methods have little impact on secondary environmental pollution. The microbial anaerobic degradation method has certain advantages over the microbial aerobic degradation method. The phytotoxicity of anaerobic degradation products was higher than that of aerobic degradation products, and other properties were improved than that of the aerobic degradation products.
In summary, the environmental risk characteristics of PCB degradation products were increased in different degrees under different degradation pathways, which, for the future, indicates that the environmental risk characteristics of PCB degradation products should not be neglected.
The Estimation of the Environmental Risk Characteristics of Environmentally Friendly PCB Transformation Products in Plants
In this study, the environmental risk of the PCB phytodegradation products was the highest. Environmentally friendly PCB derivatives refer to those PCB molecules whose functions remain unchanged and environmental risk characteristics (such as the representative characteristics of persistent organic pollutants) are improved by the design method of molecular modification. Considering the phytodegradation pathway as an example, some environmentally friendly PCB derivatives were designed in different studies In summary, the environmental risk characteristics of PCB degradation products were increased in different degrees under different degradation pathways, which, for the future, indicates that the environmental risk characteristics of PCB degradation products should not be neglected.
The Estimation of the Environmental Risk Characteristics of Environmentally Friendly PCB Transformation Products in Plants
In this study, the environmental risk of the PCB phytodegradation products was the highest. Environmentally friendly PCB derivatives refer to those PCB molecules whose functions remain unchanged and environmental risk characteristics (such as the representative characteristics of persistent organic pollutants) are improved by the design method of molecular modification. Considering the phytodegradation pathway as an example, some environmentally friendly PCB derivatives were designed in different studies [16,32,33], and their parent molecules (low migration environmentally friendly derivative P1 and the parent molecule PCB-52, low bioconcentration environmentally friendly derivative P2, and the parent molecule PCB-189, and low toxicity environmentally friendly derivative P3 and the parent molecule PCB-209) were selected for analyzing the environmental risks. The specific path inference of the molecules is shown in Figure 5. The higher the parameter value of migration is, the lower the risk of physical environment will be. The other four environmental risk characteristic parameters showed contrary characteristics. As shown in Table 3, the ranges of phytotoxicity, estrogen toxicity, bioconcentration, persistence, and mobility of the phytodegradation products of the three PCBs (PCB-52, PCB-189, and PCB-209) were −63.71% to 34.98%, −13.84 to 29.36%, −74.97% to 16.03%, −184.02% to 11.43%, and −61.55 to 18.42%, respectively. Though the environmental risk of most of the PCB degradation products has been reduced, the environmental risk of some products is still increasing. The target organism of phytotoxicity, estrogen toxicity, and bioconcentration of the PCBs is focused on the human body. The increased phytotoxicity represents that the transformation products of PCBs, for example, they may enter the human body through the food chain and, finally, increase the threat to human health [34]. The increase of estrogen toxicity also represents that the transformation products of PCBs interfere with the health of the human endocrine system and affect the normal expression of estrogen [35]. The bioconcentration also represents the enrichment ability of the transformation products of PCBs in the human body. The greater the enrichment degree, the stronger the harm to the human body [36]. The persistence effect of the transformation products of PCBs is also implied in the human body and organism in the environment. The long-term existence of the transformation products of PCBs will damage the health of the human body or organism exposed to the environment of PCB metabolites [37], and the migration effect of the transformation products of PCBs also includes the human body or organism far away from the contaminated environment, which represents the long-distance mobility of the transformation products of PCBs in the atmosphere. The research shows that, in Arctic seabirds and Greenland sharks, PCBs were detected at certain concentrations [38,39], indicating that, once the transformation products of PCB conversion products flow into the environment, they will have long-distance migration and cause risks to the environment health. Therefore, as compared to the parent compounds of PCBs, the migration characteristics of phytodegradation products of the three PCBs showed the highest increase in environmental risks, and the variation range was up to 61.55%, which indicates that, when the migration ability of PCB degradation products is improved once they flow into the environment, there is a risk of long-distance migration. The potential risk of long-range migration of the transformation products of PCBs cannot be completely overcome by controlling their parent's migration capacities.
The variations of phytotoxicity, estrogenic toxicity, bioconcentration, persistence, and migration of the three environmentally friendly PCB derivatives (P1, P2, and P3) of the phytodegradation products were estimated to range from −18.33% to 21.81%, −21.60 to 40.81%, −23.06% to 60.65%, −49.06% to 13.65%, and −22.46 to 117.32%, respectively. Similarly, the environmental risk of some degradation products was observed to increase. As compared to the parent molecules of the environmentally friendly PCB derivatives, their phytodegradation products showed the best bioconcentration performance. The results showed that the risk of bioconcentration of the PCB degradation products was increased. The bioconcentrations of PCBs in breast milk in urban areas in China were 2.66-3.90 pg/g [36] and, in the adipose tissue of Belgians, were 490-ng/g lipid weight [40]. The accumulation of PCBs in the human body increases with age and, hence, can indirectly cause visceral [41], endocrine [42], and reproductive diseases [43]. Therefore, the control of the bioconcentration ability of environmentally friendly PCB derivatives should not be neglected. Figure 6 is an effect diagram that represents the changes in the environmental characteristics of PCB conversion products. In Figure 6, the size of the sphere represents the activity value of each molecule. Comparing the size of the sphere, the final phytodegradation product of P1 represents the low mobility derivative of PCB-52 ( Figure 6). As compared to the final phytodegradation product of PCB-52, the migration of the final P1 phytodegradation product is still lower. This result is found to be consistent with the design concept [16,32,33]. In addition, the estrogenic toxicity and the migration of the final P1 phytodegradation product also showed a significant improvement as compared to the final PCB-52 phytodegradation product. The improvement in estrogen toxicity and migration were estimated as 19.52% and 156%, respectively. As compared to the final degradation product of low bioconcentration derivative P2, the final PCB-189 degradation products showed no improvement in terms of the bioconcentration properties and improvement in the estrogen toxicity and migration properties. It was observed that the phytotoxicity of P3 was improved compared to PCB-209, but the environmental risk of the final product was increased. The environmental risk of P3 was higher as compared to PCB-209 in biotoxicity, but the environmental risk of the final product was significantly improved by 26.14%. In addition, the migration of the final P3 product was improved up to 37.68%.
showed that the risk of bioconcentration of the PCB degradation products was increased. The bioconcentrations of PCBs in breast milk in urban areas in China were 2.66-3.90 pg/g [36] and, in the adipose tissue of Belgians, were 490-ng/g lipid weight [40]. The accumulation of PCBs in the human body increases with age and, hence, can indirectly cause visceral [41], endocrine [42], and reproductive diseases [43]. Therefore, the control of the bioconcentration ability of environmentally friendly PCB derivatives should not be neglected. Figure 6 is an effect diagram that represents the changes in the environmental characteristics of PCB conversion products. In Figure 6, the size of the sphere represents the activity value of each molecule. Comparing the size of the sphere, the final phytodegradation product of P1 represents the low mobility derivative of PCB-52 ( Figure 6). As compared to the final phytodegradation product of PCB-52, the migration of the final P1 phytodegradation product is still lower. This result is found to be consistent with the design concept [16,32,33]. In addition, the estrogenic toxicity and the migration of the final P1 phytodegradation product also showed a significant improvement as compared to the final PCB-52 phytodegradation product. The improvement in estrogen toxicity and migration were estimated as 19.52% and 156%, respectively. As compared to the final degradation product of low bioconcentration derivative P2, the final PCB-189 degradation products showed no improvement in terms of the bioconcentration properties and improvement in the estrogen toxicity and migration properties. It was observed that the phytotoxicity of P3 was improved compared to PCB-209, but the environmental risk of the final product was increased. The environmental risk of P3 was higher as compared to PCB-209 in biotoxicity, but the environmental risk of the final product was significantly improved by 26.14%. In addition, the migration of the final P3 product was improved up to 37.68%. In summary, the environmental risks of the final degradation products of environmentally friendly PCB derivatives P1 and P2 showed improvements, agreeing with the modification results. However, some degradation products still showed an increase in environmental risks, indicating that the environmental risk control of the PCB degradation products and their environmentally friendly derivatives cannot be neglected. The potential environmental risk of PCBs cannot be completely controlled by the theoretical modification of single environmental characteristics. Therefore, the environmental risks of the In summary, the environmental risks of the final degradation products of environmentally friendly PCB derivatives P1 and P2 showed improvements, agreeing with the modification results. However, some degradation products still showed an increase in environmental risks, indicating that the environmental risk control of the PCB degradation products and their environmentally friendly derivatives cannot be neglected. The potential environmental risk of PCBs cannot be completely controlled by the theoretical modification of single environmental characteristics. Therefore, the environmental risks of the transformed products of the environmentally friendly PCB derivatives are also required to be considered.
The Validation of the Total Score and Its Estimated Value of PCBs and Their Products Containing Different Chemical Structure
The phytotoxicity value and estrogen toxicity value of parent PCBs are derived from the total score after docking with the corresponding enzymes. Taking the phytotoxicity and estrogen toxicity as examples, we calculated the total score of the PCBs and their metabolites containing different chemical structures (such as −OH, −SO 2 CH 3 , etc.) in the manuscript and analyzed the correlation and relative error between the total score and the estimated value by the 3D-QSAR model ( Table 4). The results showed that the correlation coefficient r between the total cost of 33 molecular estrogen toxicities and their predicted values was 0.547, which met the correlation coefficient test standard (i.e., when p = 0.001, the correlation limit value r 0 is 0.539). However, the correlation coefficient r between the total cost of the phytotoxicity of 33 molecules and their estimated values was only 0.369, the correlation was relatively lower, which only met the correlation coefficient test standard when p = 0.05 (the correlation limit value r 0 is 0.339). Most of the relative errors were within the allowable range, only one-third of the molecules having a relative error more than 10%. The above results indicate that, indeed, the 3D-QSAR model constructed by the parent PCBs data is slightly lesser accurate in estimating PCBs with different chemical structures and their metabolites. Most of the relative errors are negative, indicating that the 3D-QSAR model has indeed underestimated the toxicity of PCBs with different chemical structures and their metabolites, which should actually be higher than those estimated values. However, since the purpose is to measure the environment risk of PCB metabolites on a relative scale, the overall trend of toxicity of the PCB metabolites is consistent with the estimation in this study. The overall analysis of the results is reasonable in the manuscript.
Conclusions
In this paper, the transformation pathways of PCBs (phytodegradation, microbial degradation, biometabolism, and photodegradation) were derived. The constructed 3D-QSAR models were used for estimating the POP characteristics (toxicity (phytotoxicity and biotoxicity), bioconcentration, migration, and persistence) of PCBs and their transformed products. In addition, for the environmental risk evaluation of PCBs and their environmen-tally friendly derivative transformation products, the plant degradation pathway with the highest environmental risk increase was selected. The environmental risk of some PCBs and their derivative degradation products was observed to be increased, which indicated that the environmental risk control of PCBs and their environmentally friendly derivative degradation products could not be neglected. The potential environmental risk of PCBs cannot be completely controlled by theoretical modification considering single environmental characteristics. Therefore, the environmental risks of the transformed products of environmentally friendly PCBs are also required to be considered.
Supplementary Materials:
The following are available online at https://www.mdpi.com/article/10 .3390/toxics9090213/s1: Table S1: Estimated statistics of the environmental risk characteristics of PCBs and their transformation products. | 2021-09-28T06:23:05.682Z | 2021-09-01T00:00:00.000 | {
"year": 2021,
"sha1": "9772a0d27f30e9e8f600a6660cbdcfcbdf931975",
"oa_license": "CCBY",
"oa_url": "https://www.mdpi.com/2305-6304/9/9/213/pdf",
"oa_status": "GOLD",
"pdf_src": "PubMedCentral",
"pdf_hash": "1f0a2a7e4f4d65d15cd32d741e038e199799d51d",
"s2fieldsofstudy": [
"Environmental Science"
],
"extfieldsofstudy": [
"Medicine"
]
} |
21707322 | pes2o/s2orc | v3-fos-license | Intravenous glucocorticoid for pain control after spinal fusion
Abstract Objective: Postoperative pain was a common symptom after spinal surgery. This meta-analysis aimed to assess whether intravenous glucocorticoids has a beneficial role in reducing pain in patients following spinal fusion. Methods: We systematically searched PubMed, Embase, the Cochrane Central Register of Controlled Trials, Web of Science, and Google databases, from inception to March 2, 2018. Randomized controlled trials (RCTs) that comparing intravenous glucocorticoids with control treatment for spinal fusion were included. A meta-analysis was performed to generate pooled risk ratio (RR) and weighted mean difference with corresponding 95% confidence interval (CI) for discontinuous outcomes (the occurrence of nausea and infection) and continuous outcomes (visual analog scale [VAS] at 12, 24, and 48 h; total morphine consumption; and the length of hospital stay), respectively. Results: Eight clinical trials involving 918 patients (glucocorticoid group = 449, control group = 469) were finally included in this meta-analysis. Compared with control, intravenous glucocorticoids had significantly reduced VAS at 12, 24, and 48 hours with statistically significance (P < .05). Intravenous glucocorticoids can decrease the occurrence of nausea (RR = 0.42, 95% CI 0.29–0.62, P = .000; I2 = 0.0%) and the length of hospital stay. No difference was noticed in the occurrence of infection between glucocorticoids intravenous and control (P > .05). Conclusion: Existing evidence indicated that intravenous glucocorticoids have a beneficial role in decreasing early pain and the occurrence of nausea after spinal fusion surgery. In consideration of the limitation in current meta-analysis, more high-quality RCTs were needed to identify the optimal dose of glucocorticoids in spinal fusion patients.
Introduction
Spinal interbody fusion has become a widely accepted technique for addressing pathology in spine. [1,2] However, several patients will underwent acute pain and chronic pain after surgery. [3] This resulting in a major challenging to manage. Adequate pain control after a spinal surgery is a prerequisite to enable early mobilization, which leads to improved functional recovery and enhance patient satisfaction. [4] Multiple pain control was usual in modern surgical spine care. [5] Glucocorticoids have attracted increasing attention as possible analgesic adjuvants in the treatment of acute postoperative pain. [6,7] However, the benefit versus harm of intravenous glucocorticoids in spinal infusion patients is still undetermined. Thus, we conducted a meta-analysis from randomized controlled trials (RCTs) to conclude whether intravenous glucocorticoids was associated with a reduction of pain intensity and postoperative nausea and vomiting (PONV) after spinal fusion.
Materials and methods
This meta-analysis was conducted in accordance with the recommendations of the Cochrane Handbook for Systematic Reviews of Interventions [8] and was written in accordance with the Preferred Reporting Items for Systematic Reviews and Metaanalyses (PRISMA) checklist. [9] 2.1. Search strategy and study selection Electronic databases (PubMed, Embase, the Cochrane Central Register of Controlled Trials, Web of Science, and Google database) were searched from their inception until October 29, 2017. Free text and MeSH terms "dexamethasone," "betamethasone," "triamcinolone," "prednisone," "pain," "postoperative," "preoperative," "analgesia," and "opioid" were used individually and in various combinations with AND or OR. No language restriction was used. An attempt to identify additional studies not found by the primary search methods was made by reviewing the reference lists from identified studies. Meta-analysis was collected from published data and thus ethical review or approved was not necessary.
Data extraction and outcome measures
Two authors (FQ and KQS) independently extracted the author; publication year; the number of patients in intervention and control groups; the proportion of male patients and the mean age of the patients; the dose of glucocorticoids; and comparison, outcomes, and duration of follow-up. Any disagreement was resolved by discussion. Different types of glucocorticoids were converted to equivalence to dexamethasone: 0.75 mg dexamethasone = 4 mg methylprednisolone = 5 mg prednisolone = 20 mg hydrocortisone. [10] The outcomes were VAS at 12, 24, and 48 hours; the occurrence of nausea; the length of hospital stay; and the occurrence of infection after spinal fusion surgery. If the data were presented in figures or other forms, we used GetData Graph Digitizer software to extract relevant data. [8] 2.4. Risk of bias assessment and quality of evidence assessment Two authors (YJ and ZJY) independently evaluated the risk of bias of included RCTs and written in Reviewer Manager 5.3.0. [8] The assessment criteria included the following 7 domains: random sequence generation, allocation sequence concealment, blinding of participants and personnel, blinding of outcome assessment, incomplete outcome data, selective outcome reporting, and other biases. All domains were evaluated as "low," "high," or "unclear" according to the Cochrane Handbook for Systematic Reviews of Interventions (version 5.3.0) [8] and the risks of bias were drawn by the Review Manager 5.3.0 software (The Nordic Cochrane Centre, The Cochrane Collaboration, Copenhagen, Denmark). Kappa values were used to measure the degree of agreement between the 2 reviewers. [11] Two reviewers (GC and KRS) independently evaluated the quality of evidence assessment in accordance with the Grading of Recommendations Assessment, Development and Evaluation (GRADE). [12] Each items were classified as high, moderate, low, or very low. [12,13]
Statistical analysis
For VAS at 12, 24, and 48 hours; the length of hospital stay; and the length of hospital stay after spinal fusion surgery, the weighted mean difference (WMD); and 95% confidence interval (CI) were calculated. For dichotomous outcomes (the occurrence of nausea and infection), we calculated the risk ratio (RR) and 95% CI. Heterogeneity was considered to be statistically significant if the I 2 value was >50%. A fixed-effects model was applied if the I 2 value was <50%. All statistical analyses were conducted using Stata 12.0 (Stata Corp, College Station, TX). The subgroup analysis was conducted based on the dose of glucocorticoids (≥10 mg [high dose] or <10 mg [low dose]) and total knee arthroplasty (TKA) or total hip arthroplasty. The relationship between glucocorticoid dosage and the occurrence of PONV was explored using SPSS software (SPSS Corp, Los Angeles). The correlation coefficient (r) was used to assess the relationship between glucocorticoid dosage and the occurrence of PONV. A P value <.05 was considered statistically significant.
Search results and quality assessment
In the initial search, 356 articles were selected for full-text screening. Then, we used Endnote X7 (Thomson Reuters Corp, Los Angeles) software to remove the duplicate articles (48 articles were removed). Next, Then, according to the inclusion criteria, 209 articles were removed after reading the titles and abstracts. Finally, we included 8 RCTs [14][15][16][17][18][19][20][21] with 918 patients (glucocorticoid group = 449, control group = 469, Fig. 1) in this metaanalysis. The general characteristic of the included RCTs can be seen in Table 1. The sample ranged from 19 to 146. The mean age ranged from 36.8 to 55.2. We converted glucocorticoid equivalent to the dose of dexamethasone and the dose ranged from 3 to 80 mg. Risk of bias summary and risk of bias graph can be seen in Figures 2 and 3, respectively. Compared with control treatment, intravenous glucocorticoid can reduce VAS scores at 24 hours (WMD = À7.46, 95% CI À11.17, À3.74, P = .000, I 2 = 83.7%, Fig. 4, GRADE: very low) after spinal fusion surgery.
Length of hospital stay. A total of 3 studies (307 patients)
were included in the meta-analysis of length of hospital stay. Compared with placebo, intravenous glucocorticoids were associated with a significantly decreased length of hospital stay (WMD = À0.95, 95% CI À1.47 to À0.43, P = .000; I 2 = 44.4%, GRADE: very low; Fig. 6).
Discussion
This is the first meta-analysis that comparing intravenous glucocorticoid for pain control after spinal fusion. After strictly search, we finally included 8 RCTs and final results indicated that intravenous glucocorticoid has positive role in reducing pain intensity and morphine consumption. What's more, intravenous glucocorticoid was associated with a reduction of the length of hospital stay and the occurrence of nausea. For the safety of intravenous glucocorticoid, it will not increase the occurrence of infection.
There were a total of 3 major strength in current meta-analysis: we performed a comprehensively search of the electronic databases; we calculated the final outcomes rigorously (use random-effect model of fixed-effect model according to the heterogeneity); this meta-analysis was compliance with the PRISMA guidelines and the recommendations of the Cochrane Collaboration. Final results indicated that intravenous glucocorticoid can decrease the VAS at 12 and 24 hours and by 8.41 points, 7.46 points, and 13.99 points, respectively, with clinical significance. Liu et al [22] identified that intravenous glucocorticoids may be as an effective and safe method to reduce postoperative pain in patients following TKA. Meng and Li [23] found that intravenous dexamethasone could significantly reduce postoperative pain scores following total joint arthroplasty (TJA).
Current meta-analysis indicated that intravenous glucocorticoid has a positive role in reducing pain intensity and postoperative nausea in spinal infusion. Lee et al [24] revealed that dexamethasone 8 mg may be valuable for preventing patient-controlled analgesia-related nausea in patients undergoing major orthopedic surgery. However, Liu et al [25] reported that 10 mg dexamethasone given intravenously during induction in major gynecological surgery provided only minimal pain reduction. Wittayapairoj et al [21] hypothesized that dexamethasone might only be beneficial for less extensive procedures and have a relatively small influence on extensive and invasive procedures.
Current meta-analysis revealed that intravenous glucocorticoid will not increase the occurrence of infection when compared with control group (P > .05). Richardson et al [26] conducted a retrospective analysis of 6294 patients who underwent TJA and found that a single intravenous dexamethasone dose resulted in no statistically significant difference in the rate of infection after TJA. Toner et al [27] conducted a meta-analysis of 56 clinical trials, and the evidence did not find any safety concerns with respect to the use of perioperative glucocorticoids and subsequent infection, hyperglycemia, or other adverse outcomes. Waldron et al [28] revealed that blood glucose levels in the glucocorticoid group were higher at 24 hours than saline group with statistically significance. In the present study, results shown that intravenous glucocorticoids will not increase the blood glucose level compared with the control group. However, blood glucose alterations were specifically mentioned in only 4 trials; more trials should focus on this important side effect.
Our review and meta-analysis has several limitations. Included studies with patients were treated with different types of glucocorticoids and thus have a large heterogeneity for the outcomes. Final outcomes were with large heterogeneity and
Conclusions
In conclusion, the present meta-analysis demonstrated that intravenous glucocorticoids can alleviate pain and reduce the incidence of nausea without sacrificing safety. Considering the limitations of the current meta-analysis, the conclusions regarding infection and blood glucose levels should be interpreted cautiously; more RCTs are warranted before making final recommendations. | 2018-05-21T21:28:04.247Z | 2018-05-01T00:00:00.000 | {
"year": 2018,
"sha1": "8a669bdbf444928dc055126efae53a4aa1ccbddf",
"oa_license": "CCBYNC",
"oa_url": "https://europepmc.org/articles/pmc5976326?pdf=render",
"oa_status": "GREEN",
"pdf_src": "PubMedCentral",
"pdf_hash": "8a669bdbf444928dc055126efae53a4aa1ccbddf",
"s2fieldsofstudy": [
"Medicine"
],
"extfieldsofstudy": [
"Medicine"
]
} |
16933254 | pes2o/s2orc | v3-fos-license | Research, Part of a Special Feature on Exploring Opportunities for Advancing Collaborative Adaptive Management (cam): Integrating Experience and Practice Integrating Collaboration, Adaptive Management, and Scenario- Planning: Experiences at Las Cienegas National Conservation Area
There is growing recognition that public lands cannot be managed as islands; rather, land management must address the ecological, social, and temporal complexity that often spans jurisdictions and traditional planning horizons. Collaborative decision making and adaptive management (CAM) have been promoted as methods to reconcile competing societal demands and respond to complex ecosystem dynamics. We detail the experiences of land managers and stakeholders in using CAM at Las Cienegas National Conservation Area (LCNCA), a highly valued site under the jurisdiction of the Bureau of Land Management (BLM). The CAM process at Las Cienegas is marked by strong stakeholder engagement, with four core elements: (1) shared watershed goals with measurable resource objectives; (2) relevant and reliable scientific information; (3) mechanisms to incorporate new information into decision making; and (4) shared learning to improve both the process and management actions. The combination of stakeholder engagement and adaptive management has led to agreement on contentious issues, more innovative solutions, and more effective land management. However, the region is now experiencing rapid changes outside managers' control, including climate change, human population growth, and reduced federal budgets, with large but unpredictable impacts on natural resources. Although the CAM experience provides a strong foundation for making the difficult and contentious management decisions that such changes are likely to require, neither collaboration nor adaptive management provides a sufficient structure for addressing the externalities that drive uncontrollable and unpredictable change. As a result, LCNCA is exploring two specific modifications to CAM that may better address emerging challenges, including: (1) creating nested resource objectives to distinguish between those objectives that may be crucial to maintaining ecological resilience from those that may hinder a flexible response to climate change, and (2) incorporating scenario planning into CAM to explore how climate change may interact with other drivers and alter options for the future, to identify robust management actions, and to prioritize ecological monitoring efforts. The experiences at LCNCA demonstrate how collaboration and adaptive management can be used to improve social and environmental outcomes and, with modifications, may help address the full range of complexity and change that threatens to overwhelm even the best efforts to sustain public lands. INTRODUCTION Federal public lands comprise nearly 650 million acres and almost 30% of the U.S. This number nearly doubles when including lands under state or local jurisdictions. The core challenge for public land managers is to sustain in perpetuity the public benefits these …
INTRODUCTION
Federal public lands comprise nearly 650 million acres and almost 30% of the U.S.This number nearly doubles when including lands under state or local jurisdictions.The core challenge for public land managers is to sustain in perpetuity the public benefits these lands provide, from resource harvest to biodiversity protection, watershed function to recreational opportunities.This mandate often places public land managers in the middle of political and legal battles between various interests (local/national, corporate/private, motorized recreation/ wilderness advocates, extractive uses/preservationists) who perceive themselves to benefit differentially from alternate management priorities.Because ecosystem management involves decision making within extremely complex natural and social dynamics, the outcomes of management actions are highly unpredictable.Federal land managers must attempt to meet this mandate with declining annual budgets that currently range from US$3.79 per acre (Bureau of Land Management 2009, U.S. Senate 2009a) to US$32.73 per acre (National Park Service 2012).A handful of staff trained in natural resources are often responsible for a thousand square miles or more.
Traditional management paradigms that expect agency staff to single-handedly produce optimal outcomes for all people over all time spans are poorly suited to the reality of these challenges, yet these approaches continue to dominate agency cultures (Shindler and Cheek 1999).In contrast, collaborative decision-making approaches are being increasingly promoted by agencies and stakeholders as a way to navigate competing demands (Johnson 1999, U.S. Government 2004, Bureau of Land Management 2009, U.S. Senate 2009b).Similarly, adaptive management approaches that recognize uncertainty of outcomes and provide mechanisms for responding to changes observed on the ground have begun to be applied more broadly as an effective way of working with ecological complexity (Holling 1978, Walters and Holling 1990, Gunderson 1999, Johnson 1999, Folke et al. 2005, Williams et al. 2009).Many practitioners have attempted to combine
BACKGROUND Location
Fifty miles southeast of the metropolitan area of Tucson, Arizona (population: one million), lies the 300,000-acre Cienega watershed, a broad, 4000 ft.high grassland valley suspended between four forested "sky island" mountain ranges (Fig. 1).Much of the upper watershed is under the jurisdiction of BLM and designated as a National Conservation Area.Las Cienegas National Conservation Area (LCNCA) includes some 50,000 acres of public land administered by BLM plus an additional 50,000 acres of State Trust Land on which BLM holds the state grazing leases.Management of LCNCA therefore influences land health across much of the upper watershed, which is vital to the Tucson metropolitan area for flood control and aquifer recharge (Keith 1981, Knight 1996, Fonseca 2008).
Cienega Creek flows north along the bottom of the valley into the Tucson basin.At the south end of the watershed, in an area commonly called the Sonoita Valley, a low divide separates surface water flow between Cienega Creek and the upper watersheds of Sonoita Creek (tributaries of the Santa Cruz River) and the Babocomari River (a tributary of the San Pedro River).All three creeks have sections that flow year-round, rare sources of perennial surface water in a semiarid landscape (Pima County Board of Supervisors 1999, Pima Association of Governments 2000) and an indicator of both near-surface groundwater and a relatively healthy watershed that is able to capture and slowly release rainfall (Bota 1997, Fonseca 2008).
LCNCA supports several threatened and endangered species and five of the rarest plant communities in the Southwest: cienega wetlands, cottonwood-willow riparian forests, sacaton grasslands, mesquite bosques, and semidesert grasslands.In conjunction with lands managed by the U.S. Forest Service, Pima County, the Department of Defense, and numerous private landowners with conservation easements, the LCNCA's grasslands and woodlands maintain connectivity among several of the region's sky island mountain ranges and play a vital role in regional wildlife linkages (Beier et al. 2006).The Cienega Watershed and the Sonoita Valley are also renowned for their archaeological and more recent western cultural heritage (Swanson 1951, Dowell 1978, Eddy and Cooley 1983, Majewski 2004).The landscape's natural resources continue to support a thriving rural community concentrated around the town of Sonoita and scattered in pockets of private land throughout the valley.Management of the LCNCA is multiuse and includes active livestock grazing as well as a variety of recreational activities.
History of collaborative adaptive management at LCNCA
The Sonoita Valley has a long history of public engagement.Local residents advocated for the land exchange that eventually brought private lands into public ownership as the BLM Empire-Cienega Resource Conservation Area in 1988.Prior to this, the lands faced an uncertain future that most likely included development for housing or water extraction for mining.Following the 1988 land acquisition, BLM initiated a traditional planning process to develop the required land use plan.The process stalled after several years because of a combination of changing agency priorities, limited public participation opportunities, and increasing polarization and http://www.ecologyandsociety.org/vol18/iss3/art43/Because this initial planning effort frustrated stakeholders who felt they were unable to meaningfully participate, in 1995 the BLM Field Manager decided to initiate a collaborative process for developing the site's resource management plan (RMP), encouraging more extensive public engagement, guided by the principles of ecosystem management (Grumbine 1994, Kaufmann et al. 1994, Christensen et al. 1996, Szaro et al. 1998).BLM employed a neutral facilitator and worked directly with participants to design the planning process, which included a series of open public meetings.Initial meetings led to the formation of the Sonoita Valley Planning Partnership (SVPP), an ad hoc group with participants from more than a dozen communities in southeastern Arizona and representatives of conservation organizations, grazing interests, recreational user groups, and federal, state, and local agencies.SVPP participants embraced the collaborative approach, regularly attending monthly SVPP meetings and more frequent working group meetings.Many SVPP members were part of a successful public campaign that resulted in Congressional designation of National Conservation Area status for the area in 2000.
Later in the planning process, the collaborative group opted to incorporate an adaptive management approach to build in management flexibility and address uncertain outcomes of management actions.The jointly developed RMP (Bureau of Land Management 2003) incorporates this collaborative adaptive management (CAM) approach.This science-based adaptive management program is designed to provide decision makers with reliable and timely information on resource conditions and trends.
The general structure of the LCNCA CAM process, developed incrementally by participants based on evolving needs at the site (timeline summarized in Fig. 2), closely parallels descriptions in theoretical literature (Williams et al. 2009;Fig. 3).LCNCA uses shared goals and measurable objectives to guide management actions, an ecological monitoring program to gather information about key resources and to measure progress in meeting RMP objectives, and a biological planning process to incorporate new information into decision making and provide a forum for regular stakeholder engagement.Twice a year, stakeholders meet at LCNCA to discuss results of the monitoring and proposed management actions, as well as research results that might inform these actions.Implementing management actions is largely the responsibility of BLM and the grazing permittee, but other stakeholders contribute substantially with staff and volunteer time as well as grant-funded projects.
CORE ELEMENTS OF CAM AT LCNCA
The combination of stakeholder involvement and adaptive management has yielded many benefits at this site, benefits that could not have been attained by either approach in isolation.Each of the following four core elements reflects the philosophy, lessons, and evolution of CAM at LCNCA and involve both stakeholder participation and learning about ecological dynamics.
Developing shared goals and measurable objectives
Having shared goals and measurable objectives has helped participants move beyond entrenched positions, to problem solve together, and use science as the arbiter of what works.Initially, stakeholders involved in the SVPP planning process focused on their values and concerns about the Sonoita Valley, just as BLM focused on what uses were allowable.Resulting discussions about appropriate land use and resource allocations frequently dominated meetings and at times were quite contentious, as participants advocated for their individual, or their organization's, interests.It soon became apparent that the focus needed to shift from allowable uses to the desired conditions of resources that supported these uses.
Leadership from BLM staff played a large role in this shift, and represents a departure from the agency's traditional focus on permitted uses toward a primary focus on resource conditions.Through a delegated system of authority, BLM line officer decision making has traditionally been organized around approving permitted uses.Las Cienegas planning staff recognized that the agency and stakeholders were unlikely to overcome stalemates unless both sides could bring their common goals to the forefront, and that this meant considering resource conditions ahead of permitted uses.Support from a Field Office Manager who valued creative problem solving endorsed the shift to emphasizing resource condition, which quickly became the foundation upon which both collaboration and adaptive management could grow.This shift in focus led participants to craft a shared vision, followed by a negotiated agreement on the primary resource goals: maintenance of healthy riparian areas and native grassland systems, and associated water, vegetation, wildlife, and cultural resources.These broad goals support many recreational opportunities, livestock grazing, and other public land uses.The agreement to give priority to resources over uses was pivotal because it allowed the group to move forward in developing specific objectives for the Sonoita Valley and LCNCA, and to ultimately reach agreement on a preferred set of alternative management strategies for resolving issues and achieving those objectives.
Moving from broad shared goals to the development of more specific and quantifiable resource objectives included several steps.SVPP invited technical experts to give a series of educational presentations on the resources and management activities within the area, which helped participants develop a common understanding about these resources.For example, presentations on semidesert grasslands function provided understanding about how to achieve a healthy and diverse vegetation community.Following these educational presentations, working groups drafted objectives for each of the goals using best available science, e.g., data on baseline resource conditions, current state of knowledge on resource and system dynamics, and in some cases ecological models.The group conducted additional field assessments and gathered data to support the development of objectives.
Ultimately, the SVPP revised and finally agreed on the specific, ecologically based, and measurable resource objectives that became the foundation for the CAM process, and for the legislation that created Las Cienegas National http://www.ecologyandsociety.org/vol18/iss3/art43/Conservation Area and Sonoita Valley Acquisition Planning District in 2000 (U.S. Senate 2000).In accordance with the Las Cienegas National Conservation Area Establishment Act, the Las Cienegas RMP included the SVPP's desired future conditions and management strategies as the agency's preferred alternative.
Solid science: gathering relevant and reliable information
Having reliable information on status and trends of important natural resources has both enhanced stakeholder confidence and improved the link between monitoring and management decisions.BLM began collecting baseline information and establishing monitoring soon after acquiring the lands now within LCNCA, but the program lacked objectives, a plan to guide it, and actual use of the data.In 2000, BLM and stakeholders held an ecological monitoring workshop that generated numerous suggestions (summarized in Bureau of Land Management 2003) but no clear direction forward.The RMP calls for an ecological monitoring program, but BLM initially had trouble adequately resourcing the program to effectively inform adaptive management efforts.In 2004, BLM and stakeholders asked The Nature Conservancy (TNC) to help the group evaluate and refine monitoring targets, protocols, and implementation.Evaluations of monitoring program areas have addressed four main questions: q Can existing monitoring show whether resource objectives are being met?
q Are results usable by decision makers?
q Do monitoring protocols have adequate power to detect change around critical ecological thresholds?
q Is the proposed monitoring feasible and can it be consistently applied?
LCNCA's upland monitoring protocols provide the most developed examples of how evaluations have refined monitoring (Gori andSchussman 2005, Gori et al. 2010).A review of data being collected on range conditions showed that some measurements were not tied to RMP objectives; other measurements did track objectives but had insufficient sampling intensity to detect important changes around levels believed to be important ecological thresholds (Bestelmeyer 2006, Groffman et al. 2006); and some key ecosystem drivers were not being measured at all.In response, the monitoring protocols were changed to increase sample size for key parameters, decrease effort spent on parameters not tied to objectives, and add periodic tracking of drivers, all while preserving comparability with previous data (Gori et al. 2010).Some modifications took time to be accepted by all parties, but field visits demonstrating that data was capturing important changes boosted acceptance.
Evaluating how managers and stakeholders used monitoring data proved to be as important as evaluating data content.
Mismatches between decision making and information accessibility prompted changes in when data was gathered and how it was shared.For example, the rancher typically made decisions on how many cattle to buy and sell in late October.
Making sure that the biological planning group received monitoring results well before these shipping dates improved the group's ability to respond quickly to conditions on the ground.However, even when received in advance of decisions, unanalyzed, minimally summarized data inspired little dialogue.Summarizing and analyzing monitoring results in ways that highlighted changes in key parameters, such as percent bare ground, encouraged the group to discuss areas of concern and suggest options for addressing those concerns (Simms et al. 2006).Such analyses showed, for instance, that areas with high shrub cover often failed to meet perennial grass cover objectives.Visiting such sites in the field and discussing this data in the context of ecological models prompted the group to advocate for implementing more prescribed fire and other vegetation treatment actions proposed in the RMP.
Ecological models (Fig. 4) have been very important in these efforts to refine monitoring, revise objectives, and interpret results with stakeholders.State-and-transition models developed by the Natural Resources Conservation Service (Bestelmeyer et al. 2009) have been especially useful at this site for enhancing communication among stakeholders with varying backgrounds.These models have helped identify ecological thresholds and drivers of change, such as the point at which loss of ground cover tends to dramatically increase soil erosion rates or the role of fire in maintaining open grasslands.Models have occasionally been modified to better meet needs at this site (Tiller et al. 2012).The generalized state-and-transition model has also prompted many discussions about causes and uncertainties in grassland dynamics observed at the site, a shared learning result that has also been observed elsewhere (Knapp et al. 2011a(Knapp et al. , 2011b)).
By modifying measurement methods and timing, the group has been able to tie monitoring more closely to resource objectives, key ecological drivers and thresholds, and agency decision-making cycles.Improved usability of data has, in turn, boosted support for monitoring.Biological planning participants now expect to have and discuss monitoring results at meetings.This serves as an accountability mechanism to ensure consistent data collection and analysis, and further solidify the site's shift in emphasis from allowable uses to maintaining desired resource conditions.As demonstrated elsewhere (e.g., Serrat-Capdevila et al. 2009), putting relevant and reliable science at the table benefits both social dynamics and ecological problem solving.
Incorporating new information into decision making
By bringing managers and stakeholders together to evaluate new information, the biological planning process provides a crucial mechanism for exploring and responding to changes on the ground.This process convenes participants on a http://www.ecologyandsociety.org/vol18/iss3/art43/Fig. 4. A conceptual model of southern Arizona semidesert grasslands.This model is generalized from numerous models developed for specific ecological sites; value ranges reflect differences in thresholds among specific soil types and plant communities.Such models may also be useful during scenario planning as a tool to understand how changing drivers may alter ecosystem states.
recurring basis to evaluate resource conditions, based on recent monitoring results, research, and field visits, and discuss upcoming decisions and actions.This process has proven to be an effective way of engaging stakeholders in an adaptive management loop for livestock grazing (Simms et al. 2006, Gori et al. 2010).Managers have also sought to broaden the scope of the process beyond its initial focus on livestock grazing management.
BLM and stakeholders have continued to modify the biological planning process to better coordinate and review monitoring results, promote continued high level of stakeholder involvement, and ensure that timely science and an appropriate review process inform BLM decisions.As part of improving the biological planning process, the BLM engaged an independent facilitator, Southwest Decision Resources, through an agreement with U.S.Such shared learning has helped build agreement on solutions to contentious issues and has better prepared the group to respond to emerging challenges by educating participants about regional trends and dynamics.For instance, when the SVPP first began meeting, some stakeholders were adamantly opposed to a continuation of grazing at LCNCA, while participating ranchers wanted to continue their livelihoods and managers were required to consider multiple use regulations.
Through field trips, joint fact-finding, meetings, consultation with subject matter specialists, and a discussion of concerns, those in opposition to grazing recognized that this use could be compatible with RMP objectives for ecosystem health if managed responsibly.Monitoring of range conditions continues and has more recently documented episodic grass cover declines coinciding with prolonged drought conditions.Discussions on field trips with outside resource experts and among stakeholders have revealed similar declines across the region and spurred efforts to test methods for quantifying grass mortality.Including the grazing permittee in these field trips revealed that earlier discussions of drought impacts had alerted him to watch for grass mortality, and he had already adjusted his livestock rotation to avoid areas of higher mortality to promote their recovery.
Although tension remains around participants' desire to know as much as possible about ecological dynamics and the limitations on resources to conduct monitoring and research (Cienega Watershed Partnership, State of the Watershed, unpublished report 2011), participants continue to look for new ways to encourage shared learning among participants, learn more from the work of others, and increase resources available for monitoring and research on the LCNCA.
Accomplishments and ongoing challenges
More than 15 years of sustained commitment at LCNCA has progressively improved the processes that bring stakeholders together to pursue shared goals, learn more about system dynamics and effects of these actions, and work effectively together to respond to conditions on the ground.Ongoing investments made by both agencies and stakeholder groups testify to the benefits of both good relationships and tangible outcomes on the ground.Additional challenges, however, remain.Many of these are consistent with critiques of adaptive management in the literature (Walters 1997, Moir and Block 2001, Stankey et al. 2003).Some agency decisions and programs are better suited to stakeholder feedback and the biological planning cycle than others.It is not always clear or well documented how decision makers balance new ecological information with other factors.
Limited monitoring resources make it impossible to track all relevant aspects of ecological dynamics, and multiple use management complicates design of experimental research.Like many practitioner sites, LCNCA does not always reach the bar set by adaptive management theory of conducting rigorous experimental testing of multiple hypotheses at the same time (Walters and Holling 1990).However, the LCNCA experience shows that practical, user-created versions of CAM can provide ecological and social benefits even if they do not meet the strict definition laid out in theory, as long as participants continue to strive for improvement.
Beyond these ongoing challenges, stakeholders have recognized future challenges that are largely external to LCNCA, including an uncertain political and budgetary landscape, climate change, and other regional developments.These externalities will challenge the current CAM framework (York and Schoon 2011), which is designed to address issues that are internal to the LCNCA system and controllable through various actions and tools at the manager's disposal.
To maintain ecosystem resilience and ensure that this collaborative institution can survive future political, social, and environmental changes and more effectively address externalities will require innovative and flexible responses, new tools, and an evolution of the CAM process, all while retaining the four core elements that have made the process work thus far.
THE CHALLENGE POSED BY CLIMATE CHANGE
Over the past decade, there has been growing awareness that climate change may significantly alter ecological functioning across the desert Southwest, including sites like LCNCA.Numerous models robustly predict that over the coming century this region will become increasingly warm and dry, with winter-time precipitation falling dramatically and temperatures rising 3-5°C (Christensen et al. 2007, Diffenbaugh et al. 2008, Seager and Vecchi 2010).Temperatures across the desert Southwest have already risen by more than 1°C over the past several decades, and much of the region has been mired in a decade-long drought, perhaps indicative of future precipitation regimes (Seager et al. 2007).
This anticipated shift in temperature-precipitation regimes has been coupled with a number of more rapid changes, including increasing development, intensifying water-use, and the spread of non-native species.been profound (Barnett et al. 2008).The combination of these changes with a changing climate will tax the already stressed ecosystems across the Southwest (Serrat-Capdevila et al. 2007, Marshall et al. 2010, Powell 2010).
As a result, the desert Southwest is becoming a focal point for discussions on preparing for and responding to climate change in a way that reduces impacts to natural and human communities.The exact trajectory that climate change will follow is unknown even on the global scale, and on a regional or local scale is still poorly understood.How species and resources respond to changing climatic conditions is an expanding area of research (Parmesan 2006, Allen et al. 2010, McDowell et al. 2011).Despite many recent discoveries, however, surprises abound (Williams and Jackson 2007), and it remains difficult for managers to apply this growing body of knowledge at individual sites.How societies will respond to a changing climate is also unknown, and any response is likely to significantly impact ecological systems.Other threats, such as urban sprawl, energy development, and mineral extraction, often occur with limited long-term planning considerations; these threats are tied to fluctuations in the national economy, making predictions on the local-scale difficult.Land management agencies face increasing political and financial uncertainty because of political gridlock.
Preparing for and responding to climate change will therefore be complicated by vast uncertainties (Peterson et al. 2003, Millar et al. 2007, Julius and West 2008, Baron et al. 2009, Lawler et al. 2010).Such uncertainties will make it difficult for managers to succeed at sustaining, in perpetuity, the benefits of public lands.Others have noted the constraints these externalities place upon collaborative institutions that are often geared to respond to issues that are largely internal to an ecosystem (York and Schoon 2011).
Increased awareness among LCNCA stakeholders and managers about the potential impacts of climate change has grown out of conversations with climate experts and modelers, communications with other public land managers, resource scientists, and conservation organizations, monitoring established by the CAM process, and direct observation of changes within the Sonoita Valley and the surrounding region.
The increased awareness about potential climate change impacts, coupled with other unpredictable and largely uncontrollable changes occurring in the region has resulted in the acknowledgement at LCNCA of the need to consider how to anticipate and respond to these changes (Table 1).Some decisions are likely to be exceedingly difficult (Craig 2010), not only because they will be contentious, but also because the impact of these changes, and how these changes will merge and affect each other, is highly uncertain.
Many authors have advocated adaptive management and collaboration as key tools for providing the flexibility, learning, and public support needed to prepare for and respond to climate change and other rapid changes (Baron et al. 2009, Heller and Zavaletta 2009, Lawler et al. 2010).The experience with CAM at LCNCA gives the site a head start in addressing these emerging challenges.Focused ecological monitoring and adaptive response loops enable participants to observe changes as they occur and provide a mechanism for discussing implications of these changes on management decisions.Stakeholder involvement, broad expertise, and a tradition of shared learning enhance the group's ability to contemplate future changes.Having cultivated shared goals, trusting relationships, and developed essential problem-solving skills, the CAM process provides a foundation for making the difficult and contentious management decisions that uncertain changes are likely to require.
BUILDING ON CAM
Despite the evident benefits of CAM elements outlined above, stakeholders recognize that additional tools may be required to address these emerging challenges.Further, making adjustments to existing activities may not be enough to buffer ecosystems and the services and benefits they provide to http://www.ecologyandsociety.org/vol18/iss3/art43/human communities from the effects of these changes.For these reasons, LCNCA stakeholders engaged experts at the 2010 Collaborative Adaptive Management Network (CAMNet) conference to help think through both the benefits and limitations of CAM processes in preparing for and responding to rapid and uncontrollable change.
CAMNet discussions, and a subsequent report for LCNCA about how to address climate change uncertainty in collaborative decision-cycles (J.Caves 2010 Scenario Planning at Las Cienegas: Decision Support Tools and Futures Planning, unpublished manuscript), generated two main ideas that participants have continued to explore at LCNCA since 2010: re-evaluating management objectives through a climate change lens, and using scenario planning tools to explore additional types of uncertainty created by these rapid changes.The report's recommendations and the ideas generated at CAMNet continue to be discussed at biological planning meetings.These discussions raised questions about whether existing objectives will be adequate, achievable, or even desirable in the future.However, few managers or stakeholders were ready to simply abandon existing objectives.Participants wanted tools that would help them rigorously and transparently explore which objectives might be especially important in the future, and which might only promote inflexible attempts to recapture a bygone past.
Similarly, the observation that so many of these changes appear to be beyond local manager and stakeholders' control has prompted ongoing discussions about the inherent limitations of conventional adaptive management.Discussions of scenario planning as a tool for addressing how such uncontrollable drivers may interact to impact the landscape have resonated with participants, and they have begun blending these tools into the CAM approach.
Nested objectives: from core to conditional Measuring progress toward specific, ecologically-based targets for desired resource conditions has been a cornerstone of LCNCA CAM; yet, this creates a surprising conflict when faced with a changing climate.The LCNCA RMP objectives were derived from shared goals using best available science that captured collective understanding of the functions, processes, and compositions of current and historical ecosystems.However, with changes in temperature and precipitation already being observed, ecosystem processes and composition cannot be assumed to remain stationary (Milly et al. 2008), nor are they likely to only change within historic range of variation (Williams and Jackson 2007).Given the uncertainty of future changes, the existing specific, numerically driven RMP objectives may become obsolete despite the best efforts of BLM and stakeholders.Others have recognized this shortcoming; an analysis of management plans across BLM special management units rated LCNCA low on being prepared to deal with climate change, precisely because the RMP has such specific resource objectives (Koopman 2010).Despite the drawbacks of managing toward objectives that specify ecosystem parameters based upon historical conditions, discarding these measurable objectives overall would leave the LCNCA CAM process without essential benchmarks.
Evaluating existing RMP objectives from a climate change perspective may help resolve this dilemma.Under a shifting climate, some RMP objectives may be more achievable than others, and some may contribute more to overall shared goals of healthy watersheds and communities in the future.To accommodate both specific, historically based objectives and objectives aimed at maintaining ecosystem resilience into the future, RMP objectives can be organized in a nested structure (Fig. 5): core objectives that emphasize long-term watershed functioning, for example, would be surrounded by specific objectives based upon historical indicators of watershed health components such as erosion thresholds, and subsequently, by desired conditions for individual species or assemblages.In this example, the outer objectives are seen as being more dependent on assumptions of stationarity and therefore less controllable, while the core objectives represent those components necessary to maintain ecological resilience in the watershed.This nested structure suggests that as climate shifts and ecosystems respond, some conditional objectives could be discarded and others retained, while the core objectives are intended to be achieved under any future scenario.
The process of employing this nested objective structure, and any subsequent decision to discard conditional objectives, will need to continue the LCNCA tradition of stakeholder engagement.Such an effort may be contentious, but encouraging open participation should continue to be viewed as an opportunity to educate one another about the relationships among objectives, to generate objectives that encompass an array of environmental and social factors, and to improve support for whatever core objectives are ultimately chosen.The scenario planning approach described below could guide such open discussions of what constitutes core objectives, and could add rigor to evaluations of conditional objectives.
Incorporating scenario planning
Scenario planning is designed to consider a variety of possible and plausible futures rather than decide upon a particular, accurate future prediction (Mahmoud et al. 2009).Using these various scenarios, stakeholders can determine which decisions, objectives, and monitoring targets are most relevant under a range of future conditions.Further, scenario planning can specifically address surprises and unanticipated threats by providing a structure to make decisions based upon these scenarios (Peterson et al. 2003, see their Fig. 1).As a result, climate change can be explicitly considered with other, unrelated impacts, such as mineral extraction, recreation, and development to understand linkages between these changes and produce a web of future trajectories.To investigate options for integrating scenario planning into the LCNCA CAM process, the group invited scenario planning experts to community science forums.Based on these discussions, the group outlined three steps for effectively merging scenario planning and CAM.
q Construct scenarios to characterize possible futures and identify sources of uncertainty; q Use the scenarios to review objectives and to identify both robust and contingent actions; q Revise ecological monitoring to better cover the sources of uncertainty.
These steps, which have only recently been initiated at LCNCA, will require continued active collaboration among stakeholders and public land agencies.Indeed, scenario planning augments this collaboration, by providing a structure with which to collectively learn about, discuss, and act upon the highly uncertain and uncontrollable changes already facing the area.
Construct scenarios
An ongoing challenge in CAM has been a coherent method to explore the uncertain, uncontrollable factors that may impact the landscape (Williams et al. 2009; Cienega Watershed Partnership, State of the Watershed, unpublished report 2010; Table 1).Scenario construction creates a framework to more explicitly explore uncertainties and how they impact the watershed by developing models that simulate how an ecosystem responds to these uncertainties (Mahmoud et al. 2009).Such models can greatly aid in understanding which changes occurring in the Cienega watershed may have a greater impact on LCNCA.For example, future uses of State Trust Lands that are adjacent to much of LCNCA, and over which BLM and stakeholders have little control, represent a major uncertainty.To capture this and other externalities, stakeholders have asked that scenario construction include agency budgets and societal investment as major components, as has been done with the National Park Service (Weeks et al. 2011).Constructing scenarios that incorporate these uncertainties with shifting temperature and precipitation regimes will document how these potential changes interact to produce varying ecosystem responses.
These models could improve the decision-making process at LCNCA in a number of important ways and help the LCNCA CAM process survive potentially large social and ecological changes.First, models could enhance shared understanding of how changes in and to the region may interact to impact LCNCA.Second, models can be used to explore the critical, yet potentially contentious decisions that may have to be made under different scenarios.Third, models may help prioritize actions and monitoring, both spatially and temporally, to maximize efficient use of resources.Finally, models provide another mechanism to fully capture the range of knowledge various stakeholders possess about the LCNCA system.
Identifying robust actions
A direct outcome of scenario planning is the identification of management actions that, ideally, will help meet the core objectives of improving ecological resiliency regardless of future socioeconomic or environmental conditions.Given the increasingly constrained budgets of land management agencies, these "robust" actions gain additional importance, allowing LCNCA to maximize ecological benefits with a minimum of wasted effort (Lempert and Schlesinger 2000).
Identifying robust actions as early as possible allows participants to more quickly respond to rapid change and builds capacity among participants to continue using scenario planning tools when needed.As important, scenario planning http://www.ecologyandsociety.org/vol18/iss3/art43/should identify actions already occurring or planned to occur that only meet core objectives under certain scenarios and, therefore, are considered contingent rather than robust.Additionally, these scenario planning exercises could be used to evaluate the utility of the nested objective structure and inform when conditional objectives might be dropped or modified.
LCNCA has already begun focusing on some robust actions based upon knowledge generated through the CAM process: (1) building resilience into floodplains through restoration and enhancement activities, including restoring riparian sacaton grasslands and reducing erosion in arroyos; and (2) working with partners to continue protecting landscape connectivity, particularly along elevational and latitudinal gradients through purchases of land and easements of target areas that protect key water resources.Protection efforts by BLM and partners have already secured east-west connectivity between "sky island" mountain ranges (Pima County Board of Supervisors 2011).Some landscape connectivity initiatives have made use of the unique and forward-thinking provision in the LCNCA Establishment Act that created the Sonoita Valley Acquisition Planning District.This provision provides for future acquisitions of lands or easements by BLM within the District and for their addition to LCNCA without further legislative action.Additional investments are gradually securing northsouth connectivity between two more ranges.These connected lands include terrain managed by BLM, U.S. Forest Service, National Park Service, Pima County, Fort Huachuca, Arizona State Parks Department, TNC, and private landowners.These actions improve ecological resilience by simultaneously addressing many of the changes and challenges (see Table 1) facing the Cienega Watershed.
Prioritizing ecological monitoring efforts Although evaluations described above are improving how LCNCA monitoring tracks ecosystem change relative to existing objectives and previously recognized thresholds, these reviews have not yet addressed possible novel ecosystem dynamics or shifting thresholds.Scenario planning may identify critical aspects of the ecosystem that will be sensitive to threshold-level changes that monitoring could examine to determine if the system is moving into realms described by one scenario or another.This evidence could, in turn, prompt the group to shift management direction as they deem some objectives no longer achievable or appropriate, moving from managing for resilience to managing for change (Craig 2010, Lawler et al. 2010).
Some monitoring protocols are already being modified to better track climate-related parameters.For example, the LCNCA recently installed an array of more accurate rain gauges to help understand drought impacts across the landscape and to document the magnitude of storm events that may be increasing in intensity and variability (Christensen et al. 2007).Stakeholders are working to integrate existing climate stations into monitoring efforts and determine what additional information needs to be collected across the site.TNC and BLM staff have also begun monitoring groundwater changes to detect impacts of drought and human activities, as well as improve modeling of the basin's dynamic water resources.
THEORETICAL LESSONS
In addition to highlighting key practical lessons for implementing and modifying CAM, the experiences at Las Cienegas provide insight into a theoretical framework for addressing the full range of complexity that faces natural resource systems.We suggest that such complexity can be broken into ecological, social, and temporal elements (Fig. 6), and illustrate how this trio of complementary tools, i.e., collaboration, adaptive management, and scenario planning, are each suited to addressing particular aspects of this complexity.
Fig. 6.Three types of complexity face land managers: temporal, ecological, and social.Each of these complexities can be addressed using complementary tools: adaptive management, collaboration, and scenario planning.
Adaptive management is well suited to responding to slowmoderate changes and to using the scientific process to address ecological questions.Collaboration addresses ecological complexity by increasing the breadth of knowledge available to land managers and stakeholders, while also improving outcomes where there are local and/or internal conflicts.Scenario planning provides a structure to aid land managers and stakeholders in anticipating more rapid, external, or large-scale changes that may impact the natural-resource system.http://www.ecologyandsociety.org/vol18/iss3/art43/Adaptive management is designed to harness the scientific process to understand and work with ecological complexity.Its ability to address temporal complexity in an ecosystem, i. e., unpredictable changes through time, is largely responsive because participants monitor results and modify subsequent actions.Collaboration brings in the ecological knowledge of stakeholders to augment land managers' expertise.
Collaboration also folds societal dynamics, including economics, into decision making; as stakeholders address local and internal conflicts, their participation can yield increased support for contentious decisions.Scenario planning fills a critical gap by providing a structure with which to understand and anticipate externalities that are beyond the control of land managers or stakeholders.Such externalities include societal issues at the state and national levels, including changes in policy or budgets, as well as rapid global and regional ecological externalities such as climate shifts.In this sense, scenario planning allows participants to anticipate and plan for the effects of such externalities rather than simply respond to external forcings as they occur.
In applying this combination of tools, we have also proposed an innovative structure to "nest" descriptions of desired resource conditions in a way that reconciles adaptive management's needs for measurable objectives, which are often based on past conditions, with the recognition that climatic changes can create novel ecosystems.Allowing for flexibility in objectives creates a need for additional rigor to ensure that the objectives being used continue to track and promote progress toward agreed-upon goals.Scenario planning can provide that rigor by helping participants explicitly identify the conditions under which each set of objectives would continue to support ecosystem resilience versus where they may hinder responses to inevitable change.
Finally, we return to the concept of using the four core elements to implement CAM.Our experience at Las Cienegas has demonstrated that these elements can create a structure and philosophy that emphasize continued improvement of the CAM process.These observations suggest that using these core elements will be as valuable for designing and applying modifications to CAM as for developing such inclusive, responsive processes to begin with.
CONCLUSIONS
Years of experience at this site confirm the value and relevance of collaborative adaptive management approaches.The four core elements that have emerged from experience have served the site well, allowing for flexible management actions that strive to achieve shared goals, ensuring that monitoring informs actions, creating mechanisms for collaboration, and encouraging continued shared learning.As a result, managers and stakeholders have achieved several important milestones, including: the adoption of a resource management plan with broad community support; a suite of partners that are invested in implementing the plan; a regular and effective ecological monitoring program that tracks changes on the ground; and the ability to work collaboratively to adapt management decisions to suit changing conditions.Managers and stakeholders at other BLM sites such as the Agua Fria National Monument have noted the benefits of this approach, and are looking to our CAM process as an example that they can adapt and use at their own sites.This foundation will be crucial for adapting to global climate change, the effects of which are already being seen across the desert Southwest.Managers and stakeholders in this watershed have long attempted to work across multiple jurisdictions within the broader landscape, addressing emerging stressors by emphasizing landscape connectivity and resilience.More recently, BLM and stakeholders at LCNCA have begun to enhance CAM by incorporating flexibility into the RMP objectives through nesting objectives and adding scenario planning.These modifications will explicitly prepare managers and stakeholders to understand how multiple external drivers may interact with ecosystem responses, and identify robust actions and monitoring efforts needed to respond to and detect the rapid and uncontrollable changes facing the Cienega Watershed.
The evolution of CAM at this site parallels a growing recognition nationwide that public lands can no longer be managed as isolated parcels; rather, protection of these critical lands and resources must address ecological, social, and temporal complexity that often spans land boundaries and traditional planning horizons.Public lands across the country face similar pressures that will require new planning tools to sustain the benefits of these lands.The LCNCA experience demonstrates that collaborative adaptive management can prepare managers and stakeholders to face these challenges, but that additional tools, currently being implemented at this site, may be necessary to fully address the range of complexity and change faced by public lands.Fortunately, these tools are all broadly applicable.Such an approach promises to help managers and stakeholders effectively navigate the complexity and change that threatens to overwhelm even the best efforts to sustain public land resources.
Responses to this article can be read online at: http://www.ecologyandsociety.org/issues/responses.php/5749
Fig. 3 .
Fig. 3.The collaborative adaptive management process at Las Cienegas National Conservation Area.At most stages of the cycle, stakeholders are actively involved through participation in the semiannual biological planning meetings, volunteering, or participation on a technical team (TT).The four core elements are essential for implementing this loop.We propose incorporating scenario planning as part of the CAM cycle, drawing upon the nested objectives to further refine the design of proposed actions.BLM = Bureau of Land Management; SH = Stakeholder.Note: the Permittee is considered a stakeholder and member of the TTs.
Fig. 5 .
Fig.5.An example of nested resource management objectives.The concept of nested objectives is a theoretical innovation designed to reconcile adaptive management's needs for measureable objectives, which are often formulated based upon past experience, with the threat that climate change could move this site into an ecological state with no historical analog.At the center of this diagram are core objectives that are crucial to multiple societal goals and that are intended to be met despite changes in climate or other conditions.Moving outward from the core, objectives become increasingly specific and the ability to meet them becomes increasingly dependent on assumptions of stationarity.Therefore, outer objectives may be outside of managers' control and might be considered conditional, to be modified as they become unachievable or conflict with other objectives.Within each circle are examples of shared goals and resource management objectives.
Forest Service Tonto National Forest and Arizona Game and Fish Department) asked BLM http://www.ecologyandsociety.org/vol18/iss3/art43/staff,stakeholders, and the facilitator from Las Cienegas to help them apply CAM tools to a new management challenge at their own site.Work with the AFNM aims to foster development of the same four core elements at this new site, while allowing the site's stakeholders and managers to tailor both the process and the results to their own needs.Early progress suggests that learning from LCNCA experience is helping this site more quickly solidify agreement on shared goals.Testing new monitoring approaches and coordinated planning mechanisms at AFNM may benefit Las Cienegas as well, developing shared learning opportunities at higher institutional and spatial scales.In addition to the four core elements described above, a few other enabling factors are important: leadership from a few key participants and ongoing commitment from many others; BLM institutional support and flexibility; and willingness of all parties to vest authority in people dedicated to group problem solving.Although most participants have contributed their time without direct financial compensation, the availability of funding to partners has also catalyzed much progress.Cost-share funding from BLM to partners has provided seed money and/or stop-gap funding for implementation of many projects, including design, collection, and analysis of monitoring data; installation of erosion control structures; habitat improvements for endangered species; and creation of youth stewardship groups.
Others have noted the benefits of this approach.In 2011, managers at the Agua Fria National Monument (AFNM) and two partner agencies (U.S. ecological, and social benefits of CAM, and reconcile different mandates among land agencies (Meridian Institute 2010; Cienega Watershed Partnership, State of the Watershed, unpublished report 2010).
Table 1 .
Numerous changes observed in the Sonoita Valley are categorized by the amount of control local managers and stakeholders have over these changes.As control of their causes and consequences decreases, nested management objectives and scenario planning become increasingly important.Changes marked with † represent changes that stakeholders have indicated should be considered components of any scenario planning exercise.
Population growth in the American Southwest has been staggering, rising from 3 million in 1900 to over 50 million people in 2000 (MacDonald 2010).Water usage from 1950 to 2000 has nearly doubled (Koniecski and Heilman 2004), and hydrologic changes have http://www.ecologyandsociety.org/vol18/iss3/art43/ | 2018-05-08T18:11:57.306Z | 2013-01-01T00:00:00.000 | {
"year": 2013,
"sha1": "6af4137f1f4256fb65c36bf847d19b1fe72ca164",
"oa_license": "CCBY",
"oa_url": "https://www.ecologyandsociety.org/vol18/iss3/art43/ES-2013-5749.pdf",
"oa_status": "GOLD",
"pdf_src": "Crawler",
"pdf_hash": "6af4137f1f4256fb65c36bf847d19b1fe72ca164",
"s2fieldsofstudy": [
"Environmental Science",
"Sociology"
],
"extfieldsofstudy": [
"Engineering"
]
} |
246946014 | pes2o/s2orc | v3-fos-license | Post-void bladder ultrasound in suspected cauda equina syndrome—data from medicolegal cases and relevance to magnetic resonance imaging scanning
Objective Post-void residual (PVR) scans of less than 200 ml are increasingly being used to rule out the likelihood of cauda equina syndrome (CES) and to delay emergency MRI scanning in suspected cases. This study was done to review a series of 50 MRI confirmed cases of CES and to test the hypothesis that a PVR of less than 200 ml was unlikely to be present. Methods Fifty consecutive medicolegal cases involving CES were audited. Records were reviewed to see if PVR scans were done. MRI scans were reviewed, clinical and radiological diagnosis reviewed, and treatment recorded. Results Out of 50 CES cases, 26 had had PVR scans. In 14/26 (54%) the PVR scan was ≤ 200 ml. In one case, the CES diagnosis was in question leaving 13/26 (50%) cases where there was a clear clinical and MRI diagnosis of CES despite the PVR being ≤ 200 ml. All 13 were classified as incomplete cauda equina syndrome (CESI) and all proceeded to emergency decompression. Conclusions This study is the first in the literature to demonstrate that there is a significant group of CES patients who require emergency decompression but have PVRs ≤ 200 ml. The results demonstrate the existence of a significant group of CESI patients whose bladder function may be deteriorating, but they have not yet reached the point where the PVR is over 200 ml. Given the accepted understanding that CESI is best treated with emergency decompression, such patients are likely to have worse outcomes if MRI scanning and therefore surgery is delayed. We recommend the following: PVR is recommended as an assessment tool in suspected CES. A PVR of ≤ 200 reduces the likelihood of having CES but does not exclude it; clinical suspicion of CES should always lead to an MRI scan. Further investigation of PVR as a prognostic tool is recommended.
Introduction
The CES is a condition that can lead to severe disabling symptoms causing long-term social and medical morbidity. Early diagnosis and treatment of CES can prevent harm. The failure to diagnose and treat CES before there is permanent and/or severe neurological injury is important for all patients and is also important medicolegally. There is no universally agreed definition of CES. Many symptoms and signs are quoted as "red flags" for CES but none reliably predict cauda equina (CE) compression on MR imaging [1][2][3][4][5]. This leads to high rates of negative MRIs in patients who have suspected CES [6]. Bladder ultrasound is a cheap, noninvasive assessment of bladder function, which is widely available in emergency departments. The residual volume of urine present in the bladder post-void (PVR) has been proposed as an accurate assessment of the probability of a patient having CES [6] [7]. Katzouraki et al. have stated that if the PVR is ≤ 200 ml, and there are no clinical signs of CES, the probability of a negative MRI is 98.7%, and such patients do not require emergency MR imaging [7]. Deyo et al. [8] in 1992 went even further and stated that "the predictive value of a negative test (no urinary retention) would be almost 0.9999".
Whilst we value the assessment of objective measures to diagnose cauda equina syndrome and support the widespread use of bladder scanning, we wish to document our experience that cauda equina syndrome requiring emergency decompression can still be present when a PVR is less than 200 ml.
Methods
We retrospectively reviewed the two senior authors' 50 most recent medicolegal reports that concerned patients who were litigating in relation to CES. The reports were prepared for both claimants and defendants. We identified those cases where the PVR was recorded. In cases where the PVR was ≤ 200 ml, we recorded the following: age, sex, bladder symptoms, urinary and/or bowel incontinence at any time, subjective and clinician-tested impairment of perineal sensation, reduced anal tone, level of compression on MRI, confirmation of diagnosis by a radiologist and surgeon, the clinical decision to perform emergency decompression, and the timing of surgery. We used the same criteria as Katzouraki et al. [7] for a positive MRI scan, namely "a large lumbosacral disc prolapse occupying most of the canal cross-sectional area sufficient to compress the CE…".
All cases have been anonymised and have no identifying data in accordance with the World Medical Association Declaration of Helsinki statement of ethical principles for medical research [9].
Results
These are summarised in the diagram (Fig. 1) and table (Table 1) below. Of 50 medicolegal cauda equina syndrome cases, 26 had records of PVR being measured. In 14 of these, the PVR volume was 200 ml or lower. In one case, there was doubt about the diagnosis, and we therefore excluded it from analysis, leaving 13 cases where there was a clinical diagnosis of CES, confirmed by MRI, and where emergency surgery was performed. In all 13 cases, the cause of cauda equina compression was a lower lumbar disc herniation, eight being at the L4/5 level and five at the L5/S1 level. In all cases, the MRI scan was done within 24 hours of the PVR assessment. All cases were classified as CESI, as there was executive control of bladder emptying, i.e. voluntary voiding was possible and took place before the PVR was measured. In 11 of these cases, surgery was performed within 24 hours of the MRI scan, and in two, there was a delay (2 days and 3 days) despite the diagnosis being clear retrospectively. In many of the cases, there were aspects of treatment that were potentially negligent; however, this aspect of management is not the subject of this review and did not affect the data collected.
Discussion
CES is a constellation of clinical symptoms and signs, not all of which have to be present to make a diagnosis of CES, and many are not present when CES is diagnosed. The syndrome ranges from mild symptoms of CE irritation to severe neurological and visceral injury [10]. Published definitions of CES vary with different symptoms and signs being emphasised by different authors [11]. Early diagnosis and treatment is imperative to achieving good outcomes by preventing further neurological injury and to permit neurological recovery particularly in patients with CESI treated within 48 h [12]. Unfortunately, no symptom or sign or combination accurately predicts CE compression on MR imaging [2][3][4], which leads to large numbers of negative MRIs performed so that a diagnosis of CES is not missed. A retrospective and then a prospective study of PVR in the assessment of potential CES patients have been performed [6,7]. The prospective study was of 260 suspected CES patients. A positive MRI was defined as an MRI showing "a large lumbosacral disc prolapse occupying most of the canal cross-sectional area sufficient to compress the CE…" The mean canal occlusion was 76.5% (95% confidence interval 72-81%). Emergency MR imaging was performed in 226 patients, 34 (15%) had a positive MRI. Thirty-four patients had normal perineal sensation, normal voluntary anal contraction (VAC) and a 7 . In a subsequent letter 12 , the authors said that such patients can be managed on a routine basis "using the local radiculopathy pathway". Similar symptoms and signs of CES were found in both MRI groups and did not discriminate between the MRI positive and negative patients. In the subsequent letter, it was noted that 18 patients were catheterised for both painful and painless urinary retention or two PVRs of > 200 ml [13]. Ninety-seven percent of MRI positive patients had had one or more episodes of urinary incontinence but were CESI at the time of diagnosis. CES has been divided into CESI and CESR. The distinction between CESI and CESR is that CESR is a late stage of CES with often poor outcomes, whereas CESI is associated with better outcomes particularly if treated when symptoms are modest or treated rapidly [12]. CESI has been defined as "a patient with urinary difficulties of neurogenic origin, including altered urinary sensation, loss of desire to void, poor urinary stream and the need to strain in order to micturate" [14]. This implies that there is still executive control of the bladder in the CESI patient. CESR has been defined as "painless urinary retention and overflow incontinence where the bladder is no longer under executive control" [14].
Bladder sensation is subjective, and the perception of bladder sensation is influenced by many intrinsic and extrinsic factors [15]. Examination of perineal sensation and anal tone is operator dependent. Katzouraki et al. [7] emphasised the quantitative nature of the measurement of PVR, which in general is true, but bladder scanning is operator dependent, it must be performed immediately post-void, and it can be inaccurate in patients with abdominal scarring, pregnancy, or uterine prolapse [16]. If the PVR can be measured, the patient must be able to void, and therefore these patients are not CESR [13]. In the study of Katzouraki et al. [7], most patients did not have CESR despite most reporting one or more episodes of urinary incontinence. PVR ≥ 200 ml is a sign of incomplete bladder emptying not CESR and is consistent with retention of executive control of the bladder. An ideal PVR is 0 ml, but in young adults, < 50 ml is normal; a PVR of up to 100 ml can be normal in older adults; > 200 ml is incomplete bladder emptying [15]. Anecdotally, we were aware of patients with PVRs of ≤ 200 ml who had a large compressive disc prolapse. We were also aware that the paper [7] has been used to suggest that a PVR ≤ 200 ml implies there is no risk of CES even if there are symptoms and/or signs of CES. This issue has become important in medico-legal litigation. In the case of Jarman vs Brighton and Sussex NHS Trust [17], it was found that there was no requirement for MR imaging in a woman with symptoms but no signs of CES and a PVR of 48 ml despite the fact that it was agreed (on the basis of subsequent progressive symptoms and signs, a positive MRI and urgent surgery) that she did have symptomatic CES at the time there was a PVR of 48 ml.
Our study was not prospective, and all our cases were self-selected on the basis of suspected malpractice; nevertheless, it was a relatively well-documented retrospective study of recent medicolegal cases. Out of the 26 patients who had post-void scans, i.e. they could void and were therefore CESI, we found 13 cases where the PVR was less than 200 ml, and the diagnosis was confirmed on MRI. Our patients are not exactly the same as the patients reported by Katzouraki et al. 7 . Case 12 had bladder symptoms but normal perineal sensation and anal tone with a positive MRI. Case 11 had bladder symptoms and subjective perineal numbness that was not confirmed on examination and normal anal tone but a positive MRI. This case would also not have satisfied the criteria for an MRI scan. Both patients (that is 2/13 or 15%) would not have satisfied the criteria for MRI of Katzouraki et al. [7]. This is also the position of the medicolegal case of Jarman [17]. These cases represent a small subset of CES cases who have symptomatic CES without signs, normal or near-normal bladder emptying with a positive MRI who do require surgery. They are similar to a previously described subset of CES patients, CES early (CESE) [18]. Early surgery is appropriate in these cases, and if the low PVR had delayed an MRI scan and had led to delayed surgery, the outcome of these patients could well have been worse than with early surgery. In our study, a further five patients had reduced perineal sensation with normal anal tone and a positive MRI where the PVR was ≤ 200 ml, which emphasises that the PVR is not a substitute for clinical assessment and a PVR ≤ 200 ml does not rule out CES where there are positive signs. Worryingly, in our medicolegal practice, we are increasingly seeing doctors say that, because the symptoms and signs of CES are not diagnostic and because the PVR is a quantitative measurement, a PVR ≤ 200 ml indicates that the patient does not have CES even in the presence of objective signs. If it was thought that a PVR ≤ 200 ml did not require an MRI (which is not what Katzouraki et al. 7 recommended), all 13 of our cases would have been missed. We believe that in some centres, PVR is being used in an uncritical way. PVR must be considered in conjunction with the clinical symptoms and signs, not in isolation. The PVR is a potentially useful tool and further study will be important because it could be that low PVRs correlate with better bladder outcomes.
The addition of PVR to clinical assessment in the management of potential CES patients is welcome, but it must be used in conjunction with, not as a substitute for, an accurate clinical history and examination. There must be a stage in many CES cases where bladder symptoms precede incomplete bladder emptying, and this is the ideal time to make a diagnosis because outcomes are likely to be excellent. In a young patient with symptomatic lumbar degenerative disc disease and no pre-existing bladder problems, symptoms of bladder dysfunction and/or perineal sensory loss raise the question of CES even where there are no objective signs. We support the conclusions of Katzouraki et al. [7], but we believe the recommendations should be expanded to take into account patients who have symptoms but no signs of CES (Table 2). CESI patients who have objective signs of CES (including reduced perianal sensation and/or reduced VAC and/or reduced anal squeeze and/or tone should have emergency MR imaging and if the MRI is positive, emergency surgery, whatever the PVR. Any CESI patient with a PVR > 200 ml should have MR imaging regardless of objective signs given the high prevalence of CES as established by Katzouraki et al. 7 . If the MRI is positive, and there are no objective signs, these patients can probably have surgery performed urgently and that would include first thing on the following day's emergency list. If there are symptoms but no signs and a PVR ≤ 200 ml, emergency MR imaging is not required, but we believe there should be an early nonemergency MRI to detect the small proportion of patients who have symptomatic CES only, who would benefit from surgery before they deteriorate. We would recommend MR imaging within 24 hours in these patients. This policy will not reduce the number of MRIs that are required, but it will move some into the normal working day, which is an advantage. Large numbers of these MRIs will be negative but that is the price that has to be paid for preventing potentially severe long-term harm in a small number of patients. The urgency of MR imaging in the CESR patient is more complex because it is generally accepted that more urgent surgery after CESR does not lead to better outcomes. Nevertheless, it is the case that neurological deterioration in CES is typically continuous and progressive [19,20], and objective signs of CES continue to progress after CESR [21]. It is probably prudent to perform urgent MR imaging and then surgery on the next morning's emergency list in CESR patients.
Author contribution All authors contributed to the study's conception and design. Material preparation, data collection, and analysis were performed by all authors. The first draft of the manuscript was written by Nicholas Todd, and all authors commented on previous versions of the manuscript. All authors read and approved the final manuscript.
Data availability All data used in this study is kept in GDPR compliant secure files and can be retrieved for review.
Declarations
Ethics approval and consent to participate Both medicolegal offices are compliant with GDPR regulations on data security. All cases have been anonymised and have no identifying data in accordance with the World Medical Association Declaration of Helsinki statement of ethical principles for medical research.
Competing interests
The authors declare no competing interests.
Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http:// creat iveco mmons. org/ licen ses/ by/4. 0/. | 2022-02-19T14:40:35.468Z | 2022-02-19T00:00:00.000 | {
"year": 2022,
"sha1": "d03ebfa1dff4fce9c9db0a3d7dadfa1694ab8b2b",
"oa_license": "CCBY",
"oa_url": "https://link.springer.com/content/pdf/10.1007/s00264-022-05341-0.pdf",
"oa_status": "HYBRID",
"pdf_src": "Springer",
"pdf_hash": "9931b3b624e54a236c121d6dc00c3cbe71a1a095",
"s2fieldsofstudy": [
"Medicine"
],
"extfieldsofstudy": [
"Medicine"
]
} |
11594623 | pes2o/s2orc | v3-fos-license | Differential upregulation in DRG neurons of an α2δ-1 splice variant with a lower affinity for gabapentin after peripheral sensory nerve injury
Summary A minor α2δ-1 splice-variant, (ΔA+BΔC), with reduced affinity for gabapentin, is differentially upregulated in DRG neurons following ligation. This could influence the efficacy of gabapentin.
a b s t r a c t
The a 2 d-1 protein is an auxiliary subunit of voltage-gated calcium channels, critical for neurotransmitter release. It is upregulated in dorsal root ganglion (DRG) neurons following sensory nerve injury, and is also the therapeutic target of the gabapentinoid drugs, which are efficacious in both experimental and human neuropathic pain conditions. a 2 d-1 has 3 spliced regions: A, B, and C. A and C are cassette exons, whereas B is introduced via an alternative 3 0 splice acceptor site. Here we have examined the presence of a 2 d-1 splice variants in DRG neurons, and have found that although the main a 2 d-1 splice variant in DRG is the same as that in brain (a 2 d-1 DA+B+C), there is also another a 2 d-1 splice variant (DA+BDC), which is expressed in DRG neurons and is differentially upregulated compared to the main DRG splice variant a 2 d-1 DA+B+C following spinal nerve ligation. Furthermore, this differential upregulation occurs preferentially in a small nonmyelinated DRG neuron fraction, obtained by density gradient separation. The a 2 d-1 DA+BDC splice variant supports Ca V 2 calcium currents with unaltered properties compared to a 2 d-1 DA+B+C, but shows a significantly reduced affinity for gabapentin. This variant could therefore play a role in determining the efficacy of gabapentin in neuropathic pain.
Ó 2013 International Association for the Study of Pain. Published by Elsevier B.V. All rights reserved.
Introduction
Voltage-gated Ca 2+ channels of the Ca V 1 and Ca V 2 families contain 3 subunits: the pore-forming a1 subunit, together with 2 auxiliary subunits, b and a 2 d, both of which increase the functional expression of the channels [4,11,13,20,21]. The a 2 d subunits are each the product of a single gene (CACNA2D1-4), encoding an a 2 d preprotein, which is posttranslationally processed into a 2 and d [22]. We have recently shown that a 2 d subunits can form glycosylphosphatidylinositol-anchored proteins [19], which are constitutively endocytosed and reinserted into the plasma membrane via the recycling endosomes [5,48]. We have identified that the mechanism whereby a 2 d-1 and -2 subunits enhance plasma-membrane expression of calcium channels involves the metal ion-dependent adhesion site (MIDAS) motif in their von Willebrand factor A (VWA) domain [12,31], and it is also important for the enhancement of presynaptic vesicular release [31].
The a 2 d-1 subunit has a widespread distribution; both mRNA and protein are found in neuronal tissue, heart, skeletal, and smooth muscle [3]. In dorsal root ganglion (DRG) neurons, a 2 d-1 is the main a 2 d subunit expressed [5,16,36]. The a 2 d-1 protein is upregulated following various types of peripheral nerve injury [5,39,49], and this upregulation is essential for the rapid development of the subsequent behavioural mechanical hypersensitivity seen in animal models [41]. By contrast, Ca V 2.2 mRNA and protein are not reported to be consistently upregulated following sensory nerve damage [1,36,49], suggesting that upregulated a 2 d-1 protein enhances Ca V 2.2 trafficking and presynaptic function.
The a 2 d-1 and a 2 d-2 subunits bind to the gabapentinoid drugs, gabapentin and pregabalin. These were developed as antiepileptic drugs and are also widely used in the treatment of various forms of neuropathic pain [46]. The a 2 d-1 subunit has been shown to represent the target for these drugs in the alleviation of hyperalgesia in experimental models of neuropathic pain [24]. We have found that gabapentin and pregabalin reduce the trafficking of a 2 d subunits both in vitro and in vivo [5,30,48], which is likely to represent one of their main mechanisms of action. Furthermore, a 2 d-1 has been reported to interact with thrombospondins, a family of extracellular matrix proteins, and this may influence its trafficking as well as the effect of gabapentinoid drugs [23].
Alternatively spliced isoforms of a 2 d-1 in different tissues have been observed previously [10,22]. It was reported that the mouse cacna2d1 gene has 3 alternatively spliced regions (A, B, and C), and 5 splice variants were identified [3] (Fig. 1A). In the present study we have examined the hypothesis that there may be differential upregulation of specific splice variants of a 2 d-1 following peripheral nerve damage, and that this might potentially contribute to either the state dependency [14,25] [17] was also included to identify transfected cells from which electrophysiological recordings were made. Transfection was performed as described previously [40].
In control experiments where a 2 d was omitted, the ratio was made up with empty vector.
Isolation of detergent-resistant membranes (DRMs)
This procedure was performed as previously described [19,32]. Briefly, confluent cells from 6 175-cm 2 flasks (72 hours after transfection), or pelleted homogenate from rat whole brain, were resuspended in 1.5 mL 2-(N-morpholino)ethanesulfonic acid (MES)-buffered saline containing 1% (2% for brain tissue) Triton X-100 (Perbio, Tattenhall, Cheshire, UK) and left on ice for 1 hour. After the addition of an equal volume of 90% (w/v) sucrose, the sample was overlaid with a 10-mL discontinuous sucrose gradient and centrifuged at 140,000 Â g for 18 hours at 4°C. One-mL fractions of the gradient were harvested from the top to the bottom of the tube, washed free of sucrose by dilution in MES-buffered saline and subsequent centrifugation. The detergent-resistant membranes (DRMs) were prepared from pooled fractions 4, 5, and 6. The resulting pellet was resuspended in 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) 10 mM pH 7.4 and complete protease inhibitor cocktail (Roche Diagnostics GmbH, Mannheim, Germany). The isolated DRM fractions were collected for Western blot analysis and used in the 3 H-gabapentin binding assay.
Immunoblotting
Western blotting was performed as described previously [40]. The following primary antibodies were used: anti-a 2 -1 (1:1000; mouse monoclonal, Sigma-Aldrich, St. Louis, MO, USA) and antiflotillin-1 (1:2000; mouse monoclonal, BD Biosciences, Franklin Lakes, NJ, USA). The secondary antibody used was goat anti-mouse coupled to horseradish peroxidase (Bio-Rad Laboratories, Inc., Hercules, CA, USA). Protein bands were quantified using Image J software (rsb.info.nih.gov). All a 2 d-1 splice variant expression lev-els were corrected for background and normalized to DA+B+C protein expression on the same gel.
3 H gabapentin binding assay
Binding of 3 H-gabapentin to DRM fractions from transfected tsA-201 cells was carried out in a final volume of 250 lL at room temperature for 45 minutes. DRMs (4 lg protein per tube) were incubated with various concentrations of 3 H-gabapentin [45] (30-50 Ci/mmol, American Radiolabeled Chemicals, St. Louis, MO, USA) in 10 mM HEPES/KOH pH 7.4, then rapidly filtered through glass fibre (B grade, Whatman) filters presoaked with 0.3% polyethyleneimine. Filters were washed 3 times with 3 mL ice-cold 100 mM Tris/HCl pH 7.4 and counted in a scintillation counter. Concentrations of 3 H-gabapentin >10 nM were achieved by adding nonradioactive gabapentin and correcting the specific binding by the dilution factor [12]. Nonspecific binding was determined in the presence of 100 lM nonradioactive gabapentin. Data points were determined in triplicate, and data were analysed by fitting specific binding to the equation for a rectangular hyperbola.
Spinal nerve ligation (SNL)
A total of 17 male Sprague-Dawley rats (Central Biological Services, University College London, London, UK) weighing 130-150 g at time of surgery were employed for this study. All experimental procedures were approved by the UK Home Office and followed the guidelines of the International Association for the Study of Pain [51]. Selective spinal nerve ligation (SNL) surgery was conducted as previously described [34]. Briefly, the left L5 and L6 spinal nerves were isolated and tightly ligated with 6-0 silk thread under isoflurane anaesthesia (50% O 2 : 50% N 2 O). Haemostasis was confirmed and the wound was sutured. After surgery the animals were allowed to recover and housed at a maximum of 5 per cage. Food and water were available ad libitum. The foot posture and general behaviour of the operated rats were monitored throughout the postoperative period, and the development of mechanical hypersensitivity was confirmed at 7 days post surgery in the affected limb ipsilateral to the ligation, as previously described [5].
Enrichment of small and large DRG neurons using density gradient centrifugation
In order to obtain sufficient material, both L5 and L6 DRG were extracted from the ipsilateral and contralateral sides of 2 rats, either naïve animals or 7 days after SNL. Nerve roots were trimmed and the isolated ganglia were incubated in 5 U/mL of collagenase (Sigma-Aldrich) in 2 mL of Hanks balanced saline solution in a shaking water bath at 37°C for 30 minutes, and then triturated. The cell suspension was sedimented at 4°C for 5 minutes at 200 Â g. The pellet was resuspended in culture medium composed of Dulbecco's Modified Eagle's Medium: Nutrient Mixture F-12 (Gibco, Invitrogen, Life Technologies, Grand Island, NY, USA), penicillin (100 U/mL), streptomycin (100 lg/mL), L-glutamine, (2 mM), nerve growth factor (100 ng/mL, Sigma-Aldrich), and 10% foetal calf serum. Four mL of DRG neurons in culture medium was filtered through a 100-lm cell strainer (BD Biosciences) and layered onto 5 mL of a solution of 40% Ficoll (Histopaque-1077, Sigma-Aldrich) and 60% phosphate-buffered saline containing 4.2 mM NaHCO 3 . The final pH was adjusted to 7.3-7.4 with HCl. The preparation was centrifuged at 100 Â g for 15 minutes at 4°C, and DRG neurons were separated according to their size into a low-density fraction (LDF) and a high-density fraction (HDF) [28]. The 2 fractions obtained were diluted to 10 mL with culture medium and then centrifuged at 200 Â g for 5 minutes at 4°C. The LDF and HDF pellets, enriched in viable small and large neurons, respectively, were . Leu-Glu-Ala, which is the end of a 2 -1) was submitted to Phyre2 [33] for structure prediction (http://www.sbg.bio.ic.ac.uk/phyre2). The amino acid numbering includes the N-terminal signal sequence of a 2 d-1. Region I ($amino acids 491-607) was predicted with $99% confidence, and modelled on a number of bacterial CSDs, including the extracellular domain of the Bacillus subtilis CSD (mmhk1s-z2) and the putative sensory box/ggdef protein from Vibrio parahaemolyticus. Region II ($amino acids 687-886) was modelled with a predicted $98% confidence on a separate subset of CSDs, including the C4-dicarboxylate transport sensor protein dctb, and the mcp_n and cache domains of methyl-2 accepting chemotaxis protein from Vibrio cholerae. The regions between domains I and II and beyond II are not modelled with high confidence. The approximate positions of the two spliced regions B and C are indicated. They are both likely to be in exposed loops, B being within the first chemosensory domain, and C being between the 2 domains. A is not present in this model. obtained and immediately used for RNA extraction, or plated on 13-mm-diameter poly-D-lysine-coated (10 mg/mL; Sigma-Aldrich) glass coverslips. Cells were incubated at 37°C (95% air, 5% CO 2 ), and observed in bright field after 2-5 hours to measure the density and diameter of the neurons in each fraction. Only phase-bright cells with a typical DRG morphology (large, clear nucleus with prominent nucleolus) were considered for statistical analysis. Larger cells were often slightly elliptical in cross-section, and the diameter was taken as the mean of the major and minor axes. Quantification of neurofilament-200 (NF-200) mRNA, a well-established marker for medium to large neurons [26,29] was employed to confirm the successful separation of neuronal fractions.
Quantification of splice variants and NF-200 by quantitative polymerase chain reaction (PCR) and capillary electrophoresis (CE)
RNA isolation and reverse-transcription polymerase chain reaction (RT-PCR) was carried out as follows. Total RNA was extracted from individual L4 or L5 pulverized frozen DRG 7 days after SNL or sham surgery, and pelleted LDF and HDF after density gradient centrifugation. RNA was isolated using the RNeasy Protect Mini Kit (Qiagen, GmbH, Hilden, Germany). RNA concentrations and purity were determined spectrophotometrically. RT was performed on 1 lg of total RNA using Superscript III reverse transcriptase (Invitrogen, Carlsbad CA, USA), using random hexamer primers (Promega, Madison WI, USA) and RNaseOUT (Invitrogen).
In order to quantify the a 2 d-1 splice variant expression pattern in rat tissue ( Primers were designed either manually or using the open-source software Pri-mer3. The techniques were optimized in a series of preliminary experiments for cycle number and starting quantity to ensure points of measurement were acquired during the linear phase of amplification ( Supplementary Fig. 1, and data not shown).
Capillary electrophoresis (CE) was carried out following dilution with deionized water of aliquots of RT-PCR products, and further analysed by laser-induced fluorescence (LIF). A detailed description and validation of the CE/LIF technique used for quantitative analy-sis of RT-PCR products was reported previously [43]. One lL of sample was diluted with 12 lL HiDi-Formamide (Applied Biosystems, Foster City CA, USA), and 0.5 lL of GeneScan 400HD ROX Size Standard (Applied Biosystems). Amplified PCR products were separated on an ABI 3100 Avant Genetic Analyzer (Applied Biosystems) running a 50-cm capillary with 3100 POP-6 polymer (Applied Biosystems). Each sample was injected in triplicate from separate wells on the plate. The amplified products were sized and quantified in GeneMapper v3.5 software using the Local Southern method. This analysis method was chosen for its reciprocal relationship between fragment size and mobility. The relative abundance of mRNA was determined as the ratio of integrated peak area for each PCR product relative to that of coamplified TBP in order to allow a direct comparison between different preparations. Then, data from the ipsilateral side were normalized to their respective contralateral side. The percentage of the total transcript represented by the minor DRG isoform (DA+BDC) was calculated as 100⁄ pa DA+BDC /(pa DA+B+C +pa DA+BDC ) [44]. Data were analysed using Microsoft Excel (Microsoft Corporation, Redmond, WA, USA) and SPSS statistical software (SPSS Inc, Chicago IL, USA) or GraphPad Prism 4 (GraphPad Software, San Diego, CA, USA). Statistical significance was determined using the nonparametric Mann-Whitney U test and one-way analysis of variance with a suitable post hoc analysis. P values <0.05 were considered as statistically significant. All data are presented as mean ± SEM for the indicated number of experiments.
Distribution of different splice variants of a 2 d-1 in rat tissue
Splice variants of a 2 d-1 containing the 3 alternatively spliced regions (A, B, and C) were identified previously in mouse tissue [3]. In that study it was reported that regions A and B were part of the same exon, with alternative 3 0 splice acceptor sites, which spliced region A in or out [3]. However, the currently available human, mouse, and rat genomic sequences from Ensembl, ENSG00000153956 (human), ENSMUSG00000040118 (mouse), and ENSRNOG00000033531 (rat), indicate that regions A and B are in separate exons, with region A in rat being encoded by exon 18a, and B representing an alternative 3 0 splice acceptor site (start site) of exon 19. A diagram of potential splice variants is given in Fig. 1A.
The following cDNAs were assembled by standard molecular biological techniques in the rat a 2 d-1 backbone: +A+B+C, +A+BDC, DA+B+C, DADB+C, DADBDC, and DA+BDC. All constructs gave rise to products of the expected size with the PCR primers used in this study ( Fig. 1B and data not shown). We then examined their distribution in adult rat tissues. DADBDC was found to be the main splice variant in rat heart, but a number of other splice variants were also identified by the much more sensitive CE/LIF, demonstrating that this method can be used for identifying and quantifying a 2 d-1 splice variants (Fig. 1B, C). We found +A+BDC in skeletal muscle and DA+B+C in cerebral cortex (Fig. 1B), as shown previously for mouse tissues [3]. We also found the same brain splice variant, DA+B+C, in DRG (Fig. 1B).
3.2. Location of the spliced regions within a 2 d-1 The a 2 d subunits contain 2 domains with homology to bacterial extracellular chemosensory domains (CSDs) or Cache domains [2], which are downstream of the VWA domain. Bacterial extracellular CSDs bind a variety of nutrients and are generally associated with an intracellular histidine kinase signalling complex [15]. The plant ethylene receptor ETR1 is also a member of this family [15].
In a 2 d-1, the first of these CSDs is situated between amino acids $491-607 [21]. Fig. 1D gives the approximate locations of the spliced regions, and Fig. 1E [33] illustrates a homology model of the 2 CSDs, showing that regions B and C are situated within a loop in the first CSD of rat a 2 d-1 (I), and in the linker between CSD I and II, respectively. The model in Fig. 1E was generated using a sequence from which region A was absent, but A would be situated just upstream of region B. It is possible that within a 2 d subunits, these domains might be important for their ability to bind small ligands, including gabapentin [21].
Determination of 3 H-gabapentin binding affinity for a 2 d-1 splice variants
We then examined whether there were differences in 3 H-gabapentin binding affinity between the different a 2 d-1 splice variants ( Fig. 2). All determinations were performed on concentrated DRM fractions following expression in tsA-201 cells, since we have shown previously that there is a large increase in apparent affinity of both a 2 d-1 and a 2 d-2 for 3 H-gabapentin in DRMs [18,30].
To ensure that the radioligand binding experiments were not influenced by differential expression of a 2 d-1 protein between the different splice variants used, the relative expression level in DRMs of each splice variant was analysed by Western blot. All splice variants partitioned similarly into DRM fractions, and their level of expression was also found to be similar ( Fig. 2A, B). We found that both DADBDC (one of the splice variants found in heart) and DA+BDC showed significantly lower affinity for 3 H-gabapentin (K D values of 211 and 153 nM, respectively), compared to the major brain and DRG splice variant DA+B+C (Fig. 2C, D). Both these splice variants with lower affinity lacked region C, whereas all the splice variants containing region C, including DA+B+C, showed a higher affinity for 3 H-gabapentin, with K D values between 80 and 102 nM (Fig. 2C, D). Thus, there was a nearly 3-fold difference in affinity between the splice variants with the highest and lowest Gabapentinoid drugs are used to treat neuropathic pain, so we wondered whether there were changes in splice variant expression in DRG following SNL, which could potentially influence the effi- The y-axis indicates the fluorescence signal peak height, which corresponds to the expression level of the respective splice variant. mRNA levels of DA+B+C and DA+BDC are higher in the side ipsilateral to SNL (B) than in the contralateral side (A). Note that the expression of the housekeeping gene (TATA-box binding protein) remains at a constant level on both sides. (C) SNL (7 days) leads to increased expression of the major a 2 d-1 (DA+B+C) splice variant in L5 DRG. Data are expressed as percentage of relative peak areas of the ipsilateral side normalized to the respective contralateral side. The relative increase is shown in L4 DRG from sham-operated rats (black bar, n = 6), L4 DRG from L5/L6 SNL rats (white bar, n = 11), L5 DRG from sham-operated rats (grey bar, n = 6), and L5 DRG after SNL (cross-hatched bar, n = 7). There was no significant difference in L4 DRG between SNL and sham-operated animals. Error bars represent SEM. Analysis of variance (ANOVA), F = 9.012, P < 0.0001, and Gabriel post hoc analysis: ⁄⁄ P < 0.01. (D) A pronounced upregulation of the a 2 d-1 splice variant DA+BDC was observed in ipsilateral L5 DRG 7 days after SNL. Data are expressed as percentage of relative peak areas of the ipsilateral side normalized to the respective contralateral side. The relative increase is shown in L4 DRG from sham-operated rats (black bar, n = 6), L4 DRG from SNL rats (white bar, n = 11), L5 DRG from sham-operated animals (grey bar, n = 6) and L5 DRG after SNL (cross-hatched bar, n = 7). Although DA+BDC mRNA levels are 2.4-fold higher in SNL L4 compared to L4 sham-operated animals, this was not statistically significant (P = 0.814). Error bars represent SEM. ANOVA, F = 11.12, P < 0.0001, and Gabriel post hoc analysis: ⁄⁄⁄ P = 0.001. (E) Comparison of the percentage of total transcript represented by DA+BDC splice variant in DRG ipsilateral to SNL, in L4 from sham-operated rats (black bar, n = 6), L4 DRG from SNL rats (white bar, n = 11), L5 DRG from sham-operated animals (grey bar, n = 4), and L5 DRG after SNL (cross-hatched bar, n = 8). The data show the pronounced shift in favour of the short splice variant in L5 after SNL. Data are expressed as percentage of relative peak areas for DA+BDC mRNA transcripts normalized to the sum of DA+B+C and DA+BDC peak areas. Error bars represent SEM. ANOVA, F = 9.39, P = 0.000, and Gabriel post hoc analysis: ⁄⁄ P < 0.01, ⁄⁄⁄ P < 0.001. cacy of these drugs. We chose 7 days after SNL as the time point for our analyses because we found previously that the increase in a 2 d-1 mRNA was not significantly different between 7 and 14 days after SNL, and we also noted less variability in the increase in a 2 d-1 mRNA at 7 than at 14 days [5].
In order to detect and quantify any minor a 2 d-1 splice variants in DRG, we used CE/LIF, and confirmed the presence of the major splice variant DA+B+C both in unaffected contralateral DRG (Fig. 3A), and in ligated DRG after SNL (Fig. 3B). The relative abundance of DA+B+C mRNA was quantified by CE/LIF after 27 cycles of RT-PCR (Fig. 3C). The DA+B+C-transcript level in ligated L5 showed a 3.6-fold increase, compared to the contralateral side. It was also significantly higher in L5 DRG following SNL, compared to L5 sham-operated animals, and compared to L4 SNL DRG and L4 DRG from sham-operated rats. There was no significant difference in a 2 d-1 levels in L4 DRG between SNL and sham-operated animals (Fig. 3C). Surprisingly, we also observed a novel splice variant in DRG neurons, DA+BDC a 2 d-1, which was particularly evident following the upregulation of a 2 d-1 mRNA that occurs after L5/L6 SNL (Fig. 3B).
DA+BDC was identified in DRG both by DNA sequencing following agarose gel separation (unpublished results), and by the size of the product on CE/LIF after RT-PCR (Fig. 3A, B). The mRNA level of the DA+BDC splice variant was quantified by CE/LIF after 29 cycles of RT-PCR (Fig. 3D). The expression level of DA+BDC was found to be $10 times higher (1083%) on the ipsilateral side, compared to the respective contralateral side in L5 DRG. Moreover, DA+BDC mRNA levels were significantly higher compared to L5 sham-operated animals, and L4 SNL and sham-operated rats. Although DA+BDC mRNA levels were 2.4-fold higher in SNL L4 compared to L4 sham-operated animals, this was not statistically significant (P = 0.814). Thus, the proportion of DA+B+C was significantly increased in L5, ipsilateral to SNL compared to sham-operated L5, but this was not the case for L4, which was not ligated. The DA+BDC splice variant represented 5.1 ± 0.5% of the total a 2 d-1 mRNA in shamoperated L5, and 8.4 ± 0.4% in SNL L5 DRG (Fig. 3E).
3.5. The relative abundance of DA+B+C and DA+BDC in different DRG classes DRG neurons are heterogeneous in morphology and the size of their somata relates to different functional subtypes. DRG with small and medium-sized somata form mainly nonmyelinated C fibres and myelinated Ad fibres, which conduct pain sensation, whereas nonnociceptive Ab fibres have larger somata [35]. a 2 d-1 is expressed in every sub-type of DRG neuron [5], but to date, the distribution of a 2 d-1 splice variants in DRG subpopulations is unknown. For the most prevalent DRG calcium channel a1 subunit, Ca V 2.2, it has been found that there is a differential distribution of a particular splice variant in small DRG neurons [1,6], and this splice variant is downregulated following SNL in rats [1]. Therefore, we wished to determine whether there was differential distribution in small and large DRG neurons of the a 2 d-1 DA+BDC splice variant, which showed reduced gabapentin binding affinity and a more pronounced upregulation following SNL, compared to the predominant a 2 d-1 splice variant DA+B+C.
To obtain populations enriched with small or large neurons, DRG neurons extracted from L5 and L6 of 2 SNL rats were separated according to their size, as described in Materials and Methods. The LDF was enriched in smaller neurons, while the HDF was enriched in larger neurons (Fig. 4A). The mean diameter for neurons in the LDF was 10.0 ± 0.2 lm (n = 418), with a pronounced peak at 5-10 lm. In the HDF, the smallest neurons (<15 lm in diameter) were almost absent, and the neuron diameter was up to 50 lm, with a peak at 21-25 lm. The mean diameter was 24.5 ± 0.7 lm (n = 260). As expected, NF-200 mRNA, a marker for large myelinated DRG neurons [42], measured by CE/LIF, was sig-nificantly higher in the HDF (13.9 ± 3.8 arbitrary units (AU)) compared to the LDF (1.8 ± 0.4 AU) (Fig. 4B), confirming that the HDF contains mainly larger neurons expressing NF-200.
We then used RT-PCR and CE/LIF to quantify the relative abundance of a 2 d-1 DA+B+C and DA+BDC in the small and large DRG neuron fractions after SNL. In agreement with the previous experiments, both DA+B+C (Fig. 4C) and DA+BDC (Fig. 4D) transcript levels were significantly increased on the ipsilateral side compared to the contralateral side after SNL. We further analysed whether the differential upregulation of DA+BDC compared to DA+B+C mRNA found in whole DRG (Fig. 3E) was present in both small and large neuron fractions. This was calculated as the percentage increase on the ipsilateral compared to the contralateral side, of the 2 transcripts in the small neuron fraction (white bars) compared to the large neuron fraction (grey bars). This analysis was performed to provide a direct comparison with the values found in whole DRG (Fig. 3C, D). We found that the increase of DA+BDC mRNA following SNL was significantly greater compared to the corresponding DA+B+C increase, only in the small neuron fraction, and not in the large neuron fraction (Fig. 4E). Thus, although upregulation following SNL of the 2 DRG transcripts DA+B+C and DA+BDC occurs in both small and large DRG neurons (Fig. 4C, D), the increased upregulation of DA+BDC compared to DA+B+C following SNL occurs preferentially in the small DRG neuron fraction containing nonmyelinated nociceptors (Fig. 4E).
Electrophysiological properties of the 2 splice variants present in DRG
It was important to determine whether a 2 d-1 DA+BDC was a functional splice variant, and we therefore compared the properties of Ca V 2.2 calcium channel currents co-expressed with b1b and either a 2 d-1 DA+BDC or DA+B+C. Ca V 2.2 was used because it is the main calcium channel in DRG neurons. We found that the properties of the currents formed from these combinations were very similar in terms of their ability to increase Ca V 2.2 current density compared to the absence of a 2 d (Fig. 5A-C), their ability to increase the inactivation rate of the currents (Fig. 5D, E), and their ability to hyperpolarize the steady-state inactivation of the currents (Fig. 5F). Since Ca V 2.1 is also present in DRG neurons, we also examined calcium currents formed by this channel. Similar results were obtained, in terms of the effect of the a 2 d-1 splice variants on current amplitude (Supplementary Fig. 2A-C), voltage-dependence of inactivation ( Supplementary Fig. 2D), and kinetics of inactivation ( Supplementary Fig. 2E).
Genomic arrangement of a 2 d-1 giving rise to splice variants
The cacna2d1 gene encoding mouse a 2 d-1 was found to contain 3 alternatively spliced regions within the a 2 moiety (A, B, and C) [3]. It was originally reported that regions A and B were part of the same exon, with alternative 3 0 splice acceptor site, which spliced region A in or out [3]. In this scenario, splice variants of a 2 d-1 should not exist in which region A is expressed without B.
However, our analysis of the mouse genomic sequence has found that A is encoded by exon 18a, and B is formed as a result of utilizing an alternative 3 0 splice acceptor site for exon 19. In confirmation of this, we have found splice variants in rat heart in which A is expressed without B (Fig. 1C).
In skeletal muscle, a 2 d-1 +A+BDC was the only splice variant detected [3]. We did not identify any minor splice variants in skeletal muscle in our study. We also found that the +A+BDC splice variant bound 3 H-gabapentin with high affinity, in agreement with previous findings of high-affinity binding of 3 H-gabapentin to native rat skeletal muscle a 2 d-1 [27]. Our results confirm that the lack of effect of gabapentin on skeletal muscle function is not a result of its inability to bind to the skeletal muscle a 2 d-1 isoform.
In mouse heart, a 2 d-1 DADB+C was previously found to be the most prevalent splice variant, although other splice variants were also observed, specifically DA+B+C, DADBDC, and DA+BDC [3]. In this study we found that DADBDC was the predominant isoform in rat heart tissue, but we also found 6 other splice variants from L5/L6 DRG, ipsilateral and contralateral to SNL. Data are expressed as DA+B+C or DA+BDC relative peak areas. Transcript levels in small (white bars) or large (grey bars) DRG neurons prepared from L5/L6 DRG, both contralateral (hatched bars) and ipsilateral (solid bars) to SNL. DA+B+C and DA+BDC transcript levels were significantly higher on the ipsilateral side compared to the contralateral side in both the small neurons (4.0 ± 0.6-fold for DA+B+C and 9.2 ± 2.4-fold for DA+BDC), and in the large neurons (4.0 ± 0.7-fold for DA+B+C and 7.4 ± 2.4-fold for DA+BDC). Error bars represent SEM. Statistical analyses were performed using 2 sample unpaired Student's t-test, for the data indicated. (E) Comparison of the upregulation in ipsilateral compared to contralateral L5 and L6 DRG of the 2 DRG a 2 d-1 transcripts, 7 days after SNL. In the small neuron fraction (white bars), the extent of upregulation of DA+BDC (right, n = 6) is significantly greater than that of DA+B+C (left, n = 7). In contrast, there is no significant difference between the upregulation of the 2 transcripts in the large neuron fraction (grey bars). Data are expressed as relative peak areas for the ipsilateral side normalized to the respective contralateral side. Error bars represent SEM. Statistical analyses were performed using unpaired Student's t-test. , or Ca V 2.2/b1b (j, n = 12). Current amplitude was normalized to whole-cell capacitance and plotted against membrane potential. Data are fitted using a modified Bolzmann function, as previously described [12]. (C) Peak current density was À103.56 ± 13.9 pA/pF in presence of DA+B+C (black bar) and À116.63 ± 13.9 pA/pF in presence of DA+BDC (grey bar). These values were both significantly higher than the current density measured in the absence of a 2 d-1, which was 9.6 ± 2 pA/pF (white bar). Error bars represent SEM. Statistical analysis was performed using 1-way analysis of variance (ANOVA) and Bonferroni post hoc analysis, ⁄⁄⁄ denotes P < 0.001). (D) Representative current traces in response to a long depolarizing voltage step (0.9 second) to 0 mV for Ca V 2.2/b1b, co-expressed in tsA-201 cells either without a 2 d (left), or with a 2 d-1 DA+B+C (middle) or a 2 d-1 DA+BDC (right). 1 mM Ba 2+ was used as a charge carrier. Holding potential was À90 mV. Traces are normalized to their peak. (E) a 2 d-1 DA+B+C and DA+BDC significantly accelerated the inactivation of currents compared to no a 2 d.
The decay phase of individual current traces at 0 mV was fitted with a single exponential function, and the mean time constant (s) of inactivation was 204.3 ± 10.3 ms for DA+B+C (black bar, n = 10), 186.4 ± 15.8 ms for DA+BDC (grey bar, n = 14), and 248.0 ± 23.6 ms for Ca V 2.2/b1b without a 2 d (white bar, n = 10). Statistical analyses were performed using 1-way ANOVA, Bonferroni post hoc analysis, ⁄ P < 0.05. (F) Steady-state inactivation curves for I Ba evoked by a test pulse to +20 mV after a 10-second conditioning prepulse of between À100 and 0 mV. Ca V 2.2/b1b/a 2 d-1 DA+B+C (d, n = 15), Ca V 2.2/ b1b/a 2 d-1 DA+BDC (D, n = 13), Ca V 2.2/ b1b (j, n = 10). Error bars represent SEM. Data were fitted with a single Boltzmann equation, and the mean voltages at which the channel is 50% inactivated were À61.3 ± 1.2 mV, À60.0 ± 1.9 mV, and À50.7 ± 1.9 mV, respectively. (Fig. 1C). It is of interest to note that the DADBDC splice variant has a significantly lower affinity for 3 H-gabapentin, compared to the major brain splice variant (Fig. 2). This result correlates with an early finding that the specific binding of 3 H-gabapentin to heart membranes is lower than that in brain membranes, although this may additionally represent a smaller number of binding sites [27]. In brain, the only splice variant found previously was DA+B+C [3], and its presence was confirmed in our study. We found that this was also the main splice variant in DRG neurons. Following SNL in rats, we found previously that the level of a 2 d-1 mRNA was increased >500% in L5/L6 DRG neurons, ipsilateral compared to contralateral to the ligation, and this was accompanied by a similar increase in a 2 d-1 protein [5]. We confirmed that result here, for the main splice variant DA+B+C, where a 3.6-fold increase was observed. However, by CE/LIF we were also able to observe the presence in DRG of a second, minor splice variant DA+BDC, whose level was increased over 10-fold following SNL. Therefore, this splice variant, which has a lower binding affinity for 3 H-gabapentin, was differentially upregulated in SNL. In the experiments described here, DA+BDC increased from 5.1% of the total transcript in sham-operated L5 DRG to 8.4% in L5 ipsilateral to SNL.
Involvement of spliced regions of a 2 d-1 in 3 H-gabapentin binding
To date, several sites in a 2 d-1 have been established to be important for its ability to bind gabapentin [9,50]. The triple arginine motif, situated just upstream of the VWA domain in a 2 d-1, is essential for high-affinity 3 H-gabapentin binding, and also for the ability of gabapentin to alleviate hyperalgesia [24], and to inhibit calcium currents when applied chronically [30]. One of the other regions identified by Wang et al. [50] is located just downstream of region B, and another is just upstream of region C.
In the present study, we found that both the DA+BDC and DADBDC splice variants possessed significantly lower affinity for 3 H-gabapentin, compared to the other splice variants tested. However, there was no significant difference in the gabapentin binding affinity to a 2 d-1 +A+B+C, containing region A, compared to its absence in DA+B+C. Similarly, for the presence or absence of region B, the 3 H-gabapentin binding affinity was similar for a 2 d-1 DA+B+C and DADB+C. These data indicate that A and B are not directly involved in the binding of gabapentin. This suggests that it is primarily the absence of region C in a 2 d-1 DA+BDC and DADBDC that is critical for the reduced 3 H-gabapentin binding affinity of these 2 splice variants. The sequence of region C in rat a 2 d-1 is SKKGKMK, which is positively charged, as is the triple arginine motif that is essential for 3 H-gabapentin binding.
It is notable that the affinity of gabapentin for a 2 d-1 is much greater (ie, a lower K D ) than the clinically relevant concentrations. One potential factor contributing to this discrepancy is that an endogenous ligand may occupy the gabapentin-binding site on a 2 d-1 in vivo, and compete with gabapentin. This also accounts for the finding that purification of the a 2 d proteins results in a marked increase in apparent affinity for gabapentin, as the endogenous ligand is removed during purification [8,18].
Efficacy of gabapentin following SNL
It has been demonstrated that the effect of gabapentin is state dependent, in that in most circumstances it is able to inhibit neuropathic pain responses in experimental animals, while having no effect on physiological nociception [14,24,25,41]. Alterations in gene expression occur following neuropathic nerve damage, which lead to changes in primary afferent inputs into the spinal cord, including the upregulation of a 2 d-1 in these terminals [5]. It has been demonstrated that gabapentin blocks calcium currents acutely in DRG from mice overexpressing a 2 d-1, but not in control animals [36], and this model may mimic the neuropathic condition. Our results indicate that the upregulation of specific splice variants of a 2 d-1 in SNL does not contribute to determining the state dependence of the efficacy of gabapentin, since no splice variants with an increased affinity for gabapentin were identified in DRG neurons following SNL. Thus, the state dependence of gabapentin is likely to depend on the overall elevation of all a 2 d-1 splice variants.
Biophysical properties of a 2 d-1 splice variants found in DRG
It is thought that increased presynaptic calcium currents, resulting from the upregulation of a 2 d-1, contribute to the hyperexcitability of DRG that underlies hyperalgesia and allodynia [36]. If the alternative splicing of a 2 d-1 were to modify the electrophysiological properties of a 2 d-1-containing channels, the consequences of altered function would be augmented following differential upregulation of the a 2 d-1 DA+BDC splice variant, as a consequence of nerve damage. However, we have demonstrated here that the ability of a 2 d-1 DA+BDC to elicit calcium currents is not affected by deletion of region C. a 2 d-1 DA+BDC enhances calcium currents resulting from the co-expression with either Ca V 2.2 or Ca V 2.1 to the same extent as a 2 d-1 DA+B+C. This result indicates that the differential upregulation of a 2 d-1 DA+BDC in DRG neurons does not contribute to triggering hyperexcitability to a greater extent than the main DRG splice variant.
Changes in calcium channel splicing following sensory nerve damage
It is of interest to compare our results with those for the exon 37a variant of Ca V 2.2, which is selectively expressed in DRG neurons, and conducts larger calcium currents than the exon 37b variant [1,6]. Despite being present overall at $7% of the main splice variant 37b of Ca V 2.2, it was found selectively in small DRG neurons also expressing VR1 [1,6]. Interestingly, the 37a splice variant was downregulated to $2% in DRG following SNL [1], whereas there was no change in the main Ca V 2.2 splice variant containing exon 37b.
In contrast, in our study there was a greater increase of a 2 d-1 DA+BDC compared to the main splice variant a 2 d-1 DA+B+C following SNL, and this differential increase occurred particularly in the nonmyelinated small DRG neuron fraction. This, together with our finding that DA+BDC had a significantly lower affinity for gabapentin, and an equivalent ability to enhance neuronal calcium currents, could be highly relevant to the response to gabapentin in patients with chronic neuropathic pain. The rat a 2 d-1 DA+BDC mRNA sequence used in this study has 95% amino acid homology with the human sequence, and C region is identical in both species. Therefore, it is reasonable to speculate that human a 2 d-1 DA+BDC (accession number P54289 isoform 5) is expressed in DRG neurons and undergoes similar upregulation in conditions leading to the development of chronic neuropathic pain. It is, further, tempting to speculate that differences in the extent of upregulation of a 2 d-1 DA+BDC in humans could potentially contribute to the variable efficacy of gabapentinoid drugs [37,38].
Conclusion
We have identified a novel a 2 d-1 splice variant DA+BDC, which is expressed in DRG neurons and is differentially upregulated com-pared to the main DRG splice variant a 2 d-1 DA+B+C, particularly in small nonmyelinated DRG neurons following SNL. This splice variant supports Ca V 2 calcium currents with unaltered properties, but shows a significantly reduced affinity for gabapentin. It could therefore play a role in determining the efficacy of gabapentin in different forms of neuropathic pain. Furthermore, targeting this splice variant for drug discovery could increase the therapeutic efficacy of gabapentinoid drugs in the future. | 2016-10-19T14:40:27.821Z | 2014-03-01T00:00:00.000 | {
"year": 2014,
"sha1": "5ebbd2d3c757cff6a56c3100b73cfa4797b73343",
"oa_license": "CCBY",
"oa_url": "https://europepmc.org/articles/pmc3988960",
"oa_status": "GREEN",
"pdf_src": "Elsevier",
"pdf_hash": "5ebbd2d3c757cff6a56c3100b73cfa4797b73343",
"s2fieldsofstudy": [
"Biology",
"Medicine"
],
"extfieldsofstudy": [
"Medicine"
]
} |
52992290 | pes2o/s2orc | v3-fos-license | Iatrogenic pseudoaneurysm after bevacizumab therapy in patients with metastatic colorectal cancer: Two case reports
Pseudoaneurysms are extremely rare in patients with metastatic colorectal cancer (mCRC) treated with FOLFIRI and bevacizumab in the first-line setting. We herein present two rare cases of iatrogenic pseudoaneurysm developing in patients with mCRC after administration of FOLFIRI and bevacizumab. The first patient was a 57-year-old man who was admitted to our institution with intermittent massive bloody discharge following treatment with the 9th cycle of FOLFIRI combined with bevacizumab. Colonoscopic examination revealed sizeable bloody clots in the rectum, but no active bleeder was identified; however, the patient's hemoglobin level was found to be persistently decreased, so an angiography was performed. The angiography revealed a pseudoaneurysm with contrast extravasation from a branch of the left internal iliac artery. Embolization of the bleeding vessel was performed, and the post-embolization angiography revealed no active bleeding. The second patient was a 65-year-old man who observed blood in the stool following treatment with the 5th cycle of FOLFIRI combined with bevacizumab. The angiography revealed a pseudoaneurysm in the superior rectal artery, and intravascular embolization with coils was performed. The patient was discharged without any signs of recurrent bleeding. Following a review of the relevant English literature, to the best of our knowledge, this is the first report on the formation of a bevacizumab-related pseudoaneurysms in mCRC patients. The aim of this study was to alert clinicians to the possibility of this rare complication.
Introduction
Combining conventional systemic chemotherapy with bevacizumab, an angiogenesis inhibitor, is currently recommended as first-line treatment for patients with metastatic colorectal cancer (mCRC), as it achieves a median overall survival of 22.7 months (1).
Bevacizumab was developed by Ferrara et al as a recombinant humanized antivascular endothelial growth factor monoclonal immunoglobulin G antibody (2) and it has been approved by the US Food and Drug Administration as an adjuvant agent in the treatment of mCRC. However, bevacizumab therapy has been associated with certain adverse events, such as bleeding (3%), gastrointestinal perforation (2%), arterial thromboembolism (1%), hypertension (5.3%), proteinuria (1%) and wound healing complications (1%) (3)(4)(5)(6). Furthermore, hemorrhagic complications, such as pulmonary hemorrhage and hemoptysis, have been reported, mainly in patients with lung cancer, whereas gingival and vaginal bleeding have also been reported, although less commonly (3). In 2015, a retrospective study by Collins et al (7) reported that bleeding occurred in 41% of mCRC patients; however, the incidence of grade 3 bleeding was found to occur in only 1.6% of the cases, and there were no reported cases of grade 4 or 5 bleeding.
We herein present two cases of pseudoaneurysm with massive lower gastrointestinal bleeding in patients with mCRC during treatment with FOLFIRI and bevacizumab.
Case reports
Case 1. A 57-year-old male patient was diagnosed with rectal carcinoma with liver metastases. The patient had a previous history of hypertension, which was well-controlled with medication, but no previous history of malignancy. Colonoscopy revealed a polypoid tumor with superficial ulceration located 8 cm proximal to the anal verge (Fig. 1A); biopsy of the rectal tumor was suggestive of moderately differentiated adenocarcinoma. Abdominal computed tomography (CT) scans revealed a rectal tumor (Fig. 1B, circle) with concomitant multiple liver metastases ( Fig. 1C and D, arrows) and regional lymph nodes metastases; the disease was determined to be clinical stage cT3N2aM1a (stage IVa). Neoadjuvant concurrent chemoradiotherapy (CCRT) was administered in accordance with treatment guidelines, and FOLFIRI [irinotecan 120 mg/m 2 as a 120-min IV infusion, leucovorin (LV) 200 mg/m 2 as an IV infusion over 2 h, and 5-fluorouracil (5-FU) 2,800 mg/m 2 as an IV infusion over a 46-h period biweekly] combined with bevacizumab (5 mg/kg) was administered as a first-line chemotherapeutic regimen. After a treatment period of 3 months, CT scans were used to evaluate the response to neoadjuvant therapy; the scans revealed simultaneous shrinkage of both the liver metastatic lesion and the primary rectal tumor.
Six days after the 9th cycle of bevacizumab injections, the patient visited our emergency room after having passed a massive bloody clot from his anus. The physical findings on initial presentation included a blood pressure of 124/78 mmHg, heart rate of 130 beats per min, and hemoglobin levels of 8.6 g/dl. Contrast-enhanced abdominal CT scans revealed no evidence of gastrointestinal tract or vascular abnormalities (data not shown). The patient received a blood transfusion and was admitted to the hospital with a tentative diagnosis of lower gastrointestinal bleeding.
On colonoscopic examination performed after admission, there was blood in the sigmoid colon and rectum; however, there was no sign of active bleeding in the colon or rectum. The patient's hemoglobin level decreased to 6.2 g/dl, even after the blood transfusion, and an angiography was scheduled as a further diagnostic measure. The angiography revealed a pseudoaneurysm with contrast extravasation in a branch of the left internal iliac artery ( Fig. 2A). Active bleeding from the pseudoaneurysm was observed; therefore, transcatheter embolization was performed. A Tornado coil and Nester coil (both from Cook Inc., Bloomington, IN, USA) were inserted in this branch of the left internal iliac artery, and ~3 ml of Avitene mixture (Davol Inc., Woburn, MA, USA) was infused together with a contrast medium. Post-embolization angiography revealed no signs of further bleeding (Fig. 2B), and the patient's hemoglobin level progressively increased to 9.6 g/dl. The patient was eventually discharged after 2 weeks of uneventful hospitalization, but he ultimately succumbed to progressive metastatic multiple liver lesion deterioration 1 year after this event.
Case 2.
A 65-year-old male patient was diagnosed with rectosigmoid carcinoma and associated liver and lung metastasis. The patient had a previous history of hypertension and type 2 diabetes mellitus, which was well-controlled with medication, but no previous history of malignancy. Colonoscopy revealed an ulcerated tumor located 14-18 cm proximal to the anal verge (Fig. 3A); biopsy was suggestive of adenocarcinoma. Abdominal CT scans revealed a rectosigmoid tumor (Fig. 3B) with concomitant multiple liver metastases and bilateral lung metastasis; the disease was determined to be clinical stage T4aN2aM1b (stage IVb). CCRT was used in accordance with treatment guidelines, and FOLFIRI combined with bevacizumab was administered as a first-line chemotherapeutic regimen. After a treatment period of 3 months, CT scans were performed to evaluate the response to neoadjuvant therapy; the scans revealed mild progression of the rectosigmoid cancer, with pericolic fat infiltration.
Eight days after the 5th cycle of bevacizumab injections, the patient visited our emergency room after having observed a large amount of blood in the stool. On initial physical examination, the blood pressure was 97/66 mmHg, the heart rate was 104 beats per min, and the hemoglobin level was 7.8 g/dl. The patient received a blood transfusion and was admitted to our hospital with a tentative diagnosis of lower gastrointestinal bleeding.
Contrast-enhanced abdominal CT scans revealed a small pseudoaneurysm (0.5 cm), and an angiography was scheduled as a further diagnostic measure. The angiography revealed a pseudoaneurysm with contrast extravasation in the superior rectal artery (Fig. 3C). Active bleeding from the pseudoaneurysm was observed; therefore, transcatheter embolization with coils was performed. Straight coils (2x1 and 5x1 mm) were inserted in the pseudoaneurysm feeder, and post-embolization angiography revealed no further bleeding (Fig. 3D). The patient was eventually discharged after 2 weeks of uneventful hospitalization, but he eventually succumbed to progressive metastatic multiple liver and lung lesion deterioration 9 months after this event.
Discussion
Radiological imaging plays an impor tant role in primary diagnostics, staging, evaluation of treatment response, follow-up, and even for minimally invasive interventions. CT and MRI are included in national and international guidelines and structured reporting is strongly recommended (8).
Bleeding complications associated with bevacizumab therapy may be attributed to the anti-angiogenic effect of this agent, which inhibits endothelium growth, thus resulting in vessel wall breach and the formation of pseudoaneurysms. In the present cases, the exact pathophysiological mechanism involved in pseudoaneurysm development remains unclear. However, abdominal CT scans of the patient performed prior to bevacizumab therapy revealed no radiological signs of aneurysm formation.
Angiogenesis inhibitors have rapidly emerged as first-line treatment in cancer therapy, usually in combination with cytotoxic chemotherapy. The most frequent toxicity encountered with the use of bevacizumab is hypertension, which occurs in up to 32% of the patients (9). Patients with hypertension are most commonly treated with oral agents, such as diuretics or calcium channel blockers; however, a small number of patients do not respond to this treatment, and bevacizumab must be discontinued (10,11). Our patient had hypertension prior to treatment; the condition did not worsen with the concomitant administration of bevacizumab and was well-controlled with the use of antihypertensive medication.
The exact pathophysiological mechanism involved in the development of the pseudoaneurysm in our cases has not been fully elucidated. However, a possible correlation between the injections of bevacizumab and the formation of a common iliac or superior rectal artery aneurysm may be reasonably hypothesized; thus, it cannot be excluded that bevacizumab may have been a potential trigger. The aim of the present study was to alert clinicians to this rare adverse event associated with the use of bevacizumab.
Based on this observation, it is recommended that patients selected for anti-angiogenic treatment are closely monitored through imaging, and whenever a gastrointestinal massive bleeding from a pseudoaneurysm occurs or a pseudoaneurysm increases in size, it should be managed with selective embolization.
In conclusion, to the best of our knowledge, cases of pseudoaneurysm formation in mCRC patients under bevacizumab therapy have not been previously reported. We herein describe two such cases, and although there is no direct evidence of an association between bevacizumab therapy and the formation of pseudoaneurysms, the need for its consideration during anti-angiogenic therapy must be emphasized. When a pseudoaneurysm with bleeding becomes apparent and is life-threatening, transcatheter embolization is the preferred treatment. | 2018-09-18T02:47:34.840Z | 2018-09-06T00:00:00.000 | {
"year": 2018,
"sha1": "e61658d8a673cd0c336446143104f60b6ddde2c5",
"oa_license": "CCBYNCND",
"oa_url": "https://www.spandidos-publications.com/10.3892/mco.2018.1712/download",
"oa_status": "GOLD",
"pdf_src": "Anansi",
"pdf_hash": "e61658d8a673cd0c336446143104f60b6ddde2c5",
"s2fieldsofstudy": [
"Medicine"
],
"extfieldsofstudy": [
"Medicine"
]
} |
229416016 | pes2o/s2orc | v3-fos-license | Deep-bed filters as post-treatment for ozonation in tertiary municipal wastewater treatment: impact of design and operation on treatment goals
Ozonation followed by biological post-treatment is an established technology for abatement of organic micropollutants (OMP) from municipal wastewater. Although the necessity of biological post-treatment for oxidation by-product (OBP) removal is widely accepted, there is still discussion about the appropriate design and operation. The presented pilot-study investigates the impact of filter material and contact time on the removal efficiency of bulk organics, OMP, and OBP in three different deep-bed filters operated in parallel as post-treatment after ozonation (biological activated carbon (BAC) filter, dual-media filter sand/ BAC and dual-media filter sand/anthracite). The use of BAC instead of non-adsorptive filter material resulted in higher removal of DOC and dissolved oxygen which indicates increased biological activity. Moreover, both BAC containing filters showed additional removal for a number of OMP even at high treated bed volumes of > 50000 whereas no removal was observed in the sand/anthracite filter. Analysis of N -nitrosodimethylamine (NDMA) and several carbonyl compounds revealed a clear formation of these biodegradable OBP during ozonation. A strong correlation was found between carbonyl formation and the specific ozone dose. Removal of OBP in the sand/BAC and the sand/anthracite filter was tested at different empty bed contact times (EBCT). While NDMA was efficiently removed independent of EBCT changes, there was a slightly negative impact of shorter EBCT on the reduction of carbonyl compounds. Furthermore, it was demonstrated that the integration of enhanced phosphorus removal into post-treatment is feasible with relatively low efforts by inline coagulant dosing (FeCl 3 ) in the filter influent. Deep-bed filtration is the most common technology for biological post-treatment after ozonation in tertiary wastewater treatment. This long-term pilot- study shows that the choice of filter material and empty bed contact time are important for an optimised removal of organic contaminants and that an integration of enhanced phosphorus removal in post-treatment by coagulant dosing is feasible without drawbacks.
Introduction
Anthropogenic organic micropollutants (OMP) are detected in surface waters worldwide in the range of ng L −1 to μg L −1 . 1 Introduction of OMP into surface waters can be attributed to a large extent to the discharge of municipal wastewater treatment plant effluents. 2 Only a part of the wide spectrum of chemicals is sufficiently removed by conventional activated sludge systems. 3,4 Despite their low concentrations, OMP can pose a potential risk for aquatic ecosystems. 5,6 Moreover, they can be critical if receiving waters serve as resources for drinking water production. Natural barriers in the drinking water treatment train like managed aquifer recharge cannot guarantee full removal of OMP. 7 Ozonation for removal of OMP from municipal wastewater has been studied extensively in the last years and it developed to an established technology with several full-scale applications already implemented, mainly in Switzerland and Germany. 8,9 It is generally accepted that ozone treatment requires biological post-treatment in order to degrade potentially toxic and/or carcinogenic oxidation by-products (OBP) formed in the oxidation process. However, there is still discussion about the appropriate design and operation of such a post-treatment step. Numerous systems have been studied for post-treatment such as fixed bed bioreactors, 9 moving bed bioreactors, 9,10 constructed wetlands, 11 integrated solutions with ozonation as an intermediate step in the activated sludge process 12 or deep-bed filters. Latter option is most commonly applied and uses either nonadsorptive filter materials such as sand 8,9 or granular activated carbon 9,13 (GAC).
The above-mentioned post-treatment options have an impact on several different water quality parameters but a uniform approach for assessment of these processes has still not been established. Considering the concept of biological post-treatment after ozonation, efficient removal of biodegradable OBP can be regarded as the minimum requirement for a suitable solution. Typical representatives of biodegradable OBP are carbonyl compounds which are mainly formed by oxidation of bulk organic matter and often associated with toxic effects. 14,15 Also, potentially carcinogenic nitrosamines such as N-nitrosodimethylamine (NDMA) can be formed during ozone treatment in presence of specific precursors like secondary and tertiary amines. 16 However, NDMA can be degraded in biological-post-treatment. 9 Beyond OBP removal, treatment goals of post-treatment can differ depending on the site-specific requirements (e.g. targeting additional OMP removal). While OMP abatement by ozonation is a selective process, an additional purpose of post-treatment can be to complement the ozone action mode and widen the spectrum of abated substances. Relevant additional removal of OMP has only been reported for GAC containing deep-bed filters that were either operated as primarily adsorptive treatment steps or as biological activated carbon (BAC) filters. 9,13,17 This also applies to transformation products of individual OMP which are often only slightly modified (e.g. formation of N-oxides) and thus do not exhibit an improved biodegradability compared to their parent compounds. 18,19 For post-treatment with BAC filtration, substantial OMP removal was still observed at high treated bed volumes (BV) of approx. 50 000 leading the authors of the study to consider the involvement of biotransformation processes. 9 Long-term water quality gains without a frequent exchange of the costly GAC material would make BAC filters a promising compromise between non-adsorptive filters and primarily adsorptive GAC filters.
The combination of ozonation and subsequent biological treatment also reduces concentrations of bulk organic matter by first enhancing its bioavailability in the oxidation process and subsequently degrading the bioavailable carbon source in the post-treatment. While the removal of the biodegradable/ assimilable fraction of organic matter (usually measured as BDOC or AOC) after ozonation is an important aim for drinking water applications in order to guarantee biological stability in the water distribution network, 20 its relevance in advanced wastewater treatment is the prevention of oxygen consumption in the receiving water. Moreover, organic bulk parameters can be used as a simple indicator for efficiency comparison of different post-treatment technologies.
A common treatment goal in advanced wastewater treatment is an enhanced phosphorus removal, typically realized as a coagulation/filtration step. If back-washable deep-bed filters are applied, enhanced phosphorus removal can be integrated with relatively low efforts by installing a coagulant dosing station in the filter influent. 21 The present pilot-study investigates ozonation followed by a single-media BAC filter, a dual-media sand/BAC filter, and a dual-media sand/anthracite filter (non-adsorptive), all operated in parallel. Both dual-media filters are equipped with coagulant dosing in the filter influent for additional phosphorus removal. The removal efficiency for OBP (carbonyl compounds and NDMA), OMP, and bulk organics with different filter materials (non-adsorptive anthracite vs. BAC) and different operational conditions (influence of contact time) is the main focus of this study. In addition, the operation of the post-treatment as a coagulation/filtration step for enhanced phosphorus removal is evaluated.
Secondary effluent quality
The pilot-plant was installed at the municipal WWTP Schoenerlinde, north of Berlin, which represents a conventional secondary treatment. After mechanical pretreatment, aerated sand/grease trap, and primary sedimentation, wastewater passes activated sludge treatment with pre-denitrification. Phosphorus is removed by enhanced biological treatment, complemented by coagulant dosing in the activated sludge process. Final treatment takes place in secondary clarifiers. The secondary effluent quality for relevant parameters is summarised in Table 1. The selected OMP are typical representatives of compounds with a fast (diclofenac), moderate (benzotriazole), and slow (metformin) reaction with ozone. An overview of all monitored OMP and their reactivity with ozone is given in Table S1. †
Pilot-plant
As shown in Fig. 1 the pilot-scale treatment of secondary effluent consists of an ozonation unit (flow rate approx. 7 m 3 h −1 ) followed by different deep-bed filtration steps for posttreatment that are operated in parallel.
Based on previous experiences a specific applied ozone dose of 0.7 mg O 3 /mg DOC was targeted for efficient OMP degradation. 9,22 Ozone dose control via online measurement of the UV absorbance at 254 nm (UVA 254 ) in the influent and effluent was applied. 23 Ozonation was operated with a target UVA 254 reduction of 47% since this value was determined to correspond with the target ozone dose in Schoenerlinde secondary effluent. 24 Online ozone measurement in the influent and the offgas enables the ozone unit to calculate the actual amount of dosed ozone via mass balancing. The calculated specific ozone dose was 0.65 ± 0.09 mg O 3 per mg DOC (arithmetic mean ± standard deviation) and hence, slightly lower than originally targeted. The ozone generator (GSO40, Xylem Inc.) is supplied with oxygen by three pressure swing adsorption units (Topaz Ultra, Chart Industries Inc.). Ozone is dosed into a recirculated sidestream by a venturi injection system and subsequently mixed with the mainstream. Two stainless steel ozone reactors connected in series, with a volume of 2 m 3 each, enable a hydraulic retention time of >15 min for reaction of ozone. The maximum treatment capacity of the ozone unit is 15 m 3 h −1 .
Ozonation effluent is distributed to the inlets of the three identically constructed deep-bed filter columns with a diameter of 0.3 m each. An overflow keeps the water level in the supernatant of each filter and hence the filtration pressure constant. The first filter represents a widely used solution for biological post-treatment after ozonation, a single-media filter, filled with GAC (BAC filter). The other two filters additionally aim at the enhanced removal of phosphorus, besides biological post-treatment. Hence, they are operated as coagulation/filtration steps with prior ferric chloride (Stockmeier Chemie, Germany) dosing. Due to the increased solids load originating from coagulation, the filters are designed as dual-media filters, with sand in the lower layer for safe particle retention. While in one filter the upper layer consists of anthracite (S/A filter), GAC with comparable grain size is used in the other one (S/BAC filter). BAC and S/ BAC filter were commissioned with fresh GAC and operated within this study until approx. 53 000 BV (corresponds to approx. 1.5 years of pilot-operation).
For coagulation in the dual-media filters S/A and S/BAC, a stock solution (0.19 kg Fe per L) was dosed inline in front of static mixers into the filter influents. Since ortho-phosphate (o-PO 4 -P) was not monitored online, a constant coagulant dose of 1.8 mg Fe per L was applied and could not be adapted to variations of phosphorus concentrations, as it would be the case at full-scale. The dose was calculated for annual mean phosphorus concentrations in the secondary effluent from 2016 (o-PO 4 -P = 0.2 mg L −1 , P tot = 0.4 mg L −1 ) based on the desired molar ratio of 4.4 mol Fe per mol o-PO 4 -P. As phosphorus concentrations during the study period were higher than in 2016 (Table 1), the molar ratio fell below the target value of 4.4 mol Fe per mol o-PO 4 -P for several samplings.
Filter backwash was carried out daily on weekdays, first with air (2 min, 60 m h −1 ) and then with water (8 min, 60 m h −1 ). At the beginning of each filtration cycle filtration rates were adjusted with variable area flow meters to approximately 10 m h −1 during phase 1 and 5 m h −1 during phase 2 in order to reach target EBCT of 7.5 and 15 min, respectively. Actual EBCT and BV were calculated based on cumulative filtrate volumes measured by water meters in the filter effluents. Calculation of EBCT and BV relates to the upper filter layer only. Due to inattention during filter backwash, ∼15% of the GAC layer from the BAC filter (at ∼9000 BV) and ∼8% from the S/BAC (at ∼35 000 BV) filter were flushed out. After these incidents, EBCT and BV calculations were adjusted accordingly. Details on filter media and operational parameters are given in Table 2. The discrepancy between target EBCT and actual EBCT is based on deviations of the variable area flowmeter and the water meter measurement.
Short-term changes of the described operational parameters were done for the experiments on impacting factors of OBP formation and removal presented in chapter 3.3.
Sampling
Regular sampling campaigns were conducted to monitor the treatment efficiency of the pilot system over time. To compensate diurnal concentration changes, samples were taken as 24 h composite samples, using 10 L glass bottles. Automatic samplers with a cooling system were used for ozonation influent and effluent. Filter effluent samples were collected with a multichannel peristaltic pump and bottles were stored in cooled boxes during sampling.
Special sampling campaigns were conducted for OBP that required temporary adjustment of operational parameters. Different specific ozone doses were tested (each dose for approx. 2 h), in order to investigate the ozone dose dependency of carbonyl formation. In another campaign, filtration rates were varied (each filtration rate for one day), to examine the influence of EBCT on OBP degradation. Sampling campaigns for OBP were carried out as grab samplings in order to minimise storage times and hence potential biotransformation of the target compounds in the sample. Furthermore, copper sulfate was added as a biocide to aldehyde/ketone samples due to their high biodegradability.
For all samplings, a time offset according to the hydraulic retention time of the respective treatment process was considered in order to receive corresponding influent and effluent samples. Analysis of OMP was carried out with high performance liquid chromatography followed by tandem-mass spectrometry (HPLC-MS/MS) according to DIN 38407-F47 and DIN 38407-F36. For industrial chemicals benzotriazole and tris(1-chloro-2propyl) phosphate (TCPP) automatic sample preparation was carried out by Thermo Fisher Scientific (Dreieich, Germany) EQuan Max online solid-phase extraction with a Hypersil Gold™ aQ column (12 μm, 2.1 × 20 mm) and an injection volume of 1 mL. The subsequent LC-MS/MS system consisted of an UltiMate 3000 HPLC (analytical column: ACQUITY UPLC HSS T3, 1.8 μm, 2.1 × 50 mm, Waters, Eschborn, Germany) and a Q Exactive™ high resolution mass spectrometer (Thermo Fisher Scientific, Dreieich, Germany). All other analysed OMP where measured on a Waters (Eschborn, Germany) ACQUITY HPLC system (analytical column: ACQUITY UPLC HSS T3, 1.8 View Article Online μm, 2.1 × 100 mm, Waters, Eschborn, Germany) with direct injection (injection volume: 50 μL) followed by Xevo TQ-S tandem mass spectrometer (Waters, Eschborn, Germany). Water with 0.1% formic acid and methanol were used as eluents in all applied LC methods.
Oxidation by-products.
Aldehydes (formaldehyde, acetaldehyde, propanal, butanal, hexanal, heptanal, nonanal, glyoxal, methylglyoxal) and acetone were determined by gas chromatography (Fisons Instruments 8000) followed by electron capture detectors (GC-ECD). A derivatisation process using O-(2,3,4,5,6-pentafluorobenzyl)hydroxylamine (PFBOA) was carried out before GC-ECD analysis. Results were confirmed by parallel measurement with gas chromatography (SCION GC-436) coupled with a TQ mass spectrometric detector (BrukerDaltonics, Germany). Detailed information on the analytical method for carbonyl compounds is provided in the supplementary information SI2. For the ozone dose dependency experiment all above-mentioned carbonyl compounds were measured while for the filtration rate experiment only formaldehyde, acetaldehyde, propanal, butanal, glyoxal, methylglyoxal, and acetone were investigated. In the following chapters, the sum of all measured carbonyl compounds will be addressed as total aldehydes, although acetone is not an aldehyde but a ketone.
NDMA samples were pre-treated in a solid-phase extraction step (combination of octadecyl-modified silica gel and carbonaceous sorbent) before analysis with gas chromatography followed by quadrupole MS according to a literature protocol. 16 LOQ for NDMA was 5 ng L −1 .
Data processing
When the effluent concentration of a process was below LOQ for a certain compound its removal or relative effluent concentration (c/c 0 ) was only calculated if the influent concentration was at least 5 times the LOQ. Otherwise, the data was not taken into account to minimise errors based on false assumptions. If the influent concentration condition was fulfilled removal or relative effluent concentration was calculated using ½ LOQ as effluent concentration. Thus, the highest possibly committed error amounts to ±10% removal or relative effluent concentration, respectively.
All boxplot figures are defined as follows. The lower and upper boundary of the box represents the 25th and 75th percentile, respectively. The intermediate line in the box marks the median value. Minimum and maximum values are illustrated by the whiskers, as long as they are not excluded as outliers. Values are defined as outliers if their distance to the upper or lower boundary of the box is bigger than 1.5 times the interquartile range (IQR: distance between 25th and 75th percentile). 25 3 Results and discussion
Organic bulk parameters
The results for organic bulk parameters DOC and UVA 254 under steady-state conditions are shown in Fig. 2. It was found that after an initial breakthrough of both parameters in the BAC containing filters a constant level of removal was reached from approx. 10 000 BV onwards (Fig. S1 †). All data before that were excluded from the box-plots in Fig. 2. The observed behaviour is typical for BAC filter operation which can be divided in the first phase of adsorption (breakthrough), the second phase of concurrent adsorption and biodegradation (transition from breakthrough to steadystate), and the third phase of biodegradation (steady-state). 26 Other pilot and full-scale studies on BAC filtration of wastewater treatment plant effluents with and without prior ozonation confirmed the establishment of biological degradation processes in the filters. 17,21 DOC and UVA 254 exhibited different behaviour during ozonation. While mean DOC reduction was marginal (5%), UVA 254 was reduced by 44% on average. Low DOC removal confirms that reaction with ozone at applied doses mostly leads to partial oxidation and not to mineralisation of bulk organic matter. The strong decrease of UV 254 can be mainly attributed to the oxidation of aromatic compounds, which typically absorb light in this range of wavelengths. The direct reaction of ozone specifically attacks these electron-rich moieties via ozonide formation and subsequent cleavage of the C-C bond. 14 Aldehydes, ketones, and carboxylic acids are typical products of this reaction 14 and their formation was confirmed by several studies on ozonation of drinking water or wastewater. 15,[27][28][29] They can therefore be considered oxidation by-products of the bulk organic matter that need to be removed by subsequent biological filtration. 30 The formation of these compounds and their fate during posttreatment were also investigated in this study and will be discussed separately in chapter 3.3.
Laboratory results for UVA 254 showed that the target UVA 254 reduction of 47% during ozonation was not completely reached. This might be explained by deviations between the UVA 254 probes used onsite for online-monitoring and the photometer used for laboratory analysis. Despite regular cleaning, online probes are more prone to biofouling due to constant wastewater exposition. Furthermore, influent and effluent are measured with two separate probes which produces additional potential for errors. The overestimation of UVA 254 removal by the online probes was also in line with the slight ozone underdosing (0.65 instead of 0.7 mg O 3 per mg DOC).
The post-treatment steps BAC, S/BAC, and S/A removed DOC on average by 11%, 19%, and 15%, respectively. The better performance of the dual-media filters S/BAC and S/A compared to BAC is likely to result mainly from coagulant dosing in the influent. It is well known that, besides removing phosphate from water, the coagulation process with ferric chloride also causes a reduction of DOC. 31,32 The slightly higher EBCT in the upper layer of the dual-media filters during both phases of operation and the extra contact time in the sand layer might have also led to additional biodegradation of DOC. Despite nearly identical operational conditions, DOC removal in the S/BAC filter was more pronounced than in the S/A. This indicates that biological (Fig. 2) and COD (Fig. S3 †) were analogous to DOC and hence, confirm the described findings. With dissolved oxygen (DO) concentrations mainly above 20 mg L −1 after ozonation, redox conditions were strictly aerobic. Hence, the predominant biodegradation mechanism for organic matter is aerobic respiration, using oxygen as the final electron acceptor. Anaerobic respiration processes with other electron acceptors such as nitrate, ferric iron, or sulfate are negligible under the described conditions. 33 DOC removal in the filters based on biodegradation should therefore be closely connected to DO consumption (ΔDO). Indeed, average ΔDO in the S/BAC filter was 1.8 mg L −1 higher than in the S/A (Fig. S3 †), which supports the assumption of higher biological activity on BAC than on anthracite.
In accordance with these results, other studies also observed better removal of biodegradable organic matter on BAC than on non-adsorptive filter materials. [34][35][36] Multiple explanations for the benefits of BAC have been reported. The porous structure of GAC media provides a significantly higher specific surface area and increased roughness of the surface, compared with compact grains of anthracite or sand. 35,36 The rough surface facilitates the attachment of microorganisms and hence, microbial colonization. 36 The high specific surface area of GAC plays an important role for the adsorptive removal of organic contaminants, but it is not fully available for biofilm growth. Typically, a high portion of the total surface area is formed by mesopores (2-50 nm) and micropores (<2 nm), which are not accessible for bacteria, due to their size. 37 Since the grain size distribution is the same in the S/BAC and the S/A filter, it can be assumed that the total surface area available for biofilm growth is also similar. However, there is a strong interaction between sorption processes on GAC and the attached biofilm that promote an efficient removal of organic compounds. While microorganisms on non-adsorptive media only have access to the organic substrate that intrudes the biofilm by diffusive transport from the water, biomass attached to GAC can additionally degrade organic matter adsorbed to the outer activated carbon surface. 26 Thus, the biofilm on GAC has an enhanced availability of organic substrate. Moreover, biodegradation of adsorbed compounds regenerates adsorption sites on the GAC surface and thereby prolongs its adsorptive lifetime. 26 These biodegradation processes are usually incomplete and lead to the formation of smaller biotransformation products. Due to their reduced size, these molecules are then capable of diffusing into the micropore system of the activated carbon and adsorb there again. If the concentration on the outer GAC surface decreases sufficiently, the biotransformation products in the micropores are desorbed and transported back to the outer surface, where a further degradation by the biofilm takes place. 26 3.2 Organic micropollutants 3.2.1 Ozonation. The abatement of the monitored OMP during ozone treatment is shown in Fig. 3. Depending on the reactivity of individual OMP with ozone performance ranged from degradation below detection limit to no degradation at all. Ozone reacts selectively with molecules that contain electron-rich moieties such as activated aromatic systems, deprotonated amino groups, organosulfur compounds, or olefins. 38 These compounds are characterised by high second-order reaction rate constants (k O 3 ) with ozone that usually result in efficient abatement. Also, the unselective and fast reaction of OH-radicals formed by ozone has an impact on the degradation of OMP. 38 The reactivity of a compound with OH-radicals is expressed by the second-order reaction rate constant k OH .
As suggested by previous studies, it is useful to categorise OMP based on their k O 3 values as fast reacting (k O 3 > 10 4 M −1 s −1 ), moderately reacting (k O 3 = 10 2 -10 4 M −1 s −1 ) and slowly reacting compounds (k O 3 < 10 2 M −1 s −1 ). 22,39,40 Table S1 † shows the second-order rate constants and the respective categorisation for the investigated OMP. Indeed, all fast reacting OMP (4-formylaminoantipyrine (FAA), diclofenac, carbamazepine, clarithromycin, sulfamethoxazole) were degraded by more than 90% on average during ozonation. For olmesartan and 4-OH-diclofenac, k O 3 values were not found in literature. However, it is likely that they are >10 4 M −1 s −1 since the mean abatement of the compounds was also above 90%. In the case of 4-OH-diclofenac, a similar behaviour as diclofenac can be expected due to its almost identical chemical structure (one additional hydroxy group). However, it is likely that they are >10 4 M −1 s −1 since the mean abatement of the compounds was also above 90%. In the case of 4-OH-diclofenac, a similar behaviour as diclofenac
View Article Online
can be expected due to its almost identical chemical structure (one additional hydroxy group). The degradation efficiency for OMP with a moderate reactivity with ozone (metoprolol, candesartan, benzotriazole, bezafibrate, gabapentin) ranged from over 80% on average for metoprolol down to approx. 40% for gabapentin (k O 3 = 2.2 × 10 2 M −1 s −1 ). In contrast to fast reacting OMP, their abatement is strongly influenced by the reaction with OHradicals, besides the direct reaction with ozone. 41 The k OH values of the compounds are similar, ranging from 7.3 × 10 9 M −1 s −1 to 9.1 × 10 9 M −1 s −1 . Hence, it could be observed as expected that for most of the OMP, degradation efficiency correlated with their k O 3 values. Only bezafibrate stayed behind the expected reduction. With a k O 3 of 5.9 × 10 3 M −1 s −1 , an abatement similar to candesartan (k O 3 = 5.6 × 10 3 M −1 s −1 ) would be assumed. A comparable study confirmed a similar degradation for bezafibrate and candesartan during ozone treatment. 9 The discrepancy in the present study might be explained by the small number of samplings that could be used for evaluation of the respective treatment efficiency (n = 9), due to low influent concentrations often close to LOQ.
The abatement during ozonation for slowly reacting OMP (valsartan, primidone, mecoprop, acesulfame, metformin, TCPP) lay between approx. 70% on average for valsartan and below 0% for TCPP. The concentration increase of TCPP could not be explained within this study. To the best of our knowledge a formation of TCPP during ozone treatment has not been reported yet and is therefore unlikely. For the slowly reacting compounds, the reaction with OH-radicals is the dominating degradation mechanism. 41 Consequently, the abatement in this group of OMP was determined to a large extent by their k OH values (k OH for TCPP was not available), and not with k O 3 values. Compounds with high k OH values can even be degraded efficiently (e.g. valsartan with k OH = 10 10 M −1 s −1 ), despite their low direct reactivity with ozone. Consequently, a clear separation of moderately and slowly reacting OMP in terms of abatement efficiency is not possible. One approach for a more precise prediction of degradation based on second-order reaction rate constants, is a further sub-division of slowly reacting compounds into k OH < 5 × 10 9 M −1 s −1 and k OH ≥ 5 × 10 9 M −1 s −1 . 41 For the remaining compounds gabapentin lactam, oxypurinol, tertbutryn, and valsartan acid, literature data on reaction kinetics was not available. Based on their medium abatements between 40% and 80%, a high reactivity with ozone (k O 3 > 10 4 M −1 s −1 ) can be excluded. However, a further classification as moderately or slowly reacting OMP is not possible, as described above.
Among the compounds with lower elimination efficiency were gabapentin and oxypurinol. This result is of particular concern because the two compounds can be critical for drinking water production downstream of the WWTP. Due to their persistence in the environment, both have been found in different compartments of the water cycle including ground and drinking water. [43][44][45] There is limited data on toxicity for oxypurinol and gabapentin. However, health-related indication values for drinking water that were defined by the German Environment Agency, have been temporarily exceeded in one of Berlin's waterworks in the past. 43 Hence, efficient removal of these compounds during advanced wastewater treatment is an important goal, especially for oxypurinol with its high secondary effluent concentration of 35 μg L −1 . Fig. 4 shows the relative effluent concentrations (c/c 0 ) of 6 selected OMP over treated BV in the different filters. The selection of the OMP was made based on their behaviour in the BAC filters. They represent a removal by combined adsorption and potential biotransformation (Fig. 4a-c), a biotransformation from parent compound to transformation product (Fig. 4c and d), a purely adsorptive removal (Fig. 4e), and no removal at all (Fig. 4f). The remaining 5 investigated OMP are depicted in Fig. S2 † and can also be categorised with these types of behaviour.
Relative effluent concentrations of the S/A filter remained at approx. 100% over the whole testing period for all monitored OMP, demonstrating that no relevant additional removal took place. These findings are in accordance with other comparable studies where post-treatment in nonadsorptive filters did not contribute to OMP removal. 9,13,35 In contrast, other studies that worked with longer contact times did report biodegradation in tertiary filtration under oxic conditions (without previous ozonation) for benzotriazole, TCPP, gabapentin, and metoprolol. 21,47,48 Hence, there is potential for additional OMP removal during post-treatment under appropriate conditions. It is likely that the EBCT of approx. 16 min in the S/A filter was insufficient for biological processes.
Another reason that could hinder biotransformation of OMP, is the formation of readily biodegradable organic matter during ozonation which is preferentially degraded by microorganisms. Carbon limitation can substantially promote OMP removal in biological systems, as long as the removal mechanism is not based on a co-metabolic reaction. 49 At the beginning of the operation, GAC containing filters BAC and S/BAC showed very similar breakthrough behaviour for most OMP. Low initial relative effluent concentrations gradually increased with advancing BV. Typically, breakthrough curves in GAC adsorbers keep rising until they asymptotically converge towards complete breakthrough (c/c 0 = 100%). If biotransformation processes are involved besides adsorption, breakthrough curves should stabilise at a level of c/c 0 < 100%. 26 The breakthrough behaviour of combined adsorption and potential biotransformation was observed for metoprolol, oxypurinol, gabapentin, benzotriazole, primidone, valsartan, and TCPP (Fig. 4a-c and S2a-d †). For most substances, the change of target EBCT from 7.5 min to 15 min at ∼33 000 BV for the BAC filter and 28 000 BV for the S/BAC filter is clearly visible as a drop in the breakthrough curve. Extension of EBCT improves kinetic conditions for both, adsorption and biodegradation. The steady OMP removal from approx. 30 000 BV onwards until the end of the testing period (∼53 000 BV) points at the involvement of biological transformation processes besides adsorption during BAC filtration. In that sense, the first 30 000 BV can be considered as an acclimation period for OMP removal. The levels of steady-state abatement varied a lot between the compounds. Well adsorbing compounds metoprolol, oxypurinol, and benzotriazole exhibited the highest removal. Excluding all data below 30 000 BV, median abatements of these OMP in the S/BAC filter amounted to 69%, 76%, and 91%, respectively. Benzotriazole and metoprolol are known to be biodegradable under oxic conditions and behaved similarly in another study, where they were still well abated in a loaded GAC filter with 50 000 BV subsequent to ozone treatment. 9 In contrast, oxypurinol degradation was only reported under strongly reducing conditions during river bank filtration. 50 A pilot study in drinking water treatment also observed steady removal of oxypurinol in a GAC adsorber with >35 000 BV but assumed that it was based on adsorption. 43 Further operation of the filters will reveal if breakthrough of oxypurinol will continue at a later stage or if removal will stay steady due to biotransformation. As explained in section 3.2.1, the removal of oxypurinol is an important treatment goal, due to its potential negative impact on drinking water production, downstream of the WWTP. Considering only data after acclimation, the combined treatment with ozone and S/BAC would reach an efficient removal of 86%, whereas the combination of ozone and S/A would result in 42% removal. Among the moderately and poorly adsorbing OMP gabapentin, valsartan, primidone, and TCPP, steady-state abatement was lower. Median removal efficiencies in the S/BAC filter above 30 000 BV for these compounds were 13%, 18%, 24%, and 32% respectively. Biological removal of gabapentin in BAC filters could be expected as its biodegradability in different kinds of filter systems has been documented in a number of studies. 21,35,42,46,48 However, the achieved abatement stayed behind the results of GAC filters with similar EBCT. 21,44 Once again, higher concentrations of biodegradable organic carbon formed during prior ozone treatment could be one explanation for the comparatively low gabapentin removal. Moreover, one study suggested that high OMP influent concentrations also contribute to an enhanced removal, as increased gabapentin abatement was observed after a concentration rise in the influent. 21 A recent study revealed that gabapentin lactam is the major biological transformation product of gabapentin and that it exhibits a higher persistence to biodegradation than the original compound. 51 Indeed, there was no evidence for gabapentin-lactam biodegradation in BAC filters and a slight formation was noticed after the acclimation period (Fig. 4d). Valsartan reached full breakthrough in the S/BAC filter with shorter EBCT and started to be steadily removed after extending EBCT. This behavior clearly points at biotransformation of valsartan in the GAC filter bed, which is confirmed by another study on tertiary GAC filtration. 21 However, it is likely that part of the removed valsartan is biologically transformed into valsartan acid. 52 Due to high valsartan acid concentrations in the filter influent compared to valsartan (more than 10 times higher), the analytical accuracy does not allow for relating partial valsartan removal to valsartan acid formation. Removal of valsartan acid did not take place after the initial adsorptive phase of filter operation anymore, which demonstrated that valsartan acid was persistent to further biotransformation. This is in disagreement with several studies that reported efficient biodegradation of valsartan acid under oxic conditions. 42,46,50 While two studies worked with hydraulic retention times in the range of days in soil column experiments, 46,50 the third study achieved ∼50% biodegradation with only 15 min EBCT in a GAC adsorber fed with drinking water. 42 Thus, contact time cannot be the only driver for biological valsartan acid removal but other factors such as carbon source availability seem to play an essential role. Several studies found that the compound primidone was not biodegradable, neither in GAC filters 21 nor in natural systems, 53 whereas significant removal was reported during infiltration in the hyporheic zone of a wastewater impacted river under oxic/suboxic conditions. 54 Hence, biological transformation of primidone is possible and potentially explains the observed removal in the S/BAC filter. Inconsistent results for biodegradation of TCPP in tertiary wastewater treatment were found in literature. While removal of approx. 50% (median) was observed in a retention soil filter subsequent to a WWTP, 47 very low biological abatement of TCPP was found in BAC filtration as post-treatment after ozonation. 9,55 Contact time seems to play an important role since TCPP had already converged towards complete breakthrough during the first phase of operation (target EBCT 7.5 min) and steady removal established only after doubling EBCT.
Among the OMP with combined adsorption and potential biotransformation, compounds with a higher adsorption affinity to GAC also reached a more efficient steady-state removal. As discussed in section 3.1 there is a close interaction between adsorption and biotransformation on the GAC surface. The immobilisation of OMP by adsorption makes biotransformation processes on the GAC surface independent of contact time. 26 Thus, it is consistent that better adsorption comes along with better biotransformation for not readily biodegradable compounds. Also, it explains why biotransformation of OMP was observed in the BAC filters, but not in the S/A filter. A prediction of the behaviour based on distribution coefficients log D 7.4 was not possible. For instance, metoprolol was removed significantly better than primidone, although their distribution coefficients indicate the opposite (log D 7.4 (metoprolol) = −0.4, log D 7.4 (valsartan) = 1.1). Other factors and chemical properties such as the molecule size also play an important role. Since the biofilm strongly reduces convective transport to the GAC surface, diffusion is the dominant mass transport process. 26 The diffusive transport preferentially permits small molecules to pass the biofilm and adsorb onto the GAC. 26 Typical behaviour of a purely adsorptive removal was observed for candesartan, gabapentin lactam, and valsartan acid ( Fig. 4d and e and S2e †). After a gradually decreasing removal efficiency at the beginning of the pilot operation, full breakthrough was reached at 20 000-30 000 BV. In the case of gabapentin lactam, even a slight formation was observed after full breakthrough, most likely due to biotransformation of other sartans. 52 Metformin was the only compound that was not removed at all in the BAC filters from the beginning on (Fig. 4f). It is known that biotransformation of metformin by activated sludge treatment is possible, leading to the formation of guanylurea. 56 However, the short contact time in the filters compared to conventional activated sludge treatment is most likely the reason, why metformin was not affected by BAC filtration. A biotransformation of metformin, immobilised on the GAC surface by adsorption, can also not be expected, since it is extremely polar (log D 7.4 = −5.6) and does not adsorb in relevant amounts.
Formation of OBP during ozonation and their fate during post-treatment
Aldehydes, ketones, and carboxylic acids are typical OBP formed by the reaction of ozone with both, OMP and effluent organic matter. 15,28 Since concentrations of effluent organic matter (mg L −1 range) are typically several orders of magnitude higher than of OMP (ng L 1 -μg L −1 range) formation of these compounds is expected to be higher compared with other transformation products that result from the reaction with specific precursors. Indeed, a sampling campaign at different specific ozone doses revealed a clear, ozone dose dependent concentration increase of carbonyl compounds (Δc) during ozonation, as depicted in Fig. 5 and S4. † The carbonyl formation varied a lot between the investigated compounds. At a specific ozone dose of 0.67 mg O 3 per mg DOC, it ranged from <1 μg L −1 for propanal and methylglyoxal up to >10 μg L −1 for formaldehyde and heptanal. Other studies confirmed formaldehyde as a major carbonyl by-product from wastewater ozonation. 15,28 As shown in Fig. 5 and S4, † the formation of some of the compounds correlates well with the applied specific ozone dose (R 2 > 0.8 for formaldehyde, acetaldehyde, butanal, heptanal, nonanal, and glyoxal). Since there is an excess of effluent organic matter and the reaction is expected to be limited by the availability of ozone the observed trend for aldehyde formation in dependence of the ozone concentration seems logical. However, for other investigated carbonyl compounds (propanal, hexanal, methylglyoxal, acetone) the correlation was not observed or not as pronounced (Fig. S4 †). Interestingly, the best correlation with the ozone dose (R 2 = 0.98) was found for the total aldehyde concentration (sum of all measured carbonyl compounds) which reveals that the overall potential for aldehyde formation at a given ozone dose is valid even though unpredictable shifts between concentrations of single compounds might occur. These results are in accordance with literature where a strong correlation of total aldehydes and the specific ozone dose was observed (R 2 = 0.99). 15 The influence of the ozone dose on NDMA formation was not investigated within this study because it has been previously reported that there is no correlation. 8,9,22 The variation of specific precursor concentrations over time has a higher impact on NDMA formation than the ozone dose.
The formation of OBP during ozone treatment and their removal during post-treatment in dual-media filters applying different EBCT or filtration rates, respectively, were investigated. As shown in Fig. 6, a total aldehyde formation of up to approx. 40-100 μg L −1 was observed at specific ozone doses between approx. 0.7 and 1 mg O 3 per mg DOC (the increased ozone doses were not intended but due to fouling on the UVA 254 probes for ozone dose control). With an EBCT of approx. 15 min (corresponds to a filtration rate of 5 m h −1 ) the carbonyl compounds were removed by 88% and 87% in the S/ BAC and the S/A filter, respectively. When filters were operated at higher filtration rates and hence lower contact times, a decrease of removal was observed. An EBCT of approx. 7 min (corresponds to filtration rate of 10 m h −1 ) resulted in a reduction of total aldehydes concentration by 74% and 75% in the S/BAC and S/A filter, respectively. A further decrease of EBCT down to 5 min (corresponds to filtration rate of 13 m h −1 ) again lowered the removal performance in the S/BAC filter a degradation of 68% was observed, while in the S/A filter removal decreased to 51%. The results demonstrate that changes of EBCT within the tested range affect total aldehyde removal efficiency in the deep-bed filters. In full-scale applications, deep-bed filters are often operated with EBCT that are constantly changing as a function of diurnal flow variations of the WWTP (especially with rain events). Thus, it might be relevant to take decreased removal performance at low EBCT into account for the dimensioning of deep-bed filters in posttreatment applications. Many studies on biological filtration after ozonation have been conducted with typical EBCT of ≥10 min. 9,13,17,57 The lower end of EBCT conditions that occur in full-scale wastewater systems during rain events and dry weather peaks were rarely investigated. 58 More research in that field is needed in order to provide solid information on a wider spectrum of compounds for improved dimensioning of posttreatment filters in the future. The effect of filter material played a minor role for carbonyl compound degradation. Both filter materials, BAC and anthracite, proved to be suitable for removing well degradable carbonyl compounds, although at the lowest EBCT the S/BAC slightly outperformed the S/A filter, likely due to higher biological activity. As depicted in Fig. 7, NDMA concentrations of up to approx. 40 ng L −1 were found after ozone treatment while influent concentrations were always below LOQ. Posttreatment with both dual-media filters removed the formed NDMA again, reaching concentrations below LOQ for almost all samplings. Hence, a high removal performance was achieved independent of the choice of filter material or EBCT within the investigated range. However, another study observed a strong decline of NDMA removal in a BAC filter and an anthracite filter after reducing EBCT from 10 min to 2 min, 58 which shows that a further decrease of EBCT could have a negative effect on performance.
Combination of nutrient removal and post-treatment
As depicted in Fig. 8, ozonation did not have a significant effect on P tot or TSS. The marginal decrease of both parameters can be attributed to sedimentation effects in the ozone contactors. With filter influent concentrations (= O3_eff) of P tot up to 1 mg L −1 , the constant coagulant dose of 1.8 mg Fe per L resulted in molar ratios below the target value of 4.4 mol Fe per mol o-PO 4 -P in several samplings. However, the dual-media filters exhibited efficient removal. The 75th percentile concentrations of P tot in both filters effluents were <0.1 mg L −1 . A reliable compliance with an effluent concentration of 0.1 mg L −1 is feasible, if the coagulant dose is controlled as a function of the current phosphorus concentration in the filter influent. In full-scale coagulation/filtration systems this is state-of-the-art. The BAC filter also slightly reduced P tot concentrations although coagulant dosing was not applied. Here, the removal is linked to the retention of phosphorus containing TSS in the filter bed. As also shown in Fig. 8, all filters reached TSS effluent concentrations of mostly <1 mg L −1 . A similar study with a GAC containing coagulation/filtration step without previous ozonation also reached effluent concentrations predominantly below 0.1 mg L −1 P tot . 21 It can be concluded that the use of GAC as filter material is equally suitable for coagulation/filtration when compared with typically applied materials like sand or anthracite. The results also show that the ozone process does not adversely affect the coagulation/ filtration step. Neither does the coagulation process have negative effects on the filters' function as biological posttreatment considering the results for OBP, OMP, and bulk organics.
Results for nitrogen species NH 4 -N and NO 3 -N are shown in Fig. S12 and S13. † Residual NH 4 -N in the secondary effluent was not affected by the ozone treatment but further removed during deep-bed filtration, most likely by nitrification. NO 3 -N concentrations remained constant throughout the whole treatment process. Nitrification of NH 4 -N does not result in a visible concentration increase of NO 3 -N, since NH 4 -N concentrations are approx. 100 times lower than for NO 3 -N and the effect is marginal. The high DO concentrations after ozonation do not allow for a NO 3 -N reduction in the filters by denitrifying bacteria since their development requires anoxic redox-conditions. A recent study on an ozone/BAC treatment system demonstrated that
Conclusions
The present study demonstrates that design and operational aspects have to be taken into account for an optimised posttreatment with deep-bed filters after ozonation. The use of BAC instead of non-adsorptive filter material like anthracite is more suitable for promoting metabolic activity and hence, improves aerobic degradation of organic matter. Furthermore, post-treatment with BAC filters allows for additional removal of several OMP, whereas non-adsorptive filters do not contribute to OMP removal. Since OMP removal in the BAC filters remained relatively stable even after 50 000 treated BV the involvement of biological processes besides sorption is assumed. The filters of this study will be further operated in order to investigate their long-term performance and possibly verify the assumption. The primary function of biological post-treatment after ozonationthe efficient removal of biodegradable OBPcan be achieved independent of the choice of filter material. A change of EBCT (5-15 min) or filtration rate (5-13 m h −1 ), respectively, has a limited impact on the efficient removal of OBP within the tested range. However, the slight decrease of total aldehydes removal at short EBCT suggests that a further reduction of EBCT should be avoided. Tertiary treatment applications that additionally aim for enhanced phosphorus removal can integrate an inline coagulant dosing in the influent of the deep-bed filter without affecting its function as biological post-treatment.
Conflicts of interest
There are no conflicts to declare. | 2020-11-26T09:05:52.817Z | 2021-01-01T00:00:00.000 | {
"year": 2021,
"sha1": "f99293f6c87b616cde69b2f8872c7a1a79d1486b",
"oa_license": "CCBYNC",
"oa_url": "https://pubs.rsc.org/en/content/articlepdf/2021/ew/d0ew00684j",
"oa_status": "HYBRID",
"pdf_src": "ScienceParsePlus",
"pdf_hash": "e1a214b5daab9fdda5178bfb37a0a309ab04e966",
"s2fieldsofstudy": [
"Engineering"
],
"extfieldsofstudy": [
"Environmental Science"
]
} |
234219694 | pes2o/s2orc | v3-fos-license | Analysis of the influence of heat source well parameters on pumping temperature of groundwater source heat pump
In this paper, MATLAB software is used to simulate the influence of parameters of different heat source wells on pumping temperature in groundwater source heat pump system. The absolute value of horizontal permeability coefficient of aquifer will not affect its pumping temperature, but when the permeability coefficient ratio increases from 1 to 10, the pumping temperature decreases by 1.1°C; When the specific heat capacity increases from 2000 kJ / m3 · °C to 3000 kJ / m3 · °C, the pumping temperature decreases by 0.8 °C; When the degree of thermal dispersion increases from 1m to 3m, the pumping temperature decreases by 0.4°C.
Introduction
Due to the rich water resources in Tibet, and the groundwater source heat pump system uses almost constant temperature groundwater as the cold and heat source, and the energy consumption is only 70% -80% of that of the conventional underground pipe system [1]. Therefore, it is feasible to popularize groundwater source heat pump system in Tibet. According to the different pumping and backwater modes, groundwater source heat pump system is mainly divided into two categories: different well recharge structure and pumping and irrigation same well structure [2]. The groundwater source heat pump system with pumping and irrigation in the same well reduces the number of wells drilled, and greatly reduces the construction cost and operation and maintenance costs compared with the system with different wells. In summer, the underground water source heat pump system discharges the excess heat in the room to the constant temperature underground water, which produces the indoor refrigeration function; in winter, the underground water source heat pump system extracts the heat higher than the outdoor in the groundwater and puts it into the room, thus producing the indoor heating effect. So as to achieve the purpose of seasonal energy storage and renewable energy recycling.
In this paper, based on the experimental data of the groundwater source heat pump system in the same well, the influence of the parameters of the heat source well on the pumping temperature of the groundwater source heat pump system is simulated by using MATLAB software.
Where:ν is the groundwater overflow factor, s -1/2 ·m -1/2 , When it is connected with the underground aquifer as an aquiclude, 0 = ν ; s is the drawdown of groundwater in aquifer, mH2O; t is the running time, s; r, z is the component of cylindrical coordinates, m; r K is the horizontal permeability coefficient of aquifer, m/s; z K is the vertical permeability coefficient of aquifer, m/s; B is the thickness of underground aquifer, m; B' is the thickness of aquitard, m.
The boundary conditions of outer shaft lining are as follows: Where: Qw,p is the pumping flow of the heat source well, m 3 /s; Qw,r is the reinjection water flow of heat source well, m 3 /s; Kh is the comprehensive permeability coefficient of heat source well, m/s; b1 is the distance between the bottom of the pumping filter section of the heat source well and the roof of the aquifer, m;bs1 is the length of the pumping filter section in the heat source well, m; b0 is the distance between the pumping and return water filter sections in the heat source well, m; bs2 is the length of filter section of return water in heat source well, m; b2 is the distance between the top of filter section of return water of heat source well and the roof of aquifer, m.
Implementation of the model
In this paper, when using MATLAB software, its specific calculation parameters are set as follows: The aquifer thickness of the groundwater source heat pump system with pumping and irrigation in the same well is set as 40m, the radial flow range of groundwater in the heat source well is set as 200m, the unit water storage coefficient of aquifer in the heat source well is set as 1.0×10 -6 m -1 , and the influence range of thermal dispersion caused by groundwater velocity fluctuation in aquifer is set as 1m. The whole model is uniformly set as non-uniform grid model. The impermeable layer thickness of the roof and bottom plate of the aquifer where the heat source well is located is set as 10m, the calculation range of the temperature field in the aquifer is set as 100m, the accuracy of the temperature field of the aquifer is set as 10 -5 , and the accuracy of the groundwater flow equation is set as 10 -7 . The number of grids is 152 × 84 (horizontal × vertical). Each time step is set as 3 hours, and the total calculation time is set as 72 hours (comparison test time). The physical and thermal parameters of the well are shown in Table 1.
Model validation
Due to the different pumping flow of the groundwater source heat pump system in the test, the temperature difference between the pumping and return water of the heat source well will be different (the pumping flow will increase, and the temperature difference between the pumping and return water will decrease), as shown in Table 2. Now in order to verify the correctness of the model established in this paper, the temperature difference between the pumping and return water of the heat source well in this paper is set as 2.5 ℃, 2.1 ℃, 1.8 ℃ and 1.6 ℃ respectively. The model calculation results are compared with the experimental results, as shown in Fig. 1-4. It can be seen from figure 1-4 that under different working conditions (condition 1-condition 4) simulated in the established model, the simulated value of the groundwater source heat pump system is in good agreement with the experimental measured value. Taking the simulation condition 1 as an example, the measured value of pumping temperature is 17.9 ℃, the simulated value is 18.7 ℃, the absolute error is 0.8 ℃, the error is only 4.5%, and the error range is less than 5%; The measured value of return water temperature is 20.4 ℃, the simulated value is 21.3 ℃, the absolute error is 0.9 ℃, the error is only 4.4%, and the error range is less than 5%. Therefore, the correctness of the model is verified by the above data. The reason why the temperature of pumping and return water in the model is higher than that in the test is mainly because the influence of factors such as overflow and seepage between aquifers is ignored. The summary of the model and experimental measurements is shown in Table 3.
Influence of horizontal permeability coefficient
In this section, under condition 1, only the absolute value of the horizontal permeability coefficient in the aquifer of the heat source well is changed from 10 -3 to 10 -5 (that is, the soil structure of the aquifer is 100 times closer) to simulate the change of pumping temperature of the heat source well. The results are shown in Figure 5.
Fig. 5 Relationship between pumping temperature and horizontal permeability coefficient
It can be seen from Figure 5 that when the absolute value of horizontal permeability coefficient of aquifer in heat source well changes, its pumping temperature hardly changes. This is because in the same aquifer soil structure, under the condition that the permeability coefficient ratio (horizontal permeability coefficient / vertical permeability coefficient) is constant, simply reducing the absolute value of horizontal permeability coefficient will also reduce its vertical permeability coefficient. At the same time, the mixing ratio of raw water and reinjection water in the pumping area of heat source well will be reduced according to the same proportion. Therefore, the absolute value of horizontal permeability coefficient of aquifer in heat source well will not affect the pumping temperature of the well, but reducing its absolute value will only increase the difficulty of groundwater recharge.
Influence of permeability coefficient ratio
In this section, under condition 3, only the permeability coefficient ratio of aquifer in the heat source well is changed, that is, the value of horizontal permeability coefficient to vertical permeability coefficient is changed from 1 to 10 to simulate the change of pumping temperature of the heat source well. The results are shown in Figure 6. Fig. 6 Relationship between pumping temperature and permeability coefficient It can be seen from Fig. 6 that the pumping temperature of the heat source well will gradually decrease from 21.5 ℃ to 20.4 ℃ in the process of changing the permeability coefficient ratio from 1 to 10, and the drop range is about 1.1 ℃. This is because when the horizontal permeability coefficient is much greater than the vertical permeability coefficient, it means that the underground water inflow of the aquifer is large and sufficient, which increases the proportion of the original water and return water. Therefore, for the ground water source heat pump system, it is better to select the place where the groundwater is sufficient and the horizontal permeability coefficient is far greater than the vertical permeability coefficient, or set the middle partition board in the recharge area of the heat source well to reduce the vertical permeability coefficient.
Influence of specific heat capacity of volume
Under the condition of condition 3, only the volume specific heat capacity of the aquifer in the heat source well is changed, that is, the heat extraction potential of the aquifer itself is changed, and it is gradually increased from 2000kJ/m 3 ·℃ to 3000kJ/m 3 ·℃, so as to simulate the change of pumping temperature of the heat source well. The results are shown in Figure 7. It can be seen from Fig. 7 that the pumping temperature of the heat source well is almost inversely proportional to the volumetric specific heat capacity of the aquifer. When the volumetric specific heat capacity of the aquifer changes from 2000kJ/m 3 ·℃ to 3000kJ/m 3 ·℃, the pumping temperature drops by about 0.8 ℃. This is due to the increase of the specific heat capacity of the aquifer, which enhances the heat storage capacity of the aquifer and improves the heat capacity of the aquifer where the heat source well is located. Therefore, the heat source well of groundwater source heat pump system should choose the place with high rock proportion as far as possible.
Influence of thermal dispersion
In view of the fact that the thermal dispersion of groundwater in the heat source well will directly reflect the mutual thermal influence phenomenon between heat source wells [4] , this section still chooses to change the thermal dispersion of groundwater only under condition 3, that is, to change the thermal dispersion range of groundwater caused by velocity fluctuation from 1m to 3m, so as to simulate the change of pumping temperature of the heat source well. The results are shown in Figure 8. It can be seen from Fig. 8 that with the increase of thermal dispersion, the pumping temperature of the heat source well decreases in equal proportion. In the process of thermal dispersion from 1m to 3m, the pumping temperature drops by about 0.4 ℃. This is due to the increase of thermal dispersion, which makes the disturbance of groundwater increase and the average of heat increase, which leads to the increase of effective heat transfer and the increase of heat influence range of recharge water. Therefore, the pumping area of the heat source well of the ground water source heat pump system should be set at the place where the underground water flow rate is fast.
Conclusion
(a) The absolute value of horizontal permeability coefficient of aquifer does not affect the pumping temperature of heat source well, but the permeability coefficient ratio does. When the permeability coefficient ratio increases from 1 to 10, the pumping temperature drops by about 1.1 ℃. Therefore, the place where the horizontal permeability coefficient is far greater than the vertical permeability coefficient should be selected as far as possible.
(b) The volume specific heat capacity of aquifer will affect the pumping temperature of the heat source well. When it increases from 2000 kJ/m 3 ·℃ to 3000 kJ/m 3 ·℃, the pumping temperature will decrease by about 0.8 ℃. Therefore, the place with high rock proportion should be selected as far as possible.
(c) The increase of thermal dispersion of groundwater will reduce the pumping temperature of the heat source well. When the thermal dispersion changes from 1m to 3m, the pumping temperature will decrease by about 0.4 ℃. Therefore, it is better to select the place where the groundwater velocity is fast. | 2021-05-11T00:04:38.904Z | 2021-01-07T00:00:00.000 | {
"year": 2021,
"sha1": "71015a80fc1e3b93bf775b574f1c70e256ad5251",
"oa_license": null,
"oa_url": "https://doi.org/10.1088/1755-1315/631/1/012047",
"oa_status": "GOLD",
"pdf_src": "IOP",
"pdf_hash": "c1e77f633dad6a21dd9df486079742ca82d4b9e9",
"s2fieldsofstudy": [
"Engineering",
"Environmental Science"
],
"extfieldsofstudy": [
"Physics",
"Environmental Science"
]
} |
236036537 | pes2o/s2orc | v3-fos-license | Growth rate-dependent flexural rigidity of microtubules influences pattern formation in collective motion
Background Microtubules (MTs) are highly dynamic tubular cytoskeleton filaments that are essential for cellular morphology and intracellular transport. In vivo, the flexural rigidity of MTs can be dynamically regulated depending on their intracellular function. In the in vitro reconstructed MT-motor system, flexural rigidity affects MT gliding behaviors and trajectories. Despite the importance of flexural rigidity for both biological functions and in vitro applications, there is no clear interpretation of the regulation of MT flexural rigidity, and the results of many studies are contradictory. These discrepancies impede our understanding of the regulation of MT flexural rigidity, thereby challenging its precise manipulation. Results Here, plausible explanations for these discrepancies are provided and a new method to evaluate the MT rigidity is developed. Moreover, a new relationship of the dynamic and mechanic of MTs is revealed that MT flexural rigidity decreases through three phases with the growth rate increases, which offers a method of designing MT flexural rigidity by regulating its growth rate. To test the validity of this method, the gliding performances of MTs with different flexural rigidities polymerized at different growth rates are examined. The growth rate-dependent flexural rigidity of MTs is experimentally found to influence the pattern formation in collective motion using gliding motility assay, which is further validated using machine learning. Conclusion Our study establishes a robust quantitative method for measurement and design of MT flexural rigidity to study its influences on MT gliding assays, collective motion, and other biological activities in vitro. The new relationship about the growth rate and rigidity of MTs updates current concepts on the dynamics and mechanics of MTs and provides comparable data for investigating the regulation mechanism of MT rigidity in vivo in the future. Graphic Abstract Supplementary Information The online version contains supplementary material available at 10.1186/s12951-021-00960-y.
Introduction
Microtubule (MT) cytoskeletal filaments play an important role in supporting extended cellular structures, including axons and dendrites, because their flexural rigidity is 2-3 orders of magnitude higher than that of other cytoskeletal filaments, such as actin and intermediate filaments [1,2]. In vivo, the mechanical stiffness of MTs can be dynamically controlled according to their intracellular function [3,4]. In the in vitro reconstructed MT-motor systems, motor-driven MTs can be used as active matter in collective motion or transport of molecular cargoes as nanoscale shuttles or bio-robots [5][6][7][8][9][10]. As an intrinsic mechanical property of MTs, flexural rigidity determines the fluctuation of the free tip of MTs during the gliding assay and affects MT gliding behavior and orientation [10][11][12][13][14].
Owing to the crucial significance of MT rigidity in biological functions and engineering applications, numerous measurements on flexural rigidity or persistence length of MTs have been reported using a variety of measurement methods , such as thermal fluctuations [13,17,19,20,22,26,32], optical tweezers [18,23,28,38], and atomic force microscopy [29]. From these studies, it was found that MT flexural rigidity can be modified by changes in the polymerization conditions, presence of MT-associated proteins (MAPs), and post-translational modification state of the tubulin dimer [40]. However, the reported values of flexural rigidity vary substantially between several studies, as listed in Additional file 1: Table S1. For example, the flexural rigidity of MTs that are polymerized in the presence of guanosine-triphosphate (GTP) and stabilized by paclitaxel, have been reported to vary between 0.03 and 3.2 × 10 −23 N·m 2 -two orders of magnitude in range [14][15][16][17][18][19]. These discrepancies hamper our understanding of the regulatory mechanisms in MT rigidity, thereby challenging the precise manipulation or regulation of MT rigidity and impeding further investigation on the role of flexural rigidity on routine activities, including gliding motility and collective motion.
To address this issue, we developed a new method to evaluate the MT rigidity by improving the conventional measurement methods from two aspects. One is the localization precision of the shape of MTs during image analysis. Most previous studies measured MT flexural rigidity based on the deformation generated by an external force [17][18][19][20][21][22][23][24][25]. The deformation was measured by skeletonizing the MTs captured in fluorescence or differential interference contrast images and determining their positional shifts. The precision of the skeletonization method varies from 0.02 to 1 pixel, depending on the method. Lower precisions cause a larger measurement error in MT deformation. The other aspect is the variable parameters of polymerization that are not reported in published papers, such as the growth rate of the filaments. As the growth rate is sensitive to experimental conditions such as tubulin concentration, protein source, polymerizing temperature, and the binding of MAPs [41][42][43][44][45][46][47][48], the variation in growth rate causes inconsistency in the measured flexural rigidity.
To demonstrate our method, we investigated the effects of localization precision and growth rate on flexural rigidity. We evaluated MT flexural rigidity at low-level and nanometer-level localization precision using simulation analysis. Further, we modified the growth rate of MTs by altering tubulin concentration and measured flexural rigidity using nanometer-level localization precision. A new relationship between the growth rate and flexural rigidity of MTs was revealed that MT rigidity decreases and goes through three regimes with growth rate increases, which enables us to develop a potential quantitative method for designing the flexural rigidity of MTs.
To test the feasibility of this method, we chose the gliding motility assay as a platform to observe how growth ratedependent flexural rigidity influences the collective motion of MTs, as flexural rigidity has been proven to influence the gliding behaviors and trajectories of MTs [10,[12][13][14]. Furthermore, we utilized machine learning to recognize, categorize, and verify the distinctive patterns of softer MTs and stiffer MTs.
Our study established a more accurate method of measuring MT rigidity by highlighting the importance of localization precision and growth rate in the measurement process. Furthermore, we developed a standard quantitative method to study the influences of MT rigidity on their routine behaviors by adjusting the growth rate of MTs.
Reagent and protein preparations
All the reagents were purchased from Sigma-Aldrich, unless stated otherwise. Tubulin was purified from porcine brains after two cycles of polymerization and depolymerization [49], resulting in a final tubulin concentration of 282.7 µM. Recycled tubulin was prepared from tubulin by an additional cycle of polymerization and depolymerization to remove any non-polymerized tubulin. Fluorescently labeled tubulin was prepared by adding 50 M or 15 M excess succinimidyl ester-conjugated tetramethylrhodamine (C-1171, Invitrogen, USA) or Alexa Fluor ™ 488 (A-20000, Invitrogen), respectively, to tubulin. His6-tagged kinesin-1 (1 ~ 465aa) was purified from Escherichia coli using a nickel-nitrilotriacetic acid affinity resin according to a previously described method [50]. The prepared proteins were stored in liquid nitrogen until use.
MT fluctuation model
We developed a model using our previous method to simulate the thermal fluctuations of MTs with specific length (L) and defined flexural rigidity ( κ set ) [10]. MTs that were assumed to be tethered at one end behaved as a cantilever beam in this model. The immobilized end was set as the origin of the orthogonal coordinate system. The shape of the MT-free segment, y(s), was expressed by the superposition of sine, cosine, hyperbolic sine, and hyperbolic cosine waves, W n (s/L) [51].
where, s is the path length from the tethered end along the MT, and q n is 1.875 (n = 1), 4.695 (n = 2), 7.855 (n = 3), and (n − 0.5)π (n ≥ 4) [51]. Equating thermal energy with MT bending energy calculated from MT deformation enables us to derive MT flexural rigidity, κ, with the variance of amplitude in each nth mode, var(a n ) [52].
Here, k B is Boltzmann's constant, T is the absolute temperature under experimental conditions (298 K), and a n is the nth mode amplitude, which follows a Gaussian distribution with a mean of zero and variance of var(a n ). The thermal fluctuation model of MTs was generated by setting L to 5, 10, and 30 µm, and κ set to 0.03, 0.3, and 3 × 10 −23 N m 2 , within the range of previously measured rigidities for GTP-polymerized, paclitaxel-stabilized MTs [14][15][16][17][18][19].
The measured flexural rigidity, κ meas , was calculated from the rounded coordinates at different localization precisions. First, the xy-coordinates of the shape of MTs in the model were rounded to the nearest multiple of the given localization precision: 1 nm (≈ 0.01 pixels), 10 nm (≈ 0.1 pixels), and 100 nm (≈ 1 pixel). For example, y = 236.8 nm was converted to y = 237 nm under a localization precision of 1 nm, y = 240 nm under 10 nm, and y = 200 nm under 100 nm. Thereafter, MT was divided into N + 1 points with the converted coordinates of (x′ k , y′ k ). The n th mode amplitude of the converted shape, a′ n , was Fourier inverse transformed from Eq. (1) and solved using the midpoint method as follows: where, Δs′ k is the length of the infinitesimal segment calculated by and (2) The κ meas was measured under each localization precision by substituting a′ n and L′ into Eq. (3). Flow chambers were constructed by bonding the Au-stripe-patterned SiO 2 substrate (see Additional file 1: Figure S1 for fabrication details) and a coverslip (C218181, Matsunami Glass, Japan) with 10-µm thick double-sided tapes (7070W, Teraoka Seisakusho, Japan). We introduced 2 mg mL −1 streptavidin and incubated thrice for 3 min for non-specific binding on the Au-patterned substrates. After washing the flow chamber with BRB80 (80 mM PIPES, 1 mM EGTA, and 1 mM MgCl 2 ), the partially biotinylated MTs were immobilized via biotin-streptavidin binding by incubation for 5 min (Fig. 1A). Free MTs were washed with BRB80 containing an O 2 scavenger system (8.0 µg mL −1 catalase, 25 mM d-glucose, 20 µg mL −1 glucose oxidase, 1% β-mercaptoethanol, 20 mM DTT, and 20 µM paclitaxel). The flow chamber was then sealed with clear nail polish to prevent drying or leakage.
Measurement of flexural rigidity
MTs were observed under an IX73 inverted epifluorescence microscope (Olympus, Japan) with an excitation filter (GFP/DsRed-A-OMF, Opto-Line International, Inc., USA), a complementary metaloxide-semiconductor camera (ORCA-Flash 4.0 V2, Hamamatsu Photonics, Japan), image-splitting optics (W-VIEW GEMINI, Hamamatsu Photonics) with a bandpass emitter (FF01-512/25-25 and FF01-630/92-25, Semrock, USA), dichroic mirror (FF560-FDi01-25 × 36, Semrock), and oil-immersion objectives. The magnification and exposure time were set to 100 × (NA 1.4) and 100 ms, respectively. To observe MT thermal fluctuation, the frame rate and recording period were set to 2.5 frames s −1 and 200 s, respectively. We used an ND25 filter equipped with a shutter (VMM-D3, Uniblitz, USA). Optical images were stored as sequential image files in TIFF format using the HCImage software 1B and Additional file 2: Movie S1, Additional file 3: Movie S2, Additional file 4: Movie S3, Additional file 5: Movie S4). The MT shapes were determined using the MT tracking software, fluorescence image evaluation software for tracking and analysis (FIESTA) [53] with sub-pixel resolution (0.02 pixels, ~ 2.2 nm) via 2D Gaussian fitting (Additional file 1: Figure S2). Multiple significance tests were performed on all data using Steel-Dwass tests at a critical value of p < 0.05, and normality or log-normality was tested among outlierremoved data using Lilliefors tests at a critical value of p > 0.05. All curve fittings were performed using the leastsquares method in MATLAB (R2019b).
Growth rate measurement using TIRF microscopy
A flow chamber was constructed by bonding two coverslips (C218181 and C024361, Matsunami Glass) with 50-µm thick double-sided tapes (400P50, Kyodo Giken Chemical, Japan). First, 50 μg mL −1 neutravidin was introduced and incubated for 5 min at 25 °C. After washing the channel thoroughly with BRB80, 1% Pluronic solution was introduced into the flow cell for 5 min at 25 °C and washed twice with BRB80.
The partially biotinylated seed MTs were introduced and selectively immobilized onto substrates via biotinneutravidin binding with a 5 min incubation period. After washing the free seed MTs, a mixture of non-labeled and tetramethylrhodamine-labeled tubulin, MgSO 4 , and GTP was introduced into the flow cell. The final tubulin concentration was modified to 20, 30, 35, 40, 50, 75, 100, 150, and 200 μM, similar to the flexural rigidity measurements. The flow cell was sealed with clear nail polish and placed on a 37 °C heater installed on the stage of the microscope.
An EMCCD camera (iXon Ultra 897, Andor Technology, UK) and an inverted microscope (IX 71, Olympus) were used for fluorescence observation of the MT elongation process. Laser light sources (DPGL-2050F, Suwtech, China) with a wavelength of 532 nm were used as the excitation light source. The laser beam path was adjusted such that the excitation light was in the total reflection state. A fluorescent mirror unit (U-MWIG 3, Olympus) was used to separate the excitation light and fluorescence. To measure the elongation rate, 500 fluorescence images were captured with an exposure time of 100 ms, binning 1 × 1, using a 60 × objective lens (APON 60 XOTIRF, Olympus) for total internal reflection fluorescence (TIRF) microscopy. Image data were acquired in the TIFF format using the image acquisition software (Andor iQ 3, Andor). From the obtained sequential fluorescence images, the elongated lengths of the MTs were measured and the elongation rates were calculated using ImageJ (National Institutes of Health). Multiple significance tests were performed on all data using Steel-Dwass tests with a critical value of p < 0.05.
In vitro gliding assay
The coverslips (C218181 and C024361, Matsunami Glass) used for the gliding assay were treated with piranha solution at 60 °C for 10 min, rinsed with deionized water and then dried with nitrogen gas. The central area (5 × 5 mm) of the coverslip was exposed to plasma with the surrounding area covered by tape. Two PIPES-based buffer solutions were used in the assay: (i) casein buffer, with 0.3 mg mL −1 casein dissolved in the BRB80 and (ii) motility buffer, BRB80 with 5 mM ATP, 0.3 wt% methylcellulose, and O 2 scavenger system.
The gliding assay was conducted by introducing MTs onto kinesin molecules with motility buffer (Fig. 5A). First, 2 mg mL −1 streptavidin was introduced and incubated for non-specific binding to the substrate. After washing the chamber with BRB80 solution, biotinconjugated kinesin-1 (0.154 mg mL −1 ) was added and incubated for 5 min and immobilized on the glass via biotin-avidin binding. After washing with casein buffer, MTs and motility buffer were introduced into the chamber. All gliding assays were performed at 25 °C. The gliding motion assays were observed using an inverted epifluorescence microscope (IX73, Olympus) with an excitation filter (GFP/DsRed-A-OMF, Opto-Line International, Inc.) and 60 × oil-immersion objectives. The exposure time was set at 50 ms with a frame rate of 1 frame s −1 and a recording period of 300 s. The addition time of the motility buffer was set as 0 s, and the whole observation time for each assay was 60 min. The captured images were stored as sequential image files in TIFF format using the HCImage software (Hamamatsu Photonics).
MT pattern analysis using machine learning
A deep convolutional neural network (CNN) model including pre-trained ResNet50 top layers, a global average pooling layer, and a final dense layer activated using the Softmax function was constructed to classify the two MTs (Fig. 5B) [54]. We captured 1400 MT pattern images and 700 images each from softer-MT and stiffer-MT groups from 10 independent experiments for the dataset (840 images for the training process, 360 images for the validation process, and 200 images for the test process). All the images were cropped and resized to 224 × 224 pixels as input images for the model. The ground truth of the images was manually labeled. The model was trained by monitoring the validation loss, and the parameters were updated using the Adam optimizer. The model was trained for 20 epochs with a batch size of 32, and a final validation accuracy of 98% was obtained (Additional file 1: Figure S5). The calculated probabilities for each class were used for MT classification, and the sum of the probabilities was 100%. Tensorflow2.0 (www. tenso rflow. org) and Keras API (keras.io) were used during the entire process.
A score class activation map (Score-CAM) was introduced to visually interpret the prediction strategy of the CNN classifier by obtaining the weights of each activation map on the predicted class [55]. For the deep learning model f (), we used input X and output Y: Y = f (X).
The activation of the input X 0 generated in the convolution layer l is indicated as A l , and the kth channel activation of A l is A k l . Further, A k l is extracted, normalized, and upsampled to an input size of M k l . M k l was used as a mask to be projected onto the original image X 0 . For a given baseline X b , the contribution S c k of A k l to the output class c is calculated as follows [55]: where, • indicates the element-wise product and α c k is the corresponding weight of A k l to the target class c.
The heatmap L c score−CAM is generated by the linear combination of activation maps A k l and their weights α c k on target class c.
Here, the activation function ReLU was used to select the features with positive importance scores.
Flexural rigidity of MTs under different localization precisions
The flexural rigidity of MTs was measured from the rounded coordinates at different localization precisions (Fig. 1). Figure 1C-E show the converted shapes of MTs under the localization precision of 1, 10, and 100 nm with MT length L = 10 µm and defined flexural rigidity κ set = 0.3 × 10 −23 N m 2 . The shapes in 500 frames were superimposed with different colors. Under low localization precision, the rounded MT shapes were discontinuous and substantially deviated from the original shapes. The shape and approximate value of κ for an MT of L = 10 µm and κ set = 0.3 × 10 −23 N m 2 was calculated using Eq. (1), as shown in Fig. 2A, B. The shapes of MTs are represented by the blue line, whereas the fitted curves are indicated by the red lines and red dots with localization precision of (A) 1 nm and (B) 100 nm. As the localization precision decreases, the thermal fluctuation of MTs cannot be traced precisely, and the accuracy of the approximation drops remarkably. The discontinuous shape under 100 nm localization precision cannot be represented well by Eq. (1), thereby leading to a significantly larger fitting error than that under 1 nm localization precision. Given that k B T is constant at a specific temperature, the flexural rigidity, κ, can be derived from the slope between q n 4 and L 4 / var (a n ) through Eq. (3) [52]. Figure 2C and D show the relationship between q n 4 and L 4 /var (a n ) with localization precision of (C) 1 nm and (D) 100 nm on a logarithmic graph. When the localization precision is high, q n 4 and L 4 /var (a n ) are linearly related regardless of the number of modes (Fig. 2C). However, when the localization precision is low, q n 4 and L 4 /var (a n ) maintain a linear relationship only in the low-order mode (n ≤ 4) (Fig. 2D). This is because thermal fluctuation cannot be precisely tracked in the higher-order (q n ) mode, in which the deformation of the shape of MTs is smaller than the localization precision.
Nanometre-level localization precision eliminates the measurement error
The relative measurement error of κ due to the digitization of the shape of MTs was determined from 1-κ meas /κ set . Figure 3 shows the relative measurement error of flexural rigidity under each condition of L (5, 10, and 30 μm), κ set (0.03, 0.3, 3 × 10 −23 N·m 2 ), and localization precision of (A) 1 nm, (B) 10 nm, and (C) 100 nm. The measurement error was found to be affected by the localization precision, MT length, L, and the defined flexural rigidity, κ set . Lower localization precision, smaller contour lengths, and larger κ set will all result in larger measurement errors. Under low localization precision (10 and 100 nm), the flexural rigidity is largely underestimated, especially for short and/or stiff MTs, by overestimating thermal fluctuations.
These results help explain previous discrepancies reported in the literature. For instance, Venier et al. [24] reported a three-fold smaller κ than Valdman et al. [35]. Both studies adopted almost the same conditions (tubulin concentration, nucleotide, MT length, and paclitaxel stabilization) but different localization precision for determining MT shape (1 pixel for Venier et al. [24] and 0.1 pixels for Valdman et al. [35]). As indicated in Fig. 3C, the pixel-level localization precision of Venier et al. [24] may have caused the underestimation of κ. The same can be said for the difference in reported κ between Hawkins et al. [21] and Lopez et al. [36]. Although most of the conditions were the same, Hawkins et al. [21] skeletonized the MT image with 1-pixel localization precision and reported a 3.5-fold smaller κ than Lopez et al. [36] with 0.1-pixel localization precision.
Our previous studies have shown that MT flexural rigidity is independent of L [10]; however, the current results on simulated filaments indicate that the contour length, L, influences the measurement accuracy of flexural rigidity. Venier et al. [24] and Mickey and Howard [17] used similar polymerization conditions and pixellevel localization precision, but different lengths of the MTs in each study. Venier et al. [24] analyzed shorter MTs (8-15 µm) than Mickey and Howard [17] (24-68 µm) and obtained a 2.8-fold smaller κ. This is possible because the small deflections of short MTs caused an underestimation of κ (Fig. 3C).
The results also indicate that the flexural rigidities of MTs with a large κ are more likely to be underestimated, especially at low localization precision. Mickey and Howard [17] reported that κ increased about 1.3 times with tau protein bound. Whereas, Felgner et al. [18] used a similar ratio of tau to tubulin and reported that the κ increased approximately 2.5 times when measured using optical tweezers. The discrepancy in the increase of κ between the two groups is probably caused by the underestimation of κ due to the low pixel-level localization precision used by Mickey and Howard [17]. In addition, Cassimeris et al. [19] co-polymerized XMAP215, another type of MAPs, with tubulin and did not find any change in κ under pixel-level localization precision. This could be caused due to a very low precision that hinders the detection of changes in κ because of the larger measurement error. Thus, it would be interesting to measure the effects of XMAP215 with improved localization precision. It is noteworthy that, when the localization precision is at 1 nm, the relative measurement error is less than 5% regardless of the value of L or κ set (Fig. 3A and Additional file 1: Figure S3A). Thus, the measurement error caused by L and κ can be significantly reduced by improving the localization precision. Therefore, nanometre-level localization precision is indispensable for the precise and accurate measurement of flexural rigidity for MTs of various lengths and stiffnesses. Further, by improving the localization precision to the nanometre level, it is possible to obtain new findings by reinvestigating the influences of these factors on κ.
Dependence of MT flexural rigidity on the growth rate
The measured values of flexural rigidity and growth rates of MTs polymerizing with tubulin concentrations of 20-200 µM are summarized in Additional file 1: Table S2. The relationship between the growth rate and flexural rigidity of the MT is shown in Fig. 4B. The growth rate was found to significantly increase with higher tubulin concentrations within the range of 30-150 µM. The slope of the growth rate was 0.1 µm min −1 µM −1 , which is approximately tenfold faster at 150 µM than that at 30 µM. No significant differences in growth rates were observed between 20 µM and 30 µM as well as between 150 µM and 200 µM using the Steel-Dwass test (p > 0.05, Additional file 1: Table S3). Therefore, the whole variation process of growth rate could be divided into three phases according to their statistical differences (Additional file 1: Table S3), with tubulin concentration values of ≤ 30 μM, 30-100 μM, and > 100 μM, which are illustrated by a red, yellow, and blue background, respectively (Fig. 4B).
Meanwhile, flexural rigidity was found to be dependent on the growth rate. When the growth rate was less than ~ 1.4 μm min −1 , that is, the tubulin concentration was lower than 30 μM, the flexural rigidity remained similar to that observed between 20 and 30 μM (no statistical significance of MT rigidity at 20 and 30 µM was detected, Additional file 1: Table S4). Once the MTs elongate faster than 1.4 μm min −1 , the flexural rigidity tends to decrease as the growth rate increases. At a growth rate of ~ 10.1 µm min −1 (tubulin concentration higher than 100 μM), the flexural rigidity becomes independent of the growth rate.
The dependence of the flexural rigidity on the growth rate can be explained by the number of defects generated and repaired during polymerization. As reported by Schaedel et al. [56], faster growth tended to generate lattice defects on MTs, which reduced the MT flexural rigidity. We found that when the tubulin concentration was too low to facilitate fast MT polymerization (20-30 µM), MTs show high bending stiffness, most likely due to the formation of fewer lattice defects. At a low growth rate, MTs repair their tubulin defects while elongating, thereby resulting in higher flexural rigidity with fewer defects [56].
Fast growth of MTs can result in accumulation of defects that cannot be repaired. Thus, in this regime (30-100 μM), higher number of defects in MTs will result in softer filaments. MT rigidity was found to decline monotonically as the growth rate increased in the region (Fig. 4B), which is consistent with the results of Janson and Dogterom [25,26].
As the tubulin concentration during polymerization was increased to over 100 µM, the growth rate increased and then reached the upper limit value (no significant differences in the growth rate at 150 and 200 µM were observed in Additional file 1: Table S3). Correspondingly, no change in rigidity was observed (Fig. 4B, no significant difference of MT rigidities at 100, 150, and 200 µM were detected in Additional file 1: Table S4), which indicates that there should be a certain upper limit number of lattice defects accumulating on MTs. Once the quantity of tubulin defects exceeds a certain threshold, fatal structural damages will occur to break MT or cause MT depolymerization. As a result, when MTs grow fast to exceed the upper threshold value (~ 10.1 µm min −1 ), the number of tubulin defects of the surviving MTs remains constant at the upper limit. Therefore, no additional tubulin defects would be generated, the flexural rigidity of MTs Results from previous studies display discrepancies in their reported rigidity values owing to the different polymerization speeds. For instance, Valdman et al. [35] and Lopez et al. [36] performed experiments using the same measurement and conditions (localization precision, nucleotide, MT length, and paclitaxel stabilization) except for tubulin concentration (Valdman et al. [35], 20 μM; Lopez et al. [36], 50 μM). These publications were from the same group, and therefore, there is a likelihood that the tubulin type was identical. However, the measured κ reported by Valdman et al. was approximately 1.4fold larger than that reported by Lopez et al. Although the growth rates were not quantified or reported, it is conceivable that the growth rate observed by Valdman et al. [35] would be smaller than that observed by Lopez et al. [36] because of the difference in the tubulin concentrations. Therefore, we highly recommend future measurements of flexural rigidity to include a measurement of the growth rate for the tubulin polymerization methods used in this study.
Furthermore, our results reveal the relation between MT growth rate and rigidity with a wider range of tubulin concentration (20-200 µM) than that in previous studies (4.5-28 µM) [25,26,57]. For instance, the growth rate and flexural rigidity of MTs polymerizing with a higher tubulin concentration (> 100 µM, blue area in Table S2), respectively, which corroborate the reported data of MTs in vivo (growth rate of 12-24 µm min −1 and flexural rigidity of 0.3-0.4 × 10 −23 N·m 2 ) [58,59]. Interestingly, although the reported tubulin concentration in cells (4-24 µM) is much lower than that used here (> 100 µM), growth rates of MTs in cells are still comparable with or even faster than that observed in our results [60,61]. The effect of tubulin concentration on the growth of MTs could be compensated by various kinds of MAPs in cells, such as the plus-end tracking proteins, CLASP, CLIP-170, XMAP215, and EB1 [4,62]. However, the influences of these MAPs on MT rigidity are still unclear. Combined with our results, further experiments on the relationship between growth rate and rigidity in the presence of MAPs will induce an agreement in understanding the factors that influence rigidity in vivo.
Moreover, our results provide a quantitative link between growth rate and flexural rigidity for bare MTs, which suggests a potential method for designing flexural rigidity of MTs in vitro. Flexural rigidity is an important parameter that affects the direction of MT gliding. MTs modified with different flexural rigidities have been widely applied in nano-and bioengineering applications, including molecular shuttles, active self-organization, and collective motion [8][9][10]. Although the flexural rigidity of MTs can be modified by various MAPs, nucleotides, and MT-stabilizing reagents [16-21, 25-27, 36], they are also known to influence the molecular structure and biophysical functions of MTs [4,36,62,63]. For instance, Janson and Dogterom [25,26] polymerized MTs with/without oxygen-scavenging system (4 mM dithiothreitol, 0.2 mg ml −1 catalase, 0.4 mg ml −1 glucose oxidase, and 50 mM glucose) and found the oxygen-scavenger may promote the polymerization speed of MTs from 1.5 to 2.72 µm min −1 and soften MTs from 2.73 to 1.74 × 10 −23 N·m 2 (Additional file 1: Table S1). Their studies suggested that regulating growth rate could become a potential method to control MT rigidity, however, no specific implementable scheme to realize this idea so far. Here, the newfound relationship of growth rate and MT rigidity controlled by tubulin concentration addressed this issue. Compared with these conventional modification methods [16-21, 25-27, 36], our growth rate-dependent method is controlled solely by the concentration of tubulin. Therefore, the method not only simplifies the preparation of MTs, but also avoids uncertain influences on MTs, which will widen the applicability of the flexural rigidity for engineering applications.
Growth rate-dependent flexural rigidity influences the collective motion in the MT motility assay
Two groups of MTs were used to perform the MT gliding assays. Softer-MTs were polymerized at 100 μM tubulin concentration with the growth rate of 10.10 ± 0.46 μm min −1 and κ of 0.27 ± 0.10 × 10 −23 N·m 2 , whereas stiffer-MTs were polymerized at 30 μM tubulin concentration with the growth rate of 1.37 ± 0.07 μm min −1 and κ of 0.80 ± 0.34 × 10 −23 N·m 2 ( Fig. 4B and Additional file 1: Table S2).
Distinct patterns of collective motion emerged in the two groups of MTs during the gliding assays. Initially, MTs in both groups were uniformly distributed with random orientations, which could be treated as an isotropic phase (0 min, Fig. 5C). Next, the gliding MTs continually collided with their neighboring MTs in the presence of a depletion force induced by methylcellulose. The gliding directions and orientations of the MTs were tuned by frequent collisions. The collective motion gradually arose in both groups and formed stable nematic patterns after 50 min. Softer MTs tended to form local steam, whereas stiffer MTs formed stable bundles (50 min, Fig. 5C).
MT patterns formed (at 50 min) by the two MT groups were categorized into two classes, softer-MTs and stiffer-MTs, using the trained deep CNN classifier. The 200 MT pattern images were categorized using the trained CNN classifier with an accuracy of 96.5% (100% accuracy for softer-MTs and 93% accuracy for stiffer-MTs) (Fig. 5D). The types of MTs could be predicted and sorted into their corresponding classes according to their patterns, which suggested certain general differences in patterns between the two groups.
The differences in patterns between the two groups were revealed by analyzing the classification strategy of the CNN classifier. Score-CAM was introduced to visually explain how the classifier categorized the MT patterns into the two groups (Fig. 5E) [55]. The more important the area or feature in the image for making the final categorization decision by the classifier, the higher the score gained and the stronger the responses in the CAM. For the images sorted into softer-MT groups, a uniformly high activation responded and overlaid the entire image area, which indicated that a large-scale local stream with a relatively even distribution was detected (Fig. 5E). In contrast, for the images sorted into the stiffer-MT group, only the bundle area was highly activated in the heatmap, which indicated that a distinguished uneven structure (MT bundle) was identified (Fig. 5E). The differences in the Score-CAM patterns between the two groups were in accordance with our observed results.
The collective motion of MTs has been proposed to emerge due to a depletion force between filaments, which reduces filament crossover, promotes alignment, and leads to a transition of filaments from an active, isotropic state to locally aligned polar patterns [64]. According to the Vicsek model, identical objects, including particles or filaments moving at a constant speed, tend to interact locally by aligning with neighbors [65]. A similar opinion was proposed that self-propelled objects, such as gilding MTs, show progressively larger probabilities to align with their neighbors [66]. Recently, an important supplement to these hypotheses stated that MTs align and are actively transferred from their original bundle to another antialigned bundle [67]. All the theories indicated that alignment is a critical step in the formation of collective motion, and the formation of MT bundles is a dynamic process. As an apparent and key factor affecting the gliding and alignment behavior of the filaments, MT flexural rigidity should directly influence the phase transition process of collective motion.
The two groups of MTs enable us to infer that MTs with different κ perform differently on the occasions of collision, alignment, and diffusive separation. Compared with the stiffer-MTs, the softer-MTs are more flexible in changing their gliding directions. Although the bundles of softer MTs were formed by the depletion force, the MTs inside the bundle could be easily dispersed in the next collision or disturbed by another anti-aligned bundle. In contrast, the bundles formed by stiffer-MTs are long-standing and tend to merge with neighboring bundles, which results in a relatively large bundle. Moreover, the performance differentials of defined softer MTs and stiffer-MTs demonstrated that the method for designing flexural rigidity is feasible.
Conclusions
In summary, an accurate method of measuring MT flexural rigidity was developed by improving the localization precision up to nanometer scale. Based on this improved methodology, we further discovered that flexural rigidity is directly affected by growth rate and goes through three phases among a wide range of tubulin concentrations (20-200 μM), which revealed a new relationship between dynamics and mechanics of MTs and deepened the understanding of the regulatory mechanism of MT flexural rigidity in vivo. These findings not only offer reasonable explanations for long-standing discrepancies in previously measured flexural rigidities, but also provide a convenient method for the production of MTs with predefined stiffness.
Consequently, as shown with the example of MT collective motion, the proposed method would promote the development of potential nanobiotechnology applications by eliminating methodological inconsistencies among assays and providing a controllable experimental environment. Additionally, owing to the simplicity and ease of manipulation, the method for designing flexural rigidity of MTs by altering tubulin concentrations will be a useful quantitative tool to investigate the biological functions of MT rigidity or study the influences of MT flexural rigidity on their routine behaviors in vitro, including gliding motility assays, collective motion, and other biological activities.
Besides tubulin concentration, the regulations of the dynamics and mechanics of MTs in vivo involve many other factors, including MAPs and post-translational modifications. Our results revealed a new quantitative relation between the growth rate and flexural rigidity of MTs by solely modifying the tubulin concentration. It provides comprehensive and comparable data of growth rate and MT rigidity with that in vivo and can be a datum reference for investigating the regulation mechanism of MT mechanics and the functions of specific MAP in the future. | 2021-07-19T13:39:38.260Z | 2021-07-19T00:00:00.000 | {
"year": 2021,
"sha1": "c83d5f2f183a37533b98bfa2c8d719ae0fc20be7",
"oa_license": "CCBY",
"oa_url": "https://jnanobiotechnology.biomedcentral.com/track/pdf/10.1186/s12951-021-00960-y",
"oa_status": "GOLD",
"pdf_src": "PubMedCentral",
"pdf_hash": "c83d5f2f183a37533b98bfa2c8d719ae0fc20be7",
"s2fieldsofstudy": [
"Biology"
],
"extfieldsofstudy": [
"Medicine"
]
} |
237934225 | pes2o/s2orc | v3-fos-license | Use of Sertraline in Hemodialysis Patients
Depression and anxiety are the most common psychiatric disorders in end-stage renal disease (ESRD) patients treated with hemodialysis (HD) and may correlate with lower quality of life and increased mortality. Depression treatment in HD patients is still a challenge both for nephrologists and psychiatrists. The possible treatment of depressive disorders can be pharmacological and non-pharmacological. In our article, we focus on the use of sertraline, the medication which seems to be relatively safe and efficient in the abovementioned population, taking under consideration several limitations regarding the use of other selective serotonin reuptake inhibitors (SSRIs). In our paper, we discuss different aspects of sertraline use, taking into consideration possible benefits and side effects of drug administration like impact on QTc (corrected QT interval) prolongation, intradialytic hypotension (IDH), chronic kidney disease-associated pruritus (CKD-aP), bleeding, sexual functions, inflammation, or fracture risk. Before administering the medication, one should consider benefits and possible side effects, which are particularly significant in the treatment of ESRD patients; this could help to optimize clinical outcomes. Sertraline seems to be safe in the HD population when provided in proper doses. However, we still need more studies in this field since the ones performed so far were usually based on small samples and lacked placebo control.
Introduction
Depression and anxiety are the most common psychiatric disorders in end-stage renal disease (ESRD) patients treated with hemodialysis (HD). Their prevalence is increasing in recent years [1], reaching the rate of 20-40% for depression [2,3] and 20-52% for anxiety [4], depending on the methodological approach. According to the studies, these psychiatric disorders may correlate with lower quality of life, increased hospitalization rate, suicidal behavior, hemodialysis nonadherence, and increased mortality [5]. Despite the number of studies on this issue, major depressive disorder (MDD) in ESRD patients is still underdiagnosed, and its treatment is not optimal [6] [ Table 1]. Anxiety disorders seem to be overlooked even more often [7], which is probably associated with a significant overlapping of symptoms with depression [8]. As initiation of dialysis is a large change in everyday functioning with a potential to induce or worsen psychiatric symptoms, screening for depression [9] and anxiety is suggested at the beginning of renal replacement treatment.
The possible treatment of depressive and anxiety disorders can be pharmacological and non-pharmacological. The first choice drugs for both of the above are selective serotonin reuptake inhibitors (SSRIs), including citalopram, escitalopram, fluoxetine, fluvoxamine, paroxetine, and sertraline Table 2 [10,11]. While their administration is widely considered to be safe and effective in ESRD patients, further studies are still needed in this field [12]. Non-pharmacological interventions may include psychotherapy, mindfulness meditation, and frequent HD [13].
In our article, we focus on the use of sertraline due to several limitations regarding the use of other SSRIs in ESRD patients. Fluoxetine and fluvoxamine are considered to be less efficacious [14]. Moreover, fluvoxamine has a significant impact on liver metabolism of other drugs. Administering citalopram and escitalopram bears the risk of increasing QTc interval in doses larger than 20 mg and 10 mg, respectively. These doses have been set as maximum for elderly patients [15,16], limiting therapeutical options with these drugs in many ESRD patients. While there have recently been voices raising doubts about decisions limiting the use of citalopram and escitalopram [17], these restrictions still stand. In view of the above, sertraline may seem like a relatively safe and efficient medication. However, before administering, one should consider several side effects particularly significant in the treatment of ESRD patients. These adverse effects, along with specific benefits, are discussed below in Table 3 and and Figure 1. Table 1. Comparison of depression in ICD-10 and DSM-5 classifications.
Nomenclature
Depressive Episode [18] Major depressive disorder (MDD) [19] Main symptoms • Depressed mood to the degree that is definitely abnormal for the individual, present for most of the day and almost every day, largely uninfluenced by circumstances • Loss of interest or pleasure in activities that are normally pleasurable • Decreased energy or increased fatiguability [18] • Depressed mood most of the day, nearly every day, as indicated in the subjective report or in observation made by others Diagnostic criteria At least two main symptoms and additional symptoms in a total number of at least four [18] At least one main symptom and additional symptoms in a total number of at least Duration of symptoms At least two weeks [18] At least two weeks [19] Severity of symptoms Clinical differentiation: • Mild Depressive Episode (at least two main symptoms and additional symptoms with a total number of at least four) • Moderate Depressive Episode (at least two main symptoms and additional symptoms with a total number of at least six) • Severe Depressive Episode (all three main symptoms and additional symptoms with a total number of at least eight) [18] The symptoms ought to cause significant impairment in social, occupational or another important area of functioning [19] SARI-serotonin receptors antagonist with serotonin reuptake inhibition; SMS-serotonin modulator and stimulator; SNRI-serotoninnorepinephrine reuptake inhibitors; SNRISA-serotonin-norepinephrine reuptake inhibitor and serotonin receptors antagonism antidepressant with potent antipsychotic D2 receptor blockade/antagonism; TCA (tricyclic antidepressants). Table 3. Effects of sertraline use in hemodialysis patients.
Sertraline-General Properties of the Medication
Studies evaluating sertraline's safety and efficacy in ESRD patients performed so far usually had small samples and lacked placebo control [39]; therefore, in further parts of this article, references are also made to data from the general population. The European
Sertraline-General Properties of the Medication
Studies evaluating sertraline's safety and efficacy in ESRD patients performed so far usually had small samples and lacked placebo control [39]; therefore, in further parts of this article, references are also made to data from the general population. The European Renal Best Practice (ERBP) guidelines suggest that trials with SSRIs in patients who meet criteria for moderate Major Depressive Disorder (MDD) should last for 8-12 weeks, after which the effect of the treatment should be evaluated [43]. This is rather inconsistent with the psychiatric clinical practice when the first evaluation for depressive and anxiety patients is usually done after 4 weeks with considerations of temporizing, increasing the dose, switching to another antidepressant, or combining several drugs in patients with minimal response or safety issues [44,45].
Before starting the treatment with sertraline, no specific medical tests are required [46]. To minimize side effects, psychiatrists usually start with a subtherapeutic dose of 25 mg for one week with an increase to 50 mg after this period. These means of precaution should be especially considered in patients with coexisting anxiety, in advanced age, and those more susceptible to side effects. While doses up to 300 mg have been used, the maximum dose is considered to be 200 mg. ERBP guidelines do not suggest any need to change the dosing in stages 3-5 of chronic kidney disease (CKD), but some studies suggest that smaller doses of sertraline may be required in ESRD patients, yet the post-hemodialysis supplementation is suggested to be unnecessary [47]. The effect of the treatment can be usually seen after 4-6 weeks. The drug should be taken daily. While most psychiatrists and patients prefer it to be taken in the morning, some patients may experience sedation, and an evening dosage would be more appropriate. The general rule is that sertraline should be taken every day in the same regimen (time of day, dose splitting, with or without food). Due to a fairly long half-life time (66 h in the general population when including its less active metabolite, norsetraline) [48], missing a single dose should not result in any withdrawal side effects. This is also the reason why this drug is relatively easy to discontinue. Sertraline is a moderate inhibitor of CYP2B6 and CYP2D6 with a dose-dependent effect of increasing other drugs metabolized through these pathways [49]. Effect on other CYP450 subunits is not clinically significant, and its interactions are less potent than these of fluoxetine, fluvoxamine, or paroxetine.
Sertraline is considered to be safe to use. The adverse effects that seem to be more common while using sertraline compared to other SSRIs are diarrhea and activation [50]. In some cases, drowsiness, headache, or sexual dysfunctions may appear [50]. One should also keep in mind the antiplatelet effect of sertraline, which, while in some cases beneficial [51], may be an issue in patients with greater bleeding risk [52]. Modification of antidepressant treatment needs to be considered in some patients [53]. Regularly observed practice is also an adjustment of antiplatelet therapy dose (for instance, by reducing acetylsalicylic acid dose from 150 mg to 75 mg) after adding sertraline. This course of action, however, has no support in current literature.
Adverse effects during sertraline administration are usually mild and can be alleviated by changing the treatment regimen (lowering the dose, changing the time of administration, splitting the dose into BID (two times a day) or TID (three times a day), adding low doses of trazodone for sexual dysfunctions or insomnia, etc.) [54,55].
The general rule during antidepressant treatment is that we should struggle to achieve full remission and continue administration of the drug for the maintenance period of at least 6 months before any attempts of down-titrating or discontinuing the medication [56].
Impact on Cardiovascular System
The relation between chronic kidney disease (CKD) and cardiovascular disease is complicated and bidirectional. On the one hand presence of CKD is the risk factor for developing cardiovascular disease (CVD). In the meta-analysis that enrolled more than 100.000 participants, reduction in both eGFR and albuminuria was associated with allcause mortality and cardiovascular mortality independently of each other and traditional cardiovascular risk factors [57]. On the other hand, cardiovascular disease remains the major cause of morbidity and mortality in patients with end-stage renal disease (ESRD). In general, the prevalence of cardiovascular diseases among ESRD patients who received HD was 70.6%, being also high (>50%) among younger patients between 22 and 44 years of age, according to US Renal Data System 2018 [58]. Besides the existence of traditional coronary artery disease (CAD) risk factors, such as diabetes and hypertension, patients with CKD are also exposed to other non-traditional, uremia-related cardiovascular disease risk factors, including inflammation, oxidative stress, and abnormal calcium-phosphorus metabolism. HD patients can be asymptomatic or manifest atypical symptoms of a chronic coronary syndrome (CCS), mimicking symptoms of fluid overload or intradialytic hypotension (IDH). Furthermore, patients undergoing dialysis due to CKD were frequently excluded from coronary artery evidence-generating clinical trials [59], which can also influence outcomes in this population. Taking into account the abovementioned limitations, the proper diagnosis and therapy of ESRD patients with CVD can be limited both by the safety issues and the insufficient data. Depression is associated with increased disease burden and with a higher risk of all-cause mortality and cardiovascular mortality worldwide [60,61]. The metaanalysis that included a total of 293 studies with 1,813,733 participants from 35 countries confirmed the presence of association between depression and excess mortality [60].
The effect of antidepressant treatment on the cardiovascular system may vary depending on the agent used and type of disease. SSRIs can cause side effects such as QTc prolongation [62] or bleeding [63], but they can also have some desired effects like reduced platelet reactivity [23,24], Table 3. SSRIs are relatively safe and are the first-line therapy for patients with heart failure [64], although the efficacy of antidepressant therapy on the outcomes of patients with HF is controversial [65]. SSRIs are also the first-line treatment for pharmacological management of MDD in CAD patients since they do not have the cardiotoxic adverse effects of tricyclic antidepressants [66]. Due to its low risk of drug-drug interactions, adverse effect profile, and potential for beneficial antiplatelet activity, sertraline could be considered the first-line choice antidepressant for patients with CAD [67].
Patients dependent on maintenance hemodialysis are at high risk of arrhythmia due to the comorbidities, high level of polypharmacy, and electrocyte disturbances. SSRIs can cause prolongation of QTc interval, with citalopram and escitalopram having the greatest impact [62,68] with the effect increasing with the dose [69]. Other SSRIs, including sertraline, are associated with QTc prolongation but seem to be safe when provided in the recommended doses (Table 3). In the retrospective cohort study, Assimon et al. compared two groups of SSRIs: those with higher potential for prolonging the QT interval and those with lower potential (citalopram and escitalopram versus fluoxetine, fluvoxamine, paroxetine, and sertraline, respectively). The authors observed that the initiation of a higher versus lower QT-prolonging-potential SSRI was associated with a higher risk of sudden cardiac death in a cohort of patients on hemodialysis. Elderly patients, females, patients with conduction disorders, and those treated with other non-SSRI QTc-prolonging medications were the most endangered ones [20]. The results emphasize the importance of individualization of the therapy. More studies are still needed to clearly understand the safety profile of SSRIs in the dialysis population.
The hemostatic disorders in the ESRD population are complex, and patients can experience two opposite complications: bleeding diathesis and thrombotic tendency. One of the mechanisms causing the bleeding diathesis is platelet dysfunction and impaired platelet-vessel wall interaction. Dialysis reduces the risk of hemorrhage by improving platelet abnormalities induced by toxins, but it can also contribute to bleeding by the platelet activation during the hemodialysis sessions [70]. Antidepressants with potent serotonin reuptake inhibitor (SRI) activity increase the risk of bleeding through different mechanisms, and the upper gastrointestinal (GI) tract is the commonest site of SRI-related abnormal bleeding [63]. The meta-analysis by Jiang et al. that included a total of 22 studies (6 cohort and 16 case-control studies) involving more than 1,073,000 individuals revealed that SSRI use was associated with an almost 2-fold increase in the risk of developing upper GI bleeding, especially among patients at high risk for GI bleeding (concurrent use of nonsteroidal anti-inflammatory or antiplatelet drugs) [21]. CKD is associated with increased risk of bleeding in both operative and non-operative patients [25,71]. The large populationbased study by Iwagami et al. demonstrated that while the relative risk of GI bleeding associated with SSRI exposure was constant, the excess risk of GI bleeding associated with SSRIs markedly increased among patients with decreased kidney function [22], Table 3.
On the other hand, one of the potential mechanisms contributing to excessive CV (Cardiovascular) risk is increased platelet reactivity [72] and excessive stickiness of endothelial cells to platelets [70] in both CKD [72] and MDD populations [73]. Some data suggest that treatment of MDD with SSRI may reduce platelet aggregation and activation markers [23][24][25]. In the recently published, randomized, double-blind trial, Jain et al. analyzed the whole blood platelet aggregation (WBPA), plasma levels of E-selectin and P-selectin on treatment with sertraline vs. placebo. The study group consisted of 175 participants with CKD, and the observation period lasted for 12 weeks. The results revealed the increased adenosine diphosphate (ADP)-induced platelet aggregability in CKD patients compared to controls with normal kidney function, regardless of the presence of comorbid MDD. Treatment with sertraline did also not affect platelet function. These findings suggest that increased platelet activation may not be a major pathological mechanism by which depression leads to worse cardiovascular outcomes in patients with CKD. Future studies should include positive MDD controls without CKD to confirm these findings [74].
Intradialytic Hypotension
Intradialytic hypotension (IDH) is a common complication of hemodialysis (HD), which worsens the patient's outcome. It is reported to occur in about 20% of HD sessions [75,76]. The definition of IDH is not standardized and differs between various sources. According to the National Kidney Foundation Disease Outcomes and Quality Initiative (KDOQI), IDH is a drop in systolic blood pressure (SBP) or mean arterial pressure (MAP) associated with symptoms such as vertigo, weakness, abdominal or chest pain, muscle cramps, nausea, and paleness [77]. On the other hand, Flythe et al. analyzed various definitions of IDH. They showed that an absolute nadir SBP < 90 mmHg or <100 mmHg (depending on pre-HD SBP), regardless of the symptoms, was most potently associated with mortality [78]. Thus, nadir-SBP based definitions are gaining popularity in the literature [79].
IDH is proved to be an independent risk factor for mortality in HD patients [80]. HDinduced myocardial stunning may contribute to a higher prevalence of cardiovascular events in this population [81]. IDH may also have negative effects on other organs, such the brain and the GI tract [79,82].
Pathophysiological mechanisms involved in IDH development are complex. In general, hypotension during HD occurs when compensatory systems controlled by sympathetic stimulation (such as cardiac output, plasma refill, and peripheral vascular resistance) are unable to equal decreased effective plasma volume due to hemofiltration and decreased extracellular osmolality associated with sodium removal [79,82].
One of several pharmacological agents, which have been tested in the prevention of IDH, is sertraline. This SSRI has improved symptoms in refractory neurocardiogenic syncope [83]. Both of these conditions seem to have a similar pathogenic mechanism, which is the paradoxical sympathetic vasoconstrictors withdrawal owing to rapid serotonin release in the central nervous system. It is postulated that sertraline may induce down-regulation of postsynaptic serotonin receptors through intensification of serotonergic transmission and thereby moderate the potential response to the sudden increase in cerebral serotonin levels in response to hypovolemic stress [83].
Molin et al. conducted a study in which they examined the efficacy of sertraline in the prevention of IDH. The analyzed group consisted of 16 hemodialysis participants who were observed for 12 weeks. They described dialysis-induced hypotension as a drop of SBP ≥ 30 mmHg during the procedure or pre-hemodialysis SBP ≤ 100 mmHg accompanied by symptoms: weakness, cramps, dizziness, headache, blurred vision, nausea, vomiting, and malaise; any SBP ≤ 90 mmHg and/or DBP ≤ 40 mmHg regardless the symptoms; or any patient with symptoms listed above who required nursing intervention. Participants enrolled on the study had IDH in at least 50% of dialysis sessions during a 3-month period preceding SSRI use. Comparison of IDH prevalence between sertraline and placebo groups revealed no statistical difference. Nevertheless, the authors showed that the risk of reporting intradialytic symptoms was 42% higher in the placebo group [26]. However, as Georgianos and Agarwal commented in their paper, the study of Molin and colleagues may suffer from some methodological limitations, such as a small number of participants, single-blind, and no cross-over design. Furthermore, they conclude that sertraline, as a mood-enhancing agent, may be beneficial to reduce the number of uncomfortable intradialytic symptoms and nursing interventions [84].
Another clinical trial assessing sertraline in the prevention of IDH was designed by Razeghi and colleagues as a randomized, double-blind, cross-over prospective study. Inclusion criteria were similar to those mentioned in the previous paragraph. The study group consisted of 12 participants who were observed for 12 weeks total (4 weeks of sertraline, 4 weeks of washout period, and 4 weeks of placebo). They showed that sertraline therapy significantly reduced the risk of hypotension episodes by 43% and increased postdialysis systolic and diastolic BP by 8.7 mmHg and 6.0 mmHg, respectively, compared with the placebo group. Moreover, it decreased the total number of IDH interventions (although not significantly). The researchers pointed out that the positive effect of sertraline in the prevention of IDH was only seen in patients without diabetes mellitus (DM) [27].
Yalcin et al. designed two studies testing sertraline in hemodialysis patients with IDH. The first one was performed on nine patients who had been suffering from IDH (defined as at least 30 mmHg drop in SBP or SBP < 100 mmHg accompanied with symptoms) for at least 3 months. No placebo control group was included. They compared the data of the 4-week pre-sertraline period with a 4-week sertraline period. The nadir SBP and nadir DBP during hemodialysis were significantly higher, while the number of therapeutic interventions declined significantly during the sertraline part [28]. This pilot study was followed by a placebo-controlled, prospective study on 30 patients. The exclusion criteria included DM, amyloidosis, and structural heart disease in order to avoid autonomic dysfunction. The results were analogous to the pilot study [29], Table 3.
On the contrary, Brewster and colleagues did not prove any positive effect of sertraline in the prevention of IDH. A total of 18 patients enrolled on their prospective, cross-over study had IDH defined as at least three episodes of decreased SBP associated with symptoms in 50% of HD sessions over a 1-month period. Nadir SBP and DBP during HD, as well as MAP, did not differ significantly between the control phase and the sertraline phase of the study. Among possible explanations of their results, the authors enumerate population with resistant IDH enrolled on their study, the insufficient dose of sertraline, comorbidities, especially DM, which could cause diabetes-associated autonomic neuropathy, and therapy with midodrine (selective, α-1 adrenergic agonist) [30].
In conclusion, IDH is a common and dangerous complication of HD procedure. It is of great importance to find appropriate measures to face this problem. The abovementioned publications present inconsistent results, and in our interpretation, they suffer from some methodological limitations, especially low sample sizes. Despite promising results, further studies should be designed to examine the efficacy of sertraline in the prevention of IDH.
Uremic Pruritus
Uremic pruritus is a frequent and burdensome symptom affecting patients with advanced CKD. Chronic kidney disease-associated pruritus (CKD-aP) is defined as itching directly related to kidney disease, without another comorbid condition to explain itching [85]. Its severity, location, and time of onset may vary significantly, and it can occur without accompanying skin changes [86]. Due to the lack of an unambiguous definition and precise measurement tools, its frequency is difficult to determine. In the international prospective cohort Dialysis Outcomes and Practice Patterns Study (DOPPS), 13-50% of dialysis patients reported suffering from moderate to extreme pruritus differing between countries [87,88]. Furthermore, data from mentioned above DOPPS study revealed that patients with moderate to severe pruritus had significantly higher mortality rates. Uremic itch can contribute to mood disturbances and can be associated with a decreased quality of life (QoL) [87]. The study by Mathur et al. showed that the intensity of the pruritus was strongly associated with diminished health related-QoL in multiple domains, including mood, sleep, and social relations [89].
The etiology of itch in the HD population is complex. Certain patients' characteristics and dialysis parameters are correlated with the prevalence of uremic pruritus. This can include dialysis adequacy, low-flux dialyzers, hepatitis C positivity, higher CRP, calcium/phosphorus levels, older age, and underlying depression [90]. The pathogenesis of uremic pruritus is incompletely understood, though there are theories considering toxin deposition, peripheral neuropathy, immune system dysregulation, and opioid imbalance [85].
Pruritus patients suffer more frequently from depression, and the depressive state can amplify itch perception [91]. Oral antidepressants are thought to have an antipruritic effect due to their influence on serotonin and histamine levels, and they can be effective in cases not responding to other therapies and were recommended in the European Guideline for Chronic Pruritus [92].
In the systematic review by Kouwenhoven et al., a total of 35 studies evaluating the oral use of SSRIs, tricyclic antidepressants (TCAs), and/or atypical antidepressants in chronic pruritus published between 1980 and 2016 were included [93]. The majority of the analyzed studies showed the improvement of pruritus after initiation of oral antidepressant therapy. Both paroxetine and mirtazapine can be effective options in pruritus due to malignancies [94,95]. Furthermore, the cross-over randomized clinical trial by Gholyaf et al. showed that mirtazapine could be effective for UP patients who are on maintenance hemodialysis [96].
In cases of pruritus caused by cholestasis or CKD, sertraline could be an effective treatment option [31,32,97]. Two studies evaluating the use of sertraline in patients with CKD suffering from pruritus revealed a significant reduction in itching [31,32], Table 3. In an open-label trial by Shakiba et al., 19 patients undergoing HD suffering from UP were treated with sertraline in the medium dose of 50 mg daily for four months with resulted in the reduction of itch in the majority of cases [31]. In the second mentioned above retrospective cohort study conducted in the renal palliative care clinic, the treatment of sertraline in the medium effective dose of 35 mg daily was introduced in 17 patients. The study showed the effectiveness of low-dose sertraline in patients with antihistaminerefractory uremic pruritus measured using the VAS (Visual Analogue Scale) score [32]. In the double-blinded clinical study, which gathered 50 patients treated with HD and suffered from pruritus, half received sertraline in the dose of 50 mg twice a day, and half received placebo, patients were observed for the period of 6 months. The results of a decrease in itch intensity were more significant in the sertraline group compared to control. The authors emphasize the role of inflammation in uremic itching and suggest that sertraline can be an effective drug in reducing itch also due to its effect on reducing inflammatory cytokines [33].
Although the problem of CKD-aP is common, more evidence on how to optimize the treatment is still needed. The non-pharmacological approach is based on dialysis modification (using the biocompatible membranes, normalizing the substances that may aggravate itching) and phototherapy. Treatment trials of pruritus included different medications, such as antihistamine agents, cyclosporin, pregabalin, and many others. According to the previous research, gabapentin seems to be efficacious and safe in improving uremic pruritus among dialysis patients, and therefore it can be considered as a drug of the first choice [98]. The new peripherally restricted and selective agonist of kappa opioid receptors, difelikefalin, is under research and was shown to significantly reduce the itch intensity and improve quality of life compared to placebo [99].
Even though sertraline is not the first-choice therapy of the uremic pruritus, it can be considered in cases resistant to other medications, especially in the group of dialysis patients requiring antidepressant treatment at the same time. However, more studies on the effectiveness of SSRIs in UP are needed since the available evidence is insufficient.
Cytokines
Advanced CKD is often associated with chronic inflammation, which can significantly increase patient mortality [100][101][102]. Dialysis patients die 10-20 times more often from cardiovascular causes compared with the general population [103]. The probable cause of an increased CVD risk in this group is a dysfunction of vascular endothelium, which promotes the atherosclerotic process [104]. In the group of dialysis patients, an increased concentration of C-reactive protein (CRP) and pro-inflammatory cytokines such as interleukin-1β (IL-1β), interleukin-6 (IL-6), tumor necrosis factor-α (TNF-α), chemotactic factor for monocytes-1 (MCP-1), and hepatocyte growth factor (HGF) are observed, which is one of the suggested causes of damage to the vascular endothelium [105][106][107][108].
One of the proposed etiologies of depression is the inflammatory hypothesis-the presence of an inflammatory process and activation of the immune system in the peripheral and central nervous systems [109]. According to it, pro-inflammatory cytokines have the ability to modulate the synthesis, release, and turnover of monoamines, which are crucial in the regulation of mood [109]. Consequently, prolonged and severe depressive symptoms may be accompanied by an excessive concentration of pro-inflammatory cytokines such as IL-1β, IL-6, and TNF-α [110]. The study by Yamasaki et al., published in March 2020, also suggests a significant increase in the concentration of soluble interleukin-6 receptor (sIL-6R) in people diagnosed with major depressive episode [111].
Both renal failure in the course of CKD, which requires renal replacement therapy (RRT), and depression are disease processes with an increased inflammatory response [100][101][102]109,112]. However, there are few publications at the moment that assess the correlation between patient's clinical condition, degree of renal failure, concentration of pro-inflammatory cytokines in the blood serum, and the severity of depressive symptoms. Finding a relationship between them would allow determining the prognosis of the progression of the inflammatory process on the functioning of this group of patients and could indirectly answer whether antidepressant treatment resulting in the reduction of biochemical markers of inflammation could reduce the process of damage to the vascular endothelium responsible for the main cause of mortality in dialysis patients [104].
Sertraline, as well as other drugs from the group of SSRIs, is one of the most commonly used antidepressants with proven therapeutic efficacy [113,114]. At the same time, it seems to be a safe drug in people with a high risk of cardiovascular complications [115,116]. In addition, various scientific studies have shown that the use of sertraline reduces the concentration of inflammatory markers, such as CRP or pro-inflammatory cytokines-IL-1β, IL-6, and interleukin-12 (IL-12), while increasing the level of anti-inflammatory cytokines, such as interleukin-4 (IL-4) and transforming growth factor-β1 (TGF-β1) [34][35][36], Table 3. Therefore, it seems that sertraline may be a substance that, apart from its antidepressant effect, also reduces the risk of cardiovascular complications caused by chronic inflammation, which in turn may reduce mortality in patients treated with RRT [117]. However, there is still a lack of a sufficient number of publications that could prove the efficacy and safety of sertraline in CKD dialysis patients with depressive symptoms [118,119] or estimate the role of other antidepressants in this group of patients.
Sexual Dysfunction
The data on sexual dysfunctions (SD) in CKD patients are ambiguous. Some scientists state that the problem is underestimated [120,121], while others depict it as a marginal phenomenon [122]. When evaluating the severity of this issue, one needs to take into consideration that patients more often disclose sexual dysfunction symptoms while being directly asked rather than spontaneously report them [123].
In a meta-analysis by Navaneethan et al., sexual life of females and males with CKD was explored. Mainly studies using validated tools such as the International Index of Erectile Function or Female Sexual Function Index (FSFI) were taken into consideration, with a total of over 8343 patients evaluated. Erectile dysfunction was the most prevalent disorder observed in men with CKD (70%). Data from studies on women were not sufficient to perform a meta-analysis, but in general, females with CKD had a significantly lower overall FSFI score than women from the general population reporting disturbances in desire, arousal, lubrication, orgasm, and satisfaction as well as coital pain. The study highlighted that increasing age, diabetes mellitus (DM), and depression also correlate with sexual dysfunction and may generally aggravate problems with sexual life quality in patients with CKD. A high prevalence of sexual disorders was observed, especially in subjects receiving dialysis [124].
Possible pathophysiological explanations of these disorders involve toxicity of high urea level, psychological factors and endocrine disorders, such as abnormalities in the hypothalamic-pituitary axis (more specific in females), and hypogonadism (a major role in males) [125,126].
Sexual dysfunction is one of the frequent problems affecting people with CKD [127,128], but it may also be a symptom of MDD alone [129][130][131]. Furthermore, sexual disorders and depression during CKD can also develop into a mechanism of the vicious circle. Antidepressants may improve sexual functioning by alleviating MDD symptoms. On the other hand, decreased libido, delayed orgasm, anorgasmia both in men and women, erectile dysfunctions, decreased lubrication, and lower general sexual satisfaction can also be side effects of such therapy [37,130,132], especially with SSRIs.
Sertraline is considered to have a significant negative impact on sexual functioning in the general population [37,38]. Data on its potential in causing SD in the group of CKD patients are scarce, with very low quality of evidence showing a surprisingly low prevalence of SD both in AD treatment and in the placebo groups [39], Table 3. In case of medication-related sexual dysfunctions in patients not affected by CKD, we may consider changing sertraline (or other administered SSRI) to other medications with a lesser risk of causing SD (vortioxetine, bupropion) or by adding a small dose of an antidepressant with a different mechanism of action (trazodone, agomelatine) enabling us to lower sertraline dose and decrease this dose-dependent adverse effect [123,[132][133][134][135][136]. We have found no studies regarding vortioxetine in CKD. Agomelatine and trazodone do not require dose adjustment in ESRD patients, while the maximum dose of bupropion should not exceed 150 mg [43]. In patients with CKD, several other safety issues may restrict the use of medications [137], and introducing any of the above strategies requires an individual approach. Further studies are needed to evaluate the safety and efficacy of other interventions to cope with sexual dysfunctions in CKD patients.
Fracture Risk
The use of SSRIs is associated with hip fracture risk in the general population [40,41], but the impact of antidepressants on this complication in the population of hemodialysis (HD) patients is complex and not fully understood. Patients with end-stage renal disease (ESRD) treated with HD have four times higher rates of hip fracture compared with the general population [138,139], which can be related to the frequent occurrence of acidosis, mineral and bone disorders, dysautonomia, cachexia, inflammation, and other comorbid conditions. Furthermore, patients with ESRD treatment with HD have an increased risk of falls and fall-related injuries. The rate of falls was found to be 1.18-1.60 falls/year, which is significantly higher than in seniors not treated by HD [140,141].
Depressive disorders are also an independent risk factor for falls and fall-related injuries among the elderly population [142][143][144][145][146]. As mood disorders are more likely to be reported in patients with CKD than in the general population or in adults with other chronic conditions, depression may also be an important fall risk factor in this group.
Kistler et al. analyzed 16,573 individuals with CKD through The Behavioral Risk Factor Surveillance System (BRFSS) data to find an association between falls, depression, and CKD [147]. It was revealed that despite adjusting for multiple confounders, history of depression was correlated with falls and fall-related injuries. In previous studies, Kistler and colleagues already showed that patients with CKD are more likely to fall and suffer fall-related injuries compared with adults without CKD [148,149]. The factors that may be responsible for the elevated fall risk in patients with CKD are protein and energy wasting, muscle weakness [150,151], cognitive impairment, polypharmacy, hemodynamic instability, and high rates of comorbidities such as cardiovascular diseases and diabetes [152]. Kidney impairment contributes to changes in bone metabolism and leads to bone alterations and extraskeletal calcification, which may increase the risk for injury when suffering a fall [153,154]. Research available shows an effect of the entire group of SSRIs drugs without detailing the effect of sertraline on hip fractures.
Vangala et al. examined the relationship between SSRIs consumption and hip fracture occurrence among patients with ESRD treated with HD, a unique high-risk subpopulation, within which the impact of SSRIs on hip fracture risk remains unexplored [42]. They identified 4912 cases by use of the US Renal Data System (USRDS), the national registry of persons with ESKD treated with RRT (renal repacement therapy), to conduct a casecontrol study nested in the recorded person-time between 1 January 2006 and 30 September 2015 compared to 49,120 controls. SSRI use, including sertraline, was associated with increased hip fracture risk (adjusted OR (odds ratio), 1.25; 95% CI (Confidence interval), 1.17-1.35). Risk for fracture was estimated for any, low, moderate, and high SSRI use: adjusted conditional ORs were 1.25 (95% CI, 1.17-1.35), 1.20 (95% CI, 1.08-1.32), 1.31 (95% CI, 1.18-1.43), and 1.26 (95% CI, 1.12-1.41), respectively. The association between hip fracture events and SSRI use, including sertraline, has also been seen in the examination of new short-term use (adjusted OR, 1.43; 95% CI, 1.23-1.67). The stronger association with new short-term use may suggest an acute mechanism potentially related to falls. The study revealed that patients treated by maintenance HD who suffered from a hip fracture used SSRIs more commonly than controls Table 3.
As similar associations have been repeatedly demonstrated in the general population, particular attention should be paid early for any potential side effects assigned to sertraline (hyponatremia, orthostasis, QTC prolongation, and arrhythmias) that may contribute to fall risk. European Renal Best Practice Guidelines suggest that in patients with stage 3-5 CKD, treatment effect should be reassessed after 8-12 weeks to avoid "prolongation of drugs ineffective," and this view should also be taken into consideration for patients with ESRD treated with RRT.
The moderate increase in hip fracture risk associated with sertraline, similar to other SSRIs use, is worth considering, especially if patients and physicians do not perceive benefit from the medication after an appropriate duration of use.
There is evidence that sertraline, similar to other SSRIs, may decrease bone mass, and as a consequence, intensifies the risk of osteoporosis and osteoporosis-related fractures. This is significant since the dominant indication of SSRIs is depression, a condition also associated with low bone mass, osteoporosis, and nonpathological fractures. Considering that SSRIs, including sertraline, are the most prescribed antidepressants in the world, a large number of persons at risk of osteoporosis will be treated with SSRIs, which means people with an already increased risk of fractures are exposed to even greater risk. Furthermore, many of the risk factors for developing depression overlap with those for osteoporosis, such as physical inactivity, poor diet, and smoking [155].
Discussion
Depression and anxiety are the most common psychiatric disorders in the end-stage renal disease patients treated with HD and may correlate with lower quality of life and increased mortality [5]. Depression treatment in hemodialysis patients is still a challenge both for nephrologists and psychiatrists. In our article, we focused on the use of sertraline, the medication which seems to be relatively safe and efficient in the abovementioned population. However, we still need more studies in this field since the ones performed so far were usually based on small samples and lacked placebo control. In our paper, we discuss different aspects of sertraline use, taking into consideration possible benefits and side effects of drug administration (Tables 1-3), which could be helpful in optimizing clinical outcomes.
Conclusions
Depression and anxiety are the most common psychiatric disorders in end-stage renal disease (ESRD) patients treated with hemodialysis (HD). They may correlate with lower quality of life and increased mortality in the abovementioned population. Despite the importance of the problem, the knowledge of antidepressants usage in this population is still limited. In our paper, we reviewed previous studies considering the use of sertraline in hemodialysis patients, the advantages of using the drug, and the possible dangers of treatment. We believe that such a summary can be helpful for clinicians in their daily work with hemodialysis patients. | 2021-09-28T05:27:40.631Z | 2021-09-01T00:00:00.000 | {
"year": 2021,
"sha1": "f5c491bf5d051e4b1444bb2fe4c7bef6e0b6208c",
"oa_license": "CCBY",
"oa_url": "https://www.mdpi.com/1648-9144/57/9/949/pdf",
"oa_status": "GOLD",
"pdf_src": "PubMedCentral",
"pdf_hash": "f5c491bf5d051e4b1444bb2fe4c7bef6e0b6208c",
"s2fieldsofstudy": [
"Medicine",
"Psychology"
],
"extfieldsofstudy": [
"Medicine"
]
} |
259700484 | pes2o/s2orc | v3-fos-license | The Influence of Perceived Risks and Behavioral Intention: The Case of Chinese International Students
With the breakout of the COVID-19 pandemic, health risks are common, while trepidation over physical harm risks during travel has emerged, notably anti-Asian violence. Tourists tend to avoid traveling, and their perceived risks related to these harms may hinder their travel decision-making. This research aims to explore the inter-relationships among destination image, perceived risk perceptions, and behavioral intention of Chinese international students visiting San Francisco. Drawing from 252 survey responses, findings highlighted that perceived risk did not affect destination image in general; however, the levels of student traveller’s perceived risk influence the destination image’s relationship to behavioral intentions. The group with low perceived risk relies more on their affective image to determine their behavioral intention. Furthermore, this study validated that affective image could serve as the antecedent to cognitive image despite being firmly held as the cognitive image’s consequence. Managerial implications were provided for destination marketers in the post-pandemic era.
Introduction
Since early 2020, the COVID-19 pandemic's prevalent impacts have extended more than 2 years, with new variants such as Delta and Omicron spiking case rates in late 2021 and early 2022 (A.Park, 2022).The tourism industry was greatly affected, and extensive closures of international borders halted inbound tourist arrivals.The UNWTO (2022) reported that international travel in 2020 plunged to 73%, lower than the previous year's arrivals, while 2021 showed signs of recovery at a 4% increase but remained far below the pre-pandemic levels.Tourism and traveling during the pandemic are considered high-risk activities due to unprecedented public fears (Zheng et al., 2021).Governments of different countries implemented various public health measures and travel restrictions to reduce disease transmission from overseas (Studdert et al., 2020).In response, domestic tourism is foreseen as the main force to resuscitate the tourism economy (OECD, 2020).Many countries provided tourism stimulus packages and travel vouchers to boost the domestic tourism economy to encourage interstate and intrastate tourism activities (see Foo et al., 2021;NSW Government, 2021).
International students have piqued tourism researchers' interest due to their unique contributions to the economy (M.T. Lin & Liu, 2022), with host countries considering them important stakeholders and benefiting from their expenditure on tourism (King & Gardiner, 2015;Tomasi et al., 2022).International students are considered educational and quasi-domestic tourists (Dey, 2020;K. Li, 2014;Weaver, 2003), while some perceive them as international tourists.This is partly due to the fact that they stay longer as temporary residents of their host countries with different income sources, lifestyles, and financial commitments (Gardiner et al., 2013).In the United States, approximately 914,095 international students were reported to be studying during the 2020 to 2021 academic year, and 317,299 of these students originated from China, making it the largest source country (Open Doors 2021, 2021).International Chinese students are likened to Europe's 18th-century ''Grand Tourists'' who travel to enhance their social capital, and they put the same emphasis on traveling and learning Western culture while obtaining western degrees (R. Huang, 2008).Additionally, parental financial support to Chinese international students allows them to travel, especially on school breaks (Ryan & Zhang, 2007).Therefore, the Chinese youth travel market is an important travel segment for their host countries (M.T. Lin & Liu, 2022), representing a profitable target market in destinations (Burt, 2022; M. T. Lin & Liu, 2022).
Despite the ability to travel after pandemic restrictions have gradually eased, several factors still influence student travelers' destination decisions.For example, destination image plays a crucial role in determining students' travel decision-making (P.J. Chen & Kerstetter, 1999;Phau et al., 2010), while destination's reduced safety concerns are a paramount influencer in reaching students' travel goals (Babin & Kim, 2001).Along with the surge in health risks, anti-Asian violence against Asian descent and Chinese nationals also surged in many parts of the Western world, particularly in metropolitan areas of the United States (Miller, 2022;Wen et al., 2020;J. Xu et al., 2021).Chinese tourists generally perceive U.S. destinations as highly urban, with advanced economies and big cities (X.Li & Stepchenkova, 2012), but traveling to metropolizes during the pandemic may pose potential safety risks, and hazards for Chinese nationals and people with Chinese heritage in the U.S. Increased malicious anti-Asian attacks have prompted Asian-American organizations to share their concerns after the World Health Organization's official reports on COVID-19 origins, which indicated that virus circulation in Wuhan, China, since late November 2019 (AAPI Equity Alliance, 2021).Without valid scientific proof, the disease has been mistakenly profiled as the ''Chinese virus'' (Lantz & Wenger, 2023).According to a national survey, about 73% of Asian Americans in the United States feared becoming victims of hate crimes (CBS News SF Bay Area, 2021).The increased incidents have prompted the Chinese government to issue warning advisories for citizens living overseas to pay close attention to their safety (Reuters, 2022).Chinese students' perceived risks have been exacerbated by news reports on both hate crimes and health risks during the pandemic (Shi, 2021;Wen et al., 2020), triggering the public's negative risk perception and negative image perceptions toward destinations (Rittichainuwat, 2006;Shi, 2021).Therefore, by the same token, the danger of hate crimes can strongly influence students' destination perceptions and decision-making (J.Xu et al., 2021;Y. Xu et al., 2021).
Despite the increasing literature on the relationship between behavioral intentions and perceived risk perception, there is a limited discourse on perceived risks on destination image and travel behavioral intentions during the COVID-19 pandemic.Along with the health risks, the pandemic also posed an emerging travel risk in the presence of hate crimes, and few studies, if none, have discussed its ramification on tourism.Moreover, studies related to international students' risk perceptions are often neglected (Deng & Ritchie, 2018), and often, educational tourists (students) are excluded as tourists due to their long duration in the host countries (R. Huang, 2008;S. Huang & Gross, 2010).As international tourists are considered a source of destination referrals (R. Huang & Tian, 2013), understanding the impact of risk perceptions on the students' destination image and behavioral intention can convey important marketing messages for destination management organizations (DMOs).
The present study attempts to fill in these research gaps and explore the inter-relationships among destination image perceptions, perceived risk perceptions, and behavioral intention within the context of Chinese international student travelers during the pandemic, as justified by the abovementioned characteristics.This research seeks to answer the main research question: will students' perceived risk affect their destination image perceptions and behavioral intention?This research aims to: (1) propose a theoretical framework for understanding the interrelationships among perceived risk perceptions associated with COVID-19 and anti-Asian hate violence, destination image, and behavioral intention in the context of Chinese international students; (2) probe whether students' levels of perceived risk will alter their destination image perceptions and behaviors, and (3) provide managerial implications for marketers to comprehend better this consumer segment's mindset and travel behavior.
The next section provides a literature review and the hypotheses development.The outline of the methodology, presentation of findings, and discussion of implications, limitations, and future studies follow.The last section concludes the study.
Destination Image Components and Their Relationships
Destination image is described as individuals' beliefs, feelings, and multi-sensory representations of a travel destination (Son & Pearce, 2005).Tourists heavily rely on extrinsic cues of a destination when making destination choices (S.H. Park et al., 2017).A destination image is a multi-dimensional construct with interrelated components determining a destination's characteristics (Gartner, 1994).Prior research has identified bi-dimensional models of cognitive and affective images to capture destination images (Baloglu & McCleary, 1999;Martı´n & Bosque, 2008).Cognitive destination image refers to the physical destination features and elements that attract tourists (Baloglu & McClearly, 1999;Wang & Hsu, 2010), including climate, infrastructure, and accommodation facilities (Gertner, 2010).On the other hand, affective destination images refer to the tourist's general feelings and emotions related to destinations (Tan & Wu, 2016).A tourist's evaluation of a destination is usually emotional and subjective (Song et al., 2013); the more appealing the destination, the better and higher the affective evaluation of the destination (Sahin & Baloglu, 2011).
In addition to the cognitive and affective destination image, some scholars have proposed the concept of overall destination image (Beerli & Martı´n , 2004;Wang & Hsu, 2010).The overall destination image can be evaluated positively or negatively according to the attributes engendered by the destination (Baloglu & McCleary, 1999).Empirical research has also shown that the cognitive and affective image perceived by tourists can positively influence the overall destination image (C.H. Lin et al., 2007;P. Sharma & Nayak, 2019).Accordingly, the following hypotheses were proposed: The theoretical consensus was established that cognitive destination image is the antecedent of affective destination image (Agapito et al., 2013;Baloglu & McCleary, 1999).However, the relationship between cognitive and affective destination images can be bidirectional rather than uni-directional causation (Ko & Park, 2000).For instance, Zajonc (1980) stated that affect and cognition are partially independent and separate systems that can influence each other.A tourist's first emotional response to a destination may be affective (Woosnam et al., 2020).Affect may precede cognition since the affective system can facilitate individuals' decision-making by filtering and organizing future information before one's decisions (Walls et al., 2011).Hence, this study hypothesized: H3: Affective destination image positively influences cognitive destination image.
Destination Image and Behavioral Intention
Prior research held that destination images influence tourism decision-making (C.F. Chen & Tsai, 2007;Tasci & Gartner, 2007), and it can result in a positive evaluation and likelihood of visitation (Wang & Hsu, 2010).S. H. Park et al. (2017) revealed that the destination image of Japan could influence Chinese college students' visit intention to the country.A similar result is echoed in the research of Irfan et al. (2022), finding that the country's image can influence consumers' purchase decisions.A meta-analysis study conducted by Afshardoost and Eshaghi (2020), which synthesized 87 papers, found that different components of destination image played a significant role in predicting tourists' travel behavior, particularly in travel intentions.
Previous studies have validated that affective and cognitive images can influence tourists' visit intention (Noh & Vogt, 2013;Tan & Wu, 2016), while attitudinal affect and cognition contributes to tourists' travel motivation (Gnoth, 1997).However, the ability of cognitive and affective images to predict destination visit intention has remained inconsistent (Woosnam et al., 2020).For instance, M. Li et al. (2010) found that only affective destination image had a causal relationship with tourists' revisit intention rather than cognitive destination image.Nevertheless, Tan and Wu (2016) found that tourists' future visit intention is first influenced by cognitive aspects and subsequently by the tourists' affective feelings.More research is warranted to investigate the predictability of cognitive and affective destination images on tourists' travel behavioral intention.The following hypotheses were formulated: The overall destination image is a combination of cognitive and affective images that serves as a strong proxy to capture the destination image (Beerli & Martı´n, 2004).The overall destination image can directly influence tourists' future behavioral intentions, destination preferences, and loyalty (Baloglu et al., 2014;C. H. Lin et al., 2007;Zhang et al., 2014).However, previous literature has not thoroughly substantiated the impact of overall destination image on tourists' travel intention.Wang and Hsu (2010) found only an indirect influence of overall destination image on behavioral intention, while Baloglu et al. (2014) found that U.S. tourists' perception of Jamaica's overall destination image could positively influence their behavioral intention.Based on the above evidence, the following hypothesis is formulated: H6: Overall destination image positively influences behavioral intention.
Previous research revealed that overall image could play a mediating role between attitudes and behavioral intention in a study conducted among hotel consumers by H. Han et al. (2009).In the same vein, the overall destination image may mediate the relationships between tourists' cognitive and affective destination image on behavioral intention.Hanzaee and Saeedi (2011) discovered that tourists' overall destination image mediates the relationship between destination brand image and tourists' behavioral intention.Therefore, the following hypothesis is proposed: H7: Overall destination image mediates the relationships between (a) cognitive destination image and behavioral intention and (b) affective destination image and behavioral intention.
Perceived Risk Perception on Destination Image and Behavioral Intention.Perceived risk is critical in shaping travelers' decision-making (Hsieh et al., 2016;Qi et al., 2009).Perceived risk refers to the likelihood of an event occurring if the danger is beyond one's ability to control and influence tourists' travel decisions (Mansfeld, 2006).''The negative outcomes associated with perceived risk are the barriers that obstruct consumers' decisionmaking (D.J. Kim et al., 2008); influence tourist destination selection (J.Y. Han, 2005); and unsafe destinations affect intentions to return and recommend (George, 2003).Hsieh et al. (2016) further illustrate that when tourists' perceived risk increases, their travel intention will decrease accordingly.
Perceived risk can be divided into different dimensions.Dolnicar (2005) categorized young travelers' perceived risks into political, environmental, health, planning, and property risks.Health risk refers to the possibility that tourists become sick or contract lifethreatening diseases (Dolnicar, 2005;So¨nmez & Graefe, 1998), which is considered the most significant concern during trips (Deng & Ritchie, 2018;Dolnicar, 2007;Reisinger & Mavondo, 2006).Chua et al. (2021) found that the COVID-19 pandemic significantly influenced tourists' perceived risk and mental well-being, inducing uncertainty in travel decision-making.Famous tourist destinations were perceived as risky sites regarding the active spread of COVID-19 (Zaman et al., 2022).In a recent study, Zheng et al. (2021) found that the COVID pandemic triggers Chinese tourists' protective motivation and fear of the possibility of getting infected.
Physical risks, the possibility that travelers are exposed to the danger of injury, crime, and violence (Roehl & Fesenmaier, 1992;So¨nmez & Grafe, 1998), can also potentially deter tourists from visiting a destination.Reichel et al. (2007) found that physical harm is one of the apparent destination risk factors among student backpackers.Floyd et al. (2004) studied people's perceived risk perception in the aftermath of September 11 and found that people with safety concerns were less willing to travel within a year.Anti-Asian hate violence is categorized as a physical risk since many anti-Asian incidents entail physical assaults in public spaces (Lantz & Wenger, 2023).Reisinger and Crotts (2009) also discovered that tourists' health and terrorism risks were closely associated with their safety concerns and travel anxiety.Research has also shown that a destination's potential travel risks can create adverse image perceptions toward the destination (Becken et al., 2016;Kozak et al., 2007;Law, 2006).In turn, perceived risks could change tourists' cognitive and affective appraisal of a destination and travel intentions (Khan et al., 2017;Noh & Vogt, 2013).Based on the review, the following hypotheses were proposed: H8: Perceived risk negatively influences students' perceptions of the cognitive destination image.H9: Perceived risk negatively influences students' perceptions of the affective destination image.H10: Perceived risk negatively influences students' perceptions of the overall destination image.H11: Perceived risk negatively influences students' behavioral intention.Individuals' levels of risk perceptions differ based on their respective interests and concerns about a destination (Cheron & Ritchie, 1982).Previous research indicated that consumers' perceived risk could be a moderator among various variables, such as satisfaction and willingness to pay, tourists' perceived crowding and revisit intention (Casidy & Wymer, 2016;Yin, 2020).Consumers with lower perceived risk tend to have higher behavioral intentions than those with higher perceived risk.For example, consumers tend to cancel or change their travel plans once the perceived travel risks of COVID-19 increase (Minh & Mai, 2021).Tavitiyaman and Qu (2013) found that tourists with lower risks of the tsunami and SARS outbreaks had better image perceptions regarding travel satisfaction and behavioral intention than individuals with higher perceived risks.
Furthermore, Law (2006) suggested that travelers with lower risks have a higher travel preference than travelers with higher risks.Based on the abovementioned evidence, this study postulates that students with lower perceived risks may achieve different predictive results regarding the relationships between destination image and behavioral intention than students with higher perceived risks.Thus, it was hypothesized as follows (Figure 1): H12: The relationships between destination image perceptions and behavioral intention depend on students' levels of perceived risk (high vs. low perceived risk).
Study Context
The context selected for this research is San Francisco Bay Area, California, based on the following characteristics.Firstly, San Francisco is a popular tourist destination in California; and is one of the favorite destinations among Chinese tourists (China Daily, 2015;Qu & Im, 2002).Secondly, along with New York, San Franciso is one of the early epicenters of COVID-19 cases and early implementers of aggressive pandemic measures in the United States (Studdert et al., 2020).Lastly, San Franciso is reported to have a high frequency of anti-Asian incidents and violence during the pandemic (Fuller, 2021).Therefore, traveling to the San Francisco metropolis may pose certain risks for Chinese international students.
Measures
The constructs for this research were measured on the seven-point Likert scale, regarded as informative, precise, and highly acceptable for social studies (Alwin, 1997;Spector, 1992).The cognitive destination image measure was adopted from S. H. Park et al. (2017) to measure students' cognitive image of San Francisco.The measure was adopted to suit the context; for example, ''San Francisco has good quality of life; San Francisco has a prosperous tourism infrastructure, and San Francisco is a good place for shopping.''Respondents were asked about their level of agreement from 1 (strongly disagree) to 7 (strongly agree) with six items.
The affective destination image measure was adopted from Song et al. (2013) to measure students' affective feelings toward San Francisco.Respondents were asked about their level of agreement from 1 (strongly disagree) to 7 (strongly agree) with five items (i.e., ''San Francisco is a pleasant place, an arousing place; an exciting place; a relaxing place, and the tour makes me feel relaxed'').
The overall destination image scales were adopted from C. H. Lin et al. (2007) and Wang and Hsu (2010).
Respondents were asked to indicate their overall perceptions of San Francisco via two items (i.e., '' San Francisco's overall image is.'') using a seven-point Likert scale from 1 (strongly negative) to 7 (strongly positive).
Regarding student travelers' perceived risks related to COVID-19 risks such as health risks and anti-Asian hate violence, four items were adapted from previous studies (Hsieh et al., 2016;Tavitiyaman & Qu, 2013) and were modified to fit the research context.For example, ''Given the risks of being exposed to COVID-19, travelling to San Francisco is., Given the potential anti-Asian hate violence has happened in the U.S., travelling to San Francisco is.''Respondents were asked to indicate their levels of risk perception from 1 (extremely low risk) to 7 (extremely high risk).Since this study mainly concerns respondents' health and physical risks, other perceived risks (i.e., political, environmental, and financial) were not included.
The behavioral intention was measured by four items adopted from S. H. Park et al. (2017), asking respondents' likelihood to visit and recommend San Fransisco to others from 1 (very unlikely) to 7 (very likely).Items include ''I intend to make time and save money to travel to San Francisco within 24 months, and I am willing to recommend San Francisco to others.''
Procedure
This research utilized an online survey to capture the students' risk perception of traveling during COVID-19 pandemic, despite an initial plan of conducting a face-toface intercept survey.During the full-scale data collection period, which is between December 2021 and January 2022, cases of COVID-19 have surged in the United States, and any outdoor activities bear the risk of contracting COVID-19; hence, an online survey posed as an alternative method to capture respondents' perceptions which are not easily observed (Creswell & Creswell, 2017).The questionnaire was presented in English and contained several components, respondents' perceptions of cognitive destination image, affective destination image, and overall destination image of San Francisco.Subsequently, respondents were asked to rate their perceived risks and likelihood of visiting the destination.Demographics and general questions were asked toward the end of the survey.
The data collection process began with creating and programming the online questionnaire through wjx.cn, a popular Chinese survey site (Miao et al., 2020).Before launching the full-scale data collection, a pilot test was conducted at a U.S. southwestern University with 30 Chinese university students to ensure the accuracy of the measurement items.The questionnaire was reworded and revised to improve face and content validity based on the pilot testing.The online survey program generated a survey link distributed electronically through WeChat, a social networking application in China, to the screened respondents.Subsequently, each respondent forwarded the survey link to other potential participants.Participation in the study was voluntary, and respondents' information was kept confidential.However, participants opt to receive a 2 RMB WeChat Red Packet (an e-currency utilized in China) as an incentive after completing the survey.
Sampling
For this research, prospective respondents were screened using three criteria: (a) mainland Chinese nationals currently holding international student visas living in the USA for their study, (b) currently enrolled in U.S. institutions, and (c) have experienced traveling domestically within the United States.The sample was recruited using a non-probability purposive sampling technique to obtain target respondents who were willing and available during the given time (Etikan et al., 2016).More specifically, snowball sampling was employed to recruit respondents.Although non-probability sampling can lead to limited generalizability, it fits the scope of this study since snowball sampling is useful to secure a list of the unknown population (Daniel, 2011).
Using the WeChat application, an invitation to participate in the study by answering a survey was initially forwarded to one of the authors' peers who qualified for the respondent's criteria.The first batch of respondents then further distributed the invitation among their peers.Willing potential respondents were screened to match the criteria.After filtering two unusable data (i.e., straightlining answers), a total of 252 usable and completed questionnaires were retained, which is adequate for Partial Least Square (PLS-SEM) analysis.
Of the 252 respondents, 58.3% accounted for females (n = 147), 54.8% were between 25 and 35 years old (n = 138), and 61.5% were enrolled in post-graduate programs (n = 155).About 48.4% of the respondents (n = 122) had previous travel experiences in San Francisco.Around 50.7% of the respondents preferred self-driving (n = 124), and 47.2% preferred taking the plane (n = 128) as their preferred mode of travel.Furthermore, 66.7% of respondents preferred to travel with friends (n = 168), while only 12.7% preferred to travel alone (n = 32).Among respondents with travel experiences (n = 134), 78.4% (n = 105) traveled two nights and 3 days more.The respondents' demographic information is presented in Table 1.
Common Method Bias
To minimize response bias, the questionnaire was carefully worded, reviewed, and revised repetitively.
Respondents' identities were kept confidential and anonymous to reduce the comprehension of responding to survey items (Chang et al., 2010).Since the data collection made use of a single source, common method bias was assessed using Harman's single-factor approach (Podsakoff & Organ, 1986).Factor analysis via principal component analysis showed a 40.34% variance for the first factor, which is below the cut-off of 50% (Harman, 1976).Hence, the common method bias was not detected in this study.
Measurement Model Evaluation
A confirmatory factor analysis (CFA) was conducted using a maximum likelihood approach to assess the measurement model's overall model fit.The model fit for the conceptual model has reached an acceptable level with SRMR at 0.06, less than the cut-off value of 0.08 (Hu & Bentler, 1999).Table 2 shows the mean, standard deviation (SD), and factor loadings for each measurement item in the study.Factor loadings of the measurement items were all above the required threshold at 0.7 (Chin, 2010), ranging from 0.72 to 0.98.Cronbach's a and composite reliability (CR) all exceeded .7,demonstrating reliability for the constructs (Nunally & Bernstein, 1994).Moreover, the average variance extracted (AVE) exceeded the cut-off threshold at .5, demonstrating convergent validity of the internal relationships for a given factor (Hair et al., 2019).Furthermore, discriminant validity was accessed by the Fornell-Larcker criteria, confirming that the squared value of AVE for each construct was more significant than any correlations between the variables (Fornell & Larcker, 1981).See Table 3, which depicts the constructs' reliability, validity, and correlations.
Hypotheses Testing
The PLS-SEM was conducted to examine hypotheses and test the proposed relationships using Smart PLS 3.3, a common tool for prediction-driven tourism and hospitality research studies.Compared with covariance-based SEM, PLS-SEM can produce valid results with a small sample size (Rasoolimanesh & Ali, 2018).To calculate the minimum sample size for PLS-SEM analysis, G*Power 3.1.9software was utilized (Ringle et al., 2014).As a result, the results of G*Power recommended a sample of 170 as the minimum sample size for this study's model to achieve a statistical power of 0.8 (Ringle et al., 2014).Hence, the sample in this study has achieved adequate statistical power.
The results of the PLS-SEM analysis are shown in Table 4 and Figure 2. Bootstrapping with 1,000 samples was utilized to test the significance of the hypotheses.A t-value greater than 1.96 indicates the significance of a hypothesized path (Hair et al., 2020).Proposed hypotheses in this research projected that cognitive destination image (H1) and affective image (H2) positively influence the overall destination image.Results indicated that cognitive destination image with b = .18at p \ .05 and affective destination image with b = .54at p \ .001both positively and significantly influence the overall destination image, consequently supporting H1 and H2.Additionally, results strongly supported H3, which proposes that affective destination image positively influenced cognitive destination image with b = .72at p \ .001.H4, H5, and H6 propose that behavioral intention is influenced by cognitive destination image, affective destination image and overall destination image, respectively.Results found that cognitive destination image had an insignificant effect on overall destination image with b = .01,at p = .94;thus, H4 was not supported.Moreover, behavioral intention is positively and significantly influenced by affective destination image with b = .28at p \ .05;and overall destination image with b = .21at p \ 0.01, supporting H5 and H6.
This research proposes that perceived risk will negatively influence student's influences students' perceptions of the cognitive destination image (H8) and affective destination (H9) of San Francisco.The results showed that perceived risk posed to have positive effects instead of negative effects, cognitive destination image with b = .09at p = .04,and affective destination image with b = .16,at p = .03;hence, H8 and H9 were not supported.In terms of respondents' perceived risk negatively influenced students' overall destination image perceptions toward San Francisco (H10) and travel intention (H11), the results supported both hypotheses showing negative and significant relationships with b = 2.10 at p \ .05 and b = 2.14 at p \ .05,respectively.
Mediation Analysis
The Sobel (1982) test was used to examine the mediation effects of cognitive and affective destination image on behavioral intention through overall destination image (Table 5).A mediation effect is present when the indirect effect between the predictor and dependent variables is significant by the Sobel Z test (Baron & Kenny, 1986).The absolute value of the Sobel Z test score greater than 1.96 indicates a significant mediating effect at a = .05(Baron & Kenny, 1986).For cognitive destination image, the indirect effect was statistically non-significant with Z value of 1.59.Therefore, overall destination image cannot indirectly affect cognitive destination image and behavioral intention.However, the indirect effect was statistically significant for the affective destination image with Z value of 2.48.Thus, overall destination image partially mediated affective destination image on behavioral intention due to the positive relationship between affective destination image and behavioral intention.Thus, the result of the mediation analysis partially supported H7.
Multi-Group Analysis: Low and High Perceived Risk Groups
As a post-hoc analysis, a multi-group analysis (MGA) was conducted to analyze further whether the levels of perceived risk can influence students' image perceptions and behavioral intention to any extent.The sample was divided into low and high-perceived-risk groups.The median split method was used to determine the grouping (Iacobucci et al., 2015).The mean score of the four perceived risk items (based on the seven-point Likert scale) was first calculated to determine the median score.Then, the median score (5.5) was used as the dividing point to categorize the sample into low and high-perceived-risk groups.The mean of the low-perceived-risk group (n = 147) and high-perceived-risk group (n = 105) were 4.47 and 6.26, respectively.MGA was conducted to measure the conceptual framework's configural differences by comparing the proposed variables' parameter estimates and directional paths.The structural model with path coefficients for low and high perceived groups are depicted in Figures 3 and 4, respectively.According to the result, the significance of the path coefficients was not the same for the two groups.For low perceived risk groups, both cognitive (b = .22,p \ .05)and affective destination (b = .54,p \ .001)images positively influenced overall destination image; affective destination image positively influenced cognitive destination image Through comparison, there was an apparent difference between low and high-perceived-risk groups regarding parameter estimates and the significance of the hypothesized paths.The comparison of parameter estimates between the two groups is considered a special case of moderating effects (Henseler & Fassot, 2010).Therefore, H12 was supported.For the group with low perceived risk, student tourists' perceptions of affective destination image could influence their behavioral intention; cognitive and affective destination images can form international students' overall destination image perception of San Francisco.On the other hand, for the group with high perceived risk, the affective destination was not a significant predictor of behavioral intention.Yet, their cognitive destination image could not form this group's overall destination image perception of San Francisco.
Discussion
This study aims to ascertain the inter-relationships among perceived risk perceptions associated with COVID-19 and anti-Asian hate violence, destination image constructs, and behavioral intention in a conceptual model to understand better Chinese international students' travel decision-making in the U.S. The objectives of this study were all achieved.Subsequently, this study conducted a multi-group analysis to test how perceived risk plays a role in determining tourists' destination image perceptions and behavioral intention.The findings revealed that individuals with higher perceived risk are less likely to use destination images to influence their travel decisions.This result is consistent with a recently published study by Chi et al. (2022), which demonstrated that consumers' higher COVID-19 risk perceptions could increase their hesitation in travel planning.Chinese international students' hesitancy may be compounded by the potential anti-Asian violence.Examining tourists' destination image through risk perceptions can add substantial value to understanding the linkage between these concepts (Becken et al., 2016).
One of the findings in this research is the influence of affective image on cognitive destination image, despite the commonly held beliefs that cognitive destination image is the antecedent of affective destination image (Beerli & Martı´n, 2004;C. H. Lin et al., 2007).Conventional beliefs indicate that affective image is based on cognitive image; however, the affective reaction is the very first reaction that may be independent of individuals' cognition (Zajonc, 1980).Individuals' emotional experiences can influence their mental pictures (Nyer, 1997), and their emotional feelings can influence objective attributes of the cognitive image (P.Sharma & Nayak, 2019).
Moreover, in line with prior studies (C.H. Lin et al., 2007;Wang & Hsu, 2010), this study validated that cognitive destination and affective destination images can form overall destination images.Combining cognitive and affective images can reveal San Francisco's complete destination image (Hoang, 2016).This study also revealed that overall destination image can positively influence behavioral intention and mediates the effect of affective destination image on behavioral intention.Thus, overall destination image as the third component of a destination image intervening with tourists' behavioral intention should be recognized.This finding supports Chi and Qu (2008) that the overall destination image of a destination can affect tourists' destination selection, evaluation, and future travel intentions.
Unlike many previous studies (Beerli & Martı´n, 2004), this study found that affective destination image positively relates to behavioral intention rather than cognitive destination image.This result aligns with M. Li et al. (2010).Affective destination image is tourists' first automatic reaction to travel decisions (Walls et al., 2011).A positive affective association with the destination can stimulate travelers to visit the destination; in contrast, a negative affective association deters visit intention (Woodside & Lysonski, 1989).The direct effect of cognitive destination image on behavioral intention is non-significant.The result of a non-significant relationship may be due to the mediating effect of the overall destination image that reduces the strength of cognitive destination image.It can be explained that tourists' affective emotions are more important in evaluating destinations than their cognitions (S.Kim &Yoon, 2003).The results of the multi-group analysis further revealed that cognitive destination had no effects on behavioral intention in either low or high-perceived groups.However, individuals with lower perceived risk tend to rely more on their affective evaluations to determine their behavioral intention than those with higher perceived risk.Hence, this current study signals that affective destination image may be more important than cognitive destination image in determining educational tourists' travel decision-making, especially when those tourists are not risk-averse traveling to a destination laden with risks.
The study results showed that perceived risk could negatively impact students' behavioral intentions, which is in line with the findings of Qi et al. (2009).When potential risks are presented, tourists cognitively evaluate the likelihood and severity of exposure and their ability to handle the situation (Qi et al., 2009).This result is also consistent with Mitchell (1999) that consumers' purchase intention and perceived risk are negatively related.However, contrary to Khan et al. (2017) and Noh et al. (2013), the study results revealed that perceived risk could not negatively influence the cognitive and affective destination image.One possible explanation is that students' impressions of San Francisco may have already been formed through external information sources before the global pandemic and hate crime incidents.
Establishing a tourism destination image requires a longterm and stable process, and tourists' destination perceptions may not be easily altered by short-term stimulation (Hao et al., 2019).
Moreover, tourists' destination image can only change slowly over time and tend to have resiliency during a crisis (Nadeau et al., 2022).Therefore, Chinese international students' specific perceptions of the tangible and psychological aspects of San Francisco's destination image may not be easily altered directly by incidents reported on the news.This result may suggest that despite a destination laden with potential risks, the image of a destination can still be perceived positively by tourists; however, they may not necessarily set out their foot to visit the destination at the time being if they feel threatened by the perceived risks (George, 2003;Noh & Vogt, 2013).This study's authors argued that Chinese students' risk perceptions may tarnish San Francisco's reputation as a safe metropolis to live in or travel to but may not necessarily hinder its image as a tourism destination.San Francisco's tourism and scenic spots are generally rated positively and exceed Asian tourists' expectations (Qu & Im, 2002); hence San Francisco's destination image remains stable.
Theoretical Implications
This research has theoretical implications.Firstly, this research contributes to the literature by incorporating cognitive, affective, and overall destination images and perceived risk simultaneously in the same conceptual framework, which was not carried out by many studies (Noh & Vogt, 2013;Promsivapallop & Kannaovakun, 2017).Second, this research has empirically corroborated that affective image can also strongly influence cognitive destination image, despite the commonly held beliefs that cognitive destination image is the antecedent of affective destination image (Beerli & Martı´n, 2004;C. H. Lin et al., 2007).To the authors' knowledge, no research has investigated the reversal effect of affective destination image on cognitive destination.This study provides a starting point for future research to validate further the directional relationship between affective destination image and cognitive destination image.
Practical Implications
Safety concerns are damaging to a tourism destination (George, 2003).The continued impact of COVID-19 virus variants and anti-Asian violence in the U.S. metropolitan areas can still pose potential risks for certain groups of tourists.Incidents directed at Asians during the pandemic have occurred in many U.S. cities, including San Francisco, Los Angeles, and New York (Lantz & Wenger, 2023;D. Lin, 2022).Even if the pandemic is settled in the near future and tourism activities return to normalcy in the post-pandemic era, safety and health measure should still be implemented.This research's findings have several practical implications for the tourism industry.Firstly, tourists with lower levels of perceived risk rely on their affective evaluation to decide their behavioral intention.Hence, DMOs and tourism operators can enhance tourists' emotions and feelings toward the destination by providing good safety measures.Promoting an affective destination image is a strategically important technique in tourism businesses (J.Xu et al., 2017).Second, destination marketers need to establish an excellent overall destination image, which can help the destination strategize its tactics to determine its target audience, branding, and positioning.
Third, DMOs need to understand international students' travel characteristics to ensure their safety and meet their needs.It is not only for the international students' benefit but also for the prosperity of the postpandemic tourism industry.International students travel during school breaks to experience local culture and history, and their travel expenses can generate significant revenue for the host country (Payne, 2009;Sung & Hsu, 1997).Chinese students often embark on tourism activities with friends and families, especially during graduations, holidays, and school breaks (M.T. Lin & Liu, 2022;Liu & Ryan, 2011).However, attention needs to be paid since Asian students have higher risks than students from other countries (Deng & Ritchie, 2018).
Lastly, city tourism operators and DMOs need to continuously implement health and safety protocols to alleviate the impact of the potential spread of COVID-19 variants and simultaneous incidents of Asian-hate crimes.Tourism operators can coordinate with law enforcement agencies to ensure the safety of the destinations (George, 2003).Local governments need to enhance patrols in popular attractions for tourists in general (i.e., Chinatown).Good communication plans need to be implemented to update information about risky areas for tourists.Tourism operators can reduce travel risks and assist in tourists' decision-making (Tavitiyaman & Qu, 2013).
Limitations and Future Research
This research has a few limitations that need to be recognized.Firstly, limitations are found surrounding the sample and the sampling method of this research.The data was collected with a small sample size (252 respondents) on its explorative nature.Although the sample produced 80% statistical power, the small sample size may still have effects to deflate or inflate the statistical estimates (Royall, 1986).Additionally, respondents were collected from four major U.S. states-New York, Connecticut, Missouri, and Texas.Therefore, this study's use of snowball sampling likely hinders the generalizability of the findings to the population (G.Sharma, 2017).Future studies can increase the sample size with a more diverse population since the result of this study may not reflect other international students' perceptions of U.S. cities.Asian students have different perceptions than students from other parts of the world.A country-by-country analysis may be required to understand better different student tourists' perceptions (Son & Pearce, 2005).
Secondly, this study only assessed participants' perceptions of San Francisco.Therefore, the findings of this research are limited to the area of San Francisco, and there should be caution when generalizing findings to other U.S. destinations with similar situations and conditions to San Francisco.This research suggested that future studies can investigate students' perceptions toward different U.S. destinations with different conditions and conduct comparison studies.Lastly, this study was cross-sectional; tourists' perceptions may change as the world returns to normalcy.Future research can conduct longitudinal studies to compare tourists' behavior toward the destination during the pandemic with the post-pandemic period.
Conclusion
This study examined Chinese international students' behavioral intention in the U.S. by integrating destination image and perceived risks.Given the potential risks of exposure to COVID-19 and encountering anti-Asian hate crimes, Chinese students' protective motivation may be aroused, which can deter them from making travel decisions.In urban tourism destinations such as San Francisco, tourism scholars and practitioners need to recognize that perceived risk can obstruct tourists' future travel intentions (Tavitiyaman & Qu, 2013).However, tourists such as Chinese students' destination image of a destination may not necessarily be altered by their risk perceptions.Furthermore, it is worth noting that Chinese students may continue to be the main source of the international student population in the United States and other English-speaking nations (Hegarty, 2014).Young travelers are not necessarily deemed budget travelers who tend to have longer stays and higher travel expenditures (Lo & Cheng, 2011).Thus, DMOs in the United States and other western countries alike need to implement proper strategies to relegate young travelers' perceived risks and fears.
Declaration of Conflicting Interests
The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.
H1:
Cognitive destination image positively influences the overall destination image.H2: Affective destination image positively influences the overall destination image.
H4:
Cognitive destination image positively influences behavioral intention.H5: Affective destination image positively influences behavioral intention.
Figure 3 .
Figure 3. Structural model for the high-perceived risk group.
Figure 4 .
Figure 4. Structural model for the low-perceived risk group.
Table 1 .
Profile of Respondents.
Table 2 .
Mean, Standard Deviation, and Factor Loadings of Measurement Items.
Table 3 .
Reliability, Validity, andCorrelations.The bold and italic values represent the square root of AVE for the discriminant validity. | 2023-07-12T07:05:18.468Z | 2023-04-01T00:00:00.000 | {
"year": 2023,
"sha1": "2bba0d070691550aae171cb017af53e670a9d697",
"oa_license": "CCBY",
"oa_url": "https://journals.sagepub.com/doi/pdf/10.1177/21582440231183435",
"oa_status": "GOLD",
"pdf_src": "Sage",
"pdf_hash": "8ce14adcfa65787c3f58cb977a706f6df3cfc54b",
"s2fieldsofstudy": [
"Business"
],
"extfieldsofstudy": []
} |
52180173 | pes2o/s2orc | v3-fos-license | Sepsis as an important risk factor for gastrointestinal bleeding in acute coronary syndrome patients
Abstract Rationale: Sepsis is a common stressor that may decrease microcirculation in the gastrointestinal tract in patients and increase the gastrointestinal bleeding risk of stress-related mucosal disease. However, the CRUSADE (Can Rapid Risk Stratification of Unstable Angina Patients Suppress Adverse Outcomes with Early Implementation of the ACC/AHA Guidelines) bleeding risk score, recommended by authoritative guidelines for acute coronary syndrome (ACS), does not include sepsis as a bleeding risk factor. Patient concerns: The 2 cases were about ACS with hemorrhagic complications. The first patient was an 88-year-old man with hypertension, gallstones, hepatic cysts, and chest pain; the second one was a 79-year-old man with chest pain and hypertension. These 2 ACS patients had no bleeding on admission; however, both patients suffered apparent gastrointestinal bleeding immediately after the development of sepsis or severe sepsis. Diagnoses: Both patients were diagnosed as ACS with sepsis. Interventions: The first ACS patient had no use of proton pump inhibitors (PPIs) for prophylaxis prior to the diagnosis of sepsis. The second one was administered PPIs at standard oral doses. Outcomes: The first patient suffered from gastrointestinal bleeding immediately after the onset of sepsis. And oral PPIs failed to prevent upper gastrointestinal bleeding for the second patient, when severe sepsis developed. However, the second patient's gastrointestinal hemorrhage gradually stopped immediately after high doses of PPIs were administered intravenously, rather than orally. When sepsis developed again, the second patient also had no recurrent gastrointestinal bleeding under the protection of PPIs at standard oral doses. Lessons: Our report suggests that sepsis may be an important bleeding risk factor for ACS patients, and the reasonable use of PPIs to prevent gastrointestinal bleeding could be vital for ACS patients complicated with sepsis.
Introduction
Antithrombotic therapy has increasingly been used worldwide for the prevention and treatment of primary and recurrent ischemic events in acute coronary syndrome (ACS); however, this therapy is also associated with gastrointestinal bleeding complications. [1] Gastrointestinal bleeding events are accompanied by increased expenditures, as well as worse outcomes and prognosis. [2] Therefore, the minimization of gastrointestinal bleeding complications is an overarching goal in the management of ACS patients.
The European Society of Cardiology guidelines encourage clinicians to apply the CRUSADE (Can Rapid Risk Stratification of Unstable Angina Patients Suppress Adverse Outcomes with Early Implementation of the ACC/AHA Guidelines) bleeding risk score to manage the in-hospital bleeding risks of patients. When calculating the scores, baseline patient characteristics (sex, history of diabetes, and peripheral vascular disease), admission clinical variables (heart rate, systolic blood pressure, and signs of congestive heart failure), and admission laboratory values (hematocrit and calculated creatinine clearance) are key components of the CRUSADE bleeding scores. [3] In clinical practice, however, the ability of these scores to distinguish ACS patients is only moderate at different bleeding risks, [4] and the applicability of bleeding risk scores to the management of the patient remains uncertain.
Our clinical experiences suggested that patients with comorbid ACS and sepsis were more likely to exhibit gastrointestinal hemorrhage, indicating sepsis may be an important part of the risk factors for bleeding. Thus, we report 2 cases of gastrointestinal bleeding in ACS patients with sepsis, so as to provide clinical reference.
Case report one
An 88-year-old man with hypertension, gallstones, hepatic cysts, and chest pain was admitted to our hospital on November 4, 2015. On admission, the patient complained of frequent chest pain during the previous 2 years and aggravation in the previous 2 hours. The electrocardiogram results indicated that the patient had myocardial ischemia. The chest pain was gradually relieved after nitroglycerin administration. Coronary angiography verified that the patient was suffering from coronary atherosclerotic heart disease, with a left main coronary stenosis of 50%, an anterior descending branch stenosis of 50 to 75%, circumflex branch mild stenosis and complete occlusion of the right coronary artery. To treat ACS, the patient began to take drugs from the first day of admission, including isosorbide mononitrate, metoprolol, benazepril, aspirin, clopidogrel, atorvastatin, low-molecular-weight heparin, and nifedipine.
We calculated the first CRUSADE bleeding risk score for the patient immediately after his admission. He was 173 cm tall and weighed 70 kg. His blood pressure was 145/86 mm Hg, with a heart rate at 80 beats/min, and body temperature at 36.5°C. The patient did not have a history of diabetes, vascular diseases or infectious diseases, and there were no signs of bleeding, hepatosplenomegaly, or heart failure on admission. Blood biochemical examination on admission indicated leukocytosis due to stress effect of ACS. And the laboratory results revealed, white blood cell count (WBC): 9630/mL (Fig. 1A), neutrophil count: 8380/mL, percentage of neutrophil granulocytes: 87%, hemoglobin: 130 g/L (Fig. 1A), hematocrit (Hct): 36.90%, and the platelet level remained within the normal range. Besides, alanine aminotransferase (ALT: 24 U/L) and procalcitonin (PCT < 0.05 ng/mL) were also within the normal ranges, whereas increases were observed in the random blood glucose (10.7 mmol/ L), low-density lipoprotein cholesterol (LDL-C: 3.13 mmol/L), and aspartate aminotransferase (AST: 78U/L). The renal function (serum creatinine: 100 mmol/L) and N-terminal probrain natriuretic peptide (NT-proBNP: 358 ng/L) were normal. The CRUSADE bleeding risk score was 33 points on admission, indicating a moderate risk. The patient did not take any drugs, including proton pump inhibitors (PPIs), to prevent gastrointestinal bleeding. On the 3rd day, the fecal occult blood test result was negative. On the 4th day, the patient developed symptoms of fever, cough, and sputum, which were accompanied by debilitation and dizziness. On the 6th day, the patient was treated with moxifloxacin. On the 4 to 8th days, his body temperature remained abnormal with a maximum point of 38.4°C; substantial increases in the WBC (13,800/mL, Fig. 1A), neutrophils (12,390/mL), percentage of neutrophil granulocytes (89.8%), and PCT (0.38 ng/mL) were noted. The patient's condition was consistent with the diagnostic criteria of sepsis. On the 8th day, the patient presented with fresh blood stool for the first time with a 65 beats/min heart rate, 110/50 mm Hg blood pressure and a substantial decrease in hemoglobin (102 g/L, Fig. 1A), without petechiae or bruising in the skin-mucosa or subcutaneous hemorrhage. Aspirin, clopidogrel, and low-molecular-weight heparin were subsequently discontinued. A bismuth mixture, esomeprazole, thrombin, and noradrenaline were administered later; however, the stool remained black. Following termination of anticlotting drugs, on the 9th day, the patient complained of worsening chest pain, along with an observed increase in serum cardiac markers, and an emerging and complete right bundle branch block. Low doses of clopidogrel (50 mg/d) and fondaparinux sodium were administered, considering possibility of recurrent myocardial infarction. On the 11th day, the patient was referred to the cardiovascular care unit, where the anti-infective drug was substituted with imipenem. On the 13th day, the patient had a sudden loss of consciousness and limb twitching. His X-rays indicated sheet-like images in the lower field of both lungs, patchy shadows in the middle field of the right lung and suspicious patchy shadows in the left upper lung. His clinical status simultaneously worsened, with a significant increase in the WBC (16,640/mL, Fig. 1A) and PCT (6.08 ng/mL), a sharp decrease in hemoglobin (95 g/L, Fig. 1A), deterioration of cardiac function and damage of liver and kidney functions. The fecal occult blood tests remained positive since the first bloody stool during his hospitalization. At 3:12 AM of the 16th day, the patient appeared: loss of consciousness, limb twitching, and frequent paroxysmal ventricular tachycardia. His blood pressure was quite low, at 73/46 mm Hg. Ventricular tachycardia was terminated by defibrillation; however, the restoration of the sinus rhythm could not be maintained. His family members refused all rescue measures, and at 4:25 AM, the patient was considered clinical death. No autopsy was carried out afterward.
Case report two
A 79-year-old man with chest pain and hypertension was admitted to our hospital on November 2, 2015. On admission, the patient complained of chest pain that had occurred 5 hours earlier; he could not bear aggravation of the pain and exhibited symptoms of mild cough, sputum, and asthma. The electrocardiogram results showed a sinus tachycardia, and ST segment elevation in the inferior wall as well as V1-V5 leads, which suggested the potential for acute ST segment elevated myocardial infarction (STEMI) in the inferior wall and extensive anterior wall. A combination regimen of ticagrelor and aspirin was used. Coronary angiography verified that the patient was suffering from STEMI, with complete occlusion of the left main coronary and mild stenosis in the right coronary artery. A percutaneous coronary intervention (PCI) operation was immediately conducted, and a drug-eluting stent was placed in the left anterior descending branch, followed by grade-III thrombolysis in myocardial infarction (TIMI) coronary blood flow. The patient was administered drugs from the first postoperative day, including isosorbide dinitrate, aspirin, ticagrelor, atorvastatin, fondaparinux sodium, acetylcysteine, ambroxol, and furosemide. We calculated the CRUSADE bleeding risk score as 36 points, which indicated a moderate risk, the first time immediately after his admission. The patient was 160 cm in height and 65 kg in weight. His blood pressure was 122/65 mm Hg, heart rate was 65 beats/min, and body temperature was 36.3°C. This patient had a history of chronic obstructive pulmonary disease for over 20 years, and showed signs of heart failure; however, there was no evidence of bleeding or hepatosplenomegaly, and he had no previous history of diabetes, vascular diseases, or infectious diseases either. After the PCI operation, his blood pressure rapidly decreased to 64/40 mm Hg, and dopamine was immediately administered via a microinfusion pump to maintain his blood pressure. Blood examination on admission indicated leukocytosis on account of stress effect of ACS. And the results showed, the WBC: 19,030/mL (Fig. 1B), neutrophils count: 17,810/mL, percentage of neutrophil granulocytes: 93.6%, hemoglobin: 135 g/L (Fig. 1B), Hct: 40.50%, and the platelet level was within normal limits. For blood biochemical indexes, increases occurred in high sensitive C-reactive protein (CRP > 20 mg/L), PCT (45.34 ng/ml), ALT (65 U/L), AST (467 U/L), lactic dehydrogenase (LDH: 1074 U/L), LDL-C (2.03 mmol/L), and serum creatinine (115 mmol/L), whereas decreases occurred in the total protein (61 g/L), prealbumin (132 mg/L), and serum albumin (34 g/L). Increases in myoglobin (528.1 mg/L), creatine kinase-MB (CK-MB > 300 mg/L), troponin (9.290 mg/L), and NT-proBNP (20856 ng/L) were also noticed. The patient underwent a series of treatments for asthma, cough, and phlegm. On the 5th day, piperacillin/tazobactam was added, and standard oral doses of PPI (esomeprazole magnesium enteric-coated tablet, 40 mg qd po) were also administered to prevent gastrointestinal bleeding. Rosuvastatin, instead of atorvastatin, was administered to lower lipids. Meanwhile, both of spironolactone and digoxin were administered for chronic heart failure. Then, the serum www.md-journal.com markers gradually improved after admission. The levels of several biochemical indicators, such as ALT (51U/L), AST (120U/L), serum creatinine (97 mmol/L), and PCT (17.76 ng/ml), were much lower. And his blood pressure and heart rate returned to the normal level. On the 7th day, the fecal occult blood test was negative. On the 9th day, ivabradine was added. On the 10th day, serum transaminases of ALT (24U/L) and AST (36U/L), which were the representative items of liver function test, returned to the normal level. On the 11th day, benazepril was added as well. However, at 7:00 AM of the 15th day, the infection aggravated, and the patient showed symptoms of cough, phlegm, and exacerbation of asthma, with the body temperature at 38.4°C, heart rate at 78 beats/min, breaths at 20/min and blood pressure at 88/57 mm Hg. The patient began to repeatedly spit out blood from 8:10 AM of the 15th day, with a daily volume of approximately 800 mL. An emergency gastroscopy indicated erosive esophagitis and diffuse bleeding (Fig. 2). At 10:03 AM of the same day, the routine blood examination indicated a substantial increase in the WBC (17,210/mL), neutrophils (14,380/mL), and percentage of neutrophil granulocytes (83.5%), as well as a substantial decrease in hemoglobin (126 g/L). At 23:52 PM of the day, another routine blood examination was performed again, and the hemoglobin (80 g/L, Fig. 1B) exhibited a further decrease. The patient's conditions fulfilled the diagnostic criteria for severe sepsis with apparent gastrointestinal bleeding. A series of treatments were immediately initiated, and dopamine was administered via a micro infusion pump to maintain the blood pressure. To terminate the gastrointestinal bleeding, all antithrombotic drugs were stopped, and instead thrombin and noradrenaline were administered; somatostatin was administered via a micro infusion pump, and proton pump inhibitor (PPI, esomeprazole) was instantly adjusted to an intravenous, rather than oral, administration. The PPI therapy strategy during hospitalization was as follows: PPI was initially administered via intravenous pumping from high doses to low doses gradually, followed by a transition to intravenous drip, and subsequently oral maintenance treatment. Hydroxyethyl starch, succinylated gelatin and a blood transfusion were given, and the antibiotic drug was adjusted to ceftizoxime.
The fecal occult blood tests were still positive on both the 16th and 20th days after hematemesis. On the 23rd day, the patient's infection improved greatly, with a negative fecal occult blood test and a gradual increase in hemoglobin (Fig. 1B). He was then administered a single, low dose of clopidogrel (50 mg qd po) for antithrombotic therapy. On the 27th day, the single dose of clopidogrel was reverted to a standard dose (75 mg qd po). The PPI was finally adjusted to oral administration on the 33rd day. It was worth noting that the blood examination again indicated leukocytosis on the 39th day. On the 42nd day, at 7:00 AM, his body temperature was at 38.4°C, heart rate at 79 beates/min, and blood pressure at 123/59 mm Hg; an obvious increase was also noted in the WBC (12,960/mL, Fig. 1B) and neutrophils (6780/ mL). The patient's conditions indicated that sepsis had developed again. Yet there were no signs or symptoms of gastrointestinal bleeding with the protection usage of standard oral PPI. Acinetobacter baumannii was identified from the deep sputum culture, together with renal insufficiency; thus, a series of adjustments in the anti-infective therapy were implemented. Meropenem, followed by levofloxacin, and subsequently levofloxacin and minocycline were administered. The fecal occult blood tests remained negative since the 23rd day. Finally, on the 53rd day, the patient was discharged from hospital in an improved health condition.
Discussion
ACS comprises a group of cardiovascular disorders, including angina, non-ST-segment elevation myocardial infarction, and STsegment elevation myocardial infarction. The common theme that underlies these disorders is the rupture of a vulnerable atherosclerotic plaque, followed by partial or complete thrombotic occlusion of an artery. A combination of antiplatelets and anticoagulants remains the standard treatment of antithrombotic therapy for ACS; however, this therapy has been demonstrated to increase the incidence of upper gastrointestinal bleeding, possibly because of the cumulative effects of these drugs. [5,6] For ACS patients, gastrointestinal hemorrhagic complications have become an independent risk factor for subsequent mortality, which represents a hazard equivalent to or even greater than that for myocardial infarction. [7,8] Sepsis is a common stressor that has been demonstrated to decrease microcirculation in the gastrointestinal tract. [9,10] Sepsis has also been reported to be associated with an increased risk for stress ulcer related gastrointestinal bleeding during the last 20 years. [11][12][13][14][15] Furthermore, a simple regression analysis has also identified sepsis as a risk factor for stress ulcer related gastrointestinal bleeding. [10] For ACS patients, sepsis may increase the gastrointestinal bleeding risk, and once gastrointestinal bleeding occurs, it is easy to cause bacterial infection. Moreover, both sepsis and bleeding may reinforce each other, which may aggravate the situation and may even cause death in serious cases. [16] Thus, the assessment of the bleeding risk and prevention of bleeding are important objectives in the management of patients with ACS or ACS accompanied by sepsis. The CRUSADE bleeding risk score for ACS is recommended as an acknowledged and common scoring system by authoritative guidelines; [17] however, sepsis is not included. Moreover, up to date, no relative research has examined the link between sepsis and ACS bleeding risk factors. In the 2 presented cases, the ACS patients had no bleeding on admission; however, both patients suffered apparent gastrointestinal bleeding immediately after the development of sepsis or severe sepsis. The gastrointestinal bleeding might be induced by various factors, such as aspirin, clopidogrel, ticagrelor, lowmolecular-weight heparin, fondaparinux sodium, sepsis, or the cumulative effects of these factors. However, considering the characteristics of sepsis and the time point of gastrointestinal bleeding, sepsis should be at least one of the bleeding factors for both ACS patients. Does sepsis have a clinical significance as a bleeding risk factor for ACS patients? Should sepsis be included in the bleeding risk score to manage the bleeding risks for patients? How should the score be determined? More research is required to solve these problems.
In clinical practice, there is a time window for ACS patients between the onset of sepsis and gastrointestinal bleeding; thus, an early diagnosis of sepsis, an accurate assessment of bleeding risk, and timely and effective preventive measures are critical. PPIs make up the primary prevention measure that protects the alimentary tract from injury induced by antiplatelet medicines for ACS patients. [18] PPIs are also the basic precautions for stress ulceration gastrointestinal bleeding. [19] The Surviving Sepsis Campaign guidelines in 2013 favored the use of PPIs, compared with Histamine 2 Receptor Antagonists (H2RAs), for stressrelated mucosal disease prophylaxis. [20] Thus, PPIs are the preferred drugs to prevent gastrointestinal bleeding induced by sepsis in ACS patients.
Effective prophylaxis requires the correct timing and appropriate dosage of proper medication, as well as the appropriate method of administration. In the first case, the patient did not use PPIs for prophylaxis prior to the diagnosis of sepsis, and ultimately he died because of multiple organ dysfunction, which was induced by ACS, sepsis, gastrointestinal bleeding, and other risk factors. In the second case, the ACS patient was administered PPIs at standard oral doses, but it failed to prevent upper gastrointestinal bleeding when severe sepsis developed. However, the hemorrhage gradually stopped when the PPI was immediately administered at high doses intravenously, rather than orally, and the PPI was gradually adjusted to standard oral doses. When sepsis developed again, the patient had no recurrent gastrointestinal bleeding. These 2 cases suggested that ACS patients should be immediately administered PPIs to prevent gastrointestinal bleeding when sepsis occurred, and intravenous PPIs at high doses were required once severe sepsis or septic shock happened.
Conclusion
These 2 cases suggest that sepsis may be an important risk factor for gastrointestinal bleeding in ACS patients, but it is lack of direct evidence for the correlation between sepsis and gastrointestinal bleeding, so we still need large-sample clinical trials to verify the relevance and explore the mechanism. Moreover, our report indicates that PPIs could prevent gastrointestinal bleeding effectively in ACS patients complicated with sepsis, but the starting time and dosage of PPIs remain further research. | 2018-09-16T06:22:59.775Z | 2018-09-01T00:00:00.000 | {
"year": 2018,
"sha1": "b53825eda84dcfb6ad19c43dba4914fd706302a5",
"oa_license": "CCBYNCND",
"oa_url": "https://doi.org/10.1097/md.0000000000012273",
"oa_status": "GOLD",
"pdf_src": "PubMedCentral",
"pdf_hash": "b53825eda84dcfb6ad19c43dba4914fd706302a5",
"s2fieldsofstudy": [
"Medicine"
],
"extfieldsofstudy": [
"Medicine"
]
} |
219328832 | pes2o/s2orc | v3-fos-license | The effect of lipoxin A4 on E. coli LPS-induced osteoclastogenesis
Objectives The objective of the present study was to investigate the effect of lipoxin-type A4 (LXA4) on bacterial-induced osteoclastogenesis. Material and methods Human periodontal ligament cells (PDLCs) in coculture with osteoclast precursors (RAW264.7 cells) were exposed to bacterial stimulation with lipopolysaccharide (LPS) to induce inflammation. After 24 h, cells were treated to 100 ng/ml of LXA4 and 50 ng/ml of forymul peptide receptor 2 (FPR2/ALX) receptor antagonist (Boc-2). After 5 days, osteoclastic resorptive activity was assessed on calcium phosphate (CaP) synthetic bone substitute. Additionally, osteoclastic differentiation was evaluated using tartrate-resistant acid phosphatase (TRAP) staining, TRAP enzymatic activity assay, and on the expression of osteoclast-specific genes. Results We found that stimulation of in the osteoclasts with LPS-stimulated PDLCs induced a significant increase in tartrate-resistant acid phosphatase (TRAP) positive cells, higher resorptive activity, and enhanced expression of specific genes. Meanwhile, LXA4-treatment exhibited strong anti-inflammatory activity, and was able to reverse these inflammatory effects. Conclusions We conclude that (1) PDLCs are a potential target for treating bacterial-induced bone resorption in patients with periodontal disease, and (2) LXA4 is a suitable candidate for such therapy. Clinical relevance The results prove that lipoxins have a protective role in bacterial-induced periodontal inflammation and alveolar bone resorption, which can be translated into a clinical beneficial alterative treatment.
Introduction
Periodontal disease, the most common chronic disease in humans, is characterized by inflammation of the supporting tissues around the teeth [1]. Alveolar bone loss is one of the major hallmarks for disease progression, and combatting bone loss is therefore key to treating periodontal disease [2]. Normal bone remodeling is a dynamic process maintained by a balance between bone formation and resorption [3,4]. In periodontal disease, chronic inflammation disrupts the homeostatic balance between bone formation and bone resorption, in favor of bone loss [5]. There is evidence that the periodontal ligament (PDL) plays an important role in the remodeling of alveolar bone [5].
Periodontal ligament cells (PDLCs) are specialized spindle-shaped cells responsible for maintaining the integrity of the ligament that is connecting the tooth root cementum to the alveolar jaw bone [6]. However, in chronic inflammation (i.e., periodontitis), the phenotype of PDLCs changes, leading to a tissue-destructive response [7]. Microorganisms (e.g., Porphyromonas gingivalis, Fusobacterium nucleatum, and Escherichia coli) promote the PDLCs to interact with osteoclast progenitor cells (OPCs), which leads to the differentiation of these OPCs into mature osteoclasts [8,9]. During this interaction, PDLCs attract and stimulate the differentiation of OPCs towards mature osteoclasts via expression of adhesion molecules, such as adhesion molecule-1 (ICAM-1), and expression of osteoclastogenesis stimulatory factors, such as RANKL, M-CSF, and tumor necrosis factor-alpha (TNFα) [10]. In vivo, this leads to retraction of PDLCs and migration of OPCs to the bone surface; where the OPCs mature into bone-resorbing osteoclasts [11][12][13][14]. Consequently, the cascade of infection, inflammation mediated by the PDLCs, and osteoclast differentiation presents an important drug target for therapeutic agents aimed at suppressing bone loss.
Over the last decade, there has been an increasing number of studies on the anti-inflammatory effect of specialized proresolving mediators (SPMs), and especially of lipoxins (LXs) [15,16]. More recently, other effects of LXs have been observed, including the role of LXs in preventing bone resorption [17]. LXs are endogenous metabolic products derived from arachidonic acid metabolism, which have important pro-resolving and anti-inflammatory properties [18][19][20]. Lipoxin-type A4 (LXA4) binds specifically to a G proteincoupled N-formyl peptide receptor 2 (FPR2/ALXR) expressed by a variety of inflammatory cells, and which is also expressed in PDLCs [21]. The binding of LXA4 to its receptor induces a pro-resolving effect mainly by suppressing the expression of inflammatory mediators (e.g., IL-1β and IL-6) via inhibition of multiple signaling pathways, including receptor activator of nuclear factor-κB (NF-κB), which opposes inflammation [17,22,23]. The anti-inflammatory effect of LXA4 is well documented in the literature [22,24]. However, the effect of LXA4 on bacterial-induced osteoclastic differentiation during cell-cell contact between PDLCs and OPCs remains elusive.
Given the relationship between inflammation and osteoclast formation and function, we hypothesized that (1) LXA4 would have an inhibitory effect on bacterial-induced osteoclastogenesis, and (2) this effect would be reversed by FPR2/ALXR antagonist, Boc-2. To test this hypothesis, we developed a coculture model derived from a murine RAW264.7 osteoclast cell line, in direct contact with primary human PDLCs in vitro. After induction of an inflammatory phenotype of the PDLC (by the addition of E. coli lipopolysaccharide; LPS), the osteoclastic differentiation was evaluated by means of TRAP staining, TRAP enzymatic activity assay, resorption of a calcium phosphate substrate, and expression levels of osteoclast-specific genes.
Murine activator of nuclear factor-κ B ligand (mRANKL) was purchased from Peprotech (Rocky Hill, NJ, USA). All cell culture flasks and plates were purchased from Greiner Bio-one (Frickenhausen, Germany). The RNA Isolation Kit was obtained from Qiagen (Venlo, Netherlands). TaqMan Reverse Transcription kit and Fast SYBR Green Master Mix Kit were obtained from Thermo Fisher Scientific (Breda, Netherlands).
Calcium phosphate coating
A biomimetic calcium phosphate (CaP) coating was applied to glass coverslips using a two-stage process as described elsewhere [25,26]. The first stage involved immersing the glass slides in a 2.5-fold simulated body fluid (SBF) solution (1 ml/ well) at room temperature under light agitation for 3 days and daily refreshment of the solution. A total of 2.5-fold SBF was prepared by mixing Tris buffer (0.05 M; pH adjusted to 7.4 using 1 M HCl), calcium stock solution, and a phosphate stock solution together in a ratio of 2:1:1. The calcium and phosphate stock solution was prepared by dissolving the reagents specified in Table 1 in Tris buffer solution. The second stage involved immersing the glass coverslips in a calcium phosphate solution (CPS) (1 ml/well) for 1 day at room temperature under light agitation. CPS was prepared by fully dissolving each reagent in the order listed in Table 1 in MilliQ water, while ensuring that the pH did not rise above 8 using 1 M HCl. Once all the Tris was fully dissolved, the pH was adjusted to 7.4. After 1 day of soaking, the CPS was aspirated from the wells and 70% ethanol was added and left to evaporate to sterilize the CaP-coated glass coverslips. Lastly, the coverslips were gently rinsed three times with MilliQ water and dried overnight at room temperature before use in cell culture. Prior to cell seeding, the CaP-coated glass coverslips were incubated in FBS for 1 h at 37°C. Coating characterization SEM To evaluate the morphology of CaP-coated glass coverslips with 12-mm diameter (VWR B.V., Amsterdam, The Netherlands), specimens were sputter-coated with a 30nm thin chromium layer and imaged using a scanning electron microscope (SEM; Zeiss Sigma 300, Zeiss AG, Oberkochen, Germany).
X-ray diffraction A PANalytical X'Pert 3 X-ray diffractometer (Malvern Panalytical B.V., Eindhoven, The Netherlands) with a Cu Kα radiation source was employed to perform thin film X-ray diffraction (XRD) on CaP-coated glass slides using an incidence grazing angle of 2.5°. The measurements were performed at a voltage and current of 45 kV and 40 mA, respectively, with a step size of 0.02°and a count time of 1 s per step. All scans were performed between 10°and 50°2θ.
Fourier transform infrared spectral study spectroscopy Characterization of the crystal phase of the calcium phosphate coated on glass slides was investigated using FT-IR spectroscopy (Spectrum Two FT-IR Spectrometer, PerkinElmer, Waltham, USA). The samples were scanned in the 4000-650 cm −1 range with an ATR accessory.
Surface profile Characterization of the surface profile of the calcium phosphate coated on glass slides was investigated using Universal Surface Tester (UST®, Innowep GmbH, Wuerzburg, Germany). The samples were secured to the sample stage and measurements were taken using the stylus diamond tip (R = 2 μm, 60°) connected to the measurement head with velocity of 0.1 mm/s bearing the load of 1.0 mN at increments of 2.0 μm for a total distance of 5 mm.
Cell culture
All experiments were done in accordance with the national guidelines for working with human materials (Dutch Federation of biomedical scientific societies, human tissue, and medical research; code of conduct for responsible use. Available at https://www.federa.org/). The RAW 264.7 murine macrophage cell line (ATCC®TIB-71™) was purchased from the American Type Culture Collection (Manassas, VA, USA) and maintained in αMEM medium supplemented with 10% FBS and 1% PS at 37°C in a humidified atmosphere of 5% CO 2 until 80% confluence (number of passages was < 10). After informed patient consent, human periodontal ligament cells (PDLCs) were harvested from several healthy adult donors who underwent extraction of a third molar as previously described [27]. PDLCs were cultured at 37°C in a humidified atmosphere of 95% air and 5% CO 2 . The medium was replaced every 2 to 3 days until 50% confluence was reached, and then, the PDLCs were passaged before being frozen in medium supplemented with 10% dimethyl sulfoxide (DMSO; Sigma-Aldrich). After defrosting, PDLCs were passaged again and maintained in sterile 75 cm 2 culture flasks with DMEM medium supplemented with 10% FBS and 1% PS and cultured at 37°C in a humidified atmosphere of 95% air and 5% CO 2 . For all experiments, the cell passage was ≤ 5.
RANKL-induced osteoclasts
As a positive test for osteoclast differentiation, RAW264.7 cells were seeded in a 24-well plate (2 × 10 3 cells/cm 3 ) and cultured in αMEM supplemented with 10% FBS and 1% PS. At day 1, medium was refreshed with differentiation medium (50 ng/ml of murine RANKL) to induce osteoclast formation. At day 2, medium was refreshed with differentiation medium supplemented with LXA4 100 ng/ml and Boc-2 50 ng/ml, and then, cells were returned to the incubator at 37°C in a humidified atmosphere of 95% air and 5% CO 2. The medium was replaced every 2 days. After 5 days, the cells and medium were collected for TRAP staining and enzymatic assays. Parallel samples were collected for mRNA extraction.
LPS/PDLC-induced osteoclasts
Protocol for direct coculture of human PDLCs and murine RAW264.7 was adapted from a previous protocol [9]. PDLCs were cultured in a 24-well plate (1 × 10 4 cells/cm 3 ) with DMEM medium supplemented with 10% FBS and 1% PS and cultured at 37°C in a humidified atmosphere of 95% air and 5% CO 2 . After 48 h, RAW 264.7 (2 × 10 3 cells/cm 3 ) were seeded on top of PDLCs using a 1:1 mixture of basic medium (DMEM:αMEM). After 24 h, cell culture medium was refreshed with basic medium supplemented with LPS (1 μg/ml) to let PDLCs induce osteoclast formation in RAW264.7 cells, as shown in Fig. 1. After 24 h, medium was replenished with fresh medium supplemented with LXA4 (100 ng/ml) and Boc-2 (50 ng/ml), and cells were cultured accordingly as before. After 5 days, the cells and medium were collected for TRAP staining and enzymatic assays. Parallel samples were collected for mRNA extraction.
Osteoclast purification and transfer
Purification and transfer of osteoclasts was adapted from Maria et al. (2014) [26]. After induction of osteoclasts, both by RANKL and by LPS/PDLC induction, the medium was aspirated and cells were washed twice with pre-warmed sterile PBS. To detach undifferentiated cells, 0.25% trypsin (1 ml/ well) was added and the plate was returned to the incubator at 37°C for 1 min. The medium containing undifferenced mononucleated cells was aspirated, leaving behind large multinucleated cells (> 100 μm in diameter). The differentiated osteoclasts were dislodged from the plate by gently pipetting the solution, while observing under the microscope. The differentiated osteoclasts were then seeded at the exact seeding density on top of calcium phosphate-coated glass slides that had been incubated prior with FBS for 1 h at 37°C. Tissue culture plates were returned to the incubator carefully and maintained for 48 h at 37°C in a humidified atmosphere of 95% air and 5% CO 2 .
Osteoclast resorption
After incubation of osteoclast on CaP-coated glass slides for 48 h, cell culture medium was aspirated, and cells were washed twice with sterile PBS. The cells were then removed by incubating with sodium chloride (1 M) in 0.2% Triton-X-100 for 1-2 min, and subsequently washed with MilliQ water.
To stain for calcium, the CaP-coated glass slides were incubated in silver nitrate solution (5% AgNO 3 ) at RT under an UV lamp for 1 h. Then, the glass slides were washed with distilled water and the coating surface was examined by a light microscope. The osteoclast resorption pits were measured using Fiji 1.51n software ( The sequence of applied primers is given in Table 2. The expression levels were analyzed and normalized with the house-keeping gene glyceraldehyde 3-phosphate dehydrogenase (GAPDH) by calculating ΔCt (Ct gene of interest -Ct GAPDH ), and the expression of the tested gene was calculated using the 2 −(ΔCt) .
Statistical analysis
Statistical differences were evaluated using one-way analysis of variance (ANOVA) test with a pairwise multiplecomparison test (Tukey test) to assess significant difference in TRAP enzymatic activity, resorption pit formation, and gene expression between groups. All statistical analysis was performed using the GraphPad Prism v.5 (GraphPad Software, San Diego, USA). Data are expressed as mean ± standard deviation (SD). Differences were statistically significant when p < 0.05. For all assays, three independent experiments were performed using different cell batches, in which the experimental specimens were present in triplicate (n = 3).
Results
Characterization of calcium coating SEM micrographs of the CaP-coated glass slides (Fig. 2a) showed a homogeneous and uniform distribution, with a coating thickness of~5 μm as detected by a Universal Surface Tester (Fig. 2e). The XRD patterns (Fig. 2c) displayed diffraction lines characteristic of hydroxyapatite (HA), which were comparable with literature [26]. Moreover, FT-IR spectra ( Fig. 2d) ) in the HA chemical structure [28]. Also, the SEM micrographs revealed that treatment of RAW264.7 cells in just basic medium did not affect the HA coating and left the coating intact (Fig. 2b I and II), while the sheer size and number of osteoclasts (± 50−100 μm) were strongly upregulated upon treatment of RAW246.7 cells with differentiation medium (basic medium supplemented with RANKL) for up to 5 days (Fig. 2B III and IV).
Osteoclast resorption
Microscopic images showed an intact coating in response to cells cultured with medium alone (control) (Fig. 3aI). However, several resorption pits were found on the CaP coating following incubation of osteoclasts with RANKL or with LPS/PDLC stimulation (Fig. 3a II and VI). For both types of stimulation of the RAW cells, the relative area of osteoclasts was visually downregulated in response to LXA4 treatment (Fig. 3a III and VII). Conversely, the inhibitory effect of LXA4 was visibly reversed upon treatment with LXA4 together with its receptor inhibitor, Boc-2 ( Fig. 3b IV and VIII).
Furthermore, the total resorbed area (%) relative to the control (medium alone) in response to the indicated condition was measured (Fig. 3b I and II). In agreement with the visual inspection, quantitative analysis revealed no significant changes in resorption activity of the RAW cells supplemented with control medium. However, a significant increase in the resorptive capacity was observed in response to either RANKL or the LPS/PDLC challenge alone compared with control (p < 0.001 and p < 0.01, respectively). Furthermore, a significant decrease in osteoclastic resorptive activity was measured in response to treatment of both groups with LXA4 compared with RANKL or LPS/PDLC-treatment (p < 0.001 and p < 0.01, respectively). Conversely, a significant increase in osteoclast resorption activity was observed upon treatment of RANKL or LPS/PDLC-challenged cells with LXA4 and Boc-2, as compared with LXA4 treatment alone (p < 0.001 and < 0.05, respectively). Fig. 4a IV and VIII). Furthermore, TRAP activity in cell culture media was also measured to confirm the visual inspections (Fig. 4b). In agreement with morphological assessment, a significant increase in TRAP activity was observed following treatment of RAW264.7 cells with RANKL or LPS/PDLC (p < 0.0001 and p < 0.001, respectively). Moreover, TRAP activity was significantly decreased back to baseline level upon treatment of RANKL as well as LPS/PDLC-stimulated cells with LXA4 alone (p < 0.05 and p < 0.001, respectively). Conversely, the inhibitory activity of LXA4 was suppressed by treatment with Boc-2, and TRAP activity was once again significantly different compared with the control (p < 0.05 and p < 0.001, respectively). No dose-dependent inhibition was observed, as all the LXA4 concentrations investigated strongly inhibited RANKL as well as PDLCs/LPS-induced osteoclastic differentiation (Fig. 4d).
Real-time PCR
Finally, the expression of osteoclast-specific genes was analyzed to investigate the underlying mechanism by which LXA4 induces the inhibition of RANKL and LPS/PDLCstimulated osteoclastogenesis. RANKL stimulation signific a n t l y u p r e g u l a t e d t h e e x p r e s s i o n l e v e l o f 5a-c). Similarly, LPS/PDLC stimulation significantly upregulated the expression levels of osteoclast-specific genes relative to the control (Fig. 5d-f). Accordingly, the expression level of osteoclast genes was markedly higher in response to RANKL treatment compared with LPS/PDLC stimulation. Overall, the treatment of cells with LXA4 significantly downregulated the expression level of osteoclastogenesis marker genes in either case. Lastly, treatment of both types of stimulated cells with Boc-2 reversed the inhibitory effect of LXA4 back to baseline.
Discussion
Previous investigations into the pathogenesis of periodontal disease have typically centered on the role of bacterial infection. However, over the past decade, there has been an increasing interest in the inflammatory response that occurs after infection and which gives rise to osteoclastic bone resorption [29]. In view of this, it has been demonstrated that PDLCs, although not commonly regarded as inflammatory cells, might also play a role and are contributing to the differentiation of osteoclasts by their interaction with osteoclast progenitor cells [30][31][32]. In periodontal disease, bacterial colonization alters the phenotype of PDLCs by activating inflammatory signaling pathways, which promote tissue-destructive responses [7,33]. Subsequently, osteoclastic differentiation is promoted by cell-cell-mediated interactions between PDLCs and OPCs-a phenomenon facilitated by adhesion molecules (e.g., ICAM) and release of inflammatory factors (i.e., cytokines, chemokines) [29,34,35]. Cell-cell crosstalk between PDLCs and OPCs leads to the migration of TRAPpositive OPCs to the bone surface where osteoclastogenesis occurs, resulting in bone resorption [29,34,35]. In the current study, we aimed to set up a similar model, yet in an in vitro environment, to test novel types of medication, which can aid in the treatment of periodontitis. Therefore, a coculture system The periodontal microenvironment contains a wide diversity of oral pathogens which play an important role in periodontal health or disease. The periodontal biofilm is heterogenous with approximately 7000 organisms [36,37]. Although oral pathogenic bacteria, such as P. gingivalis, are commonly associated with PD, "non-oral" pathogens (e.g., E. coli) have also recently been implicated in the pathogenesis of this disease [38]. Several studies reported on the presence of non-oral bacterial species after microbiological analysis of the subgingival biofilm of PD patients [39,40]. Analysis of the subgingival biofilm samples from a large number of untreated chronic PD patients revealed elevated levels of E. coli compared with non-PD subjects [39]. Souto and co-workers revealed a positive correlation with clinical signs of PD with bacterial pathogens, including E. coli, Staphylococcus aureus, and Pseudomonas aeruginosa [40]. In primary PDLCs, an inflammatory phenotype was induced by adding bacterial E. coli LPS, which in term should trigger osteoclast formation by inducing RANKL expression in osteoblasts and other stimulatory factors (IL-1, prostaglandins, TNFα) leading up to bone resorption [41]. While oral pathogens are more pathologically relevant bacterial source, E. coli-derived LPS is more effective at inducing bone resorption in vitro because it is a strong inflammatory agonist for toll-like receptors (TLRs) [42][43][44] [45]. Conversely, P. gingivalis LPS induces a significantly lower expression of inflammatory cytokines (e.g., IL-6) compared with E. coli LPS [46]. Considering PDLCs produce a little inflammatory profile, E. coli LPS is commonly used to trigger an inflammatory immune response [47]. The subgingival biofilm of PD patients is complex with higher proportion of periodontal pathogens, including lesser known ones, and knowing the effect of different microorganisms is fundamental to our understanding of the complex mechanisms involved in this multifactorial disease. The mechanism of LPS-induced osteoclastogenesis can be explained as follows. Under inflammatory conditions, LPSstimulated PDLCs select and attract osteoclast precursors to fuse into osteoclasts by upregulating the expression of osteoclastogenesis-stimulating molecules [5]. During this interaction, LPS binds to TLR4 receptors on PDLCs and stimulates the expression of pro-inflammatory mediators, TNFα, IL-1, and PGE 2 , which play a crucial role in maturation of OPCs and bone resorption [47]. It is important to note that LPS does not promote osteoclast differentiation in the absence of osteoblasts/stromal cells (e.g., PDLCs) [48][49][50]. Thus, LPS-promoted osteoclastogenesis can be attributed to the direct cell-cell interaction between PDLCs and RAW cells and not from direct interaction of LPS [9,13]. As a control for our coculture, we also stimulated osteoclast formation artificially, as is commonly done in literature, by the addition of RANKL. The similar results between RANKL stimulation, vs. the effects obtained by LPS/PDLC stimulation, indeed make it plausible that the LPS/PDLC route is a valid representation of the actual situation. Moreover, the subsequent experiments with the CaP coating proved that the RAW cells upon such stimulation became actively matrix-degrading osteoclasts.
After setting up and validating the coculture system with the clinical situation, a suitable drug strategy was selected, for which we focused on the use of SPMs.
Among the SPMs released during inflammation, LXs are key to the resolution of inflammation [15,51]. LXA4 evokes several important protective responses in vivo, including inhibition of neutrophil recruitment, activation, and chemotaxis, via inhibition of several downstream pathways, such as NF-κB, and activator protein-1 (AP-1) [17,[52][53][54][55][56][57]. Moreove r, LXA4 ex erts a poten t antiinflammatory action by modulating leukocyte activity and promoting phagocytosis of apoptotic cells [58]. The binding of LXA4 to its receptor (FPR2/ALXR) interferes with osteoclastogenesis by suppressing the expression of inflammatory mediators (e.g., IL-1β and IL-6) via inhibition of multiple signaling pathways, including receptor activator of nuclear factor-κB (NF-κB) [59]. Inhibition of NF-κB suppresses the expression of pro-inflammatory cytokines, which counteracts inflammation-induced bone resorption [60]. The role of LXA4 in inhibition and function of osteoclasts has been recently described in the literature [17]. However, the role of LXA4 as a modulator of cell-cellmediated osteoclastogenesis remains elusive [61]. Given the lack of studies regarding the role of LXA4 in cellcell-mediated osteoclast differentiation and function and considering the importance of PDLCs in inflammationinduced osteoclastogenesis, we investigated the effect of LXA4 on osteoclastogenesis promoted by LPS/PDLC Fig. 5 Expression levels of RANKL, TRAP, and CK were determined by qRT-PCR in (a-c) RANKL-stimulated RAW264.7 monoculture, and (df) LPS/PDLC-stimulated RAW264.7. Note that LXA4 inhibited RANKL and LPS/PDLC-induced expression of osteoclast marker genes, while Boc-2 reversed this response. Expression levels were calculated in relation to internal controls (GAPDH mRNA) with a comparative Ct method. Statistical analysis was performed by one-way ANOVA with Tukey post-test, n = 100 *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001. Asterisks on top of the columns indicate significant differences from the control challenge, as model system. It was hypothesized that (1) LXA4 would inhibit osteoclast differentiation, and (2) this effect could be reversed by the receptor antagonist, Boc-2. Our data confirmed that both of these hypotheses were tested true.
In the experiments, osteoclastogenesis was analyzed using several complimentary morphological, molecular, and functional assays designed to test all aspects of osteoclast formation, activity, and function. Finally, we performed RT-PCR to fully investigate the mechanism underlying the inhibition of osteoclastogenesis. The results from these studies indicated that RANKL stimulation causes an upregulation in osteoclast differentiation from OPCs. Additionally, osteoclast formation was upregulated during cell-cell contact between PDL and RAW cells when conditioned medium containing bacterial LPS was used. The in vitro experiments demonstrated that PDLCs adapt to bacterial stimuli by upregulating the expression of osteoclastogenesis-stimulating genes, resulting in the release of pro-inflammatory mediators (i.e., cytokines and chemokines) that enhance osteoclast activity and function. Comparison of our results with literature corroborates with most of these effects. For instance, Kanzaki et al. (2001) showed that PDLCs cocultured with peripheral blood monocular cells (PMBCs) exhibited significantly more resorption pits than PMBCs cultured alone [62]. Furthermore, Bloemen et al. (2010) and Burger & Dayer (2002) showed direct that cell-cell contact increased synergistically the expression of osteoclastogenesis genes in vitro [9,63]. Similar effects were further demonstrated in vivo by Kim et al. (2005), who reported about the formation of osteoclasts, independent of RANKL signaling pathway, in response to stimulation with inflammatory mediators (TNFα and IL-1β) [64]. Considering our results in comparison with the available literature justifies the conclusion that PDLCs contribute to enhanced osteoclast formation in periodontal disease. Evidently, PDLCs can play an important role as a drug target when aiming to maintain hemostasis in the periodontium [5].
Furthermore, the results of the current study showed inhibition osteoclastogenesis in response to inclusion of LXA4 in the differentiation medium. The reduction of osteoclast formation implied a strong protective role of SPMs against inflammation-induced bone resorption, especially as it could be reversed by the addition of a specific inhibitor. Not only the number but also the function of osteoclasts could effectively be modulated; i.e., the decrease in osteoclastogenesisassociated genes was correlated with absence of resorption pits on the CaP-coated substrates. Apparently, LXA4 carries anti-inflammatory capacity after an in vitro encounter with bacterial LPS. In agreement with our data, Liu et al. (2017) showed that LXA4 treatment reduced osteoclast formation in RANKL-stimulated RAW cells [17]. Combination of our data and the literature information confirms again that that both our initial hypotheses were true.
The study has potential limitations. TRAP activity, function, and osteoclast-specific gene expression was notably lower for LPS/PDLC compared with RANKL-stimulated RAW cells. There are several explanations for this effect. Firstly, LPS may negatively affect osteoclast formation by promoting the expression of pro-inflammatory mediators (TNFα and nitric oxide), which may negatively affect cell viability (even at low concentrations) [65][66][67]. Secondly, RANKL is a strong inducer of osteoclast formation [68,69], while LPS induces formation of osteoclasts independent of the RANKL pathway by activating pattern recognition receptors, such as TLR4, which can affect activation and survival of osteoclasts [5,70]. Still, the RANKL stimulation should be considered as an artificial control, whereas the PDLCs route might be more physiologically relevant. Furthermore, murine RAW264.7 monocytes were integrated into the cell culture system because there is currently a lack of a reliable osteoclast model using a human cell line [71]. While the human monocytic leukemia cell line (THP-1) would have presented a more clinically relevant model, these cells are unable to consistently develop into multinucleated osteoclasts and are also far less responsive to LPS [72,73]. Therefore, it can be challenging to extrapolate the data obtained using this human cell line. On the contrary, murine RAW264.7 cells produce a more robust inflammatory response when challenged with bacterial LPS and have been extensively used to carry out in vitro screens for immunomodulators [74].
Conclusion
In conclusion, our findings demonstrate the importance of cell-cell signaling between PDLCs and osteoclast precursors in inflammation-induced osteoclastic differentiation. In addition, our data validate the inhibitory role of LXA4 in this process. It is important to note that the current data are the result of in vitro study and might not reflect the clinical in vivo situation. Hence, in vivo (pre)clinical experiments are needed to fully validate whether LXA4 can indeed inhibit osteoclastogenesis and prevent inflammation-induced bone resorption. Nevertheless, the results merit such further investigation and provide important considerations for the implementation of LXA4 in periodontal therapy.
Compliance with Ethical Standards
Conflict of interest The authors declare that they have no conflict of interest.
Ethical approval This article did not contain any studies with human participants or animals performed by any of the authors.
Informed consent For this type of study, formal consent is not required.
Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. | 2020-06-06T14:13:11.368Z | 2020-06-06T00:00:00.000 | {
"year": 2020,
"sha1": "530628d58f411de933b22b3fca38eb66a289efb7",
"oa_license": "CCBY",
"oa_url": "https://link.springer.com/content/pdf/10.1007/s00784-020-03385-3.pdf",
"oa_status": "HYBRID",
"pdf_src": "PubMedCentral",
"pdf_hash": "90c6373e30c42008b17724ef06c83704efc4f233",
"s2fieldsofstudy": [
"Medicine",
"Biology"
],
"extfieldsofstudy": [
"Medicine",
"Chemistry"
]
} |
51967033 | pes2o/s2orc | v3-fos-license | Identifying disrespect and abuse in organisational culture: a study of two hospitals in Mumbai, India
Abstract This paper draws on findings from a qualitative study of two government hospitals in Mumbai, India, which aimed to provide a better understanding of the institutional drivers of disrespect and abuse (D&A) in childbirth. The paper describes the structural context, in which government hospital providers can exercise considerable power over patients, yet may be themselves vulnerable to violence and external influence. Decisions that affect care are made by a bureaucracy, which does not perceive problems with the same intensity as providers who are directly attending to patients. Within this context, while contrasting organisational cultures had evolved at the two hospitals, both were characterised by social/professional inequality and hierarchical functioning, and marginalising women. This context generates invisible pressures on subordinate staff, and creates interpersonal conflicts and ambiguity in the division of roles and responsibilities that manifest in individual actions of D&A. Services are organised around the internal logic of the institution, rather than being centred on women. This results in conditions that violate women's privacy, and disregards their choice and consent. The structural environment of resource constraints, poor management and bureaucratic decision-making leads to precarious situations, endangering women’s safety. With the institution's functioning based on hierarchies and authority, rather than adherence to universal standards or established protocols, irrational, harmful practices endorsed by senior staff are institutionalised and reproduced. A deeper focus on organisational culture, embedded in the discourse of D&A, would help to evolve effective strategies to address D&A as systemic problems.
Introduction
Disrespect and abuse (D&A) of women during childbirth need to be understood not as random acts or aberrant behaviour of individual providers, but as systemic problems. 1 Various facility factors, such as overcrowding, shortages, hospital rules and policies have been implicated for accentuating D&A. 2,3 In highly stratified societies, typically, very hierarchical and undemocratic relationships form between providers and women. 4,5 Less educated women, rural women, women with a stigmatising condition like HIV, unmarried mothers, women seeking care in government hospitals have been documented to face greater levels of (D&A). [6][7][8] Presently, the discourse on D&A accommodates providers' perspectives to the extent that they provide explanations for the providers' behaviour as reported by women. Few studies have attempted to locate particular abusive, irrational, harmful practices in a specific hospital environment, with its rules, relationships and power structures. 9 For example, in Brazil, routine practice of episiotomy was embedded in a stratified health system where poor women were relegated to limited and under-resourced government institutions in which they became unwitting subjects for clinical training. 10 Various harmful and painful interventions to hasten labour (manual dilatation of the cervix and routine induction) were part of a strategy to "clear" the wards in order to cope with the high patient load in Mexico. 11 Nurses' behaviour towards poor women was embedded in the legacy of a colonial health system characterised by deeply unequal and almost coercive relationships between patients and providers in South Africa. 12 A review of research from sub-Saharan Africa concluded that individual providers' attempts to exert power and control and maintain their higher status vis-a-vis labouring women were explained by an "institution-centred, medicalised and hierarchical" model of maternity care. 13 These studies explored aspects of organisational dynamics to the extent that these produced particular practices. However, understanding the organisational context more fully would help explain how and why patterns of behaviours and actions emerge and how they get reproduced within the institution. Midwives perceived unsupportive working environment, unsatisfactory interactions with women, lack of training and the neglect of best practices as barriers to providing perinatal care. 14 Burnout and moral distress result from the interaction of social factors such as the low status of midwifery, the low wages of health workers, and organisational problems such as poor skill development and training and inter-professional conflicts. 15 Working in very constrained conditions with highly disadvantaged women engenders a fatalism about patient deaths among frontline workers 16 Official reviews inevitably end in attributing blame to lower level functionaries, further demoralising them. 17 Inevitably, these result in an organisational culture where women are devalued and D&A is normalised.
In this paper, we examine the organisational culture of two government hospitals, as reflected in providers' articulation of their beliefs, their descriptions of work and their analysis of daily situations, in order to understand the drivers of D&A in institutional obstetric care. Our analysis of provider narratives is supplemented by observation and review of documents.
Our study, focusing entirely on professional providers (doctors and staff nurses), was conducted prior to the discourse on D&A gaining currency. However, our findings still resonate with the feminist discourse on the violation of women's rights in the health system that echoes most, if not all, of the contemporary concerns. Studies which had documented women's experiences of medicalised childbirth in the Indian context had pointed to a range of problematic areas. This included the ridiculing of women's cultural beliefs and talking down to them, 18 the common use of physical force and violence, 19 coercion to accept birth-control, 20 asking for husband's consent for abortion, 21 devaluation and penalisation of women's experience of pain, 22 discrimination based on caste, 5 and a deep-rooted gender bias that produced apathy and neglect. 17 We also draw on parallel literature that criticises the over-medicalisation of childbirth and the ethics in contemporary clinical practice. This literature identifies problematic areas including the overuse of interventions such as episiotomies 10 and Caesarean sections. 23 Thus, in this study, we focus on domains of practice where violations or ethical problems had been commonly observed, namely, providerpatient interactions, cultural and social issues encountered in care-giving, management of labour pain, routine practices and procedures associated with normal vaginal deliveries, management of complications and post-delivery contraception.
Methods
The study was done in two government hospitals in the metropolitan Indian city of Mumbai between 2010 and 2013. We studied obstetric practice in one tertiary care teaching hospital attached to a government medical college (MC) and a government secondary care hospital located in an extended suburban district of the Mumbai urban agglomeration (SH). Both of these hospitals were representative of their sector in terms of organisational structure, workload and resource availability.
Two of the co-investigators (NR and PM) are practicing clinicians, the latter being an obstetrician. One of the researchers (NM) had extensive experience of government hospitals due to her prior research experience. This multi-disciplinary team had numerous discussions and debates to evolve a conceptual framework that would reflect their diverse perspectives, even as it spoke to the different disciplines that they representedsocial sciences, bioethics and medicine.
Based on these inputs, we refined the specific research question, demarcated domains of inquiry and designed data collection tools. We organised consultation processes prior to the conduct of the study. One involved international and Indian researchers and academics/researchers who undertook scientific review of the project. The other involved practising professionals from government and private hospitals in Mumbai and focused on the use of language, the approach to fieldwork, probable logistical and ethical problems that we could expect to encounter.
The study used qualitative methods, including in-depth interviews, key informant interviews and observation. Fieldwork was carried out by a team of trained social science researchers (NM, RG and three others). All the researchers were fluent in English and one or both of the dominant local languages, Hindi and Marathi. All the researchers were women, of Indian origin and, barring one, resided in Mumbai.
As a first step, we compiled information about the institutions using an observation checklist and information from key informants to draw up an institutional profile. We compiled statistics on the institutions' performance, including the numbers of deliveries conducted, C-Section rates and patient outcomes. We made note of the physical infrastructure, layout and design of the space, provisions for allied services such as pathology, radiology, pharmacy and the condition of these facilities. We also documented stated policies regarding recruitment, supervision and monitoring of staff and the challenges faced by the institution in this regard. We documented norms related to the division of labour between various categories of staff and decision-making related to patients. This profile was very useful for understanding the formal framework within which the organisation functioned. It also helped the team to identify areas where actual practice was in contradiction with stated norms.
Simultaneously, the researchers who would be involved in conducting interviews and observations had extensive informal interactions with experienced obstetricians, nurses and hospital administrators who had experience of working in similar settings, to gain an understanding of frequently used technical terms and abbreviations as well as the discursive language that marked that sub-culture, embedded in slang, jokes, "open-secrets", dictums and stock phrases. This was followed by a period of 2-3 weeks spent in the hospitals interacting with staff and observing everyday routines. Apart from gaining familiarity with the hospital's functioning, this served as an opportunity to offer clarifications and explanations to staff who were curious about our presence and, to the extent possible, reduce the possibility of our presence altering the environment.
In the government hospitals, we had access to clinics, waiting areas, staff rooms, nursing stations and antenatal and postnatal wards. These were areas accessible to the female relatives of the childbearing women. Additionally, we had been allowed to visit the labour room and operation theatre when it was not in use. As it was focused on providers, we did not study women or their relatives. We specifically avoided being present in situations or locations where we would invade the privacy of patients. Our interactions with women and their relatives were limited to responding to their queries. Some casual conversations, as which typically occur among bystanders, took place often. However, it was explicitly known to the hospital management and staff that we would not be interviewing patients.
All professional providers connected with obstetric services (obstetricians, residents, nurses, social workers and hospital administrators) in the two government hospitals were included in the sampling frame. Overall, we interviewed 16 presently employed obstetricians (residents and consultants), 5 formerly employed obstetricians, 7 nurses and 2 hospital administrators connected to the obstetrics services across these two hospitals.
Overall, we interviewed about half of obstetricians present and about one-fourth of the nurses. Written consent was obtained from each participant. A formal letter of permission signed by the Dean/ Hospital Superintendent was available and a copy was shared with each participant. However, most participants had already received information about the study from the hospital authorities, conveyed via their supervisors.
Interviews were conducted over 1-3 sessions, mostly in the wards. Each session was interrupted by the participants to attend to patients or finish paperwork. If the researchers perceived that the participant was too busy, despite having consented to be interviewed, they proactively sought an appointment on another day and or volunteered to wait. Almost all the interviews were audiotaped. We conducted their interviews in the language of the participants' choice. We shared the interview guide with each participant at the beginning of the interview. This included questions on their personal background, motivations for joining their chosen profession, training, routine practices related to conducting normal deliveries, pain-relief, post-partum contraception, complicated cases, referral and transfer, and clinical audit. We also asked them to describe the women who sought care and elicited accounts about individual women or incidents that they found challenging and rewarding.
While we waited to meet a participant, which was a period of 2-5 hours, we observed providerpatient interactions, conversations and exchanges between staff members, routines of individual providers as well as unusual events. Typically, we were present in the hospitals between 10 am and 8 pm. We made notes and included specific probes/questions in the interviews with participants related to these. These questions typically related to underlying conflicts among staff, undocumented challenges in providing care, stress and workload, adaptations to cope with specific problems, actions which aimed at building solidarity and co-operation between staff.
Interview transcripts and field notes were coded and analysed using WeftQDA. Two stage coding was undertaken. Minute coding was done to identify specific areas of discussion, e.g. patient profile, discharge procedures, pain relief. These were combined into themese.g. provider attitudes, resource management, grievance redress and supervision.
The study was approved by the Multi-Institutional Ethics Committee to which the Anusandhan Trust was affiliated. It was also approved by the ethics committee of MC. We obtained official permission to conduct fieldwork in SH, which did not have any ethics committee.
Results
The broader context As a result of high workload and adverse working conditions, delivering obstetric care was viewed as problematic in these hospitals. The participants recounted problems related to poor management, socio-cultural barriers in delivering care to patients, shortages and misallocation of resources. There was an acknowledgment that mistreatment of women in the form of shouting and physical coercion existed, although this was not necessarily perceived as abuse. Several other aspects of practice, such as use of pain relief medication, episiotomies, induction of labour and postpartum contraception, were perceived to be unproblematic.
Both hospitals offered free services and usually did not refuse admission to any woman. However, the provision of services was not located in a framework of civic rights. Despite a policy shift away from coercion, we found women were being compelled to accept birth control after delivery. There was an informal code in both hospitals that women must accept tubal ligation after two deliveries and IUD insertion after the first. The typical strategies used to pressurise women were refusing discharge, threatening not to conduct the procedure or banning her from the hospital. Typically, consent for these predetermined choices was negotiated when women were at their most vulnerable. There was no entitlement to a specific set of services and resources. The women and their relatives were largely expected to fill gaps. A study conducted in a similar context has documented women and their relatives being compelled to clean the wards and wash their own clothes. 24 While we did not observe these practices, relatives were asked to take on many tasks officially to be performed by orderlies. In both hospitals, obstetric services were spread across different floors. Laboratory and radiology services were located in different wings. Thus, relatives were required to push the gurney, transport samples, bring medicines, etc. Not only did this impose economic burdens on the family, it led to overcrowding and chaotic conditions in the wards.
Staff typically attributed these problems to the municipal corporation, which controlled vital aspects of the hospital functioning such as admission policies, charges, staffing, documentation and some aspects of clinical work. This displaced decision-making from the domain of professional to bureaucratic practice. Hospital managers either had to resort to informal means to overcome challenges or cope with existing resources.
"You have reason to get a little frustrated, agree. But you also have ways of getting it done. You have to literally explain about the requirements, and you have to give reasoning, perfect reasoning for that.
[You need] a superior who is backing you in such types of job. He should be like a godfather." (VPJ, Senior Manager, SH, male) "I cannot say we do not have enough nurses because the corporation does not have sanction for appointing more people. We have to do with whatever we have." (KSVB, Senior Faculty, MC, male) The price of seeking patronage was accommodation of local elected representatives' demand for special services or attention to particular patients, who were popularly called "note-cases", as they brought hand-written notes of recommendation. In both hospitals, there were expressions of disappointment about the top management's inability to protect the frontline staff from feeling pressurised.
"I was attending a high risk woman and another woman who was not due for another 4 to 5 hours came in and along with some local party workers, demanding that I attend to her immediately. I refused, so they threatened me and went out and soon there were more people. They called up the superintendent and complained against me. I explained, but I was compelled to attend to the other woman. They [hospital management] told me that "nothing could be done." They should have stood by me … but then, maybe had I been in their position I too would have done the same." (VAN, Senior Resident, SH, male) Specific organisational contexts and cultures in the hospitals Within this paradoxical context of arbitrary power over women and subordination to the bureaucracy, the hospitals had evolved quite differently in response to their specific contexts. SH was located at the periphery of the city. Typically, most women here were "booked patients", i.e. registered here during pregnancy and also delivered here. As the secondary care referral hospital (SH) received women from a defined catchment area, there was relatively more familiarity with women. Additionally, although it had similar provider:patient ratios to the medical college hospital (MC), the predominance of uneventful births created a more relaxed working environment.
Typical of peripheral centres, hospital management had little effective control over the specialists on the staff; there was high turnover, absenteeism and covert engagement in private practice. By administrative arrangement, a nearby private medical college placed residents here for clinical training, who became the mainstay of medical services. At any given time and particularly from late afternoon till morning, residents were the only medical staff present.
Private anaesthetists had been empanelled to provide services. Departments such as radiology and pathology did not function round the clock. This inevitably left residents in charge of making decisions, not merely based on their evaluation of the woman's medical condition, but taking into account various external constraints. There were no protocols for the management of emergencies or routine cases. This environment led them to make ad hoc decisions. Hence, sudden transfers to fairly distant tertiary care hospitals were not infrequent.
"If there is an emergency, like the baby [foetus] is in distress, we have to take in the patient even after [our shift]. But sometimes the anaesthetist is not available, or refuses to come, then we [residents] only have to hand over the patient to the other hospitals." (VAV, Senior Resident, SH, female) There were no organisational safeguards for dealing with sudden and life-threatening emergencies which were beyond the competence of residents or where there was not enough time to transfer the patient. Such incidents, when they occurred, exposed the precarious conditions of care at the hospital.
"Inverted uterus is a life threatening situation. We shifted the woman from the labor ward to the main OT. Fortunately the anaesthetist was there. We induced and immediately delivered the baby. Within 2 minutes we repositioned the uterus. Had I been not there in that shift the patient would have been dead since no one knew how to reposition the uterus." (VAM, Head of Ob/Gyn, SH, male) However, as emergencies were not frequent events, there was no attempt to discipline the medical staff more stringently. Instead, the hospital administration relied on its nursing staff to keep the hospital functional. The nursing staff were a relatively cohesive and stable group. They had a complex and ambivalent relationship with the residents. They were usually much older and skilled at performing clinical tasks. At the same time, the residents had greater clinical knowledge and could conduct surgeries. Residents were also officially responsible for conducting vaginal deliveries, although these are often conducted by nurses. This often resulted in a conflict at the frontline about the division of work and responsibility.
"Some doctors will say that he will have his lunch and only then come in for the delivery and if the patient is in 2nd stage then we will deliver her. We can't delay the delivery and put the patient at risk!" (VLD, Staff Nurse, SH, female) Also, nurses were still seen as doing less skilled and "dirty work", a phenomenon which has been documented. 25 This undermined the importance of their inputs. To illustrate, residents left it to nurses to "prepare" women for internal examination, which actually subsumed a process of getting informed consent and protecting their dignity.
"When the patient comes in the examination room, generally they are wearing "internal clothes". So telling them to remove it is really embarrassing. Of course, nurses are there [and] they will be explaining them these things … So what we tend to do is go outside and wait for the patient to get [into] position and we just do the internal examination." (VAN, Senior Resident, SH, male) However, nurses determined the working environment. Thus, wards were managed more informally. Nurses engaged in more informal interactions with women and their relatives. Thus, by and large, even medical staff at SH had much more insight into their patients' lives and described positive interactions with them.
"She [a woman] was not getting proper sleep and maybe she was not having proper emotional support at that time from the husband and the family, so she was very much frustrated and, in addition, that patient had some psychiatric problem so she … absconded." (VRB, Senior Resident, SH, male) "A few days back, a patient of mine, she got me 2 kilos of fish! I had done her LSCS [Lower Segment Caesarean Section] and had discharged her long back." (VAN, Senior Resident, SH, male) We also observed another woman who had "absconded" following her previous delivery and was pregnant again. The nurses recognised her and began to reprimand her. The woman, on her part, made attempts to cajole and humour them and she was eventually admitted.
However, while they were more informal, the nurses' treatment of women was not necessarily less arbitrary or aggressive. Shouting and the use of physical force on women during labour was openly acknowledged by the participants. As their intention was to protect women and babies, they perceived the overt violence as justified.
"Suppose the head has come out and she needs to bear down, but she is not pushing. Then we have to shout, because if she doesn't push, both will suffer. Both might die. Now people say later,'that doctor shouted, sister shouted, sister slapped'. Now, I know these things are not right, it is against human rights, [but] they do it for the good of the baby." (Matron, SH, female) The dominance of nurses, a more homogenous and familiar population of patients and a preponderance of uneventful deliveries created an environment in which women's individuality was somewhat preserved. However, the inter-professional conflict, an absence of formal frameworks for regulating clinical practice and ineffectual management created a stressful environment for the staff and posed risks for women.
Being a teaching hospital, MC had an abundance of medical staff and a greater focus on academics and research. It was also much more generously staffed and equipped. Apart from its "booked patients", this hospital received women who were referred or transferred from smaller government hospitals across the city and its periphery. MC received a much higher proportion of women experiencing complications. Several maternal deaths also took place here, mostly among transferred women. Following such events, the staff of the obstetrics department were called in for an audit, which involved the hospital staff as well as the health department bureaucracy. As a result, although a majority of women had normal vaginal births, there was a heightened focus on risk. Complementarily "saving lives" became the overarching ethic for the hospital.
"Whatever we do, we have to do to save the patient … A person who is very critical will die. [Still] I do not think anybody would have been saved outside this institute only because they had more money, or better equipment. No way!" (KSVB, Faculty, MC, male) An acute awareness of "risk" and a preoccupation with "saving lives" did translate into an importance placed on protocols. It is interesting that participants did not make any reference to international or national treatment guidelines or protocols. They referred largely to internal rules and a certain managerial process which was focused on preventing decision-making by untrained residents.
"After 4 p.m., if a critical patient comes I will see the patient and I will inform them [senior team members], they will come and see, and whatever decision is given by them I will inform to the boss [Head of the Unit] by phone [sic]. Then we describe the case, whatever investigations are available, like … this is the findings of a qualified person. The lecturer advises what is to be done. Then the HOU [Head of the Unit] may modify the decision or they may say OK you may go ahead with the same decision." (KsDe, Senior Resident, MC, female) Complementing this system of decision-making was a supervision system based on attributing blame. Repeatedly residents referred to anxiety about being wrong. In turn, they referred to correcting mistakes of their juniors, rather than mentoring. Medical staff did not collaborate with nurses, who were almost excluded from medical work and relegated to record-keeping. In turn, their supervision of hospital orderlies was also based on directives and reprimands.
"The class IV staff [hospital orderlies] is overworked at times one man will have to do the work of three men. And then when everybody will shout at him for being late, how much can the poor fellow bear? And you know what, working in this place, I have got so used to shouting while talking, that even at home, I shout when talking!" (Assistant Matron, MC, female) The focus on "saving lives" also resulted in local adaptations aimed at providing better care to selected women, whose needs were more acknowledged than usual. A system of shifts and rotations was instituted to ensure that these women who were classified as "high-risk" were treated by the same unit whenever they arrived at the hospital. While the residents unequivocally complained about long working hours and heavy workloads, they were all appreciative about this system. "They know the patients from the beginning … the patient may not have the papers when emergency occurs. But the registrar knows that so and so is the high risk patient of our unit. So that is the basic idea behind this thingthat patients are not mismanaged. He still knows that this is high risk due to this reasons." (KSDH, Senior Resident, MH, female) In contrast, the general population of women seeking obstetric care would rarely interact with the same provider more than once or twice. Thus, provider-women relationships here were 42 considerably more formal. We never heard women referred to by their name in this hospital, unlike SH. While narrating incidents, residents and nurses who had the most contact with women always described them by their medical conditions, rather than their social or family background. While a bias against certain communities, such as Muslims, was pervasive, it became more pronounced in this impersonal environment.
"My severe heart disease patient, a Muslim woman, whom we counselled and sent home after doing a MTP, telling her to not get pregnant again, she comes back with pregnancy within one month not even two, three months. Sometimes you just lose it." (KRP, Senior Resident, MC, female) In both hospitals, administering pain relief was not part of the routine process. In general, staff did not perceive any responsibility to respond medically to women's expressions of pain. While they differed in their perception about women's experience and ability to tolerate pain, participants in this hospital were more likely to judge women.
"See, pain is a very subjective phenomenon; and also depends upon the sensitivity of the woman, her ability to tolerate pain. Probably women who are more pampered by their husbands will not be able to bear pain." (KAG, Senior Resident, MC, female) The distance from women, lack of insight into their personal lives and a preoccupation with safety combined to obscure violation of women's rights. The unwritten protocols which were transmitted through the hierarchy also overwhelmed the space where reflection and discussion was required. An ethical dilemma, which required the woman's choice to also be evaluated and respected, was also rendered unproblematic and resolved mechanically.
"We then think of the mother as well as the baby. We cannot take up a patient with a haemoglobin level of 5 for C-section. There might not be time for us to wait and give her 3 or 4 bottles of blood and then do a C-section. We face a lot of dilemma these times. But we prefer having healthy mother, she can have babies later." (KRH, Senior Resident, MC, Female) An emphasis on formal structures and hierarchies was much more pronounced in the organisational culture on MC. However, rather than relying on established guidelines, they were more hierarchical, based on following directives and deferring to seniors. In the absence of supportive supervision, this system produced an environment of conflict and mistrust. An attempt was made to facilitate the care of women who faced medical risks, but staff did not, generally, perceive women's personhood.
Discussion
Using existing frameworks, 7,26 we examine the implication of organisational culture for the D&A of women. We identify drivers and their relationship to three key domains of problematic practice.
(1) Behaviours aimed at obtaining women's compliance to staff's directives -use of physical force, threats, coercion, detention etc.
As noted above, the hospitals were located in a complex structural context. On the one hand, very unequal social relationships between women and providers led to a certain "othering" of women, due to which providers felt affectively distanced and tended to blame women. In addition, exposure to formal and informal actions by external agents made them see, in general, all women and their relatives as a threat, for which they sought police protection and administrative safeguards. The consequence of both these factors was poorer rapport and less engagement with women. A paternalistic belief in their entitlement, even responsibility, to adjudicate women's best interest made overtly coercive and violent actions justifiable and morally acceptable.
However, within this largely paternalistic framework, patterns of behaviour differed. In SH, the nurses' dominant role allowed women to emerge as persons, who though subordinate could attempt to negotiate and shape relationships. Staff also experienced positive non-clinical encounters with women. Arguably, such encounters could challenge prejudice and encourage providers to view engagement with women as a part of their professional role. However, as inter-professional training was unheard of, these encounters did not gain any lasting significance.
(2) Behaviours and practices that displaced women from the focus of carenon-consented care, violation of privacy and confidentiality, being left alone, not being supported, etc.
It was evident, in both hospitals, that women were not the focal point for the design or management of services. The design of the space, the organisation of services and distribution of tasks evolved from the internal logic of the institution. Thus, male relatives required to undertake heavy work and purchases in lieu of absent orderlies crowded the hospital wards, leading to invasion of women's privacy, even while the design of the labour room made it impossible for women to have a lay companion during labour. Women could be left alone, even in the presence of skilled staff, if there was ambiguity about who was responsible for their care. Decision-making rarely involved women, but was guided either by exigencies, as in the case of SH, or by informal rules and protocols, as in MC. Women would experience these as sudden changes in treatment plans, arbitrary transfer or neglect. Contraception and abortion were offered by staff based on arbitrary rules and force was exerted to obtain compliance. The irrelevance of women to the processes of caregiving was so institutionalised that it did not even occur to staff to elicit their views. Moreover, the pressure to follow directives or to cope with inadequate conditions of care overwhelmed the space for women's expression of choice. Women faced frontline providers, who appeared arbitrary and neglectful, but were responding to invisible pressures, which they had little power to resist.
(3) Acts of omission and commission that endangered women's lives or made care-seeking more challengingroutinisation of procedures that were either harmful or not helpful, ad hoc treatment, referrals or transfers, etc.
Staff in both hospitals worked in imperfect environments, which were the result of poor management, administrative lapses and an out-dated mode of clinical practice. Frontline providers felt helpless to change the resource environment and management functioning. So they resorted to adaptation to ensure that women's lives were not lost. This included improving care for selected women by enhancing their own workload or taking ad hoc decisions to transfer women, who could, technically, be treated at the hospital. Wherever they could, they extracted resources and labour from women and their families to cover the gap in provisioning. The locus of power rested in the corporation, which did not, so to say, have to face patients every day. Administrative rules and local power dynamics dictated the flow of funds, resources and personnel, rather than a proactive attempt to alleviate the suffering of patients and improve care for them.
Evidence-based practice was conspicuous by its absence, and, thus, out-dated, potentially harmful or unnecessary practices, if they were endorsed by influential seniors, were institutionalised. The relative unimportance of updating knowledge and aligning their practice to established standards meant that residents, who were the mainstay of care, could not refer to established protocols, but had to comply with their seniors' directives. In the domain where directives were not available, they had to act intuitively within the boundaries of their competence. Even so, they remained vulnerable to blame and punitive action.
Conclusion
This study was premised on the fact that not only do violations of women's rights during maternity occur, but that providers openly acknowledge them and have explanations to offer. We explored the everyday working environment to uncover the systematic structures and processes that determine interactions between providers and women. Literature from the domain of quality of care and health systems studies has contributed to our understanding of the effect of underlying structures and processes on what happens at the point of care. 15 The discourse on D&A has helped to centre the discussion on women, rather than viewing their experience of care as a marginal aspect of quality. Going forward, addressing D&A beyond the penalisation of individuals, will require a deeper engagement with institutions and health systems to understand how social inequalities, institutional structures and processes and individual agents interact to create an organisational culture, which produces violations and is in need of change. | 2018-08-14T21:44:05.355Z | 2018-08-13T00:00:00.000 | {
"year": 2018,
"sha1": "b09a64716f9bc4d8b8c179927d6b0fb9f57219c0",
"oa_license": "CCBY",
"oa_url": "https://www.tandfonline.com/doi/pdf/10.1080/09688080.2018.1502021?needAccess=true",
"oa_status": "GOLD",
"pdf_src": "PubMedCentral",
"pdf_hash": "b09a64716f9bc4d8b8c179927d6b0fb9f57219c0",
"s2fieldsofstudy": [
"Sociology",
"Medicine"
],
"extfieldsofstudy": [
"Sociology",
"Medicine"
]
} |
16385279 | pes2o/s2orc | v3-fos-license | On the Instanton Complex of Holomorphic Morse Theory
Consider a holomorphic torus action on vector bundles over a complex manifold which lifts to a holomorphic vector bundle. When the connected components of the fixed-point set are partially ordered, we construct, using sheaf-theoretical techniques, two spectral sequences that converges to the twisted Dolbeault cohomology groups and those with compact support, respectively. These spectral sequences are the holomorphic counterparts of the instanton complex in standard Morse theory. The results proved imply holomorphic Morse inequalities and fixed-point formulas on a possibly non-compact manifold. Finally, a number of examples and applications are given.
Introduction
Given a Morse function on a compact real manifold, the Morse inequalities bound the Betti numbers in terms of the information of critical points. However, the former can not be determined by the Morse inequalities alone unless the Morse function is perfect. If the Morse function satisfies the transversality condition [45], then there is a finite dimensional complex, called the Thom-Smale-Witten complex or the instanton complex [50], which computes the cohomology groups of the manifold. (See [10] for a historical review.) The instanton complex consists of vector spaces spanned by the critical points of the Morse function (when they are isolated), graded by their Morse indices. The coboundary operators come from counting (with orientation) the number of gradient paths between critical points whose Morse indices differ by one. The latter is related to the instanton tunneling effect in supersymmetric quantum mechanics [50].
Consider a complex manifold with a holomorphic group action and a holomorphic vector bundle over the manifold on which the group action lifts holomorphically. We want to determine the Dolbeault cohomology groups (twisted by the vector bundle) as representations of the group. When the manifold is compact, the fixed-point formula of Atiyah and Bott [2] (for isolated fixed points) and of Atiyah and Singer [3] computes 1 E-mail address: swu@maths.adelaide.edu.au the alternating sum of the characters on the cohomology groups. For holomorphic Morse theory, this (equivariant) index theorem is the counterpart of the Hopf (or Lefschetz) formula. When the manifold is compact and Kähler and the group is the circle group, Morse-type inequalities were obtained by Witten [51] using a holomorphic version of supersymmetric quantum mechanics. These (equivariant) holomorphic Morse inequalities put constraints on the sizes of Dolbeault cohomology groups but do not completely determine them. In [39], a heat kernel proof was given under the additional assumption that the fixed points are isolated. In [52], these inequalities were generalized to cases with torus and non-Abelian group actions. Furthermore, it was shown that the Kähler assumption was necessary for holomorphic Morse inequalities [52], although not so for the fixed-point theorem. In [53], these inequalities were proved analytically for compact Kähler manifolds with possibly non-isolated fixed points.
In this paper, we construct the holomorphic counterpart of the instanton complex which computes the Dolbeault cohomology groups using the combinatorial data of the group action. At the same time, we investigate more closely the condition on the complex manifold for establishing a holomorphic Morse theory. Holomorphic Morse theory differs from ordinary Morse theory in a number of ways. If the circle group acts on a compact Kähler manifold in a Hamiltonian fashion, the moment map is a perfect Morse function whose critical points have even Morse indices only, which can not differ by one. Furthermore, Smale's transversality condition fails in general and the gradient paths are never isolated because of the circular symmetry. Consequently, the techniques for holomorphic Morse theory will be quite different from those for ordinary Morse theory.
We start with a complex manifold with a holomorphic action of a (complex) torus. The action of a noncompact 1-parameter subgroup is analogous to the gradient flow of a Morse function. The group action is meromorphic if, roughly speaking, all such orbits start from and end at some points in the manifold, which must be fixed points of the torus. If so, then there is a relation on the connected components of the fixedpoint set given by the direction of the flows. The central result of this paper is that if this relation is a partial ordering, then there are two (equivariant) spectral sequences converging (equivariantly) to the twisted Dolbeault cohomology groups and those with compact support, respectively. These spectral sequences will be constructed using sheaf-theoretic techniques from a filtration of the complex manifold determined by the group action. The spectral sequences, with the natural coboundary maps, are the couterparts in holomorphic Morse theory of the instanton complex in ordinary Morse theory. The information of the E 1 -terms already implies the holomorphic Morse inequalities. But unlike ordinary Morse theory, the spectral sequences do not always degenerate at E 2 . When the manifold is compact and Kähler, the partial order condition is automatically satisfied. Thus the results of [51,39,52,53] are recovered.
The rest of the paper is organized as follows. In section 2, we establish some facts about meromorphic torus actions on a compact or a suitably non-compact complex manifold. In section 3, we construct two spectral sequences converging to Dolbeault cohomology groups and those with compact support, respectively, under the partial order condition. In particular, we obtain holomorphic Morse inequalities and fixed-point formulas for a possibly non-compact manifold. We also study the condition under which the spectral sequences degenerate to cochain complexes. In section 4, we consider various examples and applications. We first present a spectral sequence calculation using the language ofČech cohomology. The application to flag manifolds yields a geometric realization of the Bernstein-Gelfand-Gelfand resolution and its generalizations. We also study the Dolbeault cohomologies and geometric quantization on non-compact manifolds.
Throughout this paper, N, R, R ± , C and C × denote the sets of non-negative integers, real numbers, positive (negative) real numbers, complex numbers and non-zero complex numbers, respectively.
Holomorphic torus actions
We first recall from [52,53] some notations of holomorphic torus actions without making the compact or Kähler assumption.
Let T be a complex torus with Lie algebra t. Let T R be the (real) maximal compact torus subgroup of T and t R = √ −1 Lie(T R ). Let ℓ be the integral lattice in t R , and ℓ * ⊂ t * R , the dual lattice. If T = C × , the multiplicative group of non-zero complex numbers, then T R = S 1 , t R = R, and ℓ = Z. In general, for any If every weight ξ ∈ ℓ * of R has a finite multiplicity r ξ , then the character char R = ξ∈ℓ * r ξ e ξ ∈ Z[ℓ * ] is well-defined. Let supp R = supp char R. As in [39,52], we write We emphasize here that the left-hand side is a notation for the formal series in Z[ℓ * ] on the right-hand side.
More generally, if R is a finite dimensional representation of T , we can write Let X be a complex manifold of dimension n. Suppose T acts holomorphically and effectively on X.
The fixed-point set X T of T in X, if non-empty, is a complex submanifold of X. Let F be the set of T acts on N α preserving the base X T α pointwise. The weights of the isotropy representation on the normal fiber remain constant within open polyhedral cones called action chambers [44]. Choose an action chamber C. Let λ C α,k = ±λ α,k , with the sign chosen so that λ C α,k ∈ C * . (Here C * is the dual cone in t * R defined by C * = {ξ ∈ t * R | ξ, C > 0}. ) We define ν C α as the number of weights λ α,k ∈ C * . Let N C α be the direct sum of the sub-bundles corresponding to the weights λ α,k ∈ C * . Then which is called the polarizing index of X T α with respect to C. In subsection 2.1, we will consider holomorphic torus actions on compact manifolds; a non-compact setting will be studied in subsection 2.2.
Meromorphic torus actions on compact manifolds
Throughout this subsection, X is a compact complex manifold with a holomorphic action of the torus T .
Then F is a finite set and each component X T α (α ∈ F ) is compact.
If T = C × , the action is meromorphic if and only if for any x ∈ X, the limits π + (x) = lim u→0 ux π − (x) = lim u→∞ exist. In this case, the holomorphic map C × × X → X can be extended to a meromorphic map P 1 × X → X.
Proposition 2.2
If the T -action on X is meromorphic, then for any x ∈ X and action chamber C, the limit π v (x) for v ∈ ℓ ∩ C depends only on C and not on the choice of v.
Proof. 1. Let X ′ be a connected component of the fixed-point set of C × v . Then X ′ ∩ X T is a closed subset of X ′ . For any x ∈ X ′ ∩ X T , let X T α be the component of X T that contains x. Since the T -action is effective and λ α,k (v) = 0 for any 0 ≤ k ≤ n − n α , we have dim X T α = dim X ′ . Therefore X ′ ∩ X T is also an open subset of X ′ . Finally, choose v 1 , . . . , v r−1 ∈ ℓ (r = dim C T ) such that {v, v 1 , . . . , v r−1 } is a basis of t R . Pick any x ′ ∈ X ′ . Since the T -action is meromorphic, the iterated limit x = π v1 π v2 · · · π vr−1 (x ′ ) exists. It is clear ; this depends only on C and not on the choice of v. ✷ We denote π v (x) by π C (x) when v ∈ ℓ ∩ C.
Remark 2.3 1. If X is a compact Kähler manifold and X T = ∅, then the T R -action is Hamiltonian [25].
Let µ: X → t * R be a moment map. For v ∈ ℓ − {0}, the 1-parameter subgroup {j v (e t ) | t ∈ R} generates the gradient flows of µ, v , along which its value strictly decreases. Therefore the limit π v (x) for any x ∈ X exists and the T -action is meromorphic.
2.
A holomorphic action on X may not be meromorphic even if X is compact and Kähler. For example, let Z act on C − {0} by k: z → 2 k z (k ∈ Z, z ∈ C − {0}) and let X = (C − {0})/Z be the quotient. Then the standard multiplication of C × on C − {0} induces a holomorphic action on X which has no fixed points and hence is not meromorphic.
In order to capture the topology of X by the fixed-point information, it is necessary to assume that the T -action is meromorphic. If so, then X has a cell decomposition according to the connected components of X T that π C maps to.
is called the Bia lynicki-Birula decomposition with respect to C.
Consider the case T = C × . If the C × -action is meromorphic, set X ± α = (π ± ) −1 (X T α ). The decompositions X = α∈F X ± α are called the plus (minus) decompositions, respectively. For example, X = P 1 is the union of C (with the standard multiplication of C × ) and {∞}. The fixed-point set is X T = {0, ∞}. We have consist of a 0-cell and a 2-cell.
The cells X C α are T -invariant. If the transversality condition is satisfied, then the decomposition (2.3) is a stratification [8,Theorem 5]. In general, this is not true even when X is Kähler. An example is the Hirzebruch surface (the blow-up of P 2 at one point) [8, Example 1]. Definition 2.5 For α, β ∈ F , we write α → β if there is x ∈ X such that π C (x) ∈ X T α and π −C (x) ∈ X T β . We write α ≺ β if either α = β or there is a chain from α to β, i.e., a finite sequence α 0 = α, a 1 , . . . , α r−1 , α r = β in F such that α i−1 → α i for all 1 ≤ i ≤ r (r > 0). Such a chain is called a quasicycle of length r if α = β.
Obviously, the relation ≺ on F depends on the choice of C.
It is easy to see that the following statements are equivalent: 1. (F, ≺) is a partially ordered set; 2. There is no quasicycle in (F, ≺); 3. There is a strictly decreasing function on (F, ≺), i.e., a function f : Consequently, (F, ≺) is a partially ordered set if one of the following is true: 1. X is Kähler; 2. ν C α > ν C β if α ≺ β and α = β; 3. The Bia lynicki-Birula decomposition is a stratification.
In each of the above cases, the moment map (projected along some direction in C), ν C · , and dim C X C · , respectively, provides a strictly decreasing function on (F, ≺).
Example 2.7 Jurkiewicz [32] constructed a smooth compact toric 3-manifold with a meromorphic T 3 -action that has 22 isolated fixed points. Choosing an appropriate action chamber, there is a quasicycle of length 6 [32]. Therefore (F, ≺) is not a partially ordered set. In [52, § 4], it is shown that there exists a T 3 -equivariant holomorphic line bundle such that the holomorphic Morse inequalities fail. This shows that the holomorphic Morse inequalities are not valid on an arbitrary complex manifold [52], though the fixed-point theorems in [2,3] requires no further assumptions. In section 3, we construct the analog of the instanton complex in holomorphic Morse theory when (F, ≺) is a partially ordered set. The existence of such a construction implies the holomorphic Morse inequalities. Moreover, the partial order condition is weaker than the Kähler condition.
Definition 2.8 Suppose X has a C-meromorphic T -action. The Bia lynicki-Birula decomposition with respect to C is filterable if there is a descending sequence of T -invariant subvarieties Notice that we allow Z p − Z p+1 to be a union of cells labeled by elements in F unrelated by ≺. In [8, Definition 2], Z p − Z p+1 is required to be a single cell. Since X C α X C β = ∅ implies α ≺ β [19, Lemma 1], the two notions are equivalent. Notice that the function α → p(α) where α ∈ F p(α) is strictly increasing on (F, ≺).
Alternatively, (2.4) can be written as We return to the simple example X = P 1 with two fixed points 0, ∞ under the meromorphic C × -action. Let So the plus decomposition of X = P 1 is filterable. For the same reason, so is the minus decomposition. The projection π C : X C α → X T α is a T -equivariant holomorphic fibration and the fiber (π C ) −1 (x) over any The closure X C α in X is analytic and contains X C α as a Zariski open set. Consequently, X C α is locally closed in X.
Proof.
If T = C × , the necessary and sufficient condition for (2.3) to be filterable was proved in [19].
Properties 1 and 2 follow from the arguments of [17]. Property 3 follows from the arguments in [18,§ IIb], where the Kähler assumption was not made. The generalization to a higher rank torus T is straightforward.
✷
The three properties of Proposition 2.9 were shown to be valid when X is a Kähler manifold [17,18,26,35] or a complete normal algebraic variety [7,8,34], prior to the work of [19]. Without any of these assumptions, one or more of the properties in Proposition 2.9 could fail [46]. Example 2.7 was originally constructed to provide a non-filterable Bia lynicki-Birula decomposition [32].
Remark 2.10
The restriction of π −C to X C α − X T α may be discontinuous and the image π −C (X C α − X T α ) may fall into more than one connected components of X T . For example, let X = P 1 × P 1 with the diagonal The reason is that the holomorphic embedding X C α → X extend only meromorphically at infinity [18,Lemma 2], where it can be discontinuous.
Notice that despite of part 2 of Proposition 2.9, a tubular neighborhood of X T α in X C α can not be identified holomorphically with that in N C α in general [17]. There is an infinite series of obstruction to this [28,22]. However, an identification is possible locally on X T α . Consider a holomorphic vector bundle E over X on which the T -action lifts holomorphically. For future applications, we also put E into a standard local form.
for y ∈ W x and t ≥ 0. Clearly, the extension is well-defined, T -equivariant and holomorphic. Next, there is a holomorphic isomorphismψ x : W x × E x → E| Wx of vector bundles, perhaps on a smaller neighborhood W x . By [17, The extension is again well-defined and is a T -equivariant holomorphic isomorphism of vector bundles. ✷
A non-compact setting
In this subsection, we consider a class of non-compact complex manifolds with holomorphic torus actions.
We hope that this class is broad enough to include many interesting examples.
Let X be a (possibly non-compact) complex manifold with a holomorphic action of the torus T .
2. there is a compact complex orbifoldX with a meromorphic T -action and a T -equivariant holomorphic embedding of X onto a Zariski open set ofX.
The simplest example is X = C with the standard multiplication by C × . The action is plus-meromorphic and X has a compactificationX = P 1 . The plus-decomposition is X = X + 0 (a single 2-cell) and there is no minus-decomposition.
Remark 2.13
Consider a C-meromorphic T -action on X. We identify X with its image inX.
1. By Proposition 2.2, which applies to the non-compact setting here, the limit π v (x) (x ∈ X) does not depend on the choice of v ∈ ℓ ∩ C and is therefore denoted by π C (x). Moreover π C (x) ∈ X T . Because X is embedded into a compact spaceX, the set F of connected components of X T is finite and each component X T α (α ∈ F ) is compact. We have the Bia lynicki-Birula decomposition (2.3) with respect to C. The action chamber C of X may be divided into several action chambers ofX; letC be one of such. Then we have X C α =XC α for any α ∈ F . For x ∈ X, the limit π −C (x) exists inX but may fall intoX − X. Therefore π −C (x) is in general not defined in X.
2. As in the compact situation, there is a relation ≺ on F . If (F, ≺) is a partially ordered set, then the properties of Proposition 2.9 for X are satisfied. In particular, X C α =XC α ∩ X is a closed subvariety in X that contains X C α =XC α as a Zariski open set. Furthermore, the Bia lynicki-Birula decomposition of X with respect to C is filterable and we have filtrations of X by closed subsets (2.4) and by open subsets (2.5).
3. If E is a holomorphic vector bundle over X on which the T -action lifts holomorphically, Lemma 2.11 also holds.
Assumption 2.14 There exists an action chamber C such that the T -action on X is C-meromorphic and the set (F, ≺) is partially ordered.
In section 3, we will establish holomorphic Morse theory on a (possibly non-compact) complex manifolds satisfying Assumption 2.14. An immediate way of obtaining such non-compact manifolds comes from Definition 2.12. We start with a compact complex manifoldX with a meromorphic T -action. Suppose the Bia lynicki-Birula decomposition ofX with respect to an action chamberC is filterable and is filtered by the closed setsX Pick any m such that 0 ≤ m ≤m − 1 and let X =X −Z m+1 . T acts holomorphically on X. Let C be the action chamber that containsC. Then the T -action on X is C-meromorphic. Moreover the Bia lynicki-Birula decomposition of X with respect to C has a filtration (2.4) by closed subsets of X. The simple example X = C falls into this category, withX = P 1 .
More interestingly, the non-compact setting here is a complex analog of the symplectic setting considered in [43,44], which we now recall. Let (X, ω) be a (possibly non-compact) symplectic manifold with a Hamiltonian action of the compact torus T R , with a moment map µ: X → t * R . The fixed-point set X T of the torus T R is a symplectic submanifold of X. Let F be the set of connected components of X T .
If in addition (X, ω) is Kähler and the T R -action preserves the complex structure on X, then there is a holomorphic T -action on X.
Proposition 2.16 Let (X, ω) be a Kähler manifold with a holomorphic T -action. Suppose that the T R -action is Hamiltonian. Then Assumption 2.15 implies Assumption 2.14.
Proof. By [44,Proposition 1.6], there is an action chamber C such that for any v ∈ C, the function µ, v on X is proper and bounded from above. Therefore if v ∈ ℓ ∩ C, the limit π v (x) exists for any x ∈ X.
Pick any v ∈ ℓ ∩ C. Since F is finite, there is a ∈ R such that µ(X T ), v > a. We construct a symplectic cut X ≥a [38]. Let C × act on X × C by u: μ is a proper function on X × C and 0 is a regular value. The symplectic quotient X ≥a =μ −1 (0)/S 1 is a compact symplectic orbifold with a Hamiltonian T R -action. Since X is Kähler, X ≥a = (X × C) s /C × holomorphically and is also Kähler [29]. Here set. Using the moment map µ, v , it is easy to show that (F, ≺) is a partially ordered set. ✷
Equivariant spectral sequences in holomorphic Morse theory
We consider a holomorphic T -action on a (possibly non-compact) complex manifold X. F is the set of connected components of the fixed-point set X T . Throughout this section, we make Assumption 2.14. Then the Bia lynicki-Birula decomposition is filterable, with descending sequences of closed sets (2.4) and open sets (2.5) in X. Let E be a holomorphic vector bundle over X on which the T -action lifts holomorphically. We want to determine the Dolbeault cohomology groups H * c (X, O(E)) (with compact support) and H * (X, O(E)) as representations of T . are T -equivariant. The spectral sequence converges T -equivariantly to the representations H * if the spaces E pq ∞ are the graded components of H * as representations of T .
Spectral sequence for cohomologies with compact support
In this subsection, we construct a spectral sequence converging to the Dolbeault cohomology groups Recall that if A ⊂ X is a locally closed subset, then for any sheaf F on X, there is a unique sheaf on X, For simplicity, we denote the sheaf O(E) by F from now on. If A is a T -invariant locally closed subset of X, then T acts on the sheaf F A and hence on the cohomology groups H * c (X, F A ).
Lemma 3.2 Under Assumption 2.14, there is a T -equivariant spectral sequence with Proof. From (2.5), we have a filtration of F by subsheaves and hence a filtration of the cochain complex Γ c (C * (F )) by This induces a spectral sequence that converges to H * (Γ (C * (F ))) = H * (X, F ), with Since the maps d pq are induced by the resolution, we get The support of F X C α is contained in the closed subvariety X C α . Therefore we have [ The result follows from (3.7), (3.8) and (3.9). ✷ Recall that π C : X C α → X T α is a holomorphic fibration with fiber C ν C α . The sheaf F | X C α is on the total space X C α . To calculate the right hand side of (3.6), we need another spectral sequence. We consider a general fibration π: Y → B over a compact base B with possibly non-compact fibers. For the time being, let F be an arbitrary sheaf on the total space Y . The cohomology groups with compact support 2. There is a spectral sequence with Proof. If A = R, then (see [36] for an analytic version) (3.14) The general case is a consequence of the Künneth formula. ✷ We now return to the situation of F = O(E).
as representations of T .
Proof. Consider the holomorphic fibration π C : X C α → X T α with fiber C ν C α and the sheaf F | X C α on X C α . For any x ∈ X T α , we want to find the stalk H q c (N C , F ) x , which depends only on an open neighborhood of (π C ) −1 (x) ⊂ X C α in X. By Lemma 2.11, we can replace X C α ⊂ X by N C α | Ux ⊂ N α | Ux and E by a trivial vector bundle with fiber E x . Moreover there is a T -equivariant isomorphism (N α (3.16) So the spectral sequence of Lemma 3.4.2 degenerates at E 2 and the result follows. ✷ its sub-bundle of any given weight is of finite rank. Therefore each weight has a finite multiplicity in the cohomology groups (3.15), and their formal characters in Z[ℓ * ] exist.
Theorem 3.7 Let X be a complex manifold with a holomorphic T -action satisfying Assumption 2.14. Let E be a holomorphic vector bundle over X on which the T -action lifts holomorphically. Then where ch T and td stand for the equivariant Chern character and the Todd class, respectively.
2. Since E pq r+1 is the cohomology of (E pq r , d pq r ), we have for a character valued polynomial Q r (t) ≥ 0. Using (3.20) recursively, we get (3.18) with Q C c (t) = r≥1 Q r (t) ≥ 0.
3. By setting t = −1 in (3.18) and using nα q=0 2. The coboundary maps {d pq r } in the spectral sequence in Theorem 3.7 or Corollary 3.8 are the holomorphic counterparts of the instanton tunneling operators in [50]. Through this spectral sequence, the cohomology groups H * c (X, O(E)) are completely determined by the combinatorial data of the T -action on X. However unlike the real case, the spectral sequence of holomorphic Morse theory does not always degenerate at E 2 . A sufficient condition for degeneracy at E 2 is If so, then the spectral sequence reduces to a cochain complex {E * 0 1 , d * 0 1 }, whose cohomology is E * 0 2 = H * (X, O(E)). This would be exactly like the Thom-Smale-Witten complex [50]. For example, if X T = F is discrete, m = n in (2.5), and F p = {x ∈ F | ν −C x = p} for 0 ≤ p ≤ n, then (3.26) is satisfied.
Spectral sequence with local cohomology groups
In this subsection, we construct an alternative spectral sequence converging to the Dolbeault cohomology groups H * (X, O(E)).
Suppose for the time being that X is any topological space containing Y as a locally closed subset and that F is any sheaf on X. We want to compute the local cohomology groups H q Y (X, F ) (q ≥ 0). Let A, L * be the sheaves on B defined by the presheaves A(U ) = Γ π −1 (U) (X, F ), L * (U ) = Γ π −1 (U) (X, C * (F )), respectively, where U is any open subset of B. Then 0 → A → L * is a differential sheaf in the sence of [27,§ II.4.1]. Let H q Y (X, F ) (q ≥ 0) be the sheaves on B defined by the presheaves H q Y (X, F )(U ) = H q (L * (U )), for any open subset U ⊂ B.
2. There is a spectral sequence with Proof. 1. This is the analog of Lemma 3.4.
Lemma 3.13
H q as representations of T .
Proof. Consider the fibration π C : X C α → X T α . For any x ∈ X T α , we want to find the stalk H q which by excision [5, § II.1, Lemma 1.1] depends only on an open neighborhood of (π C ) −1 (x) ⊂ X C α in X. By Lemma 2.11, we can replace X C α ⊂ X by N C α | Ux ⊂ N α | Ux and E by a trivial vector bundle with fiber . By Lemma 3.11.1 and Lemma 3.12, td(X T α ).
(3.44) Remark 3.16 1. The same observations in Remark 3.9.1 apply to Theorem 3.14 and Corollary 3.15. In particular, all the weights of T in H * (X, O(E)) are also of finite multiplicities. When X is non-compact, the Dolbeault cohomology groups are different from those with compact support. Therefore the results of Theorem 3.7 and Theorem 3.14 are not the same. Again, it would be interesting to have an independent analytic proof of parts 2 of Theorem 3.14 and Corollary 3.15 when X is a non-compact Kähler manifold satisfying Assumption 2.15. When X is compact, Theorem 3.7 is identical to Theorem 3.14 with an opposite action chamber. It is possible that the two theorems are dual to each other in some sense; this is also reflected by the local models in Lemma 3.5 and Lemma 3.12.
2. Remark 3.9.2 applies here as well. In particular, the complex is called the global Grothendieck-Cousin complex [30,33]. If condition (3.26) is satisfied, then the complex called the local Grothendieck-Cousin complex, is a resolution of F (see for example [33,Theorem 8.7]
Calculations inČech coholomogy theory
We interpret some of the procedures in the last section in the language ofČech cohomology theory, which is especially suitable for calculations.
For any sheaf F on X and any open cover U = {U i | i ∈ I} of X, let H * (U, F ) be the cohomology groups of theČech cochain complex where i 0 , · · · , i q ∈ I (q ≥ 0) are not equal, with the standard coboundary maps. TheČech cohomology groupš Then F V is the sheaf associated to the presheafF V andȞ * (X, We now assume that X is a compact complex manifold with a meromorphic T -action and that there is an action chamber C such that the set F of connected components of X T is partially ordered with respect to the relation ≺. Then the Bia lynicki-Birula decomposition is filterable, with filtrations of X by closed subsets We choose U to be a T -invariant, i.e., for any U i ∈ U, g ∈ T , we have gU i ∈ U. Then we have a T -equivariant filtration of theČech complex where So there is a T -equivariant spectral sequence converging to H * (U, F ) with E pq 0 = i0,···,iqF Vp/Vp+1 (U i0 ∩ · · · ∩ U iq ) and E pq 1 = H p+q (U,F Vp/Vp+1 ). Taking the inductive limit of U, we conclude that there is a T -equivariant spectral sequence with E pq 1 =Ȟ(X, F Vp−Vp+1 ) that converges toȞ * (X, F ). This is Lemma 3.2 when X is compact. However the method outlined above is more convenient for calculations, which we now illustrate.
Choose an open covering
the open cover U already satisfies H * (U, F ) ∼ =Ȟ * (X, F ). ThereforeȞ * (X, F ) can be computed by the spectral sequence associated to the filtration (4.4). According to (4.5), the spaces F p C q are given by Consequently, the spaces E pq 0 = F p C p+q /F p+1 C p+q are given by The arrows here (and below) denote the coboundary operators. Let Γ pq r be a region in t * R ∼ = R 2 = {(x, y)} such that supp E pq r = Γ pq r ∩ ℓ * . (Recall that ℓ * ∼ = Z 2 is the dual lattice in t * R ∼ = R 2 .) Then regions Γ pq 0 are given by Here the multiplicity of any weight ξ ∈ Γ pq in which case the multiplicity is 2. The regions Γ pq 1 are given by Finally, the regions Γ pq 2 are given by if c ≥ 0 and by All the weights ξ ∈ Γ pq r ∩ ℓ * (r = 1, 2) are of multiplicity 1 in E pq r . So the spectral sequence degenerates at E 2 . We recover the well-known result that the only non-trivial cohomology groups are H 0 (P More interestingly, the (holomorphic) instanton complex can be used to study the cohomology groups of vector bundles over spherical varieties, about which not all is known. (See [13] for an extension of the Borel-Weil theorem.) One notable exception is the flag manifold, which will be discussed in the next subsection.
Flag manifolds and generalized Bernstein-Gelfand-Gelfand resolutions
We show that the spectral sequence for the cohomology of a flag manifold leads to geometric realizations of the Bernstein-Gelfand-Gelfand [6] and related resolutions.
Let G be a complex semi-simple Lie group and T , a maximal torus of G. Let g, t be the Lie algebras of G, T , respectively. Let g = t ⊕ α∈∆ g α be the root space decomposition, where ∆ ⊂ t * − {0} is the root system of the pair (g, t) and g α = Ce α (α ∈ ∆). Let ∆ + be a set of positive roots and let ∆ − = −∆ + . Let n ± = α∈∆± g α . Let B be the Borel subgroup corresponding to the Borel subalgebra b = t ⊕ n + . Let W be the Weyl group of the pair (g, t). Denote by w 0 the element in W of maximal length l(w 0 ) = |∆ + |.
Recall that the Verma module of highest weight λ is the U (g)-module M λ = U (g) ⊗ U(b) Cv λ , where Cv λ is the 1-dimensional U (b)-module defined by λ ∈ t * R . M λ is free over U (n − ). As a U (t)-module, M λ is determined by char M λ = e λ α∈∆ + (1−e −α ) . When λ is a dominant weight, let R λ be the (finite dimensional) irreducible module of highest weight λ. We have a resolution of R λ by Verma modules [6] where ρ = 1 2 α∈∆+ α. This is called the Bernstein-Gelfand-Gelfand resolution of R λ .
For any w ∈ W , put n w ± = wn ± w −1 . The twisted Verma module M w λ is a U (g)-module of highest weight λ that is free over U (n w + ∩ n − ) and co-free over U (n w + ∩ n + ) [23].
For example, take g = sl(2, C) = span C {h, e, f } with commutation relations The Weyl group is W = {±1}. The twisted Verma modules of highest weight λ ∈ R are M 1 λ = M * λ and The dual is When λ is not a dominant weight, i.e., when λ ∈ N, M λ ∼ = M * λ as U (g)-modules and the isomorphism is given by When λ ∈ N, M λ and M * λ are not isomorphic U (g)-modules. The irreducible module R λ is a quotient of M λ and a submodule of M * λ . We consider the non-degenerate flag manifold X = G/B − , where B − is the Borel subgroup opposite to B. The maximal torus T acts meromorphically on X. The fixed-point set is X T = {wB − | w ∈ W }. The isotropy weights at wB − are wα (α ∈ ∆ + ). The action chambers in t are the Weyl chambers. Choose the positive Weyl chamber, denoted by "+". Then the polarizing index of wB − is ν − w = |∆ − ∩ w∆ + | = l(w) for any w ∈ W . The Bia lynicki-Birula decomposition is precisely the Bruhat decomposition X = w∈W X + w , where X + w = BwB − /B − (w ∈ W ) are the Bruhat cells [1]. These cells are also the B-orbits in X. Moreover, the relation ≺ on F ∼ = W is the Chevalley-Bruhat order [20], which is a partial ordering. Consequently, the Bia lynicki-Birula decomposition is filterable, and we have the filtration (2.4), where m = |∆ + | = dim C X.
The closed sets Z p = l(w)≥p X + w (0 ≤ p ≤ |∆ + |) are the Schubert varieties. Since Z p − Z p+1 = l(w)=p X + w (0 ≤ p ≤ |∆ + |) and ν − w = l(w), the cohomology groups H * (X, F ) with coefficients in any sheaf F can be computed by the (global) Grothendieck-Cousin complex (3.45), which becomes . (4.12) So as representations of T , H l(w) X + w (X, F λ ) is the same as the Verma module M w(λ+ρ)−ρ . In fact the above local cohomology groups are U (g)-modules. This is because the canonical resolution C * (F λ ) of F λ , on which the Lie algebra g acts, is U (g)-equivariant. (Notice however that a representation of g on an infinite dimensional space may not exponentiate to that of G.) Therefore the Grothendieck-Cousin complex (4.11) is U (g)-equivariant.
If λ + ρ is regular, the cohomology groups H * (X, F λ ), hence those of the complex (4.13), are where w λ is the unique element in W such that w λ (λ + ρ) − ρ is a dominant weight [9]. The complex (4.13) is called the generalized Bernstein-Gelfand-Gelfand resolution of R w λ (λ+ρ)−ρ [23, §2.3]. If λ + ρ is singular, then all H * (X, F λ ) = 0 [9]. When λ is a dominant weight, (4.13) is the dual of the Bernstein-Gelfand-Gelfand resolution (4.6) for R λ [33,14,15]. When w 0 λ − 2ρ is dominant, (4.13) is the Bernstein-Gelfand-Gelfand resolution for R w0λ−2ρ . Let F λ = O(L λ ). The cohomology groups H * (P 1 , F λ ) as U (g)-modules can be computed from the U (g)equivariant cochain complex be two open sets in P 1 . Then the above cochain complex becomes We define two sections s i ∈ Γ (U i , F λ ) (i = 0, 1) by s i ([z 0 , z 1 ]) = [z 0 , z 1 , z λ i ]. Then Γ (U 0 , F λ ) = span C {z k s 0 | k ∈ N} and Γ (U 0 ∩ U 1 , F λ )/Γ (U 1 , F λ ) = span C {z k+1 s 1 | k ∈ N}, where z = z1 z0 on U 0 . The actions of g on the two spaces are given by where the isomorphisms are given by z k → k! ξ k λ and z k+1 Remark 4.4 Lepowsky [37] found a Bernstein-Gelfand-Gelfand-type resolution of any irreducible U (g)module by the generalized Verma modules, which are induced from representations of a parabolic subgroup P ⊂ G. In [42], a geometric realization of this resolution was constructed using the local cohomology of the P -orbits in G/B − (rather than the B-orbits in G/P − ). Let H be the Levi subgroup of P , and h, its Lie algebra. Let ∆ H be the root system of the pair (h, t), and W H , the corresponding Weyl group. Then X = G/B − decomposes into its P -orbits according to T ′ ⊂ T , whose Lie algebra t ′ ⊂ t. Consider the (meromorphic) T ′ -action on X. The fixed-point set X T ′ = w ′ ∈W/WH Hw ′ B − /B − . Choose the action chamber C ′ ⊂ t ′ such that α, C ′ > 0 for all α ∈ ∆ + − ∆ H ∩ ∆ + .
The the Bia lynicki-Birula decomposition of X with respect to C ′ is precisely (4.20). Therefore (3.45) gives the geometric realizations of Lepowsky's resolution and similar generalizations.
Cohomology and geometric quantization of non-compact manifolds
In section 3, we obtained equivariant holomorphic Morse inequalities and equivariant index theorems for non-compact complex manifolds under Assumption 2.14. In this subsection, we apply them to establish some results on the cohomology groups and on geometric quantization.
Let X be a (possibly non-compact) complex manifold of dimension n with a holomorphic T -action satisfying Assumption 2.14. Let H pq (X) = H q (M, O(∧ p T X)), H pq c (X) = H q c (M, O(∧ p T X)) (p, q = 0, 1, . . . , n) be the Dolbeault cohomology groups of X and those with compact support, respectively. Let P (X; s, t) = n p,q=0 s p t q char H pq (X), P c (X; s, t) = n p,q=0 s p t q char H pq c (X), the character-valued Poincaré-Hodge polynomials. If the cohomology groups are finite dimensional, then h pq (X) = dim C H pq (X), h pq c (X) = dim C H pq c (X) are the Hodge numbers of X and p(X; s, t) = n p,q=0 s p t q h pq (X), p c (X; s, t) = n p,q=0 s p t q h pq c (X), the (usual) Poincaré-Hodge polynomials. Notice that if X is a non-compact Kähler manifold, the Hodge numbers or the Poincaré-Hodge polynomials do not necessarily satisfy the usual symmetry relations. For example let X = C.
Without loss of generality, we assume that the moment map µ is bounded from above. Then the C × -action is plus-meromorphic. Suppose 0 is a regular value of µ. For simplicity, we assume that the S 1 -action on µ −1 (0) is free. Then the symplectic quotient X 0 = µ −1 (0)/S 1 = X s /C × is a smooth Kähler manifold. We construct the symplectic cuts (X ± , ω ± ) as the symplectic quotients of the S 1 -action on X ×C, where the weights on C are ±1, respectively [38]. The two cuts are Kähler manifolds with holomorphic C × -actions. X + is compact and X − satisfies Assumption 2.15. The sets of connected components of X C × ± are F ± = {0} ∪ {α ∈ F | µ(X C α ) ∈ R ± }, respectively, and X C × ±,0 ∼ = X 0 , X C × ±,α ∼ = X C × α as complex manifolds [53,Lemma 4.6], which we now identify. Let N 0 → X 0 be the holomorphic line bundle associate to the circle bundle µ −1 (0) → X 0 . Then C × acts on the fibers of N 0 with weight 1. The holomorphic normal bundles of X 0 in X ± are isomorphic to N ±1 0 , respectively. Since the action of C × lifts to L, the pre-quantum line bundles L 0 → X 0 and L ± → X ± exist. We have the isomorphisms L ± | X0 ∼ = L 0 and L ± | X±−X0 ∼ = L| µ −1 (R ± ) (see for example [53,Lemma 4.9]). Proof. For α ∈ F , let td(X C × α ). The result follows. ✷ Remark 4.9 1. We can define H c (X) = n q=0 (−1) q H q c (X, O(L)) as the counterpart of (4.23) with compact support. Using However dim C H c (X) C × = dim C H(X 0 ) in general. For example, take X = C and choose the moment map µ(z) = −1 − 1 2 |z| 2 . Then dim C H c (X) C × = 1 but X 0 = ∅. 2. When (X, ω) is symplectic, the individual cohomology groups in (4.23) do not make sense, but H(X) can be defined as the index of a spin C -Dirac operator. In [21], (4.25) and (4.26) were proved for compact symplectic manifolds. (4.26) is the S 1 -case of a conjecture by Guillemin and Sternberg [29]; the cases with higher rank torus and non-Abelian group actions were proved by Meinrenken [40,41], Jeffrey and Kirwan [31], Vergne [48] and others under various generalities using localization techniques, and by Tian and Zhang [47] using an analytic approach.
3. Proposition 4.8 shows that the results of [21] holds for non-compact Kähler manifolds under Assumption 2.15. For non-compact symplectic manifolds satisfying Assumption 2.15, the validity of (4.25) and (4.26) remains open. Also it would be interesting to investigate, analytically or otherwise, whether the Tian-Zhang inequalities [47] n−1 k=0 t k dim C H k (X 0 , O(L 0 )) = n k=0 t k dim C H k (X, O(L)) C × + (1 + t)Q 0 (t) (4.35) for some Q 0 (t) ≥ 0 hold when X is non-compact and Kähler. The conjecture in [53,Remark 4.11] can also be posed in this non-compact setting.
Remark 4.10
In ordinary Morse theory, the underlying real manifold is (the bosonic part of) the configuration space of a supersymmetric system [50]. In holomorphic Morse theory, the complex manifold X, if it is Kähler, can be interpreted as the phase space of a bosonic system; this interpretation is adopted in Definition 4.7. The spectral sequence in Theorem 3.14.1 or Corollary 3.15.1 that converges to the quantum Hilbert space (4.23) is a finite dimensional model of the BRST approach in conformal field theory [24]. In [23,11], the case of flag manifolds (see subsection 4.2) was considered. Here we show that the analogy works for any quantizable Kähler manifold with a Hamiltonian S 1 -action satisfying Assumption 2.15. It would be interesting to extend the present work to infinite dimensional settings. | 2014-10-01T00:00:00.000Z | 1998-06-22T00:00:00.000 | {
"year": 1998,
"sha1": "d93a33f428df1bdd553f4b14d10ed4ef302f691d",
"oa_license": null,
"oa_url": "https://www.intlpress.com/site/pub/files/_fulltext/journals/cag/2003/0011/0004/CAG-2003-0011-0004-a007.pdf",
"oa_status": "BRONZE",
"pdf_src": "Arxiv",
"pdf_hash": "82db80f67dc6e1eb688bee364adf9428a296d9fb",
"s2fieldsofstudy": [
"Mathematics"
],
"extfieldsofstudy": [
"Mathematics"
]
} |
90598256 | pes2o/s2orc | v3-fos-license | Whole genome sequencing reveals high-resolution epidemiological links between clinical and environmental Klebsiella pneumoniae
Klebsiella pneumoniae is a gram-negative bacterial species capable of occupying a broad range of environmental and clinical habitats. Known as an opportunistic pathogen, it has recently become a major causative agent of clinical infections worldwide. Despite growing knowledge about the highly diverse population of K. pneumoniae, the evolution and clinical significance of environmental K. pneumoniae, as well as the relationship between clinical and environmental K. pneumoniae, are poorly defined. We isolated and sequenced K. pneumoniae from in-patients in a single hospital in Thailand, as well as hospital sewage, and surrounding canals and farms within a 20-km radius. Phylogenetic analysis of 77 K. pneumoniae (48 clinical and 29 non-clinical isolates) demonstrated that the two groups were intermixed throughout the tree and in some cases resided in the same clade, suggesting recent divergence from a common ancestor. Phylogenetic comparison of the 77 Thai genomes with 286 K. pneumoniae from a global collection showed that Thai isolates were closely related to the clinical sub-population of the global collection, indicating that Thai clinical isolates belonged to globally circulating lineages. Dating of four Thai K. pneumoniae clades indicated that they emerged between 50 and 150 years ago. Despite their phylogenetic relatedness, virulence factors and β-lactamase resistance genes were more numerous in clinical than in environmental isolates. Our results indicate that clinical and environmental K. pneumoniae are closely related, but that hospitals may select for isolates with a more resistant and virulent genotype. These findings highlight the clinical relevance of environmental K. pneumoniae isolates.
Background
Klebsiella pneumoniae is a clinically important gramnegative bacterium associated with opportunistic infection in patients with a compromised immune system or receiving other forms of complex medical care [1][2][3]. This species is disseminated in healthcare settings via personto-person contact, medical equipment, and contaminated environments [2,3]. The emergence of multidrugresistant K. pneumoniae is becoming an increasingly serious issue for clinical practice, largely related at the present time to isolates that express extended-spectrum β-lactamase (ESBL) enzymes that hydrolyze a broad spectrum of β-lactams [4,5]. This is compounded by the emergence of K. pneumoniae that express carbapenemases such as KPC-type β-lactamase [5][6][7] and the recent detection of colistin-resistant K. pneumoniae due to the presence of the mcr-1 gene [8]. These resistance genes are carried on mobile genetic elements that facilitate their spread within and between bacterial species, a process that is likely to result in a rise in the number of K. pneumoniae infections that are very difficult to treat. Furthermore, multidrug-resistant K. pneumoniae are associated with nosocomial outbreaks, particularly in high prevalence countries including those in East Asia [9,10].
Beyond healthcare settings and hospital patients, K. pneumoniae is ubiquitous in nature and occupies a diverse range of niches. These include environmental sources, such as soil and wastewater, mucosal surfaces and the gut of humans and animals, and food sources, such as meat [11]. Environmental K. pneumoniae is less well studied than isolate collections associated with clinical disease. Some studies have shown that K. pneumoniae of environmental origin are highly similar to clinical isolates with regards to phenotypic and some genetic features [12][13][14], but others have reported differences in virulence characteristics between the two groups [15,16]. The parallel evolution of K. pneumoniae and putative acquisition of antimicrobial resistance determinants and virulence factors in healthcare settings and the environment have led non-clinical habitats to be considered as potential reservoirs for hyper-virulent and hyper-resistant K. pneumoniae [15], although evidence to support the potential clinical importance of non-clinical K. pneumoniae is inconclusive. A recent study of the clinical relevance of meat-source K. pneumoniae showed differences in antibiotic resistance but similar virulence characteristics for isolates from retail meat and those associated with urinary tract infections in humans [16].
Here, we report the findings of an in-depth comparison of the evolution and epidemiology of ESBL-positive K. pneumoniae using a One Health approach [17,18]. We utilized the fine-scale resolution of whole genome sequencing to investigate genetic relatedness, antimicrobial resistance, and the presence of genes encoding virulence determinants in an unbiased, prospective collection of K. pneumoniae from patients in one hospital, as well as from environmental water, hospital sewage, and farm waste in the proximity of the hospital. These genomes were placed into a global context through a comparison with isolates recovered from various clinical and non-clinical sources worldwide [19]. Our results highlight the clinical relevance of environmental K. pneumoniae isolates, and demonstrate that environmental and clinical K. pneumoniae are highly related but that hospitals select for K. pneumoniae with a more antimicrobial-resistant and virulent genotype.
Study design and bacterial isolates
The bacterial collection consisted of 77 ESBL-producing K. pneumoniae isolated between 2014 and 2015. Clinical isolates (n = 48) were obtained from consecutive patients with positive samples processed by the diagnostic microbiology laboratory at Bhuddhasothorn Hospital, Chachoengsao, Bangkok, Thailand between December 2014 and April 2015. Data were collected on date of isolation and sample type, and samples were de-duplicated so that only one isolate per patient was included. Speciation and ESBL positivity were initially determined using Standard Operating Procedures supplied by the Department of Medical Science, Ministry of Public Heath, Thailand and Clinical and Laboratory Standards Institute (CLSI) guidelines (M100-S24 and M100-S25), respectively. The species was subsequently confirmed using matrixassisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS; Biotyper version 3.1, Bruker Daltonics, Coventry, UK). Antimicrobial susceptibility testing was repeated using the N206 card on the Vitek 2 instrument (bioMérieux, Marcy l'Étoile, France) calibrated against EUCAST breakpoints, and these results were used during the analysis.
Environmental and livestock-associated isolates (n = 29) were obtained through a cross-sectional survey between January 2015 and February 2015. Wastewater samples were collected from 27 canals and 11 farms within a 20km radius of Bhuddhasothorn Hospital. The farms reared pigs (n = 2), chickens (n = 6), ducks (n = 2), and both chickens and ducks (n = 1). Samples were collected from wastewater collection areas (commonly concrete gullies that drained waste from animal housing). The longitude and latitude of each sampling site were recorded using GPSMAP 60CSx (Garmin, Taiwan). A further two wastewater samples were taken from the Bhuddhasothorn Hospital wastewater treatment system (one pre-treatment and one post-treatment water sample). At each site, grab samples of 0.5 L each were collected into sterile bottles containing 9 mg of sodium thiosulfate pentahydrate (Merck, Darmstadt, Germany). All samples were transported to the laboratory on ice packs in the dark and processed within 12 h.
One mL of triplicate serial 10-fold dilutions of wastewater samples were concentrated using the filtration technique onto 0.45-μm pore size filter membranes (Merck, Darmstadt, Germany). Membranes were then placed onto the surface of ESBL Brilliance agar (Oxoid, Basingstoke, UK) and incubated for 48 h at 35°C in air. For each sampling site, up to 10 colonies suspected to be K. pneumoniae based on color (green) were picked and the presence of ESBL confirmed using the combination disc test (M100-S24 and M100-S25). Escherichia coli ATCC25922 and K. pneumoniae ATCC700603 were used as negative and positive controls, respectively. Positive colonies were speciated using MALDI-TOF MS, and antimicrobial susceptibility testing of confirmed K. pneumoniae was determined using the N206 card as described above. All isolates were stored at −80°C until further analysis.
Whole genome sequencing and pan-genome analysis DNA extraction, sequencing, and assembly of reads were performed as previously described [20]. Sequencing was performed on an Illumina HiSeq2000. Details of reads, depth of coverage, and N50 are provided in Additional file 1. The sequence reads were submitted to the European Nucleotide Archive (ENA) under accession numbers [ENA:ERP012787 and ENA:PRJEB11403]. An average coverage of 85-fold was achieved. Genomes were assembled using Velvet [21] with the pipeline and improvements found at https://github.com/sangerpathogens/vr-codebase and https://github.com/sangerpathogens/assembly_improvement. The de novo assemblies were annotated using Prokka [22], and sequence types (STs) were identified from the sequence data using a multilocus sequence typing (MLST) database (http:// bigsdb.pasteur.fr/klebsiella/) and an in-house script (https://github.com/sanger-pathogens/mlst_check).
Phylogenetic analysis and substitution rate calculation
Study genomes were contextualized against a global collection. Sequence data for 286 K. pneumoniae isolates reported previously [19] were downloaded from the ENA and combined with the 77 study genomes. As in our study, these isolates were also sequenced on a HiSeq sequencing system. Short reads for the 363 isolates were mapped against the reference genome K. pneumoniae Ecl8 (accession number: HF536482 CANH01000000) using SMALT v0.7.4 (http://www.sanger.ac.uk/science/ tools/smalt-0). An in-house tool that combined SAMtools mpileup [23] and BCFtools, as detailed in [24], was used to annotate single nucleotide polymorphisms (SNPs), after which the pairwise SNP distances were calculated from the multiple alignment to obtain the phylogenetic tree. Mapping each genome to the reference genome allowed us to identify genes that were conserved in the core genome of the study isolates and reference genome. We constructed a maximum likelihood tree using FastTree version 2.1.3 with 100 bootstraps and a midpoint root [25]. We employed FigTree (http://tree.bio.ed.ac.uk/software/figtree/), Microreact (www.microreact.org), and inhouse tools to visualize the results.
To determine the substitution rate for each clade (see the Results section for more details), reads were first mapped within each clade to the sequence obtained after concatenating contigs for the isolate with the best contigs statistics, i.e., the isolate with the highest N50 value. After mapping the reads to the new references, highdensity SNP regions (which are indicative of putative recombination events) were removed from the multiple alignments using Gubbins, which works best for closely related isolates and detects high SNP density regions based on a significantly higher number of variable sites in a sliding window across the genome compared to the rest of the genome [26]. The significance of the temporal signal was assessed according the R-squared value obtained from the root-to-tip distance versus time of isolation plot. We generated 10,000 sample sets by bootstrapping and assessed the value of R-squared against the distribution of R-squared values. Of four clades we identified on the phylogenetic tree of the Thai and global isolates, we found strong signals (>99%) for clades 1 and 3, after leaving one isolate out from either clade. BEAST version 1.7 was used to estimate the substitution rate and date the phylogenetic tree for the clades with significant temporal signal [27], using a strict molecular clock and a lognormal and uniform prior distribution models for base frequencies with constant population size. Three chains of BEAST were run for 50 million generations (sampling every 10 generations) and checked as to whether the runs had converged on similar values. Convergence was controlled using effective sample size (ESS) value (we considered a cut-off of 200 ESS for convergence). We excluded 10 million initial steps as a burn-in phase and merged the out trees with the Tree-Annotator program in the BEAST package, and chose the model in which convergence always occurred (for the clades we tested here, the strict molecular clock with a uniform prior distribution for base frequencies always converged).
Identification of antimicrobial resistance determinants, virulence factors, and plasmids
We employed the srst2 package [28], which takes short reads and maps them to reference sequences, to find antibiotic resistance genes, virulence factors, and plasmid replicons. The sequences for 79 virulence genes were obtained from the Pasteur institute data repository (http:// bigsdb.web.pasteur.fr/klebsiella/klebsiella.html, http://bigs db.pasteur.fr/perl/bigsdb/bigsdb.pl?db=pubmlst_klebsiella_ seqdef_public&page=downloadAlleles). An in-house tool was used to visualize the results. To find the possible context of the virulence (i.e., chromosomal or plasmid based), we first found the contig in which the virulence factor was located and then extracted the 5-kb sequences upstream and downstream of the gene; if this exceeded the contig size, we considered the end and start of the contig. We then performed blast searches using the NCBI nonredundant nucleotide database to find out whether the hit sequences corresponded to a chromosomal or a plasmid region. Furthermore, we assessed the significance between the presence of virulence factors in the clinical/environmental sub-populations by performing logistic regression. To this end, we took the presence/absence of individual genes as the categorical predictor parameter and the environmental/clinical status as the categorical dependent variable. Significance of association (p value < 0.05) was then assessed by considering the z-statistic, which is the regression coefficient divided by its standard error and has a standard normal distribution.
Results
A One Health approach was taken for the sampling and isolation of ESBL-positive K. pneumoniae from clinical samples in a hospital in Thailand, together with hospital sewage, environmental (canal) water, and wastewater from farms within a 20-km radius of the hospital. A phylogenetic tree of the whole genomes of the 77 K. pneumoniae isolates (48 clinical and 29 environmental, of which 24 were from canals, 3 from farms, and 2 from untreated hospital sewage) revealed a diverse population containing three major lineages, one of which contained the majority of isolates (Fig. 1). Several minor clades were also apparent, some of which showed evidence of recent expansion (Fig. 1). Clinical and environmental isolates were intermixed throughout the tree and in some cases resided in the same clade, suggesting recent divergence from a common ancestor (Fig. 1). The 3 isolates of farm origin were distributed across the tree, and clustered with canal and clinical isolates or with canal isolates alone ( Fig. 1). High genetic diversity was also reflected by the number of STs (n = 38) to which the 77 isolates were assigned. This included STs that have been isolated elsewhere in the world. For example, ST35, ST307, and ST16 were recently recovered from clinical settings in France, the USA, and the Netherlands, respectively [29][30][31]. Mapping the geographical distribution of isolates recovered from the environment demonstrated that isolates from the same sampling site were frequently very genetically diverse (Fig. 1).
To further characterize the 77 study genomes, we combined them with genomes for a global collection of 286 K. pneumoniae complex isolates [19]. The resulting phylogenetic tree revealed that Thai isolates were dispersed across the combined population. Isolates in the global collection had been defined previously based on phylogroup, and Thai isolates were observed to cluster in phylogroups KpI, KpIIa, and KpIIb, with the majority residing in KpI (Fig. 2a). This broader genetic context highlighted the presence of several clades of Thai isolates; those containing at least 4 Thai isolates (clades 1 to 4) were subjected to more detailed analysis to uncover their recent evolutionary history. These clades included 30 of the 77 total isolates. A strong temporal signal was found for clade 1 and clade 3 after removing regions of recombination. This allowed us to estimate the substitution rates, which were 8.33 × 10 −7 (95% confidence (Fig. 2b). Using the average substitution rates for clades 1 and 3, we also estimated the age of the MRCA for clades 2 and 4 to be 150 and 50 years ago, respectively (Fig. 2b). We conclude that expansion of these K. pneumoniae clones has taken place over the past few decades.
Thai isolates residing in clades 1 and 3 were intermixed with isolates from other countries in the global collection (Fig. 3). The Thai isolates in clade 1 fell into two sub-clades of clinical origin, both of which were estimated to have emerged in the past few decades. These were more distantly related to two clinical isolates from community and nosocomial infection in the global collection (Fig. 3). Clade 3 also contained two Thai subclades, one of which was composed of clinical isolates. The second sub-clade consisted largely of environmental isolates but had recently diverged from a clinical Thai isolate (Fig. 3). Global isolates in clade 3 were of hospital origin and nosocomial infections.
To generalize our findings about the epidemiological links between global and clinical and environmental Thai isolates for the isolates not included in clades 1 or 3, we excluded the isolates in the two clades and then used the average substitution rates for isolates in clades 1 and 3 to estimate divergence times between a Thai isolate and any other Thai or global isolate that was less than 250 SNPs apart (this corresponds to approximately 50 years, which is the age of the MRCA of clade 3, which is older than clade 1). This revealed that Thai isolates appeared to have diverged more recently from each other than from the global isolates (Fig. 4). The majority of recent divergences in the Thai collection occurred between isolates from the same origin of isolation, i.e., environment or hospital (17 out of 24). However, there were several cases of recent divergence involving isolates of both environmental and hospital origin, as well as evidence for recent divergence between isolates from different environmental origins, i.e., different canals (Fig. 4). Moreover, one putative transmission event may have occurred between isolates of canal and farm origin (Fig. 4). The Thai isolates involved in 25 recent divergence events between a Thai and global isolates were all of clinical origin, and 15 and 7 of these isolates were recovered from invasive and non-invasive infections, respectively (Fig. 3). Furthermore, 19 out of 25 cases occurred between ST23 isolates, a well-known hyper-virulent strain. Taken together, these findings indicate that K. pneumoniae has rapidly expanded within the environment and hospitals, Fig. 2 a Phylogenetic tree of the 77 Thai isolates placed in the context of a global collection. Each color corresponds to a country. Shaded clades refer to those described in the text. b Dating the most recent common ancestor for clades identified on the phylogenetic tree. To calculate the mean and upper and lower bounds of age root for clades 1 and 3, which lacked a temporal signal, we divided the root-to-tip distances of the root by mean substitution rates (and upper and lower bounds for 95% confidence interval) of clades 1 and 3 and that in some instances, isolates of different origins have only recently diverged. Furthermore, Thai isolates were closely related to the clinical sub-population of the global collection, suggesting that the clinical collection is a part of a global circulation of hyper-virulent K. pneumoniae (Fig. 3).
We then investigated the distribution of plasmid replicons and virulence factors in the Thai isolates. The predominant plasmid replicons were KpN3 and ColMG828 and less frequently the R plasmid, all of which are known to carry multiple resistance and virulence genes (Additional file 2: Figure S1). The majority of the global K. pneumoniae isolates also harbored these plasmid replicons (results not shown), indicating their global distribution. These replicons were present in isolates from both the environment and clinical samples (Additional file 2: Figure S1). Of the 75 virulence factors considered, 10 were present in >95% of isolates, examples being the mrk genes encoding fimbrial biosynthesis proteins and iutA encoding ferric aerobactin receptor [32]. By contrast, more than 40% of virulence genes were exclusively present in the clinical isolate collection (Additional file 2: Figure S2), including genes involved in capsule synthesis (rmpA) [33], iron transport (iro), phospholipid transport (mce), regulation and transport (multiple clb genes), and transcription regulation (kvgA). Multiple iron metabolismrelated and siderophore genes including ybt, irp, and fyuA were more common (statistical significance level for zstatistic of logistic regression: p value < 0.05) in clinical isolates and were incorporated into the chromosome. The higher number of virulence factors in clinical versus environmental isolates (Additional file 2: Figure S2), especially those involved in iron uptake, is suggestive of hyper-mucoviscous and hyper-virulent strains that may be more efficient in iron uptake and capsule production [34]. Some virulence genes were integrated into the chromosome while others were plasmid-mediated, implying that multiple mechanisms mediate their acquisition. Some virulence genes were also present in the genome of other gram-negative bacteria such as E. coli and Citrobacter koseri, indicating sharing both within and between species. A full list of the virulence factors With the exception of carbapenems, trimethoprim, tigecycline, and aminoglycosides, the Thai isolates exhibited intermediate to high resistance levels to the antibiotics tested. This finding, along with a correlation between increased minimum inhibitory concentration (MIC) values and antibiotics with a similar mechanism of action, is indicative of a limited number of effective antibiotics to treat infections (Additional file 2: Figure S3 and Figure S4). All isolates were ESBL-positive based on phenotypic testing. A genome-wide screen for known ESBLs as defined by the Comprehensive Antibiotic Resistance Database (https:// card.mcmaster.ca/) demonstrated that bla CTX-M-15 , bla SHV , bla VEB , and bla GES accounted for the ESBL phenotypes in the population. bla GES and bla VEB gene copies were found exclusively in the environmental hospital sewer isolates and clinical isolates, respectively. By contrast, bla CTX-M-15 and bla SHV had been sporadically gained by both environmental and clinical isolates. Nineteen isolates (25%) were found to be multidrug-resistant (resistant to three or more drug classes), all of which were clinical isolates. Four isolates were resistant to the carbapenem drugs. Resistance in these isolates was associated with the presence of plasmid-associated genes encoding carbapenamases, specifically New Delhi metallo-β-lactamase (NDM) (in two multidrug-resistant clinical isolates) and GES (in two nonmultidrug-resistant isolates from the hospital sewer). These genes appear to have been acquired in different lineages across the phylogenetic tree (Additional file 2: Figures S5 and S6). Isolation of Klebsiella oxytoca and Enterobacter cloacae from hospital sewers that harbor GES has been reported previously [35] and indirectly reflects its presence in the hospital population or environment. Additional file 4 provides a list of β-lactamases and ESBLs in the collection. Fig. 4 Origin, sample type, and isolate features for divergences between a Thai isolate and any other isolate from the Thai or global collection for non-clade 1 and 3 isolates over the past 50 years. This time corresponds to the formation of clade 1. To obtain the age and the upper and lower bounds for the 95% confidence interval, we divided the SNP distance by the mean substitution rates of clades 1 and 3 and the means of the lower and upper values for the 95 confidence intervals of the substitution rates estimated for clades 1 and 3. Each color corresponds to one country. The symbols ** and * signify the specific features of the Thai and the global collection, respectively. The lower bar plot shows geographical distance for Thai isolate pairs Our results indicated that isolates with an increase in MIC values and resistant phenotypes occurred in different lineages throughout the phylogenetic tree (Additional file 2: Figure S4). However, the MIC values for β-lactams (especially cephalosporins and aztreonam) were significantly higher in clinical isolates compared with environment isolates (Additional file 2: Figure S5). Consistent with this, clinical isolates carried more copies of β-lactamase genes (Additional file 2: Figure S6). Besides the chromosomally encoded bla LEN , bla SHV , and bla OKP that were present in every isolate, bla OXA , bla TEM , and bla CTX occurred throughout the tree, and these genes, in combination with other β-lactamases, were more common in clinical isolates (Additional file 2: Figure S6), which is consistent with reports from numerous countries.
No patterns were observed in the presence/absence of resistance genes for the non-β-lactam antibiotics in environmental versus hospital isolates (Additional file 2: Figure S6). The oqx efflux pump gene was present in every isolate and was not correlated with ciprofloxacin resistance. However, non-synonymous SNP densities within DNA topoisomerase IV genes (parE and parC) and point mutations in D87 DNA gyrase A, as well as in the E84 DNA topoisomerase IV genes, were exclusively present in two isolates with high MIC values for ciprofloxacin. Both mutations occurred in the quinolone resistance-determining region (QRDR) of gyrA and parC [36,37]. These point mutations were only found in clinical isolates, although both have been detected previously in E. coli isolated from aquatic environments [38]. The aminoglycoside resistance genes were noted to have been acquired on multiple occasions throughout the phylogenetic tree. Even though K. pneumoniae is reported to be intrinsically resistant to tetracyclines, the acquisition of further copies of tetracycline resistance genes occurred across the tree in both clinical and environmental isolates (Additional file 2: Figure S6).
Discussion
In this study we investigated the epidemiology of K. pneumoniae in a defined geographic area that included a general hospital and surrounding canals and farms. Our findings support the suggestion that clinical K. pneumoniae have evolved mechanisms to better adapt to survival in the clinical setting. Virulence factors were more frequent in clinical isolates and had been acquired on more than one occasion. This indicates the selection and dissemination of virulent strains in hospitals. Furthermore, the higher number of β-lactam resistance genes in clinical isolates together with higher absolute MIC values for some β-lactams for K. pneumoniae of clinical origin can be attributed to higher exposure to antibiotics, as proposed previously [39]. Given our finding of recent divergence events between clinical and environmental isolates, our results suggest that the selective pressure imposed upon clinical isolates is sufficient to result in significant changes in the genome of clinical isolates within a few decades. It has been reported previously that the prevalence of antibiotic resistance of food-borne isolates is higher for meat-source K. pneumoniae isolates than for human clinical isolates [16]. However, due to the lack of sufficient data about the strength of selective antibiotic pressure in foodanimal production versus hospitals, it is not possible to draw a definitive conclusion about antibiotic use and resistance [16].
The detection of carbapenem-resistant isolates in pretreated hospital wastewater reiterates the importance of the treatment of hospital wastewater prior to release into the environment [40,41]. Our study also highlights the role of environmental water as a potential reservoir for K. pneumoniae, where antibiotic resistant isolates may emerge. Antibiotic resistance genes present in environmental isolates were present in the founder lineage in some cases, for instance, in isolates in the mixed environmental and clinical clade 3 ( Fig. 3 and Additional file 2: Figure S3), which is consistent with contamination of such reservoirs. Resistance was also noted to have emerged in some environmental lineages against various antibiotics, for instance, ertapenem, cefoxitin, amikacin, and amoxicillin-clavulanic acid in sewage water isolates (Additional file 2: Figure S3), which presumably arises in response to antibiotics in environmental wastewater, as reported previously [42,43]. Several studies have shown the presence of highly antibiotic-resistant bacteria and resistance genes in sewage released into aquatic environments [42,[44][45][46]. In line with these findings, our results suggest that the release of untreated hospital sewage may play a role in the environmental emergence and spread of multiresistant pathogenic bacteria, and that wastewater (including hospital waste) warrants treatment to eliminate these organisms prior to release. This may be of particular importance in low-income rural areas and countries, where people are in greater contact with wastewater and may consume contaminated food and water containing high levels of antibioticresistant bacteria. Of note, wastewater is treated prior to release from Bhuddhasothorn Hospital, and no ESBLpositive K. pneumoniae were isolated from post-treated waste.
Conclusions
In this study the availability of epidemiologic information and the high resolution of whole genome sequencing allowed us to discover epidemiologic links between clinical and non-clinical K. pneumoniae. Limitations of the collection were the sparseness of the environmental collection and the lack of non-ESBL-producing strains. This happened because our isolates were selected using media that were selective for ESBL production. The isolation of K. pneumoniae from highly contaminated samples is challenging without the use of selective culture, but our approach means that the environmental collection was biased towards clinically important isolates, i.e., ESBL-producing strains. This facilitated the identification of distinct genomic patterns relating to the distribution of antimicrobial resistance and virulence factor genes in clinical isolates, but the inclusion of non-ESBL isolates, particularly from environmental sites, is required to obtain a broader assessment of clinical and non-clinical populations. The inclusion of susceptible isolates from the environment and hospital may reduce the difference in antibiotic resistance observed here between clinical and environmental isolates, although the effect on virulence gene pattern is difficult to predict. Future studies based on larger and deeper collections will be required to gain a detailed understanding of global transmission of K. pneumoniae at different geographical scales. | 2019-04-02T13:07:35.814Z | 2016-07-26T00:00:00.000 | {
"year": 2017,
"sha1": "deaba5ffde0593d08b9900970385b4e3ae8a0e4f",
"oa_license": "CCBY",
"oa_url": "https://genomemedicine.biomedcentral.com/track/pdf/10.1186/s13073-017-0397-1",
"oa_status": "GOLD",
"pdf_src": "SpringerNature",
"pdf_hash": "78eb2d9f7ebdad5c9e75f324b3e84fed4dff9bdc",
"s2fieldsofstudy": [
"Biology",
"Medicine"
],
"extfieldsofstudy": [
"Biology"
]
} |
210131289 | pes2o/s2orc | v3-fos-license | Progress in Carnot and Chambadal Modeling of Thermomechanical Engine by Considering Entropy Production and Heat Transfer Entropy
Nowadays the importance of thermomechanical engines in recognized worldwide. Since the industrial revolution, physicists and engineers have sought to maximize the efficiency of these machines, but also the mechanical energy or the power output of the engine, as we have recently found. The optimization procedure applied in many works in the literature focuses on considering new objective functions including economic and environmental criteria (i.e., ECOP ecological coefficient of performance). The debate here is oriented more towards fundamental aspects. It is known that the maximum of the power output is not obtained under the same conditions as the maximum of efficiency. This is shown, among other things, by the so-called nice radical that accounts for efficiency at maximum power, most often for the endoreversible configuration. We propose here to enrich the model and the debate by emphasizing the fundamental role of the heat transfer entropy together with the production of entropy, accounting for the external or internal irreversibilities of the converter. This original modeling to our knowledge, leads to new and more general results that are reported here. The main consequences of the approach are emphasized, and new limits of the efficiency at maximum energy or power output are obtained.
Introduction
The origin of the industrial era in the 19th century is strongly connected to thermomechanical engines whose importance in our society is well-known. Their rapid development and evolution have contributed to the foundation of thermodynamics as a new branch of science, mainly concerned with fundamental aspects, as well as numerous applications.
The famous reference case is relative to the Carnot engine [1], but it refers only to an Equilibrium Thermodynamics modelling of a totally reversible engine. Additionally, the work done by Carnot generalizes the concept of efficiency according to the First Law of Thermodynamics starting from a purely mechanical approach [2], and the concept of the Carnot cycle. The interactions between the working fluid and the surroundings during a cycle consist of a mechanical energy exchange, W, and a calorific energy exchange, Q, which are of infinite time duration for reversible conditions. This leads to a quasi-static cycle, whose mean power output of the engine, . W, is zero. Since (approximately) the 80s, a new approach has been developed including the existence of unavoidable heat transfer irreversibilities (endo-reversible machine [3]), and afterward, internal irreversibilities of the converter were also considered, but less frequently. To take account of these irreversibilities, two main forms are reported in the literature: • an entropic ratio, I [4]; • a production of entropy, that for a cycle is represented as a parameter, ∆S I [5].
In the more general case, the entropic ratio could be a function that needs to be specified. This will be considered in this paper.
The main objective encountered in the literature relative to the Non-Equilibrium Thermodynamics approach remains the power output optimization of the engine and the associated conditions (vector of state), as well as the efficiency associated with maximum power.
The present paper proposes a new modelling of an irreversible cycle focused on a necessary condition of the cycle existence, namely the heat transfer entropy. As the most important one for the cycle is the entering heat transfer entropy associated with the heat input in the converter (engine), it will be called hereafter, reference entropy, ∆S. According to the modeling assumptions that consider all cycle processes in the converter as irreversible, the total entropy production over a cycle, ∆S I is added to this reference heat transfer entropy. The coupling consequence of these two entropies in the analytical analysis of an irreversible cycle leads to new results that are reported here.
This new modelling is developed for the exo-reversible and endo-irreversible Carnot engine configuration, as the first possible extension of the primitive Carnot cycle approach.
Then, the remodeled Chambadal [29] engine configuration is described by adding the influence of the converter internal irreversibility. The optimization approach considers two ways to introduce the converter internal irreversibility, namely the entropic ratio model and the entropic production one, each of them being developed for different dependence functions of the system parameters. The consequences of this addition on the optimized power output are reported, and the limit cases-the endo-reversible Carnot engine and endo-irreversible model-are shown to be recovered.
A discussion of the overall results and an attempt to generalize the present study provide new and important upperbounds for the engine performance.
An ongoing next step will be the application of the same methodology to Curzon-Ahlborn [30] modelling of the Carnot engine by including internal irreversibilities of the converter and considering the importance of the heat transfer entropy.
Cycle Representation in T-S Diagram
The thermomechanical engine is considered as a system composed of a hot bath (heat source), converter (engine) and cold bath (heat sink), as shown in Figure 1. The coupling of the three subsystems is made by the heat transferred from the source to the working fluid in the engine at its hot side, and the heat rejected by the working fluid to the sink at the cold side, respectively.
In the Carnot engine modeling, the source and sink are two infinite bath (thermostats) at temperatures T HS and T CS respectively. The Carnot engine operates upon an irreversible cycle with perfect thermal contact (no temperature difference) with the two bathes. Thus, one can call it an exo-reversible and endo-irreversible engine. The irreversible cycle is identified in Figure 2 from state 1 (beginning of the high temperature isotherm), 2 (exit of the high temperature isotherm and beginning of the adiabatic expansion), 3 (end of the adiabatic expansion and beginning of the low temperature isotherm), and 4 (exit of the low temperature isotherm and beginning of the adiabatic compression, up to state 1). The reversible cycle (marked in red) is also represented in Figure 2 as a starting graphical cycle helping to emphasize the effect of each internal irreversibility considered in the modeling. Both adiabatic and isothermal transformations of the cycle are considered irreversible. Thus, the irreversibility on the isothermal processes is represented on the horizontal lines 12 and 34 by the corresponding production of entropy. As the irreversibility on the adiabatic processes in the cycle is also emphasized by entropy production, the graphical representation is associated to a polytropic processes represented in the T-S diagram by a corresponding dependence T = f(S). For simplicity, a linear dependence T = f(S) between states 2 and 3, and 4 and 1, respectively, replaced the real one in the analytical and graphical analysis of the cycle.
Consequently, the production of entropy on a cycle SI representing the total amount of entropy produced in the universe per cycle (= engine + environment) is expressed as: with SIH, production of entropy on the hot isotherm; SIEx, production of entropy during the adiabatic expansion; SICo, production of entropy during the adiabatic compression; and SIC, production of entropy on the cold isotherm, as shown in Figure 2. The irreversible cycle is identified in Figure 2 from state 1 (beginning of the high temperature isotherm), 2 (exit of the high temperature isotherm and beginning of the adiabatic expansion), 3 (end of the adiabatic expansion and beginning of the low temperature isotherm), and 4 (exit of the low temperature isotherm and beginning of the adiabatic compression, up to state 1). The reversible cycle (marked in red) is also represented in Figure 2 as a starting graphical cycle helping to emphasize the effect of each internal irreversibility considered in the modeling. The irreversible cycle is identified in Figure 2 from state 1 (beginning of the high temperature isotherm), 2 (exit of the high temperature isotherm and beginning of the adiabatic expansion), 3 (end of the adiabatic expansion and beginning of the low temperature isotherm), and 4 (exit of the low temperature isotherm and beginning of the adiabatic compression, up to state 1). The reversible cycle (marked in red) is also represented in Figure 2 as a starting graphical cycle helping to emphasize the effect of each internal irreversibility considered in the modeling. Both adiabatic and isothermal transformations of the cycle are considered irreversible. Thus, the irreversibility on the isothermal processes is represented on the horizontal lines 12 and 34 by the corresponding production of entropy. As the irreversibility on the adiabatic processes in the cycle is also emphasized by entropy production, the graphical representation is associated to a polytropic processes represented in the T-S diagram by a corresponding dependence T = f(S). For simplicity, a linear dependence T = f(S) between states 2 and 3, and 4 and 1, respectively, replaced the real one in the analytical and graphical analysis of the cycle.
Consequently, the production of entropy on a cycle SI representing the total amount of entropy produced in the universe per cycle (= engine + environment) is expressed as: with SIH, production of entropy on the hot isotherm; SIEx, production of entropy during the adiabatic expansion; SICo, production of entropy during the adiabatic compression; and SIC, production of entropy on the cold isotherm, as shown in Figure 2. Both adiabatic and isothermal transformations of the cycle are considered irreversible. Thus, the irreversibility on the isothermal processes is represented on the horizontal lines 12 and 34 by the corresponding production of entropy. As the irreversibility on the adiabatic processes in the cycle is also emphasized by entropy production, the graphical representation is associated to a polytropic processes represented in the T-S diagram by a corresponding dependence T = f(S). For simplicity, a linear dependence T = f(S) between states 2 and 3, and 4 and 1, respectively, replaced the real one in the analytical and graphical analysis of the cycle.
Consequently, the production of entropy on a cycle ∆S I representing the total amount of entropy produced in the universe per cycle (= engine + environment) is expressed as: with ∆S IH , production of entropy on the hot isotherm; ∆S IEx , production of entropy during the adiabatic expansion; ∆S ICo , production of entropy during the adiabatic compression; and ∆S IC , production of entropy on the cold isotherm, as shown in Figure 2.
Furthermore, for the hot temperature isotherm, the entropy production ∆S IH corresponds to: where the reference heat transfer entropy is expressed as: and it equals the entropy change in the hot bath (in module).
Similarly, for the cold temperature isotherm, the entropy production ∆S IC corresponds to: as all production of entropy terms are positive (see Figure 2). The two entropy productions ∆S IH and ∆S IC correspond to the graphical representation in Figure 3.
One outlines here that the entropy balance over the irreversible cycle is expressed as: where ∆S S represents the heat transfer entropy corresponding to the heat transferred to the sink. Note that the commutations between isothermal and adiabatic processes are assumed to be instantaneous. Moreover, one observes that while the two real isothermal processes are well identified on the diagram, it is not the same for the irreversible adiabatic processes linking state 2 to 3, and then 4 to 1. All the curves inscribed in the rectangle (2-3'-3-2'-2) satisfying the criterion of increasing entropy on the adiabatic expansion process are possible.
Plotting a linear transformation in the T-S diagram between state 2 and 3 is not the only physical acceptable solution, because in the frame of Non-Equilibrium Thermodynamics, one only has access to points 2 and 3 representing the adiabatic case.
The same discourse can be renewed for the adiabatic compression between 4 and 1. In short, all the curves inscribed in the rectangle (4-4'-1-1'-4) satisfying the criterion of increasing entropy on the adiabatic process are possible. The linear transformation in the T-S diagram between state 4 and 1 is again a possible intermediate solution.
This irreversibility approach is similar to the one of a recent publication [31]. The graphical analysis of the cycle with linearized processes between 23 and 41 illustrated in Figure 2 shows the area representing the corresponding linearized work per cycle W L as: This 'geometric' work (6) is different from the 'thermodynamic' (or mechanical) work that will be expressed in Section 2.2 by (10) since (6) depends on the linear assumption regarding the representation of the irreversible adiabatic processes between 23 and 41 as done in many references [31]. Nevertheless, it does not reflect the exact physical behavior of the endo-irreversible Carnot heat engine that could be different to the linear one. Thus, only (10), which is based on energy and entropy balances, will provide the correct expression of the mechanical work.
Analytical Model
Considering the adiabaticity (no thermal loss) and exo-reversibility assumptions for the cycle in this model, the heat transfer at the source and sink are respectively: The exo-reversibility of the heat transfer relative to the two thermostats imposes: Thus, the entropy balance on the system can be written as: where from the expression of the mechanical work per cycle from the First Law when the entropy variation corresponding to the heat transfer converted at the source is considered as reference heat transfer entropy, ∆S, ∆S H = ∆S. becomes: The first term of the second equality represents the reversible work, and the second one, the mechanical dissipation (by fluid and solid friction mainly).
The expression of W in (10) marks the presence of the internal entropy production over a cycle ∆S I that is supposed as a parameter. Its relationship with the ratio method is introduced below, by emphasizing ∆S in (10): Thus, the ratio ∆S I /∆S appears in (11) and it corresponds to the irreversibility degree d I introduced by Novikov [32]. This parameter is also related to an entropic ratio according to Ibrahim's approach [4], which represents the absolute value of the ratio of the entropy change in the cold bath to the entropy change in the hot bath and is given as: One notes that this ratio can be particularized (i.e., I H = ∆S S /∆S H ), since it represents a coefficient relative to an entropy variation corresponding to the heat exchanges at the hot and cold sides of the converter. Hence, other assignments could be given (I C , I HS , I CS ). For the adiabatic exo-reversible Carnot engine, this ratio is I = I H = I HS and moreover, it is easy to show that: and It results that the internal irreversibility diminishes the mechanical work and cycle efficiency. It remains to analyze how I or ∆S I vary with the cycle quantities. It can be reasonably assumed that ∆S I and (or) I depend on ∆S and (or) ∆T S = T HS -T CS , as the two temperatures are generally parameters of the problem for the Carnot endo-irreversible engine. Hence, two functions can be proposed, whose general forms (currently unknown in practice) are written as: If there is an optimum of W, it must satisfy the necessary condition imposed by one of the following equation sets:
The Chambadal Engine
The representation of the Chambadal engine irreversible cycle in Figure 3 shows that in addition to the internal irreversibility of the cycle that was graphically detailed in Section 2.1, the external irreversibility corresponding to the heat transfer at temperature difference at the hot side appears. Since the same components of the internal irreversibility for the endo-irreversible Carnot engine are considered here as well, the cycle representation was simplified by only preserving its shape, and the new irreversibility completing the model was emphasized. Hence, in Figure 3 case (a) corresponds to a heat transfer from a source of constant temperature T HS , while case (b) shows a finite heat capacity source with the inlet temperature T HSi . The temperature difference of the source and sink level, ∆T S , compared to the cycled fluid one, ∆T CF , that remains the same, is also relevant for the heat input in the two cases.
Note that the external irreversibility at the sink is neglected in this approach by considering the heat rejection to sink occurring at ambient temperature (T C = T CS = T 0 ). new irreversibility completing the model was emphasized. Hence, in Figure 3 case (a) corresponds to a heat transfer from a source of constant temperature THS, while case (b) shows a finite heat capacity source with the inlet temperature THSi. The temperature difference of the source and sink level, TS, compared to the cycled fluid one, TCF, that remains the same, is also relevant for the heat input in the two cases. Note that the external irreversibility at the sink is neglected in this approach by considering the heat rejection to sink occurring at ambient temperature (TC = TCS = T0). The Chambadal engine model is concerned with steady-state operation of the engine and consequently, heat rates and power output are used. We propose here to convert this modeling with reference to a cycle and the related energies-heat and work, thus leading to a different model, the quasi-Chambadal engine.
It involves a thermostat or source of finite heat capacity rate ̇ = ̇ , with an input temperature THSi. Hence, when the source is a thermostat of constant temperature, THS, the heat transfer at the hot end is expressed as: with KH = UH•AH, heat transfer conductance of the source corresponding to the steady state operation regime.
When the source has a finite heat capacity, it involves a temperature variation of the primary fluid in the corresponding heat exchanger. As the input temperature of the primary fluid THSi is generally known, by using the effectiveness -number of heat transfer units NTU method [27], one gets: Note that the two expressions of ̇ averaged on a time duration t, allow to recover the caloric energy QH, or the mass specific energy (often used) that results by dividing ̇ with the mass flow rate of the cycled fluid. Thus, a general expression of the heat transferred at the source is: where GH generally represents a finite physical dimension of the source (thus also of the system) with reference to energy here, its unit being J/K. The Chambadal engine model is concerned with steady-state operation of the engine and consequently, heat rates and power output are used. We propose here to convert this modeling with reference to a cycle and the related energies-heat and work, thus leading to a different model, the quasi-Chambadal engine.
Optimization Approach for the Entropic Ratio Model
It involves a thermostat or source of finite heat capacity rate m H c pH , with an input temperature T HSi .
Hence, when the source is a thermostat of constant temperature, T HS , the heat transfer at the hot end is expressed as: with K H = U H · A H , heat transfer conductance of the source corresponding to the steady state operation regime.
When the source has a finite heat capacity, it involves a temperature variation of the primary fluid in the corresponding heat exchanger. As the input temperature of the primary fluid T HSi is generally known, by using the effectiveness ε -number of heat transfer units NTU method [27], one gets: .
Note that the two expressions of . Q H averaged on a time duration ∆t, allow to recover the caloric energy Q H , or the mass specific energy (often used) that results by dividing . Q H with the mass flow rate of the cycled fluid. Thus, a general expression of the heat transferred at the source is: where G H generally represents a finite physical dimension of the source (thus also of the system) with reference to energy here, its unit being J/K.
Optimization Approach for the Entropic Ratio Model
The previous equation is completed in this approach by the entropy balance expressed over a cycle as: This leads to the expression of the mechanical work output as: By taking into account that I H is a potential function of T H , the mechanical work maximum value MaxW search should satisfy the necessary condition: This implies ∂I H /∂T H = I H,H = 0. Thereby, the optimum temperature at the hot-end, MaxW and the corresponding cycle efficiency respectively, result as: It is obvious that the rise of I H parameter results in an increase of the optimal temperature T H * and has the opposite effect (decrease) on MaxW and the associated cycle efficiency.
(ii) I H is a linear function of ∆T T = T H -T 0 (see Figure 3), with values ≥ 1.
One considers the following expression for I H : By combining (25) with (29), one gets: The above equation leads to the expression of the optimum temperature of the cycled fluid at the hot side expressed by: It results that irreversibility does not affect the temperature T H * , but it does affect the mechanical work and the cycle efficiency, whose expressions become: The comparison of (27) and (32) clearly shows the maximum work decrease in case ii) and by a completely different expression. The same happens for the efficiency at maximum work, when comparing (28) and (33).
Optimization Approach for the Entropic Production Model
The entropy balance takes a similar form to that of Section 2.2, namely: with T C = T CS = T 0 . One notes that the production of entropy on the cycle in (34) is not the same with (1) in the endo-irreversible Carnot engine case, since it also considers the production of entropy corresponding to the heat transfer at finite temperature difference (see Figure 3).
The mechanical work is expressed as: By using (21) in (35), one gets: The term ∆S I is a potential function of T H (also increasing one). Therefore, the MaxW search should satisfy the necessary condition: This form is simpler than the previous one, providing an equation of the second degree or more to solve.
The possible cases are as follows: (i) ∆S I is a constant parameter.
This implies ∂S I /∂T H = ∆S I,H = 0, which delivers: The expressions of MaxW and η I (MaxW) are straightforward: (ii) A linear dependence of ∆S I on the temperature difference ∆T T = T H -T CS is considered.
We assume that: Figure 3b), where from: The mechanical work optimization imposes: which leads to: It is well known that internal irreversibility always decreases W. Hence, (41) is valuable by emphasizing in the denominator two factors that modify the optimal temperature T H *: • the first, an intensive one: the second, an extensive one: The last factor is not only related to the converter through ∆S IS , but also to the hot source by the finite physical dimension G H (coupling).
This shows the interest of an entropic description of the system. Thus, the corresponding maximum power output is given by: with The associated first law efficiency is:
Quasi-Chambadal Model with Added Heat Transfer Entropy Constraint and Production of Entropy
This optimization is based on (21) from which the temperature T H is expressed as: with ∆S, reference heat transfer entropy associated to the heat input. This result is an important one, since it illustrates the coupling between the source (G H ) and the converter (∆S). The expression of the mechanical work W can be easily derived as a function of the newly chosen variable ∆S:
Conclusions
This work presents a short history of the Carnot cycle approaches developed in the frame of Equilibrium Thermodynamics, of the actual endo-irreversible Carnot engine in Irreversible Thermodynamics, and for the Chambadal model.
The analytical model developed in the frame of Equilibrium Thermodynamics integrates the irreversibility in the natural form of an entropy production, or in an entropic ratio form (with various justified and correlated definitions) and is mainly related to the irreversibility degree introduced by Novikov [32].
The mechanical work of the irreversible cycle remains linked to the form of the irreversible isotherms and adiabatics, as has been shown in Section 2.1. The irreversibilities distribution between isothermal and adiabatic processes leads to framing the actual work between zero and max MaxW, with the corresponding efficiencies pertaining to the interval between zero and the Carnot efficiency.
For the Chambadal model, the cycle approach or the one in the stationary dynamic regime come down to the same form, by introducing a characteristic property G H , which is representative of the finite physical dimensions connected to the reference heat transfer entropy ∆S, characterizing the converter.
It results in the existence of an optimum of the mechanical work either in terms of intensive variable T H , or in terms of extensive variable ∆S.
The two approaches leading to coherent results, but with different interpretations, provide a new perspective on the optimization of the Carnot engine, mainly by coupling of the hot source with the converter (G H and ∆S).
Extensions are in progress, which are based on more complete models, particularly on the Curzon-Ahlborn one, that considers the non-equilibrium at source and sink. Also, the stochastic thermodynamics approach [33,34] is intended to be used in future works to evaluate different sources of irreversibility in the corresponding heat engines exactly, without additional assumptions. It could offer an alternative to evaluate the analytical results provided by our phenomenological approach.
Conflicts of Interest:
The authors declare no conflict of interest. irreversibility of the converter IS irreversibility of the system (source-converter-sink) S source or sink, relative to temperature difference of source and sink T total, relative to extreme temperature difference of the cycled fluid 0 ambient | 2019-12-19T09:15:58.146Z | 2019-12-01T00:00:00.000 | {
"year": 2019,
"sha1": "36b8e6d108e2eeb2fc7108026be4fd6f986f369f",
"oa_license": "CCBY",
"oa_url": "https://www.mdpi.com/1099-4300/21/12/1232/pdf",
"oa_status": "GOLD",
"pdf_src": "PubMedCentral",
"pdf_hash": "d7890718fb68204d27c6602be0dd2aa69588765c",
"s2fieldsofstudy": [
"Engineering",
"Physics",
"Environmental Science"
],
"extfieldsofstudy": [
"Computer Science"
]
} |
247630242 | pes2o/s2orc | v3-fos-license | Involvement of Urm1, a Ubiquitin-Like Protein, in the Regulation of Oxidative Stress Response of Toxoplasma gondii
ABSTRACT Ubiquitin-related modifier 1 (Urm1) is a ubiquitin-like molecule (UBL) with the ability to act as a posttranslational protein modifier. Here, we characterized the Toxoplasma gondii homolog of URM1 located in the tachyzoite cytoplasm. The total loss of the TgURM1 resulted in a significant reduction in parasite invasion, replication, and virulence in mice, revealing that TgURM1 plays a pivotal role in T. gondii survival. For TgURM1, urmylation was significantly induced by oxidative stress, and mutations of the C-terminal glycine-glycine motif of TgURM1 blocked the urmylation process. Furthermore, the TgURM1 knockout strain was intolerant to oxidative stress, suggesting that TgURM1 is involved in the oxidative stress process. TgAHP1, an alkyl hydroperoxide reductase, was screened via proximity-based protein labeling techniques and proteomics and was shown to interact with TgURM1 under oxidative stress conditions. In conclusion, TgURM1 is a UBL protein involved in the response of Toxoplasma to oxidative stress. IMPORTANCE T. gondii has an intricate life cycle which involves multiple morphologically and physiologically distinct stages, and posttranslational modifications (PTMs) may be key regulators of protein expression at relevant life cycle stages. In recent years, ubiquitin-like proteins with modification functions have been discovered and studied, including Sumo, Rub1, ATG8, and ATG12. Ubiquitin-related modifier 1 (Urm1) is a ubiquitin-like molecule (UBL), which is considered to be the oldest ubiquitin-like system. In this study, we identified the Urm1 gene in Toxoplasma and explored that the urmylation of Urm1 was significantly induced by oxidative stress. Fewer studies have been conducted on ubiquitin-like proteins of parasites, and our results provide theoretical support for the research of metabolic regulation and antioxidative stress processes in T. gondii.
1. An important control strain that needs to be included to draw any inference is the complemented strain (Where the gene TgURM1 is put back in to the knockout strain and is able to restore the wildtype phenotype). Without this strain one cannot make any conclusions about the role of URM1.
Where is the western blot showing size of this protein?
3. In Fig 2C, how the invasion assay was performed? How the invasion efficiency was determined? 4. In Fig 2D, how was the replication assay was done? If the added parasites were washed after 30 min of invasion, according to the data shown, it looks like mutant parasites have defect in replication. Authors cannot conclude there is no defect in endodyogeny by using just using two markers GAP45 and IMC1 which both are markers of inner membrane complex (Fig 2H). 5. Fig 2F, how was the motility ability determined? How it was quantified? Is there a difference in the three types of motility (circular, helical and gliding)?
6. Fig 2G, how the egress assay was done and quantified? 7. The authors are working RH strain that does not form cysts. Their conclusion that the mice inoculated with the knockout strain survive because they fail to differentiate does not make any sense.
8. TgURM localizes to the parasite cytoplasm. There is no rationale provided for expression of this protein in HEK293T cells.
9. Authors are trying to identify interactors of TgURM using different approaches in sections 3.5 and 3.6. Where is the mass spec data of interactors identified by proximity-based labeling? Why there were no common proteins identified by different approaches? How then can one trust the data they have? 10. Sections 3.6 and 3.7 are very poorly written and very confusing. The authors do not provide any logical rationale why they are doing these experiments. 11. Section 3.7, line 306, which study you are referring to?
A few examples where sentences need to be corrected are as follows.
Lines 29-30
Lines 80-81 Reviewer #2 (Comments for the Author): This manuscript studied a UBL-like protein in a protozoan parasite, Toxoplasma gondii. The authors generated a straight knockout in the study and identified the defects on invasion, replication, and acute virulence in the knockout. Upon oxidative stress, the parasites perform urmylation by conjugating TgUrm1 to other proteins in the parasites. This work characterized a novel subcellular process in Toxoplasma. However, there are a few issues the authors need to address to strengthen this work. I listed them below.
Major: 1. It is unclear how TgUrm1 is tagged with 3xHA at its N-terminus in Fig. 1C. Did the Cas9 protein cut the internal region of the TgUrm1 gene for tagging? Which promoter was the tagged TgUrm1 driven? 2. I suggest the authors use the standard survival plot to document the virulence assay. The significance can be calculated using the appropriate statistical analysis.
3. In Fig. 3, the RH∆ku80 strain used in this study belongs to Type 1, which shows less tendency for cyst conversion than Type 2 strain. Therefore, the BAG1 expression did not increase significantly post alkali induction, as the authors showed in this study. Therefore, I don't feel confident that the TgUrm1 knockout displayed defects in cyst formation. Fig. 4F, the TgUrm1 knockout pretreated with diamide killed mice at the same percentage compared to non-treated TgUrm1 knockout. The authors need to run statistical analysis before concluding that oxidative stress harms the pathogenesis of the TgUrm1 knockout. 5. In Fig. 7F, a similar concern to item 4, the tBOOH-treated TgUrm1 knockout did not show virulence loss compared to nontreated TgUrm1 knockout. 6. In Fig. 4B, tBOOH did not trigger significant urmylation relative to non-treated parasites. Although both proteins interact with each other upon the presence of oxidative stress, how can the interaction be linked to urmylation? 7. If the TgUrm1 is involved in regulating the stress response, the TgUrm1 knockout would display higher ROS/RNS levels in the presence of oxidative stress than WT parasites, maybe even without oxidative stress. The authors may consider measuring the ROS/RNS levels.
In
Minor: 1. Line 24: "deficiency mutations", remove the deficiency 2. Line 182: "growth deficiency" should be growth defect 3. Lacking scale bar in IFA pictures 4. Line 207: Define Urm1-OE, overexpression of TgUrm1 in the knockout? 5. Line 210, sera were tested, not detected 6. Need scale bar for all IFA images Staff Comments:
Preparing Revision Guidelines
To submit your modified manuscript, log onto the eJP submission site at https://spectrum.msubmit.net/cgi-bin/main.plex. Go to Author Tasks and click the appropriate manuscript title to begin the revision process. The information that you entered when you first submitted the paper will be displayed. Please update the information as necessary. Here are a few examples of required updates that authors must address: • Point-by-point responses to the issues raised by the reviewers in a file named "Response to Reviewers," NOT IN YOUR COVER LETTER. • Upload a compare copy of the manuscript (without figures) as a "Marked-Up Manuscript" file. • Each figure must be uploaded as a separate file, and any multipanel figures must be assembled into one file. For complete guidelines on revision requirements, please see the journal Submission and Review Process requirements at https://journals.asm.org/journal/Spectrum/submission-review-process. Submissions of a paper that does not conform to Microbiology Spectrum guidelines will delay acceptance of your manuscript. " Please return the manuscript within 60 days; if you cannot complete the modification within this time period, please contact me. If you do not wish to modify the manuscript and prefer to submit it to another journal, please notify me of your decision immediately so that the manuscript may be formally withdrawn from consideration by Microbiology Spectrum.
If your manuscript is accepted for publication, you will be contacted separately about payment when the proofs are issued; please follow the instructions in that e-mail. Arrangements for payment must be made before your article is published. For a complete list of Publication Fees, including supplemental material costs, please visit our website.
Corresponding authors may join or renew ASM membership to obtain discounts on publication fees. Need to upgrade your membership level? Please contact Customer Service at Service@asmusa.org.
Thank you for submitting your paper to Microbiology Spectrum.
Dear editor:
I have revised the manuscript based on the comments of the reviewers, and the details are as follows: Reviewer #1 (Comments for the Author): 1. An important control strain that needs to be included to draw any inference is the complemented strain (Where the gene TgURM1 is put back in to the knockout strain and is able to restore the wildtype phenotype). Without this strain one cannot make any conclusions about the role of URM1.
Lacking of an intron in Urm1 gene, the strain in which the original TgUrm1 locus was replaced with the TgUrm1 tagged with 3xHA at TgUrm1's N-terminus, was used as the complemented strain. The complemented strain still used the endogenous promoter for transcription initiation. We have added supplementary data, including plaque, invasion, replication, virulence of complemented strain in Figure 2B-D, Figure 3A-B, and Figure 7B-G.
Where is the western blot showing size of this protein?
Western-blot result was showed in Figure 1C, and the molecular weight of HA tagged TgURM1 protein was 28 KD.
In Fig 2C, how the invasion assay was performed? How the invasion efficiency was determined?
Protocol of invasion assay and the calculation method for invasion efficiency determination were showed on lines 108-118. Fig 2D, how was the replication assay was done? If the added parasites were washed after 30 min of invasion, according to the data shown, it looks like mutant parasites have defect in replication. Authors cannot conclude there is no defect in endodyogeny by using just using two markers GAP45 and IMC1 which both are markers of inner membrane complex ( Fig 2H).
In
Protocol of replication assay and the calculation method were showed on lines 119-128. Figure 2D. Of course, our experimental results only showed that knocking out URM1 had no effect on the formation of IMC1 in progeny parasites, but it was hard to prove the relevance of knocking out URM1 on other replication processes of parasites. In addition, some sentences in the manuscript were inaccurate. In order to avoid misleading, we have deleted this part.
Fig 2F, how was the motility ability determined? How it was quantified? Is there a difference in the three types of motility (circular, helical and gliding)?
In general, gliding types of T. gondii includes circular gliding, upright twirling, and helical rotation circular. Parasites that could not complete one of the three gliding modes were defined as gliding defect. Protocol of gliding assay and the calculation method were showed on lines 129-138.
Fig 2G, how the egress assay was done and quantified?
Protocol of egress assay and the calculation method were showed on lines 139-144.
7. The authors are working RH strain that does not form cysts. Their conclusion that the mice inoculated with the knockout strain survive because they fail to differentiate does not make any sense.
In general, the RH strain does not form cysts, but with some special inducer or knocking out some special genes, the RH strain still has a tendency to form cysts. Of course, our experimental results could not fully prove the relevance of TgURM1 and cyst formation, and some sentences in the manuscript were inaccurate. In order to avoid misleading, we have deleted this part.
TgURM1 localizes to the parasite cytoplasm. There is no rationale provided for expression of this protein in HEK293T cells.
Immunofluorescence result of TgUrm1 location in HEK293T was added in Figure 4A.
Authors are trying to identify interactors of TgURM using different approaches in sections 3.5 and 3.6. Where is the mass spec data of interactors identified by proximity-based labeling? Why there were no common proteins identified by different approaches? How then can one trust the data they have?
A total of 51 proteins were identified using biotin-adjacent labeling method in the presence of Dia, while 107 proteins were identified by proteomics. Comprehensive analysis of all identification, 22 promising proteins identified by both methods were selected (Fig. 5E), which involved in oxidative stress response, immune evasion and parasite metabolism (lines 275-279).
10. Sections 3.6 and 3.7 are very poorly written and very confusing. The authors do not provide any logical rationale why they are doing these experiments.
TgAHP1 (TGGT1_286630) was first reported in our previous study "Alkyl Hydroperoxide
Reductase as a Determinant of Parasite Antiperoxide Response in Toxoplasma gondii", which was identified as a peroxisomal protein with cysteine-dependent peroxidase activity in T. gondii.
Interestingly, TgAHP1 was found in the mass spectrum data ( Figure 5E), indicating that AHP1-URM1 complexes may be detected in the presence of oxidant (lines 281-284).
We have changed some sentences and cited this reference to make the manuscript more logical. In addition, we have rewritten the Sections 3.6 and Sections 3.7 to make the manuscript more logical and complete. 11. Section 3.7, line 306, which study you are referring to?
This section referring to our previous study "Alkyl hydroperoxide reductase as a determinant of parasite antiperoxide response in Toxoplasma gondii", and we have quoted this reference.
We have rewritten the Section 3.7 to make the manuscript more logical.
A few examples where sentences need to be corrected are as follows.
Lines 29-30
We have changed this sentence to make it more logical (lines 29-30).
Lines 80-81
We have changed this sentence to make it more logical (lines 88-90).
Reviewer #2 (Comments for the Author): 1. It is unclear how TgUrm1 is tagged with 3xHA at its N-terminus in Fig. 1C. Did the Cas9 protein cut the internal region of the TgUrm1 gene for tagging? Which promoter was the tagged TgUrm1 driven?
Lacking of an intron in Urm1 gene, the strain in which the original TgUrm1 locus was replaced with the TgUrm1 tagged with 3xHA at TgUrm1's N-terminus (lines 205-207). The HA tagged TgUrm1 strain still used the endogenous promoter for transcription initiation, and the Cas9 protein cut the internal region of the TgUrm1 gene for tagging.
2. I suggest the authors use the standard survival plot to document the virulence assay. The significance can be calculated using the appropriate statistical analysis.
Thank you very much for the reviewer's suggestion. We have used the standard survival plot to document the virulence assay and the significance was analyzed using Gehan-Breslow-Wilcoxon tests, referring to the reference "Functional analysis of Toxoplasma lactate dehydrogenases suggests critical roles of lactate fermentation for parasite growth in vivo" Fig. 3, the RH∆ku80 strain used in this study belongs to Type 1, which shows less tendency for cyst conversion than Type 2 strain. Therefore, the BAG1 expression did not increase significantly post alkali induction, as the authors showed in this study. Therefore, I don't feel confident that the TgUrm1 knockout displayed defects in cyst formation.
In
In general, the RH strain does not form cysts, but with some special inducer or knocking out some special genes, the RH strain still has a tendency to form cysts. Of course, our experimental results could not fully prove the relevance of TgURM1 and cyst formation, and some sentences in the manuscript were inaccurate. In order to avoid misleading, we have deleted this part. Fig. 4F, the TgUrm1 knockout pretreated with diamide killed mice at the same percentage compared to non-treated TgUrm1 knockout. The authors need to run statistical analysis before concluding that oxidative stress harms the pathogenesis of the TgUrm1 knockout.
In
The results have been removed to Figure 7F to make the manuscript more logical, and Gehan-Breslow-Wilcoxon tests was used to analyse the significance of virulence. Fig. 7F, a similar concern to item 4, the tBOOH-treated TgUrm1 knockout did not show virulence loss compared to non-treated TgUrm1 knockout.
In
Gehan-Breslow-Wilcoxon tests was used to analyse the significance of virulence between the two strains in Figure 7F.
6. In Fig. 4B, tBOOH did not trigger significant urmylation relative to non-treated parasites. Although both proteins interact with each other upon the presence of oxidative stress, how can the interaction be linked to urmylation?
In the previous Fig 4B, tBOOH failed to trigger significant urmylation, which was due to the using of low concentration tBOOH. To make the manuscript more logical, tBOOH-induced ubiquitination was re-preformed using the unified concentration (10 -4 M tBOOH) of Figure 7, and supplementary result was showed in Figure 7A, which indicated that the treatment with 10 -4 M tBOOH also triggered significant Urm1 urmylation. Both Figure 4B and 4C in the original manuscript actually indicated that multiple oxidants could induce TURM1 ubiquitination. Thus, we removed the Figure 4B. 7. If the TgUrm1 is involved in regulating the stress response, the TgUrm1 knockout would display higher ROS/RNS levels in the presence of oxidative stress than WT parasites, maybe even without oxidative stress. The authors may consider measuring the ROS/RNS levels.
Supplementary experiments for ROS level detection were performed, and the results were added on lines 302-306 and Figure 7G. Minor:
Line 24: "deficiency mutations", remove the deficiency
We have removed the deficiency on line 24.
Line 182: "growth deficiency" should be growth defect
We have changed "growth deficiency" to "growth defect" on line 220.
Lacking scale bar in IFA pictures
We have added the scale bar in all IFA pictures, including Figure 1D, Figure 2E, Figure 4A, Figure 5C.
Line 207: Define Urm1-OE, overexpression of TgUrm1 in the knockout?
Yes, Urm1-OE is a strain, which TgUrm1 overexpressed in the knockout strain. We have defined the strain on lines 230-231.
Line 210, sera were tested, not detected
We have changed " detected " to " tested " on lines 233.
Need scale bar for all IFA images.
We have added the scale bar in IFA pictures, including Figure 1D, Figure 2E, Figure 4A, Figure 5C. Thank you for submitting your manuscript to Microbiology Spectrum. As you will see your paper is very close to acceptance. Please modify the manuscript along the lines suggested by the reviewers. As these revisions are quite minor, I expect that you should be able to turn in the revised paper in less than 30 days, if not sooner. If your manuscript was reviewed, you will find the reviewers' comments below.
When submitting the revised version of your paper, please provide (1) point-by-point responses to the issues I raised in your cover letter, and (2) a PDF file that indicates the changes from the original submission (by highlighting or underlining the changes) as file type "Marked Up Manuscript -For Review Only". Please use this link to submit your revised manuscript. Detailed instructions on submitting your revised paper are below.
Link Not Available
Thank you for the privilege of reviewing your work. Below you will find instructions from the Microbiology Spectrum editorial office and comments generated during the review.
The ASM Journals program strives for constant improvement in our submission and publication process. Please tell us how we can improve your experience by taking this quick Author Survey. Sincerely,
Björn Kafsack
Editor, Microbiology Spectrum Reviewer comments: Reviewer #1 (Comments for the Author): 1. Authors have not included any details of how the complemented strain was generated either in the materials and methods or the results section. 2. In Figure 2C and 2D, they have included the "complemented" strain but they not describe the results obtained with this strain in the results section. Authors do not provide reasons why the complemented strain was not included in the motility and egress assays shown in Fig 2E,Fand The authors addressed some questions from both reviewers. I have some concerns listed below. Please explain them.
1. For Q1 from reviewer 1, the authors mentioned that they removed the first intron and complemented the TgUrm1 gene in the original locus of TgUrm1. Please describe details for the creation of the knockout and complementation strains. It would be great to include a detailed schematic illustration. The current one in Fig 2A is too simple. 2. The authors need to provide more experimental details for their phenotypic characterizations.
3. Fig. 4A is inconclusive. I cannot tell URM1 is in the cytoplasm. 4. For Q1 from reviewer 2, please refer to the first question. Need more details. 5. For Q5 from reviewer 2, I am unsure if you can get biological significance among these very mild virulence differences using five mice.
6. For Q7 from reviewer 2, in Fig 7G, you need to include non-treated WT, knockout, and complementation strains as references. Otherwise, the treatment data are meaningless.
Overall, the authors deleted a few original observations from the manuscript during the revision, which weakens the significance of this study.
Preparing Revision Guidelines
To submit your modified manuscript, log onto the eJP submission site at https://spectrum.msubmit.net/cgi-bin/main.plex. Go to Author Tasks and click the appropriate manuscript title to begin the revision process. The information that you entered when you first submitted the paper will be displayed. Please update the information as necessary. Here are a few examples of required updates that authors must address: • point-by-point responses to the issues I raised in your cover letter • Upload a compare copy of the manuscript (without figures) as a "Marked-Up Manuscript" file. • Each figure must be uploaded as a separate file, and any multipanel figures must be assembled into one file. For complete guidelines on revision requirements, please see the journal Submission and Review Process requirements at https://journals.asm.org/journal/Spectrum/submission-review-process. Submissions of a paper that does not conform to Microbiology Spectrum guidelines will delay acceptance of your manuscript. " Please return the manuscript within 60 days; if you cannot complete the modification within this time period, please contact me. If you do not wish to modify the manuscript and prefer to submit it to another journal, please notify me of your decision immediately so that the manuscript may be formally withdrawn from consideration by Microbiology Spectrum.
If your manuscript is accepted for publication, you will be contacted separately about payment when the proofs are issued; please follow the instructions in that e-mail. Arrangements for payment must be made before your article is published. For a complete list of Publication Fees, including supplemental material costs, please visit our website.
Corresponding authors may join or renew ASM membership to obtain discounts on publication fees. Need to upgrade your membership level? Please contact Customer Service at Service@asmusa.org.
Thank you for submitting your paper to Microbiology Spectrum.
Dear editor:
We have revised the manuscript based on the comments of the reviewers, and the details are as follows: Reviewer #1 (Comments for the Author): 1. Authors have not included any details of how the complemented strain was generated either in the materials and methods or the results section.
The details of the generation of Urm1 knockout and complemented strains were shown in the Materials and Methods on lines 102-112. Figure 2C and 2D, they have included the "complemented" strain but they not describe the results obtained with this strain in the results section. Authors do not provide reasons why the complemented strain was not included in the motility and egress assays shown in
Fig 2E, F and G.
A supplemented description " The growth of defect was further confirmed, and the attenuated invasion and proliferation trends were observed in ΔUrm1 strain, comparing to wild-type RHΔKu80 strain and ComΔUrm1 strain " was added on lines 233-235 to describe the results of "complemented" strain.
There was no significant difference in the motility and egress process between the "complemented" strain and wild-type strain, and we neglected to provide the data of the "complemented" strain. We have supplemented the data of complemented strain in Fig 2E-
The authors need to provide more experimental details for their phenotypic characterizations.
Alignment details for the evolutionary tree were added on lines 492-500. Fig. 4A is inconclusive. I cannot tell URM1 is in the cytoplasm.
3.
Cytoplasmic separation was made in URM1 transfected cells, and the Western blotting was performed to confirm the cytoplasmic localization of URM1 on lines 262-264. The result was shown in Fig.4B. 4. For Q1 from reviewer 2, please refer to the first question. Need more details.
The details of the generation of Urm1 knockout and complemented strains were shown in the Materials and Methods on lines 102-112.
For Q5 from reviewer 2, I am unsure if you can get biological significance among these very mild virulence differences using five mice.
Three independent replicates were made in T. gondii pathogenicity test with five mice/group each time. Three independent replicate results were combined and re-analyzed for the significance of virulence in Fig 7F. 6. For Q7 from reviewer 2, in Fig 7G, you need to include non-treated WT, knockout, and complementation strains as references. Otherwise, the treatment data are meaningless.
In Fig 7G, the increase rate of ROS in different strains was calculated. The formula for the ROS increase rate is simplified to the following: Increase rate= [OX(+)-OX (-)]/OX(-)×100%.
Where "OX(+)" is the ROS levels of oxidant treatment group, "OX(-)" is the ROS levels of oxidant non-treatment group. The increase rates actually included the all strains (treated group or non-treated). The supplement was added on lines 577-580. Thank you for submitting your manuscript to Microbiology Spectrum. As you will see your paper is very close to acceptance. Please modify the manuscript along the lines I have recommended. As these revisions are quite minor, I expect that you should be able to turn in the revised paper in less than 30 days, if not sooner. If your manuscript was reviewed, you will find the reviewers' comments below.
When submitting the revised version of your paper, please provide (1) point-by-point responses to the issues I raised in your cover letter, and (2) a PDF file that indicates the changes from the original submission (by highlighting or underlining the changes) as file type "Marked Up Manuscript -For Review Only". Please use this link to submit your revised manuscript. Detailed instructions on submitting your revised paper are below.
Link Not Available
Thank you for the privilege of reviewing your work. Below you will find instructions from the Microbiology Spectrum editorial office and comments generated during the review.
The ASM Journals program strives for constant improvement in our submission and publication process. Please tell us how we can improve your experience by taking this quick Author Survey. Sincerely,
Preparing Revision Guidelines
To submit your modified manuscript, log onto the eJP submission site at https://spectrum.msubmit.net/cgi-bin/main.plex. Go to Author Tasks and click the appropriate manuscript title to begin the revision process. The information that you entered when you first submitted the paper will be displayed. Please update the information as necessary. Here are a few examples of required updates that authors must address: • point-by-point responses to the issues I raised in your cover letter • Upload a compare copy of the manuscript (without figures) as a "Marked-Up Manuscript" file. • Each figure must be uploaded as a separate file, and any multipanel figures must be assembled into one file. For complete guidelines on revision requirements, please see the journal Submission and Review Process requirements at https://journals.asm.org/journal/Spectrum/submission-review-process. Submissions of a paper that does not conform to Microbiology Spectrum guidelines will delay acceptance of your manuscript. " Please return the manuscript within 60 days; if you cannot complete the modification within this time period, please contact me. If you do not wish to modify the manuscript and prefer to submit it to another journal, please notify me of your decision immediately so that the manuscript may be formally withdrawn from consideration by Microbiology Spectrum.
If your manuscript is accepted for publication, you will be contacted separately about payment when the proofs are issued; please follow the instructions in that e-mail. Arrangements for payment must be made before your article is published. For a complete list of Publication Fees, including supplemental material costs, please visit our website.
Corresponding authors may join or renew ASM membership to obtain discounts on publication fees. Need to upgrade your membership level? Please contact Customer Service at Service@asmusa.org.
Thank you for submitting your paper to Microbiology Spectrum.
Dear editor:
We have revised the manuscript based on the comments. Your manuscript has been accepted, and I am forwarding it to the ASM Journals Department for publication. You will be notified when your proofs are ready to be viewed.
The ASM Journals program strives for constant improvement in our submission and publication process. Please tell us how we can improve your experience by taking this quick Author Survey.
As an open-access publication, Spectrum receives no financial support from paid subscriptions and depends on authors' prompt payment of publication fees as soon as their articles are accepted. You will be contacted separately about payment when the proofs are issued; please follow the instructions in that e-mail. Arrangements for payment must be made before your article is published. For a complete list of Publication Fees, including supplemental material costs, please visit our website.
ASM policy requires that data be available to the public upon online posting of the article, so please verify all links to sequence records, if present, and make sure that each number retrieves the full record of the data. If a new accession number is not linked or a link is broken, provide production staff with the correct URL for the record. If the accession numbers for new data are not publicly accessible before the expected online posting of the article, publication of your article may be delayed; please contact the ASM production staff immediately with the expected release date. | 2022-03-25T06:18:23.823Z | 2022-03-24T00:00:00.000 | {
"year": 2022,
"sha1": "c7148a1e8913c2f01a470ad68c63542655d49d75",
"oa_license": "CCBY",
"oa_url": null,
"oa_status": null,
"pdf_src": "PubMedCentral",
"pdf_hash": "ef6af7841731bc2604745e33b56cafadecfbc3e0",
"s2fieldsofstudy": [
"Biology"
],
"extfieldsofstudy": [
"Medicine"
]
} |
89699803 | pes2o/s2orc | v3-fos-license | Riboflavin induced tolerance against the root-knot nematode Melopidogyne graminicola in rice
The role of plant hormones such as Salicylic acid, Jasmonic acid and Ethylene in plant defense has been largely investigated particularly in dicotyledonous plants. Riboflavin has also shown effects on plant physiology but its role in plant defense/tolerance is yet to be elucidated. Rice plantlets were randomly grown in laboratory, sprayed with hormones and riboflavin 24 hours before their infection by the nematodes meloidogyne gramicola juvenile 2. Two types of infection experiments were done. The normal rice nipponbare susceptible to M. gramincola infection was used in the first while in the second series experiments four transgenics/mutants were used. The shoots length, roots length and the roots weight were measured. The numbers of galls per plant were counted under a stereomicroscope. The relative expression of the genes Osein2b, Oswrky 45, OsJAmyb and OsNPR1 was evaluated using the qRT-PCR method. It was noticed that the plantlets treated with BTH significantly developed less galls compared to control (P = 0.000<0.05). This was also observed with ETHEPHON and MeJA spray (P = https://dx.doi.org/10.4314/rj.v2i1.2D 2 Rwanda Journal, Series D, Volume 2, 2018, Life and Natural Sciences: Issue I 0.001 and P = 0.000 <0.005 respectively). The treatment with riboflavin didn’t show a significant effect (P = 0.61>0.05) in comparison with the control. However these plants grew taller enough with a strong roots system except for OsNPR1antisense line which developed more galls than others tested plants. The foliar supply of riboflavin on rice significantly upregulated OsNPR1 in leaves and roots of rice plantlets. OsWRKY45 was also upregulated in both leaves and root samples.
INTRODUCTION
Plant hormones play a vital role in plant survival and are involved in regulating plant growth, development and reproduction (Robert-Seilaniantz et al., 2011, Georgina D. Arthur et al., 2007. The role of plant hormones in plant defense has been investigated mostly in dicots like Arabidopsis (Balzan, S et al., 2014), but still a lot needs to be done in monocots (De Vleesschauwe, D et al., 2014). The dicotyledonous plant model Arabidopsis thaliana was intensely used to elucidate the production and interrelated role of salicylic acid (SA), jasmonic acid (JA) and ethylene (ET) in plant defense following pathogen attack.
It is known that rice is a staple diet in 39 countries and its demand is increasingly high as the world population keeps increasing (FOA, 1984). High yield in rice farming is compromised by many diseases and pests like the root-knot nematodes (RKNs). The problems caused by these nematodes in rice may increase in the near future as people are looking for growing rice in upland where they cultivate the so-called aerobic rice (Mew et al., 2004. It was noticed that M. graminicola is the most damaging nematode in aerobic rice fields. In their report Kamrun et al (2011) stated that there is a strong need of information from fundamental research in order to get more insight on the interaction between these nematodes https://dx.doi.org/10.4314/rj.v2i1. Moreover it is known that riboflavin (or vitamin B6) is produced by both animals and plants and, like most of other vitamins it acts as a coenzyme in the physiology of animals, plants and microorganisms (Dong, H and Beer, S.V., 2000). It has been demonstrated that this vitamin is involved in the process of antioxidation and peroxidation processes known to play a role in oxidative burst leading to hypersensitivity reactions (Jabs et al., 1996). Riboflavin induced the transcription of PR genes in arabidopsis and tobacco which triggered the systemic resistance of this plant against Peronospora parasitica and Pseudomonassyringae pv.tomato. In tobacco and tomato the action of riboflavin protected the plants against Tobacco mosaic virus (TMV) and Alternaria alternata (Dong, H and Beer S.V., 2000).
Furthermore it was proven that the use of protein kinase inhibitor K252a cancelled the effect of riboflavin in tobacco and arabidopsis. This phenomenon was also observed when riboflavin was sprayed on this plant mutated in the NIM1/NPR1 gene. However, the spray of NahG plant with riboflavin solution (10μM) induced the same resistance as in normal plants.
In light of these observations Dong, H and Beer, S.V., (2000) concluded that the foliar spray of riboflavin elicits systemic resistance in arabidopsis thaliana and tobacco and activates novel signaling process in the plant and, went further to say that riboflavin-induced signaling requires functional protein kinase which is regulated by NIM1/NPR1.
In this research, the rice variety nipponbare known to be susceptible to infection by nematodes was used to investigate the protective capacity of exogenous supply of riboflavin against the nematodes M.graminicola. To get further insight on possible connection as well as cross-talking between riboflavin, SA, JA and ET pathways in rice-nematodes interaction, another infection experiments on transgenics/ mutants rice was done. Finally the quantitative (Bailey et al., 2009); (2) Hebiba mutant. This is a Jasmonic acid pathway mutant. Hebiba was found to be defective in the gene encoding allene oxide cyclase (OsAOC) (Riemann et al., 2003); (3) NahG transgenic rice is a transgenic rice that expresses the nahG gene from a bacterium to produce salicylate hydroxylase that degrades SA into cathecol (Yang et al., 2004); (4) OsNPR1 antisense line. This is a transgenic rice whose OsNPR1 functional gene version controls the transcription of plant defense gene and it is responsive to Riboflavin (Dong, H and Beer, S.V 2000); (5) Nihonmasari rice was used as a control for Hebiba rice. (Reversat et al., 1999) was used. PVC (polymer, polyvinylchloride) tubes were filled with SAP-substrates, rice plantlets were allowed to grow on and hoagland solution was added to the seedlings as source of nutrients.
Infection experiments:
The rice seeds were geminated on a Petri dish (θ= 14 cm) on a wetted tissue paper. The seeds were incubated at 30°C for 4 days in darkness, then moved to light for 2-3 days. PVC-tubes were filled with SAP-substrates. The rice seedlings were transplanted onto SAP at the 7 th days from germination set up. The rice seedlings were sprayed with hormones at their 12 th days. These rice plantlets were infected with nematodes 48 hours after hormones sprays. Plantlets were kept in controlled rooms for 15 days after their infection. Data were then collected in this way: each plantlet was pulled out of the tube; roots were thoroughly washed with tap water to remove all the SAP. The shoots length as well as roots length were measured. Also the number of galls formed on roots was counted after staining with fuchsin as follows: the roots of each plant were tightly wrapped in a piece of well labeled mira cloth and submerged in boiling fuchsin acid staining dye (75 mg acid fuchsin, 50 ml lactate, 50 ml glycerol and 50 ml distilled water) for 2 to 3 minutes. The roots were washed with tap water, unpacked from the mira cloth, placed in 16 well-plates (5ml of distaining solution, 1:1 glycerol and distilled water with some drops of lactic acid in each well) and distained for at least two days on a shaker.
Relative expression of Osein2b, Oswrky 45, OsJAmyb and OsNPR1 genes in rice following hormones and riboflavin foliar spray
For this experiment nipponbare variety was used. 12 days-old plantlets were sprayed with riboflavin solution until run off (more or less twelve sprayer shots). Both leave and root tissues were collected 24 hours after hormone spray. From a pool of twelve treated plants, two biological replicates were formed (6 plants each), and two samples were taken (roots labeled R, and leaves labeled L). The leaves were cut above the base, roots removed from the stem and immediately deep-frozen and stored in liquid nitrogen. The samples could later on be stored at-80°C. Samples for the control plants (non-sprayed) were also taken collected.
Choice of both reference and target genes, Primer design
All primers were designed with the Primer3 program and are shown in table 1. Optimal product size was determined to 50-100bp, optimal primer size to 18-25bp, GC% to 40-60% and Tm (melting temperature) between 58-62°C. The amplicon was preferably as close as possible to the polyA tail to improve reverse transcription efficiency. Tm, selfcomplementarity and primer-dimerization were checked with Oligo Calc (Kibbe, W.A. 2007). Self dimerization and cross dimerization of each primer pair were checked again with Finnezymes Multiple primer analyzer (Finnezymes, 2010
Total RNA extraction
The working place was treated with RNase ERASE (MP Biochemicals, Brussels, Belgium) to make the space RNase-free and only RNase-free materials were used. Plant tissues were ground vigorously in a mortar and pestle. The powdered tissues were collected in 1.5ml eppendorf tubes properly labeled (filled at half), 500 l TRI reagent (Sigma-Aldrich) were added and the tubes were vortexed, kept in a pot of ice and sonicated 3 times for 10 sec, then incubated for 30 min at room temperature (RT). 100μl chloroform was added on the tubes
Quantitative real-time RT-PCR and post-run analysis
For each cDNA-sample, a mastermix (MM) with primers was prepared. The components of the MM were originating from the qPCR core kit "SYBR green I -NO ROX" (Eurogentec, 2015). 5μl of each cDNA sample was combined with 15μl of MM in a 72-Well Gene-Disc TM using the CAS-1200 TM Automated PCR Set up robot (Corbett Life Science, 2009). All PCR samples were prepared in triplicates. A 15μl no-cDNA-MM was included as negative control.
The qRT-PCR reactions were performed in the Rotor-Gene 3000 machine (Corbett Life Science) and results were generated by the Rotor-Gene 6 software. PCRs program was as follow: 5' at 95°C and 40 cycles of 45 sec at 95°C, 45sec at 60°C and 30 sec at 72°C.
Effect of hormones and riboflavin on rice infection by M.graminicola
In this research, it was noticed that there is a significant different (P = 0. Considering other agronomic parameters of tested rice, it was evident that riboflavin treated plants grew taller than other treatments and controls as exemplified by the shoots length comparison (Fig 2). It was clear that there is a significant difference in terms of shots length (P = 0.008<0.05) between the plant treated with riboflavin and the control. Moreover the rice plants treated with riboflavin developed stronger root systems (Fig. 3) where the RB-treated plants were 27% well rooted (in length) than the control. Other researchers demonstrated that riboflavin is involved in the process of antioxidation and peroxidation processes known to play a role in oxidative burst leading to hypersensive reactions. Dong, H and Beer, S.V (2000) found that Riboflavin induces the transcription of PR genes in arabidopsis and tobacco which trigger systemic resistance against Peronospora parasitica and Pseudomonass syringae pv.tomato in arabidopsis. In tobacco and tomato, riboflavin was proven to confer protection against Tobacco mosaic virus (TMV) and Alternaria alternata, respectively. However though not statistically significant it can be noticed that hebiba and OsNPR1 have developed more galls than their respective control with 12.85% and 12.26 % respectively (Fig.4).
Effect of riboflavin on pathways of targeted plant hormones
The quantification of RNAs from both leaves and roots of rice plants was done first in this series of experiments. The results (table 2) showed that the amount of RNAs (in μg/ml) obtained from leave samples was largely high than the amount of RNAs from roots where for instance COL1/COR1 was more than 3 folds, RBL1/RBR1was 2.4 folds. Reid et al., (2006) conducted a study on optimized grapevine RNAs isolation procedure and statistical determination of reference genes for RT-PCR during berry development. They extracted total RNA from grape pericarp, seeds, leaves, roots and flowers. They found that the yields were quite diverse depending on the source of the tissue. Leaves yielded the highest amount of total RNA (400-600 μg/gfw), followed by flowers, roots and pre-veraison seeds (150-300 μg/gfw), preveraison pericarp (40-120 μg/gfw), post-veraison pericarp (15-30 μg/gfw) and the lowest being from post-veraison seeds (3-10 μg/gfw). The differences observed were likely related to the developmental and metabolic properties of each distinct tissue.
In this work more RNA quantities was obtained from the rice system compared to the quantities obtained from grape tissues (Reid, 2006). This suggests that the differences observed were not only related to developmental and metabolic properties of each distinct tissue but also the species of plants.
In order to check the quality of the synthesized cDNAs, two references genes were selected. These were Eukaryotic Initiation Factor 5C (EIF 5C) and Expressed protein (EXP).
As it is shown in the figure 5, the negative control was negative, and all the house-keeping genes have been amplified with their respective fragment sizes. EIF5C was retained to be used as reference gene in qRT-PCR. As it can be seen on both gel picture (a) and (b) of the figure 5, EIF5C bands were reproduced identically in both tissues, leaves (a) and roots (b).
Regarding the relative gene expression following the foliar spray of riboflavin solution, the results showed that OsWRKY45 was upregulated in both leaf and root samples (Fig 6). The OsNPR1 was significantly upregulated in both leaves and roots.
However, Osein2b and Jamyb responded negatively to exogenous supply of riboflavin with Jamyb being highly downregulated. indicates that the effect is significant compared with the control.
Jamyb was significantly downregulated in leaves and slightly in roots while Osein2b was slightly downregulated in both leaves and roots. As expected OsNPR1 was significantly upregulated in both tissues following riboflavin treatment. The fact that Oswrky45 was upregulated by riboflavin spray confirms the synergistic action between riboflavin and SA.
This was observed on microbial pathogen on rice and it was proven on rice-nematodes interaction in this work.
The role of riboflavin as an elicitor of systemic resistance and a plant defense activator in rice as an important monocot plant was previously demonstrated by Paressa and Monica Hӧfte (2006) though they did it in rice-fungi system. They conducted study on the mechanism of riboflavin-induced-resistance and defenses response in rice against Rhizoctonia sheath diseases which are among the most important fungal diseases of rice, causing more than 50% yield losses in the world every year. These researchers found that riboflavin-IR can be linked to the induction of defense pathways leading to the formation of structural barriers such as lignin in rice plans. Their findings proved that using riboflavin as a plant defense activator can be a simple and environmentally safe strategy to control Rhizoctonia sheath diseases of rice.
Conclusion
Contrary to the JA/ET and SA effect on rice-M. graminicola interaction where the hormones protected rice ( as materialized by the reduced number of nematodes entering the roots), the treatment of rice by riboflavin didn't reduce the number of galls formed on rice seedlings.
However, it was observed that the galls formed following riboflavin treatment were reduced in size compared to other treatments. The riboflavin-treated plants grew healthier; they were more exuberant than those sprayed with hormones despite their infection with M.
graminicola. These strength and good health of the riboflavin-treated plants were proven by their long tillers and strong roots syetem. Knowing the involvement of riboflavin in oxidative burst, our results suggest that this riboflavin makes rice plants tolerant to M. graminicola infection by activating the reactive oxygen species arsenal which sequestrated the nematodes and prevented them to further growth.
Recommendation
More research works are needed to elucidate the interaction between riboflavin and other plant hormones like cytokinins and auxins as these hormones are involved in nematodes establishment in rice roots. | 2019-04-02T13:13:15.524Z | 2018-02-20T00:00:00.000 | {
"year": 2018,
"sha1": "0d274840762b537817d6d3423fc3835971f2b098",
"oa_license": null,
"oa_url": "https://www.ajol.info/index.php/rj/article/download/167110/156548",
"oa_status": "GOLD",
"pdf_src": "Anansi",
"pdf_hash": "e7fd5305043f993b7dde8cabf0c3326e12251d81",
"s2fieldsofstudy": [
"Agricultural And Food Sciences"
],
"extfieldsofstudy": [
"Biology"
]
} |
260764722 | pes2o/s2orc | v3-fos-license | Effect of ultrasonic treatment on the quality of Mianning ham
This paper investigates the optimal process for ultrasonic desalination of Mianning ham. The study analyzed various factors such as ultrasonic treatment time, temperature, and power to determine their impact on the rate of desalination of hams. A single factor test was conducted to study the rate of desalination. Further, A Box-Behnken experimental design was used to evaluate the effect of Mianning ham desalination. The design examined the impacts of ultrasound on the physicochemical properties, texture, and sensory of the ham. Response surface processing group underwent oral processing to determine the optimal ultrasonic treatment conditions with the highest acceptance level. The results show that the best conditions were: ultrasonic time 84.56 min, ultrasonic temperature 40.35°C, and ultrasonic power 150.85 W. The average desalination rate of the ham under the optimal conditions was 25.93% ± 0.69%, and the hardness was 4.48 N ± 0.62 N. Overall, this process significantly improved the desalination rate, texture, and sensory quality of Mianning ham, providing solid theoretical support for desalination processing at the back end of ham.
Introduction
Dry-cured ham is a meat product made by curing, washing, drying, and fermenting the hind legs of pork (1). One of the top ten renowned hams in China is Mianning ham, produced in Mianning County, Liangshan Prefecture, Sichuan Province, China. It is unique because it is made of hind legs from high-quality Liangshan Wujin pigs and does not contain nitrite during the fermentation process. Mianning ham is known for its tender texture, thin skin, bright red color, and unique cured flavor, which makes it easy to preserve (2).
Excessive dietary intake of salt is a global health issue, and according to the World Health Organization (WHO), adults' daily salt intake should be less than 5 g per day. However, most people consume between 5 and 12 g of salt each day. If salt consumption can be limited to the recommended levels, the WHO approximates that globally, 2.5 million deaths could be avoided yearly (3). Unfortunately, a lot of salt must be used in ham processing to boost protein properties, enhance texture and flavor, and prevent ham spoilage by microorganisms (4,5). Lowering the salt content in cured ham enhances protein hydrolysis and modifies ham texture, hue, and flavor (6).
He et al. 10.3389/fnut.2023.1199279 Ultrasonic waves can effectively reduce the curing time and accelerate salt diffusion in meat product processing through cavitation. Because of its effectiveness, ultrasonic technology applications have been extended to numerous fields, such as brining, marinating, cooking, and other food processing techniques (7). Research studies have demonstrated that ultrasonic treatment can influence the physicochemical properties of the product, including but not limited to, hardness, water retention, and flavor (8)(9)(10). However, the effects of ultrasonic processing parameters, such as time, temperature, power, and other factors, differ depending on the product itself (11). While the ultrasonic technology has been predominantly studied in the ham processing stage, its utilization in the post-ripening phase of ham production remains unexplored (12).
Oral processing, the process of food being masticated, moistened, and enveloped into boluses by saliva and then ingested, plays a crucial role in the formation of food taste (13). Oral food processing involves food dynamics, oral physiology, and sensory psychology and has emerged as a burgeoning subfield of research in food science in recent years (14). Response surface methodology (RSM) is frequently used in food processing to ascertain the most optimal processing conditions (15). This research aims to investigate the physical and chemical properties and sensory attributes of hams as performance indicators, optimize the process parameters of ultrasonic treatment time, temperature, and power using RSM, and subsequently analyze the sensory qualities of the response group following oral processing to select the most optimal process. The results will offer theoretical support for the development of new Mianning hams.
Sample
Mianning ham samples were obtained from selected representative factories in Mianning County. The ham was sliced into dimensions of 5 cm × 5 cm × 2 cm, and each sample was supplemented with 1% ultrapure water before vacuum packaging (16). Following vacuum packaging, the ham samples were subjected to ultrasonic treatment using an ultrasonic machine. The treated ham can be used immediately, or refrigerated at 4 • C after surface wiping. For subsequent oral processing experiments, the ultrasonically treated ham was boiled in water for 10 min and then cut into 5 ± 1 g cubes (17).
Experimental design 2.2.1. Single-factor test
The primary influential factors of ultrasonic treatment on ham quality are the ultrasonic time, ultrasonic temperature, and ultrasonic power. An analysis was conducted to scrutinize the effects of these three factors (ultrasonic time 40, 60, 80, 100, 120 min, ultrasonic temperature 20, 30, 40, 50, 60 • C, and ultrasonic power 100, 125, 150, 175, 200 W) on the desalination rate of Mianning ham using the desalination rate of ham as the index (11). Based on the findings from the single-factor test, response surface methodology was utilized to optimize the impact of three factors: ultrasonic time, temperature, and power in Table 1, on the physicochemical and sensory attributes of Mianning ham to enhance its quality. The experimental design employed a three-factor, three-level response surface optimization trial using DesignExpert 8.0.6 software (18). The secondary response surface analysis included linear term effects, interaction term effects, and quadratic term effects, as illustrated in Equation (1).
Y represents the dependent variable, while b 0 denotes the model constant or intercept. b 1 -b 3 represent the linear term coefficients, while b 4 -b 6 depict the interaction term coefficients. Similarly, b 7 -b 9 represent the quadratic term coefficients. A represents the ultrasonic treatment time, whereas B denotes the ultrasonic treatment temperature, and C represents the ultrasonic treatment power. Lastly, this study aims to optimize the ultrasonic treatment conditions through regression analysis and 3D response surface plots (19).
Physical and chemical testing 2.3.1. Color
The study employed a portable CR400 colorimeter with a light source of D65, an observation angle of 10 degrees, mirror component exclusion mode, and an 8 mm aperture, to determine the brightness L * , redness a * , and yellowness value b * of ham samples, measured from three distinct locations on the sample surface, with each group of samples repeated thrice (20).
pH
The pH value of the ham samples was determined by directly inserting a PH-3C-01 pH meter into the ham samples three times for each group of samples.
Moisture activity
Extracting 3 g of diced sample and spreading it evenly onto the dish designed for measuring water activity, the moisture activity value of the sample was measured using the HD-3A model moisture activity(aw) tester and recorded (21).
Cook loss
The initial weight of the ham was duly accounted for and subsequently subjected to a duration of 10 min in boiling water.
Post-cooking, the sample was dried of excess moisture and its weight was re-measured (22).
The calculation of cooking loss rate was then determined through the following formula: percentage of cooking loss = (initial weight−final weight) / initial weight.
Salt content
Begin by weighing precisely 5 g of the sample in a crucible designated for charring. Proceed to establish the ham ash volume at 100 milliliters. Next, transfer 50 milliliters of the test solution to a 250 milliliters conical flask, adding 50 milliliters of water and 1 milliliter of potassium chromate solution (5%). Finally, titrate the mixture with a solution of silver nitrate (0.1%) (23). It is through application of formula (2) that one may accurately calculate the salt content.
X is the salt content of the food (%) and V is the volume consumed by the silver nitrate titration.
Texture profile analysis (TPA)
To evaluate the physical properties of the samples, a TAXT Plus mass spectrometer was utilized. The samples were cut into 1 × 1 × 1 cm squares, and the probe was compressed twice, reducing the sample's original height by 50%, at a speed of 120 mm/min. Subsequently, the hardness (N), adhesion (N * mm), cohesion, elasticity (mm), adhesive viscosity, and chewiness (N) were calculated based on the average of three measurements for each sample (24).
Oral processing
For sensory evaluation, a balanced gender distribution of evaluators consisting of five males and five females, all of whom possess a professional background in food, were formed into a balanced team. It is essential that evaluators oral cavities be free of any abnormalities and they are in good health. They must abstain from smoking, consuming alcohol, or engaging in any other behavior that may affect sensory testing for at least 2 h prior to participating in evaluation. The evaluators within the same group should participate in experiments such as temporal dominance of sensations (TDS), oral treatment analysis, and bolus collection (25).
Bolus collection
The duration of each evaluator's chewing and subsequent swallowing was recorded as the 100% chewing time point, with the aim of investigating the variations in the physical and chemical properties, as well as the sensory characteristics of ham during oral processing. Boluses from 20, 40, 60, 80, and 100% chewing time points were collected for subsequent experiments. The boluses were either evaluated immediately after collection or preserved by wiping off any surface moisture and refrigerating at 4 • C. Masticatory parameters, such as the duration of chewing and the number of chews, were captured via camera during the oral processing stage, and consequently analyzed (26).
Water and saliva content of the bolus
The swallowing points of the bolus were selected at intervals of 20, 40, 60, 80, and 100%. Next, 5 g of the bolus sample is to be weighed and placed in the moisture tester to determine the bolus' moisture content, which is expressed as a percentage (%).
Saliva content can be calculated through the following formula, as indicated in reference (27): Saliva content = moisture content of the bolus -moisture content of the sample.
Dominance of sensations (TDS)
To that end, it is observed that TDS, a temporal sensory analysis of food products, generates a series of perceptual attributes classified as "dominant" at specific points or times in the dynamic evaluation process. This analysis determines the strength of the dominant rate of sensory attributes over time. Prior to conducting the TDS evaluation, evaluators undergo training in the appropriate sensory aspects of ham, including but not limited to, its hardness, saltiness, juiciness, gumminess, sourness, and tenderness (28). Assess the prevailing sensory characteristics perceived by the evaluators upon commencing mastication of the samples. They have ability to discern identical or contrasting sensory attributes concurrently and desist from sensing any such attributes momentarily prior to swallowing (29). Each assessor supplies three samples, each evaluation session lasting 3 min, and necessitates oral rinsing with pure water prior to every assessment.
Data analysis
The physicochemical, qualitative, and sensory data were subjected to Analysis of Variance (ANOVA) utilizing the SPSS software. Furthermore, T (Tukey's) test was employed, and the outcomes were deemed highly significant for p < 0.01, significant for 0.05 > p > 0.01, and insignificant for p > 0.05. Each metric was replicated thrice, and the results were plotted using Origin software. Additionally, Design Expert V8.0.6 was utilized for response surface analysis.
Single-factor test results
The study presents the impact of ultrasonic treatment's time, temperature, and power on the desalination rate of Mianning ham, as depicted in Table 2. The desalination rate initially increased and then decreased with an increase in ultrasonic time, ultimately stabilizing at the maximum value after 80 min. Time groups 1, 2, and 3 exhibited significant differences from time groups 4 and 5 (p < 0.05), whereas time groups 4 and 5 did not demonstrate any noticeable difference (p > 0.05). In conclusion, the singlefactor test tentatively selected the ultrasonic time of 80 min. These findings align with Zhang's study (30). With the rise in ultrasonic temperature, the desalination of Mianning ham exhibited an initial increase followed by a decrease, culminating at its zenith at 40 • C. The variations between Temp.1, 2, 3, 4, and 5 were statistically significant (p < 0.05). The one-way ultrasonic temperature test was provisionally elected at 40 • C. Upon increasing the ultrasonic Values are expressed as mean ± standard deviation; each 5 rows are grouped together, different rows of lowercase letters in the same group indicate significant differences (p < 0.05).
power, the desalination of Mianning ham displayed a gradual rise, eventually stabilizing at its optimal capacity of 150 W. Notably, there was a significant difference (p < 0.05) between power groups 1, 2, and 3 in comparison to power groups 4 and 5. However, there was no notable discrepancy (p > 0.05) between power groups 4 and 5. It is worth mentioning that the single-factor tests, for the time being, had selected the ultrasonic power of 150 W.
Response surface optimization
Response surface methodology (RSM), is a statistical technique commonly employed in food research to optimize both single and multi-factor response models. The results of the singlefactor experiments were carefully analyzed, and the Box-Behnken experimental design was used to conduct the study. The ultrasonic process was optimized to achieve the desired outcome, and the changes in the quality of Mianning ham after ultrasonic treatment were meticulously measured. The experimental protocol and data for the 17 experimental combinations of the Box-Behnken experimental design can be found in Table 3.
Water activity, pH
As depicted in Table 3, the water activity (a w ) following ultrasonic treatment was markedly lower in comparison to the control group. The aw of the ham exhibits a declining trend with an escalation in ultrasonic time, temperature, and power. The ham's muscle fiber structure is damaged by prolonged ultrasonic, prompting the salt-soluble proteins to precipitate onto the ham's surface, leading to a reduction in its water activity (31).
In the ultrasonic treatment and control groups, there was no substantial alteration in pH (p > 0.05) following ultrasound treatment.
Color
Color represents a significant physicochemical parameter in ham that carries a considerable impact on the economic efficiency of this product. Mianning ham L * , a * and b * exhibit marked differences after ultrasonic treatment when compared to the control group (p < 0.05). The response group reveals a decrease in L * with increasing ultrasonic power. The L-value of the treatment with 175 W power is lower than that in the treatment group with 125 and 150 W. As ultrasonic power increases, a * decreases and b * increases, which can be attributed to the amplification of the cavitation effect that accelerates the oxidation of myoglobin in hams by free radicals generated by water molecules, ultimately leading to a change in color (32). The L * , a * , and b * of hams decrease with an increase in ultrasound time. Prolonged ultrasound destroys the muscle structure of the ham, thus causing myoglobin to undergo destruction, giving the ham a brownish appearance. An increase in ultrasound temperature reduces the ham L * but increases a * and b * . High temperature can lead to the inactivation of some critical enzymes, which in turn reduces the enzymatic reaction and results in a change of color (33).
Desalination and cook loss
The salt content in ham is a crucial determinant influencing consumer preferences. Table 3 displays the desalination rate, which exhibits an initial increase followed by a decrease with increasing ultrasonic treatment time. Notably, the desalination rate of the 80 min treatment group was significantly higher (p < 0.05) than that of the 60 min and 100 min treatment groups. Ultrasound expedites the migration of salt from the interior of the ham to its surface. Prolonged ultrasonic treatment, however, results in an elevated concentration of salt on the surface, which in turn impedes salt precipitation. According to reference (34) desalination initially increases and then decreases. Table 3 demonstrates that the increase in ultrasonic temperature leads to the initial increase and subsequent decrease of ham desalination. This occurs because the appropriate temperature accelerates the enzymatic reaction responsible for salt migration from the interior to the surface of the ham. Elevated temperatures, conversely, deactivate particular enzymes and reduce the rate of salt ion migration. As the ultrasonic power increases, the rate of salt ion migration also increases (30).
The steaming loss rate of ham is shown in Table 3, the steaming loss of response group 13, 14, 15, 16 and 17 was significantly higher (p < 0.05) than the other treatment groups, Because ultrasonic treatment leads to a significant reduction in the salt ion content of the ham, which affects the solubility properties of the proteins in the ham and increases cooking losses (35).
Texture profile analysis
The Table 4 presents the textural profile analysis of the ham. As the ultrasonic temperature, time, and power increased, the ham hardness decreased significantly (p < 0.05). The hardness of
Frontiers in Nutrition
ham showed a negative correlation with ultrasonic time. However, prolonged ultrasonic treatment reduced the protease activity and hindered proteolysis, leading to an increase in the hardness of ham. A rise in ultrasound temperature did not entirely trigger the enzymatic activity in the ham at lower temperatures, ultimately leading to an increase in ham hardness. At higher temperatures, the loss of water in the muscle protein of ham resulted in less hardness (36). The increase in ultrasonic power and the amplified cavitation effect of ham caused a loss of internal moisture, leading to a reduction in ham hardness. The elasticity, cohesiveness, and chewiness of the ham were significantly decreased after ultrasonic treatment. The enzymes diffuse more evenly and quickly with the assistance of ultrasound, resulting in enhanced collagen and elastin hydrolysis in the ham muscle fibers. Simultaneously, the gluing effect of ham formed molecular bonds between the precipitated material and muscle, which contributed to an increase in hardness. The decrease in salt concentration limits the solubility of muscle proteins and affects binding capacity and stability (37).
Response surface optimization and modeling
Response surface modeling was executed to investigate the ultrasonic treatment of Mianning ham, and the coefficient of determination (R 2 ) along with the lack of fit were selected to examine the adequacy of the response model. The purpose of the lack of fit test is to determine whether the chosen model is in accordance with the observed data. The model is deemed appropriate if the p-value of the lack of fit is more than 0.05. The coefficient of determination (R 2 ), representing the proportion of variation due to the response surface model, should be more than 95% for a well-fitted model in experimental settings (38). Upon scrutinizing the results, it was observed that the R 2 values of the response variables, namely desalination rate and hardness, were greater than 95%, and no significant lack of fit was detected. Consequently, the model devised for the desalination rate and hardness of the ham is deemed reasonable. Table 5 displays the analysis of variance (ANOVA) for the desalination rate and hardness of ham, with A, B, and C denoting the ultrasonic treatment time, temperature, and power, respectively. The Pareto chart (Figure 1) depicts the significance of the effect of each ultrasound treatment factor, with the length of the bars representing their proportion, and the vertical coordinates ordered from top to bottom by degree of influence. The Pareto chart analysis is instrumental in determining the magnitude of the response variable's impact (39).
As depicted in Table 5, the linear impacts of the autonomous variables A, B, and C, along with the interaction effects of AB and BC, as well as the quadratic impacts of AA, BB, and CC, had a considerable (p < 0.05) influence on the desalination rate of Mianning ham. Similarly, the linear effects of the independent factors A, B and C, the interaction effects of AB and BC, and the secondary effects of AA, BB and CC significantly (p < 0.05) impacted the hardness of Mianning ham. The regression equation for predicting the value of each response variable on changing the response surface variable is as follows.
After generating the model polynomial equations for the dependent and independent variables of interest, the 2 response surface sets are optimally combined. Upon inspection of the Pareto chart ( Figure 1A) and the significance analysis in Table 5, it was discovered that ultrasonic time has the most significant impact on the desalination rate, followed by ultrasonic power and finally ultrasonic temperature. Figure 1B and Table 5 reveal the significance analysis of hardness, with ultrasound temperature, ultrasound time, and ultrasound power being the influencing factors in descending order. The R2 values for desalination rate and hardness were 99.84 and 99.73%, respectively. F-values were 479.62 and 282.06. The P-values were all less than 0.05 and the P-values for the lack of fit were greater than 0.05. This indicates that the response surface model is capable of better responding to the predicted values of the regression equation.
A three-dimensional response surface plot of the effect of each factor on the desalination rate and hardness of the ham after ultrasonic treatment was obtained using software analysis (Figure 2).
The graphical representation of the desalination rate surface plot (Figures 2A-C) is displayed. Figure 2A illustrates that as time and temperature increased, while maintaining a constant power of 150 W, the desalination rate also increased, reaching a peak value of 26.15%. Any further increase in time and temperature resulted in a decline in the desalination rate. Furthermore, Table 3 provides evidence that power has a significant impact on the desalination rate of hams. As displayed in Figure 2B, at a fixed temperature of 40 • C, the increase in time and power resulted in a decrease in the desalination rate after it reached a maximum of 26.15%. Figure 2C demonstrates that the desalination rate decreases after reaching a maximum of 26.15% when temperature and power are increased at a fixed time of 80 min. The results of the aforementioned experiments demonstrate that time has a crucial linear impact within the studied range of 60-100 min, rather than a quadratic effect. Additionally, the maximum power for the desalination rate at a time of up to 84.56 min was 150.85 W (Figure 2A), and the maximum temperature for the desalination rate at time up to 84.56 min was 40.35 • C ( Figure 2B).
As shown in Figure 2E, increasing time and power at a fixed temperature of 40 • C resulted in a constant decrease in hardness with a maximum value of 8.87 N. Table 4 shows that temperature had a significant effect on the hardness of the ham. Figure 2D shows that at a fixed power of 150 W, the increase in time and temperature causes the hardness to decrease and then increase to a maximum value of 8.87 N. Figure 2F shows that at a fixed time of 80 min, the increase in temperature causes a constant decrease in hardness with a maximum value of 8.87 N. The increase in power leads to a decrease and then an increase in hardness, with a maximum value of 8.87 N. Combined with the analysis of Figures 2D-F and Table 4, the change in temperature in the study range of 30-50 • C has an important linear, rather than quadratic, effect of time. When the temperature was 35.37 • C, the maximum response of power was 130.08 W (Figure 2D), the maximum response of time was 60.67 min (Figure 2F), and the maximum value of hardness was 8.87 N.
In summary, the optimal desalination rate process conditions after response surface optimization were 84.56 min of ultrasonic time, 40.35 • C of ultrasonic temperature, and 150.85 W of ultrasonic frequency, at which the ham desalination rate was 26.15%. The optimal hardness process conditions after response surface optimization were 60.67 min of ultrasonic time, 35.37 • C of ultrasonic temperature, and 130.08 W of ultrasonic power, at which time the ham hardness was 8.87 N. In order to select the process conditions with high consumer acceptance, the two corresponding groups mentioned above were subjected to subsequent oral sensory experiments. 3.5. Oral processing 3.5.1. Oral processing parameters The group subjected to oral processing experiments was the ultrasound experimental group, which underwent response surface optimization. The experimental group US1 was treated with an ultrasonic time of 84.56 min, ultrasonic temperature of 40.35 • C, and ultrasonic frequency of 150.85 W. On the other hand, the experimental group US2 was subjected to an ultrasonic time of 60.67 min, ultrasonic temperature of 35.37 • C, and ultrasonic power of 130.08 W. Both US1 and US2 were cooked in boiling water for 10 min, cut into cubes weighing approximately 5 ± 1 g, and subsequently processed orally.
Mastication is the regular movement of the jaw that grinds food into a bolus that can be reached for swallowing, a process known as oral processing of food. Table 6 describes the masticatory parameters as well as the salivary content during oral processing. Table 6 shows, the greater difference in swallowing time and number of chews between US1 and US2 treatment groups may be related to the salt content of the samples. As shown in Table 3, the desalination rate of US1 was significantly higher (p < 0.05) than that of US2. US1 chewed more and chewed longer than US2 and chewed more frequently. This is consistent with Omkar's study (40). The mastication rates of US1 and US2 were at 1.12 chews/s and 1.13 chews/s, indicating that ultrasound treatment did not result in a change in mastication frequency (41). Prolonged chewing leads to an increased sense of satiety, which helps consumers control food intake for weight loss (42). During the chewing process, the water content of all bolus increases. The initial moisture content was 47.86 ± 1.01% for US1 and 45.37 ± 1.15% for US2. Even though the initial moisture content of US1 and US2 were different, the difference in moisture content at the 100% chewing time point was not significant. suggesting that saliva compensates to some extent for the difference in initial moisture content during ham chewing, which is consistent with Rizo's study (43). Saliva content increases with the chewing process. The salivary content of US1 and US2 was similar at 20 and 40% of chewing time points, but US2 had higher salivary content than US1 at 60, 80, and 100% of chewing time points. It is possible that there is little difference in the perception of sensory attributes such as US1 and US2 salinity in the pre-chewing period, resulting in comparable salivary intake in the pre-chewing period. To compensate for the water content of 100% chewing time, so the saliva content of US2 is higher than that of US1 in the late chewing period (40).
Dominance of sensations (TDS)
Temporal dominance of sensations is a sensory description method that requires the evaluator to be able to continuously indicate the dominant sensation. The evaluator's "dominant sense" was defined as the sense of time to get attention. The TDS is able to collect a range of sensory properties felt at different points in time throughout the chewing process. The sensory characteristics of ham are mainly hardness, saltiness, juiciness, gumminess, sourness and tenderness (44). Figure 3 depicts the differences in the perception of Mianning ham from first chewing to swallowing US1 and US2 with different ultrasound treatments, as the different ultrasound treatments resulted in differences in ham quality. For US1 (Figure 3A), the sensory dominance within 10 s of chewing was related to hardness; the sensory dominance within 10-25 s of chewing time was juiciness; the sensory dominance within 25-35 s of chewing was softness; and the sensory dominance attribute for the last 5 s of chewing time was gumminess. The salty taste in US1 perception kept decreasing after 5 s of chewing, but the salty taste perception increased slightly again at 25-35 s of chewing time, and the sour taste also peaked at about 35 s of chewing time perception. This may occur because the chewing time is about to reach the swallowing point and the flavor is more easily perceived in the ham (45).
For US2 (Figure 3B), the overall sensory perception was similar to that of US1. However, the dominant rate of hardness is longer than US1 time as shown in Figure 3B. Because the hardness of the US1 ultrasound-treated group was lower than that of US2. US2 salty and sour flavors were perceived higher than US1. US2 sourness dominated the senses for 25-30 s. The overall perceived intensity of saltiness was higher than that of US1 because the desalination rate of US2 was lower than that of US1, which had higher degree of protein hydrolysis, resulting in a saltier and more acidic ham sample. The final swallowing phase softness and gumminess US1 and US2 showed similar perceptions. Because the water content of the food mass was similar at the time of reaching the end point of swallowing, resulting in similar softness and adhesive properties in the TDS images (46).
The response surface optimized US1 and US2 groups were analyzed for oral processing, and the chewing time and chewing frequency were higher in the US1 group than in the US2 group; the US1 group was more likely to produce satiety; and the perception of salty and sour tastes was lower in the US1 group than in the US2 group. In summary, the US1 group was more acceptable than the US2 group, so the US1 group was selected. The ultrasonic treatment conditions were ultrasonic time of 84.56 min; ultrasonic temperature of 40.35 • C; and ultrasonic power of 150.85 W.
Validation experiments
On this basis, verification experiments were conducted on the desalination rate and hardness of ham. Using the optimized Table 7, and the predicted values are similar to the validated results. It is shown that the response surface methodology is effective for modeling and optimizing the production operation of ultrasonic treatment of Mianning ham.
Conclusion
The study found that ultrasound treatment did not affect the pH of the Mianning ham, but it decrease the water activity (a w ) of the ham, particularly with higher ultrasonic time, temperature, and power. Additionally, an increase in ultrasonic power resulted in a decrease in color, as evidenced by a reduction in L * and a * , and an increase in b * . Furthermore, the L * , a * , and b * values of the ham decreased with increasing ultrasonic time. An increase in ultrasound temperature caused a decrease in L * and an increase in both a * and b * . The desalination of the ham initially increased and then decreased with increasing ultrasonic treatment time, temperature, and power. Similarly, the steaming loss of the ham first increased and then decreased with an increase in ultrasonic time, temperature, and power. Finally, the hardness, elasticity, cohesiveness, chewiness and overall texture of the ham gradually decreased as the ultrasonic treatment time, temperature and power increased.
The chewing time and chewing frequency of US1 group were higher than US2, and prolonged chewing was more likely to produce satiety. The TDS curve for US1 was lower than US2 for ham salty and sour perceptions, and US1 was a more suitable choice for consumers. The ultrasonic conditions for optimal acceptance were: ultrasonic time 84.56 min, ultrasonic temperature 40.35 • C and ultrasonic power 150.85 W. Verification experiments by this condition yielded a desalination rate of 25.93% ± 0.69% and a hardness of 4.48 N ± 0.62 N. In summary, the model obtained by optimization of ultrasonic desalination technology is suitable for the desalination process of Mianning ham. Thus, providing a theoretical basis for future desalination techniques for dry cured hams.
Data availability statement
The original contributions presented in this study are included in the article/supplementary material, further inquiries can be directed to the corresponding author. | 2023-08-10T15:16:40.096Z | 2023-08-08T00:00:00.000 | {
"year": 2023,
"sha1": "038b04ff8d11505f00454737913cc5de574e4a99",
"oa_license": "CCBY",
"oa_url": "https://www.frontiersin.org/articles/10.3389/fnut.2023.1199279/pdf",
"oa_status": "GOLD",
"pdf_src": "PubMedCentral",
"pdf_hash": "5f248a611ec8394fa06a2fc7f004d986fdf38c01",
"s2fieldsofstudy": [
"Agricultural and Food Sciences"
],
"extfieldsofstudy": [
"Medicine"
]
} |
268446889 | pes2o/s2orc | v3-fos-license | State-to-State Quantum Dynamics Study of Intramolecular Isotope Effects on Be(1S) + HD (v0 = 2, j0 = 0) → BeH/BeD + H/D Reaction
The dynamic mechanisms and intramolecular isotope effects of the Be(1S) + HD (v0 = 2, j0 = 0) → BeH/BeD + H/D reaction are studied at the state-to-state level using the time-dependent wave packet method on a high-quality potential energy surface. This reaction can proceed along the indirect pathway that features a barrier and a deep well or the smooth direct pathway. The reaction probabilities, total and state-resolved integral cross sections, and differential cross sections are analyzed in detail. The calculated dynamics results show that both of the products are mainly formed by the dissociation of a collinear HBeD intermediate when the collision energy is slightly larger than the threshold. As the collision energy increases, the BeH + D channel is dominated by the direct abstraction process, whereas the BeD + H channel mainly follows the complex-forming mechanism.
Introduction
Isotope substitution provides important insights into the dynamic mechanisms of chemical reactions.In particular, the intramolecular isotope effects that are generally represented by the resulting product branching ratio of an elementary reaction can provide more significant details about reaction dynamics and are often used to investigate bondselective processes and identify reaction pathways [1][2][3][4][5][6].Triatomic A + HD reactive systems, as the simplest example, have been extensively investigated in both experiments and in theory [7][8][9][10][11][12][13][14][15][16].It was argued on the basis of these studies that the AH/AD ratio for a complex-forming reaction that has a well on the reaction pathway is less than one, whereas the direct abstraction reactions dominated by an activation barrier usually feature an AH/AD ratio larger than one.This bias can be attributed to the mass asymmetry of the HD molecule, resulting in a biased cone of acceptance and the molecular reorientation effect.
The intramolecular isotope effects or the branching AH/AD ratios are extremely affected by the topography of the corresponding reactive potential energy surface (PES).In 1999, Skouteris et al. studied the role of the van der Waals well in the entrance channel of the Cl + HD reaction by exact quantum dynamic calculations on two different PESs and crossed molecular beam devices [17].The results showed that the shallow well (~0.02eV) can cause a strong preference for the DCl product, and the production of the DCl/HCl ratio is enhanced more than seven times for low rotational excitation HD when the very weak van der Waals interactions are included, which is also in agreement with the experimental measurements.Wu et al. studied the significance of conical intersections in the C( 1 D) + HD reaction by using a continuous supersonic flow reactor combined with the quantum dynamics and quasi-classical trajectory calculations on an adiabatic ground-state PES [18].A barrier on the reaction path induced by the conical intersection between the ∼ b 1 A ′′ and ∼ d 1 A ′′ states can regulate a special metastable-state intermediate before the long-lived complex is formed in the deep well, resulting in the CD/CH product branching ratio markedly increasing.Two unusual mechanisms with obvious nonstatistical features, namely C-H activation complex conversion and cyclic complex, were found by the trajectory analysis.In addition to the adiabatic ground-state reactive process, strong intramolecular isotope effects also exist in excited state reactions.Recently, quantum dynamics research on the nonadiabatic reaction of electronically excited Be + ( 2 P) with HD molecule revealed that the shallow wells induced by the avoided crossing on the diagonal surface bring a dramatic preference for the BeD + product [19].It can be concluded from the calculated results that the formation of BeH + favors a direct reaction process, whereas the BeD + + H channel is dominated by the complex-forming mechanism.Similar dynamic behaviors were also presented in the nonadiabatic Mg + ( 2 P) + HD reaction [20], which are consistent with the previous experimental results [21].
For the A + HD type reaction, there usually exists a dominant well or barrier that determines the preference for the production of AH or AD on the reaction path.However, the reactions of ground-state alkaline earth metal with hydrides (Be + H 2 , Mg + H 2 , and Ca + H 2 ) proceed either directly through hydrogen abstraction or indirectly via an intermediate complex [22][23][24][25][26].The complex-forming reaction paths include both an obvious well and a barrier structure, and the molecular systems were found to be linear at equilibrium, different from the T-shaped stable configuration of typical complex-forming reaction systems of CH 2 , OH 2 , and SH 2 [27].Therefore, the reactions between alkaline earth metal atoms and HD molecules may present unconventional product branching ratios and interesting dynamic mechanisms, which have not been studied until now.As the simplest example of this type of system, the BeH 2 has received an important amount of attention because of its simple electronic structures and has been considered a good testing tool for new computational methods of quantum chemistry.The structure factors, vibrational mode, and infrared emission spectroscopy of the BeH 2 molecule, and the BeD 2 and BeHD isotopomers, have been widely studied by various experimental technologies and ab initio calculations [28][29][30][31][32].However, the reaction dynamics of the Be atom with H 2 was not implemented until recently.In 2022, Yang and Chen reported a globally accurate groundstate BeH 2 PES based on a mass of ab initio points calculated at the icMRCI + Q/AV5Z level and the neural network method [33].On this new PES, the quantum dynamics of the Be( 1 S) + H 2 (v 0 = 0, j 0 = 0) → BeH + H reaction were carried out, and the dynamics results indicated that the reaction follows the complex-forming mechanism near the reactive threshold, while a direct abstraction process gradually plays the dominant role as there is an increase in collision energy.
In this work, we perform state-to-state quantum dynamic calculations of the vibrationally excited Be( 1 S) + HD (v 0 = 2, j 0 = 0) → BeH/BeD + H/D reaction using the timedependent wave packet (TDWP) method on the newly constructed PES to study the dynamic mechanisms.To initiate the reaction at the lower collision energy and analyze the microscopic dynamic behaviors and intermolecular isotopic effects in a wider energy region, the initial vibrational state of the HD molecule is set to two due to the large endothermicity (~2.45 eV) of the title reaction with the vibrational ground-state.The remainder of this paper is organized as follows: Section 2 introduces the theory and computational details; the calculated results are listed and the corresponding discussions based on these results are given in Section 3; and the conclusions of this work are displayed in Section 4.
Results and Discussion
The TDWP dynamic calculations of the Be( 1 S) + HD (v 0 = 2, j 0 = 0) → BeH/BeD + H/D reaction are carried out on the ground-state PES [33] constructed by the permutation invariant polynomial neural network model [34,35] based on high-level ab initio calculations.The used PES includes all the regions that the reaction can access and features high precision; thus, it can be used to accurately calculate the state-to-state dynamics of the Be( 1 S) + H 2 reaction and its isotopic variants.Figure 1 presents the indirect and direct microscopic pathways of the Be( 1 S) + HD reaction, which are obtained by scanning the ground-state PES with small step lengths (∆R = 0.01 a 0 , ∆∠Be-HD = 1 • ) at different coordinates (R HD -R BeH(D) ) to find the minimum energy.The indirect pathway is also the global minimum energy path, which includes a barrier and a deep well with a depth of 1.632 eV corresponding to the reactant channel.The Be atom collides with the HD molecule in the collinear direction.It passes a tiny transition state that is 2.096 eV above the energy of the reactant asymptotical region.Then, the Be atom moves along the mid perpendicular of the HD molecule together with the elongation of the H-D bond.When the system is at D ∞h symmetry, a stable H-Be-D complex is formed; finally, the product BeH/BeD molecule is generated via the dissociation of the complex at the product asymptotical region.The H atom more easily escapes the constraint of wells due to the lighter mass, so the formation of the BeD product is dominant.There is no well or barrier on the direct reaction pathway, and the energy is monotonically increasing from the reactant channel to the product channel.When the reaction process is on the direct abstraction pathway, the H atom describes a wider circle around the center of the mass of the HD molecule and is more likely to intercept the approaching Be atom.Considering the zero-point energy effect, the endothermicities of forming BeH + D and BeD + H are 1.59 eV and 1.56 eV, respectively.
ground-state PES with small step lengths (ΔR = 0.01 a0, Δ∠Be-HD = 1°) at d coordinates (RHD-RBeH(D)) to find the minimum energy.The indirect pathway is a global minimum energy path, which includes a barrier and a deep well with a d 1.632 eV corresponding to the reactant channel.The Be atom collides with t molecule in the collinear direction.It passes a tiny transition state that is 2.096 eV the energy of the reactant asymptotical region.Then, the Be atom moves along t perpendicular of the HD molecule together with the elongation of the H-D bond the system is at D∞h symmetry, a stable H-Be-D complex is formed; finally, the p BeH/BeD molecule is generated via the dissociation of the complex at the p asymptotical region.The H atom more easily escapes the constraint of wells due lighter mass, so the formation of the BeD product is dominant.There is no well or on the direct reaction pathway, and the energy is monotonically increasing fr reactant channel to the product channel.When the reaction process is on the abstraction pathway, the H atom describes a wider circle around the center of the m the HD molecule and is more likely to intercept the approaching Be atom.Cons the zero-point energy effect, the endothermicities of forming BeH + D and BeD + 1.59 eV and 1.56 eV, respectively.The total reaction probabilities as a function of collision energy for the Be( 1 S (v0 = 2, j0 = 0) → BeH/BeD + H/D at four different partial waves (J = 0, 30, 60, and displayed in Figure 2.For J = 0, the reaction threshold of the two channels is con with the corresponding endothermicity determined by the PES.There are pro oscillations on the reaction probability curves, especially near the threshold, wh attributed to the formation of a long-lived HBeD complex in the deep well of the i pathway that can support numerous bound and quasi-bound states.The thr increases and the oscillation peaks become wider as there is an increase in the because the increasing centrifugal barrier decreases the depth of the effective po well.It can be seen that the reaction probabilities of the two channels present d The total reaction probabilities as a function of collision energy for the Be( 1 S) + HD (v 0 = 2, j 0 = 0) → BeH/BeD + H/D at four different partial waves (J = 0, 30, 60, and 80) are displayed in Figure 2.For J = 0, the reaction threshold of the two channels is consistent with the corresponding endothermicity determined by the PES.There are prominent oscillations on the reaction probability curves, especially near the threshold, which are attributed to the formation of a long-lived HBeD complex in the deep well of the indirect pathway that can support numerous bound and quasi-bound states.The threshold increases and the oscillation peaks become wider as there is an increase in the J value because the increasing centrifugal barrier decreases the depth of the effective potential well.It can be seen that the reaction probabilities of the two channels present different behavior at a certain J value.For the BeD + H channel, the reaction probabilities decrease with the increasing J, but the probabilities have larger values at high partial waves for forming the BeH product when the collision energy exceeds the reaction threshold of the corresponding partial wave even though the effects of the centrifugal barrier increase.In addition, the reaction probabilities of the BeH + D channel are obviously larger than the BeD + H channel at high J values, which is because the higher zero-point energy of the BeH product makes it easier to overcome the centrifugal potential.The well is gradually smoothed by the centrifugal potential, resulting in the reaction processes via a direct abstraction pathway.
behavior at a certain J value.For the BeD + H channel, the reaction probabilities decrease with the increasing J, but the probabilities have larger values at high partial waves for forming the BeH product when the collision energy exceeds the reaction threshold of the corresponding partial wave even though the effects of the centrifugal barrier increase.In addition, the reaction probabilities of the BeH + D channel are obviously larger than the BeD + H channel at high J values, which is because the higher zero-point energy of the BeH product makes it easier to overcome the centrifugal potential.The well is gradually smoothed by the centrifugal potential, resulting in the reaction processes via a direct abstraction pathway.To show the contribution of each partial wave on the cross section of the Be( 1 S) + HD (v0 = 2, j0 = 0) reaction, the weighted opacity functions multiplied by (2J + 1) on the reaction probability at four collision energies (2.0, 2.5, 3.0, and 4.0 eV) for both of the product channels are shown in Figure 3.The BeD + H channel has a faster convergence than the BeH + D channel at a certain collision energy because the centrifugal barrier is lower when the Be atom attack occurs on the H side of the HD molecule at a given J value.All the probability distributions are dominated by a single maximum and barely present the oscillating structure, such as the C + HD reaction [18], as it corresponds to the fact that the well on the reaction path is not very deep, and the direct pathway also plays a crucial role, especially at relatively larger collision energies.For the BeD + H channel, the curves rise almost linearly with the J value and decrease abruptly near the maximum available partial wave, which is consistent with the complex-forming mechanism.The magnitude of the BeH + D channel shows a flatter increase and a more gradual drop after reaching the peak value, implying the nonstatistical dynamics behavior for the product BeH molecule.To show the contribution of each partial wave on the cross section of the Be( 1 S) + HD (v 0 = 2, j 0 = 0) reaction, the weighted opacity functions multiplied by (2J + 1) on the reaction probability at four collision energies (2.0, 2.5, 3.0, and 4.0 eV) for both of the product channels are shown in Figure 3.The BeD + H channel has a faster convergence than the BeH + D channel at a certain collision energy because the centrifugal barrier is lower when the Be atom attack occurs on the H side of the HD molecule at a given J value.All the probability distributions are dominated by a single maximum and barely present the oscillating structure, such as the C + HD reaction [18], as it corresponds to the fact that the well on the reaction path is not very deep, and the direct pathway also plays a crucial role, especially at relatively larger collision energies.For the BeD + H channel, the curves rise almost linearly with the J value and decrease abruptly near the maximum available partial wave, which is consistent with the complex-forming mechanism.The magnitude of the BeH + D channel shows a flatter increase and a more gradual drop after reaching the peak value, implying the nonstatistical dynamics behavior for the product BeH molecule.The collision energy dependence of the total ICSs of the two product channels and the product BeH/BeD branching ratio for the Be( 1 S) + HD (v 0 = 2, j 0 = 0) reaction are shown in Figure 4.The left and right ordinates represent the ICS value and the ICS (BeH/BeD) branching ratio, respectively.Compared to the reaction probability curves of a single J value, most of the oscillations are erased by summing over various partial waves.The total ICSs of the two channels increase in a monotone way with the collision energy, conforming to the characteristics of endothermic reactions.However, the two channels display different variation behaviors.The ICS curve of the BeH molecule increases almost linearly when the collision energy is below 2.90 eV, and its rising slope gradually becomes larger as the collision energy continues to increase.The ICS of the BeD + H channel is also nearly linear, increasing below 3.20 eV, and then the curve rises slowly.These results suggest that the dominance of the title reaction changes from forming the BeD product to the BeH molecule as the collision energy increases.
Molecules 2024, 29, x FOR PEER REVIEW 6 of 14 larger as the collision energy continues to increase.The ICS of the BeD + H channel is also nearly linear, increasing below 3.20 eV, and then the curve rises slowly.These results suggest that the dominance of the title reaction changes from forming the BeD product to the BeH molecule as the collision energy increases.It can be seen that the branching ratio is around one when the collision energy is slightly larger than the reactive threshold.This is because the reaction at relatively low collision energy mainly proceeds along the global minimum energy path, and a long-lived linear HBeD complex can be formed in the deep well, resulting in the two product channels being equally distributed.As the collision energy increases, the contribution of high-order partial waves increases, as shown in Figure 3, and the depth of effective potential on the reaction pathway is decreased, so the lifetime of the complex becomes shorter.As previous studies on C( 1 D) + HD [18] and Be + ( 2 P) + HD [19] reactions have shown, the shallow well can regulate the intermediate to effectively improve the AD/AH ratio.Because the H atom moves faster than the D atom, thus the Be-H bond can be elongated, leading to the complex having smaller vibrational amplitudes in the Be-D bond stretching, so the BeD molecule is formed more easily, and this effect can be enhanced for a shallower effective well.Therefore, the BeH/BeD branching ratio first decreases with the increase in collision energy.As the collision energy continues to increase, the proportion of the collision along the direct abstraction pathway increases, and the effective well on the indirect path can be smoothed by higher-order partial waves, thus the branching ratio starts to rise.In addition, the orbital angular momentum l-dependent centrifugal barrier in the BeH + D channel increases slower than the BeD + + H channel due to the larger reduced mass of the former, which can also increase the BeH/BeD ratio.The preference for forming the BeH molecule is larger than the BeD molecule when the collision energy is above 3.56 eV, suggesting that the direct abstraction process starts to dominate the title reaction.
To further understand the reaction mechanisms and the intramolecular isotope effects of the Be( 1 S) + HD (v0 = 2, j0 = 0) reaction at the state-to-state level, the rovibrationally resolved ICSs of the two product channels at collision energies of 2.5 and 4.0 eV are also calculated, as shown in Figure 5.The BeD molecule can be excited to a higher It can be seen that the branching ratio is around one when the collision energy is slightly larger than the reactive threshold.This is because the reaction at relatively low collision energy mainly proceeds along the global minimum energy path, and a long-lived linear HBeD complex can be formed in the deep well, resulting in the two product channels being equally distributed.As the collision energy increases, the contribution of high-order partial waves increases, as shown in Figure 3, and the depth of effective potential on the reaction pathway is decreased, so the lifetime of the complex becomes shorter.As previous studies on C( 1 D) + HD [18] and Be + ( 2 P) + HD [19] reactions have shown, the shallow well can regulate the intermediate to effectively improve the AD/AH ratio.Because the H atom moves faster than the D atom, thus the Be-H bond can be elongated, leading to the complex having smaller vibrational amplitudes in the Be-D bond stretching, so the BeD molecule is formed more easily, and this effect can be enhanced for a shallower effective well.Therefore, the BeH/BeD branching ratio first decreases with the increase in collision energy.As the collision energy continues to increase, the proportion of the collision along the direct abstraction pathway increases, and the effective well on the indirect path can be smoothed by higher-order partial waves, thus the branching ratio starts to rise.In addition, the orbital angular momentum l-dependent centrifugal barrier in the BeH + D channel increases slower than the BeD + + H channel due to the larger reduced mass of the former, which can also increase the BeH/BeD ratio.The preference for forming the BeH molecule is larger than the BeD molecule when the collision energy is above 3.56 eV, suggesting that the direct abstraction process starts to dominate the title reaction.
To further understand the reaction mechanisms and the intramolecular isotope effects of the Be( 1 S) + HD (v 0 = 2, j 0 = 0) reaction at the state-to-state level, the ro-vibrationally resolved ICSs of the two product channels at collision energies of 2.5 and 4.0 eV are also calculated, as shown in Figure 5.The BeD molecule can be excited to a higher rovibrational state than the BeH molecule at the same collision energy due to the smaller vibrational frequency and rotational constant for the former, which correspond to the narrower energy difference between the two adjacent vibrational or rotational energy levels.The maximum availability vibrational and rotational quantum numbers at 2.5 eV for the BeH and BeD molecules are (3,27) and (4,34), respectively.For the BeH + D channel, the envelope of rotational states is similar for each vibrational state, and the product is mainly excited to relatively low rotational states.In addition, there exists vibrational population inversion, and this behavior becomes more obvious at 4.0 eV collision energy, indicating that the BeH product is mainly formed by a direct abstraction process, and the dominance of the direct pathway significantly increases as the collision energy increases.On the contrary, the BeD product is distributed at a low vibrational state, and the ICS value gradually decreases as there is an increase in the vibrational quantum number, which is a typical behavior of the statistically dominated process.Compared to the BeH product, the BeD product prefers to populate at higher rotational states, displaying an obvious indirect reaction process.The available rovibrational state of the BeH molecule is much more than the BeD molecule at 4.0 eV collision energy, which is because the BeH + D channel is dominated by the direct pathway, and the contribution of high-order partial waves becomes larger, so a part of the energy is used to overcome the centrifugal barrier, resulting in the BeH molecule not being able to be excited to a higher rovibrational energy level.The DCSs can more intuitively reveal the microscopic dynamic mechanisms by giving the angle distributions of the product molecules.Figure 6 presents the three-dimensional plots of the total DCSs of the two product channels for the Be( 1 S) + HD (v 0 = 2, j 0 = 0) reaction as a function of collision energy.For both of the product channels, the peaks of the DCSs are distributed at two polar angles and are almost forward-backward symmetric when the collision energy is slightly larger than the threshold, implying the typical statistical nature, which is contributed by the dominance of the deep well on the indirect pathway.As the collision energy increases, the BeD + H channel still keeps the symmetric angle distribution, although a small forward or backward bias exists because of the effect of the centrifugal barrier.However, there is a clear preference for forward or backward scattering on the BeH + D channel, and this behavior is significantly enhanced at high collision energy, displaying obvious nonstatistical features.The remarkable difference in the DCSs between the two product channels suggests that the intramolecular isotope effects are significant in terms of the product angle distributions.The total DCS results further indicate that the BeH + D and BeD + H channels are dominated by the direct abstraction process and complex-forming mechanisms at most of the studied collision energies.To present more details about the angle distributions of the title reaction, Figure 7 displays the rotational state-resolved DCSs of the two product channels in the vibrational ground-state for the Be( 1 S) + HD (v 0 = 2, j 0 = 0) reaction at 2.5 eV and 4.0 eV collision energy.The symmetry of the DCSs of the two channels is broken at the rotational stateresolved levels even at collision energy, which is because the quantum state-resolved dynamics results do not follow the statistical behavior even for long-lived complex-forming reactions [36,37].The BeH molecules at low rotational states which tend to show forward scattering and backward scattering play dominant roles for the high rotational states at 2.5 eV collision energy.However, the distribution of the rotational state of the BeH product is mainly concentrated in the angle range of 10 • to 75 • when the collision energy increases to 4.0 eV.For the BeD product, apart from the obvious forward and backward peaks, strong sideways scattering also exists (60-120 • ), especially at relatively high rotational states.
Methods
The most reliable strategy for studying reaction dynamics theoretically is to perform quantum mechanics calculations on a high-quality PES [38][39][40].The quantum TDWP method [41][42][43][44][45] can not only accurately calculate dynamics data but also has a relatively small numerical cost, meaning that it has been widely applied to simple reaction systems [46][47][48][49][50][51][52][53][54][55].A detailed description of the TDWP method has been presented in the relevant literature, and we only give the essentials and main equations.The Hamiltonian of the Be( 1 S) + HD → BeH/BeD + H/D reaction in the body-fixed (BF) reactant Jacobi coordinates (r, R, θ) can be written as follows: where R is the distance from the Be atom to the center of the mass of the HD molecule, and r is the bond length of the HD molecule.J and j are the total angle momentum number of the BeHD molecule and the rotational angular momentum number of the HD molecule, respectively.μR and μr represent the corresponding reduced masses of R and r coordinates.V(r, R, θ) is the Be-HD interaction potential excluding the reference potential energy of the two-body HD molecule.The total wavefunction in the BF representation is expanded to the form of a translational-vibrational-rotational basis, written as follows: where K and M denote the projection of J in the z axis of the BF and space-fixed (SF) representations, respectively; J D ε Ω expresses the parity-adapted normalized
Methods
The most reliable strategy for studying reaction dynamics theoretically is to perform quantum mechanics calculations on a high-quality PES [38][39][40].The quantum TDWP method [41][42][43][44][45] can not only accurately calculate dynamics data but also has a relatively small numerical cost, meaning that it has been widely applied to simple reaction systems [46][47][48][49][50][51][52][53][54][55].A detailed description of the TDWP method has been presented in the relevant literature, and we only give the essentials and main equations.The Hamiltonian of the Be( 1 S) + HD → BeH/BeD + H/D reaction in the body-fixed (BF) reactant Jacobi coordinates (r, R, θ) can be written as follows: where R is the distance from the Be atom to the center of the mass of the HD molecule, and r is the bond length of the HD molecule.J and j are the total angle momentum number of the BeHD molecule and the rotational angular momentum number of the HD molecule, respectively.µ R and µ r represent the corresponding reduced masses of R and r coordinates.V(r, R, θ) is the Be-HD interaction potential excluding the reference potential energy of the two-body HD molecule.The total wavefunction in the BF representation is expanded to the form of a translational-vibrational-rotational basis, written as follows: where K and M denote the projection of J in the z axis of the BF and space-fixed (SF) representations, respectively; Jε MK (Ω) expresses the parity-adapted normalized Wigner rotation matrix.The initial wave packet in the SF representation contains a Gaussian function G(R α ), an eigenfunction of HD molecule ϕ v 0 j 0 (r α ) with the initial ro-vibrational state (v 0 , j 0 ) and the eigenfunction of total angular momentum |J Mj 0 l 0 ε⟩, written as follows: where ε = (−1) j 0 +l 0 is the parity of the system.The subscript α represents the Be( 1 S) + HD channel.The wave packet is evolved by the second-order split operator propagator [56].
During the propagation of the wave packet, R and r coordinates are multiplied by the damping functions to avoid reflection of the wave function on the boundaries.The final state-to-state Smatrix element is extracted using the reactant-coordinate-based method [57,58].The reaction probability at the quantum state-resolved level can be calculated by: The state-to-state integral cross sections (ICSs) and differential cross sections (DCSs) are obtained by using the S-matrix, written as and dσ υj←υ 0 j 0 (ϑ, E) dΩ = 1 where k v 0 j 0 is the moment in the entrance channel, ϑ expresses the scattering angle, and d J KK 0 (ϑ) represents the reduced rotation matrix element.In this work, the initial rovibrational state of the reactant HD molecule is set as v 0 = 2 and j 0 = 0.The main numerical parameters used in the TDWP calculations are listed in Table 1, which are determined by numerous tests.The partial wave is calculated up to J = 86, yielding converged ICSs and DCSs for each product channel below 4.0 eV collision energy.
Conclusions
In the present study, accurate quantum dynamic calculations on the Be( 1 S) + HD (v 0 = 2, j 0 = 0) → BeH/BeD + H/D reaction are carried out at the state-to-state level in the collision energy range up to 4.0 eV using the TDWP method on a globally accurate PES.The detailed dynamics results of the reaction probabilities, opacity functions, and total and state-resolved ICSs and DCSs of the two product channels are displayed to study the reaction mechanisms and intramolecular isotope effects.The title reaction is controlled by two different pathways, namely the indirect pathway, which includes a barrier and a deep well, and a smooth direct abstraction pathway.
There are obvious oscillations in reaction probabilities at small partial waves due to the forming of a collinear HBeD complex.The reaction probabilities of the BeD + H channel decrease with the increasing J, but the probabilities have larger values at high partial waves for forming the BeH product.The curves of the opacity functions of the BeH + D channel rise slower with the J value and display a more gradual drop after reaching the peak value.The total ICS monotonously increases with the increase in collision energy for both of the channels.The BeH/BeD branching ratio decreases firstly from around one and then rises rapidly, and the preference in generating the BeH molecule is larger than the BeD molecule when the collision energy is above 3.56 eV.The BeH molecule presents vibrational population inversion and is mainly distributed in the low rotational state, whereas the BeD molecule prefers to populate at vibrationally cold and rotationally hot states.The angle distributions are almost forward-backward symmetric when the collision energy is slightly larger than the threshold for both of the product channels.As the collision energy increases, the DCSs of the BeH molecule show an obvious forward or backward bias, while the BeD + H channel remains the symmetric angle distribution.The rotational state-resolved DCSs show nonstatistical characteristics for both of the channels.The dynamics results suggest that the BeH + D channel follows a direct abstraction process at most selected collision energies, while the BeD + H channel is dominated by the complex-forming mechanism.
Figure 3 .
Figure 3. Opacity functions of the Be( 1 S) + HD (v0 = 2, j0 = 0) → BeH/BeD + H/D reaction at the collision energy of 2.0, 2.5, 3.0, and 4.0 eV.The collision energy dependence of the total ICSs of the two product channels and the product BeH/BeD branching ratio for the Be( 1 S) + HD (v0 = 2, j0 = 0) reaction are shown in Figure4.The left and right ordinates represent the ICS value and the ICS (BeH/BeD) branching ratio, respectively.Compared to the reaction probability curves of a single J value, most of the oscillations are erased by summing over various partial waves.The total ICSs of the two channels increase in a monotone way with the collision energy, conforming to the characteristics of endothermic reactions.However, the two channels display different variation behaviors.The ICS curve of the BeH molecule increases almost linearly when the collision energy is below 2.90 eV, and its rising slope gradually becomes | 2024-03-17T16:05:05.230Z | 2024-03-01T00:00:00.000 | {
"year": 2024,
"sha1": "f449aec6e0ab2a122df6bbcb37fa544794cb35d6",
"oa_license": "CCBY",
"oa_url": "https://www.mdpi.com/1420-3049/29/6/1263/pdf?version=1710300523",
"oa_status": "GOLD",
"pdf_src": "PubMedCentral",
"pdf_hash": "1e283c43587071e16dd20ca09e7047832ff9375d",
"s2fieldsofstudy": [
"Chemistry"
],
"extfieldsofstudy": [
"Medicine"
]
} |
15745871 | pes2o/s2orc | v3-fos-license | The Behavioural Response of Australian Fur Seals to Motor Boat Noise
Australian fur seals breed on thirteen islands located in the Bass Strait, Australia. Land access to these islands is restricted, minimising human presence but boat access is still permissible with limitations on approach distances. Thirty-two controlled noise exposure experiments were conducted on breeding Australian fur seals to determine their behavioural response to controlled in-air motor boat noise on Kanowna Island (39°10′S, 146°18′E). Our results show there were significant differences in the seals' behaviour at low (64–70 dB) versus high (75–85 dB) sound levels, with seals orientating themselves towards or physically moving away from the louder boat noise at three different sound levels. Furthermore, seals responded more aggressively with one another and were more alert when they heard louder boat noise. Australian fur seals demonstrated plasticity in their vocal responses to boat noise with calls being significantly different between the various sound intensities and barks tending to get faster as the boat noise got louder. These results suggest that Australian fur seals on Kanowna Island show behavioural disturbance to high level boat noise. Consequently, it is recommended that an appropriate level of received boat sound emissions at breeding fur seal colonies be below 74 dB and that these findings be taken into account when evaluating appropriate approach distances and speed limits for boats.
Introduction
Anthropogenic disturbance is a significant catalyst of environmental change, with potentially important implications for individuals and populations [1]. There is compelling evidence supporting an association between stress in mammals, in terms of their physiology and behaviour, with human disturbance [1,2,3]. Human disturbance was reported to negatively influence the breeding success in penguins [2], while in brown bears (Ursus arctos) human disturbance was found to increase energetic expenditure as a result of behavioural modifications in the species [3]. Anthropogenic disturbance has also been associated with changes in the spatial distribution of hyena clans [4]. Fundamentally, any modifications to biologically significant activities such as an animal's hormonal state, behaviour and energy reserves may ultimately affect an animal's fitness and have detrimental effects on the population [1].
Australian fur seals, (Arctocephalus pusillus doriferus), are endemic to Australia and breed on thirteen islands located in south-eastern Bass Strait [5]. These breeding islands provide areas where seals give birth, mate and raise their young away from potential threats of terrestrial predators, including humans. During the breeding season, male Australian fur seals compete aggressively to establish and maintain territories. At this time, females give birth, mate shortly thereafter and then alternate between feeding out at sea and suckling their young on land [6]. Vocal communication, olfaction and postural displays are intrinsic to the breeding behaviour of Australian fur seals [6]. Therefore, the use of sight, smell and hearing are critically important to the species' ecological and breeding strategy.
The breeding season is an energetically costly period for Australian fur seals, where males generally fast during territorial tenure and females require energy to give birth, forage and suckle their young. Consequently, any disturbance such as anthropogenic noise from boats that induces energetically costly behaviours such fleeing or activities that interfere with communication during this critical time may detract from energy reserves that are required for breeding.
Human disturbance is a key threat to seals in Australia (EPBC Act 1999) [7] with little or no quantitative data available on the impact of anthropogenic noise on seals in the Southern Hemisphere. In Australian fur seals, there have been three studies evaluating the response of seals to boat disturbance. Two studies conducted at the haul-out sites of Steamers Head (35u109S, 150u409E) [8] and Montague Island (36u209S, 150u109E) [9] reported that the responses of Australian fur seals to boat-based approaches included increased vigilance and fleeing behaviours. Back [10] conducted controlled boat approaches at two breeding sites (Kanowna Island and Seal Rocks, 39u109S, 146u189E) and found similar fleeing behaviours, but also observed that seals at these two breeding colonies had markedly different responses, presumably due to habituation. Shaughnessy et al. [9] further commented that it is unclear as to whether the seals are reacting to boats due to changes in sight, sound or odour. As these boat approach studies did not control for the impact of noise alone, this instigated the current project, whereby we conduct quantitative studies examining the effect of noise generated from human disturbance on seals' behaviour.
Anthropogenic noise exposure has been associated with stampedes and the crushing or abandonment of pups [11]. Many studies have reported pup mortality associated with abandonment by female pinnipeds during disturbance events, such as aircraft overflights (e.g., [12,13]). Burleigh et al. [8] reported that stampedes at fur seals haul-out sites occurred in response to boat approaches at 20 m, and stampedes have also been reported at Kanowna Island due to boat approaches [14]. The challenge posed by disturbance studies is determining the appropriate or acceptable levels of disturbance. Therefore, the present study aims to investigate the behavioural and vocal response of breeding Australian fur seals at Kanowna Island to anthropogenic disturbance, through controlled noise exposure experiments.
Ethics Statement
Research for this project was conducted under Ethics Number A10/2008, Animal Welfare Committee, Deakin University.
Study site
The study examined the behavioural response of Australian fur seals on Kanowna Island (39u109S, 146u189E), Bass Strait, Australia, during the November-December 2008 breeding season over a two week period. The island has two main colonies (East Valley and Main Colony) and these comprised males, females, yearlings and pups at the time of sampling.
Kanowna Island is situated within a Marine National Park and access to the island is restricted, with boat-based approaches being permissible under seasonal contingent minimum distances of 200 m during the breeding season (November to January) and 50 m during the non-breeding season (February to October) [15].
Noise Playback Experiment
A total of 112 animals were exposed to a range of randomly selected in-air motor boat noise, played at three different sound intensities: 1) Low 64-70 dB (n = 27 animals); 2) Mid 71-74 dB (n = 49 animals); and 3) High 75-85 dB (n = 36 animals). These are perceived levels, so this is the intensity received by seals at the centre of each group under study. The range of intensity for each level is explained because of the spatial spread of animals within each group. The signals were broadcast at 5-15 m from the subjects but the sound emission was calibrated at each playback to ensure the received levels were known, see below. Thirty-two experiments were conducted on groups of seals ranging from 1 to 11 animals (average = 4 animals) and consisted of three phases: pre-stimulus (10 min), stimulus (2 min) and post-stimulus (10 min). Each group of seals, i.e. 32 groups, were used once in each experiment, so were either played the low, mid or high sound levels, and the choice of each treatment used was randomised.
The motor boat noise used in the playbacks ( Fig. 1) was prerecorded from a range of boats (n = 7). For the purpose of this study, the sound intensity emitted by each vessel was standardised to focus on examining the effects of varying sound intensities only.
Playbacks were broadcast using a MIPRO707 speaker (frequency response 60 Hz-20,000 kHz63 dB). The speaker was connected to a Sony digital tape recorder (TCD-D8; Sony Corp., Japan) and placed 5-15 m away from subjects. Several propagation tests were performed on the island to ensure that the received sound pressure was known. The sound pressure was measured with a Radio Shack Model 33-2050 sound level meter set at ''C'' weighting and fast response. Duration between two trials was at least 5 h to reduce any habituation effect by the animals in the colony.
Evaluation of responses
Four different methods were used to evaluate the responses of seals (Table 1).
1)
Behavioural scan sampling to determine behaviours prestimulus, stimulus and post-stimulus -Video recordings were used to analyse the behaviours of males, females, yearlings and pups ( Table 2). A scan sampling regime was used [16] where the behaviours of individual seals were recorded every 30 s for the duration of the experiment. Behaviours were grouped according to one of three phases: pre-stimulus, stimulus and post-stimulus and each category of behaviour was analysed separately over the three phases to determine significant changes in behaviour between the phases.
2)
Behavioural response during stimulus phase -The behavioural response of seals was examined to determine whether seals responded differently to the three received levels of motor boat noise. This experiment contained three treatments: low boat noise (64-70 dB); mid boat noise level (71-74 dB); and high level motor boat noise (75-85 dB). Individual seals behavioural responses observed during the noise playback (i.e. stimulus phase) was analysed. Each cohort's (i.e. males, females, yearlings and pups) response to the playbacks was graded on an ordinal scale from 0 to 3. The scale was as follows: 0 was no response; 1 was eye movements towards the noise source; 2 head and/or body movements towards the noise source; and 3 movements away from the noise source.
3)
The length of time seals looked at the noise source -The time a seal spent looking in the direction of the noise during the stimulus phase (2 min in duration) was recorded, and compared between individual seals over the three sound intensities.
4)
Acoustic behaviour of seals during the pre and post-stimulus phase -As males are the most vocal age group they were chosen for the sound analysis (See [6,17]) for details on analysis procedure). Vocalisations were analysed using RAVEN Pro 1.4 (Ithaca, NY) for the pre-stimulus and post-stimulus phase, but were not analysed for the stimulus phase as the recordings were of poor quality due to the boat vessel noise emitted during the stimulus phase. Twenty randomly selected bark calls from eleven males were analysed from the pre and post-stimulus phases. Only temporal features such as the repetition rate, unit and interunit duration were examined for changes in call structure with changes in noise levels.
Statistical Analysis
General linear mixed model analysis was performed in GENSTAT (Release 13.1, U.K.) to determine significant changes in behaviour between the three phases (pre-, stimulus and poststimulus). The fixed terms in the model were the phases (pre-, stimulus, and post-stimulus), the cohort (which was later removed) and the sound level (low, mid and high) with the random term as the animal ID. Each behaviour was examined separately as the dependent variable with the three phases as the independent variable. As there were no overall differences in the responses of the cohort (x 2 = 5.70, DF = 3, P = 0.127) to the three treatments (low, mid, high sound levels) it was taken out of the model.
The behavioural response during the stimulus were analysed using the ordinal logistic regression analysis using MINITAB Table 1. Description of evaluation methods to analyse the response of Australian fur seals during the noise playback experiments.
Behavioural responses during pre-stimulus, stimulus and post stimulus phases
Behaviours were grouped according to one of three phases: pre-stimulus, stimulus and poststimulus and each category of behaviour was analysed separately over the three phases to determine significant changes in behaviour between the phases.
Behavioural response during stimulus phase
Individual seals behavioural responses observed during the noise playback (i.e. stimulus phase) was analysed.
3. The length of time seals looked at the noise source during the stimulus phase The time a seal spent looking in the direction of the noise during the stimulus phase (2 min in duration) was recorded, and compared between individual seals over the three sound intensities. Version 15 (MINITAB INC, USA). The behavioural response (coded as 0 was no response; 1 was eye movements towards the noise source; 2 head and/or body movements towards the noise source; and 3 movements away from the noise source) was used as the dependent variables and was compared to the sound levels (low, mid and high) which were the independent variables included in the model. This analysis established the significance of any differences between the variables (i.e., low, mid, high) and determined a probability of each behaviour response for each treatment [see 18]. Results were considered significant at P,0.05. A uni-variate generalised linear model (GLM) was used to determine if the time a seal spent looking in the direction of the sound source (i.e. dependent variable) differed significantly between sound levels (i.e., low, mid, high) using SPSS (PASW Statistics 18, USA).
A multivariate GLM was performed to determine the difference in the dependent variable, the call characteristics (i.e., call duration, inter-unit duration and repetition rate-dependent variable) and the independent variables: the pre and post-stimulus phases, at the three different sound levels (low, mid and high). A Bonferroni correction was applied to the results of the tests to reduce the effect of increasing the probability of a Type I error.
Results
Behavioural responses during pre-stimulus, stimulus and post stimulus phases Individual behaviours (Table 2) were compared among three phases (i.e. pre-stimulus, stimulus, post-stimulus) using generalised mixed model (GLMM) analysis, with 112 individual seals used in this analysis. There were significant differences in only three behaviours: Rest (P,0.001), alert (P,0.001) and fight (P,0.001) over the three phases. The rest behaviour was lowest during the stimulus phase, whilst the alert and fighting behaviours were highest during this period. Seals were most alert during the stimulus but this decreased during the post-stimulus phase. The opposite was observed for the resting behaviours. Resting was reduced during the stimulus phase but increased during the poststimulus phase. Fighting was highest during the stimulus phase and decreased slightly during the post-stimulus phase but remained higher than the pre-stimulus phase.
Behavioural response during stimulus phase
There were significant differences between the responses of individual seals (i.e. 112 individual seals from 32 playback experiments) to the different noise levels (x 2 = 8.14, DF = 2, P = 0.017). Seals did not respond significantly differently to the low and mid sound level treatment (Z = 20.67, P = 0.504) and mid versus high sound levels (Z = 21.87, P = 0.062) but there were significant differences in the response of seals hearing the low versus high sound levels (Z = 22.83, P = 0.005). Seals reacted more strongly (i.e. moved body in the direction of the noise source or physically moved away) in response to hearing the louder sounds (Fig. 2).
The length of time seals looked at the sound source during the stimulus phase
The univariate GLM found that from 112 seals examined that the time they spent looking towards the noise source showed a strong tendency towards being significant (F = 2.99, P = 0.05). Overall seals spent more time looking at the noise source during high level disturbances.
Acoustic response during pre and post stimulus
A multivariate GLM was performed on eleven male seals comparing each of the acoustic properties analysed (i.e. unit, interunit duration and repetition rate) for both pre and post-stimulus at the three sound intensities (i.e. low, mid and high). There were statistically significant differences in male call parameters between the pre-and post-stimulus phases (F 3, 398 = 5.46, P = 0.01; Wilk's Lambda = 0.960). There were also significant differences in the calls at the different sound levels measured during the pre-and post-stimulus phases, (F 6, 796 = 14.24, P = 0.001; Wilk's Lambda = 0.816). Post-hoc tests for each call feature measured demonstrates that the unit-duration of bark calls is significantly different between the low and mid-level treatment (P = 0.002), and there are significant differences for the inter-unit duration between low and mid (P,0.001) and low and high-level (P = 0.004). Overall, both the unit and inter-unit duration got longer as the sound level increased ( Table 3). The repetition rate of barks varied significantly between treatment low and high level (P = 0.049), and mid and high levels (P = 0.011), getting faster as the sound got louder (Table 4).
Discussion
The study reports the behavioural response of breeding Australian fur seals to motor boat noise. Using controlled noise exposure experiments we were able to quantify the response of seals to three sound intensities to determine a relatively safe received level of boat noise below 74 dB. Our findings further reveal that Australian fur seals utilize vocal plasticity to cope with changes in anthropogenic noise. The results suggest seals perceive boats as potential threats with louder motor boat noise having a
Rest
The seal positions itself with either the ventral or lateral surface of its torso against the substrate. The head is raised slightly when vocalising Alert The seal is sitting in an upright posture, where the subject looks towards the noise source
Fighting
This includes, open mouth threat, chasing, lunging and biting
Locomotion
The male usually moves forward and he waves his neck from side to side, this behaviour is often accompanied with vocalisation as it moves forward, but it remains within its territory
Obstructing
The seal impedes the movement of another by using its body as an obstruction Nursing Pup is suckling its mother's teat
Nuzzling
Where the vocalising animal touches the muzzle, nape or any other part of another seals body, using its muzzle. This behaviour may occur in water or on land, and generally occurs between a male and female seal or between mother and pup doi:10.1371/journal.pone.0037228.t002 greater impact on seals, where seals displayed more aggressive and alert behaviours.
In the present study, seals reacted more strongly to hearing the louder sounds by either orientating themselves towards the boat noise or physically moving away. Seals displayed significantly different responses between low (64-70 dB) and high levels (75-85 dB), indicating that in air, noise levels above 74 dB are predicted to cause behavioural disturbance in Australian fur seals. At high levels (75-85 dB), seals displayed energetically costly behaviours. During one of the playback experiments (high level), seals began to move rapidly away from the noise, displaying a cascading effect resembling those in the initial stages of a stampede. The playback was immediately stopped to reduce the potential for seals, particularly pups, to get crushed, injured or killed. Our findings from the behavioural response of seals support the notion that received boat noise levels at 75-85 dB elicit significant aversive behaviours by seals which may potentially cause injury.
Many species of animals are heavily reliant on acoustic signals for intra-specific communication (birds: [19], for review on marine mammals see [20]. Environmental selection pressures such as background noise may affect how well vocal features such as repertoire size, spectral and temporal characteristics are communicated [21]. Vocalising animals have access to a number of signalling strategies to avoid or reduce masking by ambient noise (birds: [22]; marine mammals: [23]. Animals follow the mathematical theory of communication [24] whereby under noisy conditions animals alter their frequencies, vocalise more often, with longerand louder calls in order to communicate the same meaning and volume of information [25,26,27,28,29,30]; in marine mammals: [23,31,32,33]. Changes in vocalizations produced by Australian fur seals were evident, where seals increased their repetition rate to adjust to changes in anthropogenic noise. Seals continued to show signs of this behavioural modification during the post-stimulus phase, where vocalisations had not returned to their pre-stimulus 'baseline' calling pattern. There were also significant differences in the call structure with varying levels of noise exposure. Other studies analysing the behavioural context of the bark call in male Australian fur seals have indicated that the rate of delivery increases when seals display intra-species aggression or when trying to 'herd' females [6]. It has been suggested that varying the acoustic structure of the bark may update recipients on the emotive state of the caller [34,35] and this may be important information for researchers to understand. Changes in the acoustic structure of calls, particularly increasing the rate of delivery of the bark, indicate potential changes in emotion resembling more aggressive or heightened behaviours [36,37,38]. Therefore, when higher intensities of noise were played to seals and they produced vocalisations with faster repetition rates than would ordinarily be produced to signify aggressive responses, that this may in fact communicate a heightened sense of emotion in seals. This change in behaviour seems likely to indicate that seals recognise motor boat noise as a potential threat. The current study did not examine if the noise exposure modified the amplitude or frequency spectrum of the calls, as this could potentially be shifted. For example, North Atlantic right whales were found to adjust the volume (i.e. amplitude) of their song in increased background noise, singing louder during higher levels of background noise [26]. Another way to cope to changing noise conditions is by modifying the frequency characteristics of vocalisations. Indo-Pacific Bottlenose Dolphins produced whistles of lower frequencies with fewer frequency modulations whereas dolphins living in habitats with less ambient noise, they produced whistles at varying frequencies with greater modulations [33]. In humpback whales, males produced longer songs during LFA sonar transmissions to compensate for acoustic interference [32]. Further investigations should be done to examine the other potential changes in the vocalisations of Australian fur seals.
Access is not permitted within 50 metres of Kanowna Island between February to October (inclusive), and for the protection of breeding Australian fur seals, access is not permitted within 200 metres of Kanowna Island during the breeding season between November to January (inclusive) [15]. The results of this study suggest that a safe received level is less than 74 dB. It is important to understand that a range of factors affect the actual received levels of sound at a colony including the boat speed, engine size, wind speed and direction and other factors such as smell and sight of the boat may affect the behavioural responses. Therefore, further propagation tests are required to convert the acceptable level of 74 dB of motor boat noise into distance approaches. These propagation tests should be conducted with a range of boat sizes, engine, wind speed, wind direction and boat speeds to determine the range of variables suitable to ensure the 74 dB sound intensity is not exceeded at breeding colonies. Back [10] suggested that approach distances at naïve colonies, i.e. colonies that have not been exposed to a lot of disturbance, such as Kanowna Island should be extended to .75 m to reduce disturbance of seals by boats. Burleigh et al. [8] also recommends that an approach distance of at least 75 m is warranted from haulouts when there are fewer than 50 seals and at least I00 metres when there are 50 or more seals. In addition to studies that measure source levels of various types of boats at different speeds, noise propagation tests should be conducted to determine the minimum approach distance whereby noise levels would not exceed 74 dB, which is known to cause more aggressive and alert behaviours in breeding fur seals. | 2016-05-12T22:15:10.714Z | 2012-05-18T00:00:00.000 | {
"year": 2012,
"sha1": "611748da9cb54962bc7f5444eeeb562b5d1067b6",
"oa_license": "CCBY",
"oa_url": "https://journals.plos.org/plosone/article/file?id=10.1371/journal.pone.0037228&type=printable",
"oa_status": "GOLD",
"pdf_src": "PubMedCentral",
"pdf_hash": "cd435ab8030232f54125c58b9832199c809bd9d3",
"s2fieldsofstudy": [
"Environmental Science"
],
"extfieldsofstudy": [
"Geography",
"Medicine"
]
} |
123770425 | pes2o/s2orc | v3-fos-license | Hyperbolic-parabolic singular perturbation for mildly degenerate Kirchhoff equations: decay-error estimates
We consider degenerate Kirchhoff equations with a small parameter epsilon in front of the second-order time-derivative. It is well known that these equations admit global solutions when epsilon is small enough, and that these solutions decay as t ->+infinity with the same rate of solutions of the limit problem (of parabolic type). In this paper we prove decay-error estimates for the difference between a solution of the hyperbolic problem and the solution of the corresponding parabolic problem. These estimates show in the same time that the difference tends to zero both as epsilon ->0, and as t ->+infinity. Concerning the decay rates, it turns out that the difference decays faster than the two terms separately (as t ->+infinity). Proofs involve a nonlinear step where we separate Fourier components with respect to the lowest frequency, followed by a linear step where we exploit weighted versions of classical energies.
Introduction
Let H be a separable real Hilbert space. For every x and y in H, |x| denotes the norm of x, and x, y denotes the scalar product of x and y. Let A be a self-adjoint linear operator on H with dense domain D(A). We assume that A is nonnegative, namely Ax, x ≥ 0 for every x ∈ D(A), so that for every α ≥ 0 the power A α x is defined provided that x lies in a suitable domain D(A α ).
The singular perturbation problem for Kirchhoff equations has generated a considerable literature in the last 30 years. The state of the art has been recently presented in the survey [13], where more general nonlinearities and more general dissipative terms have also been considered. In [13] the general problem has been split into six subproblems, which we list below.
(P1) Global existence and decay estimates for the parabolic problem.
(P2) Local existence for the hyperbolic problem and local-in-time error estimates on ρ ε (t) and r ε (t).
(P3) Global existence for the hyperbolic problem.
(P4) Decay estimates for solutions of the hyperbolic problem (as t → +∞).
(P5) Global-in-time error estimates for the singular perturbation problem, which means time-independent estimates on ρ ε (t) or r ε (t) as ε → 0 + .
Subproblem (P1) is well understood (see [1,2,15,19]). The result is that problem (1.4), (1.5) has a unique global solution for every u 0 ∈ D(A) (and even for less regular data), and this solution decays at infinity as solutions of the ordinary differential equation y ′ + |y| 2γ y = 0, (1.9) which is just the special case of (1.4) where H = R and A is the identity. Also subproblem (P2) is well understood, because on a fixed bounded time interval the degeneracy of the equation plays no role. Local-in-time error estimates were proved by B. F. Esham and R. J. Weinacht in [4], then by the second author in [16], and finally by the authors in [10, Appendix A] with optimal assumptions on initial data. The typical result is that |A 1/2 ρ ε (t)| ≤ Cε when (u 0 , u 1 ) ∈ D(A 3/2 ) × D(A 1/2 ), and we know that this space is optimal if we look for estimates on |A 1/2 ρ ε (t)| of order ε, even in the linear case (see [8]). Subproblem (P3) was solved by K. Nishihara and Y. Yamada [22]. They proved that (1.1), (1.2) has a unique global solution provided that (u 0 , u 1 ) ∈ D(A) × D(A 1/2 ) satisfy the nondegeneracy assumption (1.3) and ε is small enough. It is not known whether the smallness of ε is a necessary condition. This remains the main open problem in the theory of Kirchhoff equations, both dissipative and non-dissipative, both degenerate and non-degenerate. Subproblem (P4) was first addressed in [22]. More recently, the authors in [9] and [6] provided optimal decay estimates, showing that solutions of (1.1), (1.2) decay with the same rate of solutions of the corresponding parabolic problem (see also [20,21,23,24] for the case γ = 1). The results have been recently extended in [12] to equations with weak dissipation, namely with a dissipative term of the form b(t)u ′ ε (t), where b(t) → 0 as t → +∞. Subproblem (P5) was considered by the authors in [10], with non-optimal convergence rates, and finally by the first author [5] with optimal convergence rates.
For the convenience of the reader, in section 2.1 we state all previous results needed in the sequel.
In this paper we concentrate on subproblem (P6), namely on decay-error estimates. Estimates of this type were proved by R. Chill and A. Haraux [3] in the case of linear equations, and then by H. Hashimoto and T. Yamazaki [17] for nondegenerate Kirchhoff equations. Those results were successively extended by T. Yamazaki [26,27] and by the authors [11] to nondegenerate Kirchhoff equations with weak dissipation. The nondegenerate character of the equation (namely strict hyperbolicity) seems to be essential in all previous approaches, which fail when applied to degenerate equations. This is the technical reason why subproblem (P6) resisted so far as an open problem.
In this paper we begin by showing that there is a deeper reason. Indeed we show in Example 2.2 that, without further assumptions on initial data, the expected decay-error estimates are actually false, even in the simple case where H is a two dimensional vector space. By "expected" we mean decay-error estimates such as (1.8), where the decay-rate σ(t) is the same as in subproblem (P4), and the convergence rate ω(ε) is the same as in subproblem (P2) or subproblem (P5). The rigorous verification of the counterexamples strongly relies on the asymptotic limits which have been recently found in [6].
Roughly speaking, the expected decay-error estimates are false whenever the initial condition u 1 has a nonzero Fourier component with respect to a frequency which is less than all frequencies corresponding to nonzero components of u 0 . This motivates the introduction of a special class of initial data where this cannot happen (see Definition 2.3). In Remark 2.4 we show that this requirement on initial data is easily satisfied in many concrete cases.
The main result of this paper is that in this class of initial data we do have decay-error estimates for the degenerate problem. Apart from the special assumption, the regularity we require on initial data is optimal, because it is the same which was optimal in the linear nondegenerate case. The convergence rates ω(ε) are optimal, because they are the same which appear in the local-in-time error estimates of subproblem (P2), or in the global-in-time error estimates of subproblem (P5). The real surprise lies in the decay rate σ(t). Indeed it turns out that ρ ε (t) and r ε (t) decay faster than u ε (t) and u(t) alone.
An improvement of decay rates has been observed also in [3] and [17,26], but in those cases it seems to originate from different reasons. Indeed in those examples it is essential that the operator is not coercive, while in our case we have improvement even if the operator is coercive. Roughly speaking, our improvement comes from the fact that our equation is in the same time degenerate and nonlinear. In section 2.3 below we show a simple toy model, based on ordinary differential equations of order one, which gives a flavor of this aspect. The main point, both for the improvement and for the impossibility of expected decay-error estimates for general data, is that solutions of (1.9) decay as C(1 + t) −1/(2γ) , where the constant C depends on γ, but is independent of the initial condition.
Our result requires a new approach in order to take advantage of the special assump-tions on initial data. The main idea is that in the nonlinear degenerate case Fourier components corresponding to higher frequencies decay faster. As a consequence, in the limit as t → +∞ the nonlinear terms |A 1/2 u ε (t)| 2γ or |A 1/2 u(t)| 2γ do depend on the lowest frequency only. This suggests to separate components corresponding to the lowest frequency, and this is exactly what we do in Lemma 3.4 and then in section 3.3, where we prove our basic decay-error estimate on ρ ε (t). This is the nonlinear core of the paper. After the estimate on ρ ε (t) has been established, the proof becomes more standard. We forget about components, and we regard both (1.1) and (1.4) as linear equations where we have frozen the nonlinear terms. At this point we introduce weighted versions of classical energies and we deduce all remaining integral and pointwise estimates on ρ ε (t), r ε (t), and their derivatives.
This paper is organized as follows. In section 2 we recall previous works, we state our main result, and we present some heuristics based on a toy model. In section 3 we prove our main result. In section 4 we state some open problems.
Previous results
In this section we recall some previous results needed in the sequel, adapting them to the special nonlinear term which appears in (1.1) and (1.4).
The first one answers what we called subproblem (P1) in the introduction. It can be easily deduced from the theory developed in [15]. We recall that an operator A is coercive if inf { Au, u : u ∈ D(A), |u| = 1} > 0.
Theorem A (Parabolic problem: global existence and decay estimates) Let H be a Hilbert space, and let A be a nonnegative self-adjoint (unbounded) operator on H with dense domain. Let γ ≥ 1 be a real number, and let u 0 ∈ D(A). Then we have the following conclusions.
(3) (Decay estimates) Let us assume that the operator A is coercive, that u 0 satisfies the nondegeneracy assumption (1.3), and that u 0 ∈ D(A k/2 ) for some integer k ≥ 2.
Then there exist positive constants C 1 and C 2 such that, for every positive integer j ≤ k, we have that The second result concerns subproblems (P3) and (P4). Existence and uniqueness were proved in [22] (see also [7]), while decay estimates were proved in this form in [9].
Theorem B (Hyperbolic problem: global existence and decay estimates) Let H be a Hilbert space, and let A be a nonnegative self-adjoint (unbounded) operator on H with dense domain. Let γ ≥ 1 be a real number, and let us assume that the initial condition (u 0 , u 1 ) ∈ D(A) × D(A 1/2 ) satisfies the nondegeneracy assumption (1.3).
Then there exists ε 0 > 0 for which the following conclusions hold true.
(1) (Existence and uniqueness) For every ε ∈ (0, ε 0 ) we have that problem (1.1), (1.2) has a unique global solution u ε in the space (2) (Decay estimates) Let us assume in addition that the operator A is coercive. Then there exist positive constants C 1 and C 2 such that, for every ε ∈ (0, ε 0 ), we have that The third and last result answers subproblem (P5). It follows from a more general result proved in [5] (see also [10]), where also weak dissipation terms are considered.
Let us assume that the operator A is coercive. Then the following conclusions hold true.
(1) If in addition we assume that (u 0 , u 1 ) ∈ D(A 3/2 ) × D(A 1/2 ), then there exist ε 1 ∈ (0, ε 0 ) and a constant C such that for every ε ∈ (0, ε 1 ) we have that (2) If in addition we assume that (u 0 , u 1 ) ∈ D(A 2 )×D(A), then there exist ε 1 ∈ (0, ε 0 ) and a constant C such that for every ε ∈ (0, ε 1 ) we have that Remark 2.1 Decay-error estimates can be obtained by combining Theorems A, B, and C with standard inequalities. For example, from previous results we know that Since min{x, y} ≤ x θ y 1−θ for every x ≥ 0, y ≥ 0, θ ∈ (0, 1), we have also that These estimates are in general nonoptimal, both for the decay rate, and for the convergence rate.
Notation and main result
Taking into account the decay rates of Theorem B, and the convergence rates of Theorem C, it is reasonable to expect decay-error estimates such as The following example shows that such an estimate cannot be true for all initial data, even in finite dimension.
Example 2.2 Let us take H := R 2 , and an operator A with two eigenvalues λ 2 0 < λ 2 1 , with corresponding eigenvectors e 0 and e 1 . Let us consider the solutions of (1.1) and (1.4) with initial data u 0 := e 1 and u 1 := e 0 . Let us write in components u ε (t) = u ε,0 (t)e 0 + u ε,1 (t)e 1 , and u(t) = u 0 (t)e 0 + u 1 (t)e 1 . Then it is easy to see that u 0 (t) ≡ 0. Moreover, from Theorem 3.3 of [6] we have that lim t→+∞ (1 + t) 1/γ |u ε,0 (t)| 2 = 1 λ 2 0 1 (2γλ 2 0 ) 1/γ , and in particular the limit is different from 0 and ε-independent. It follows that This example motivates the introduction of a class of initial data where components of u 1 correspond to frequencies greater than or equal to frequencies of components of u 0 . In order to state the condition in a general form, we need some basic facts from the spectral theory of operators, which we recall following [25].
Let E be the resolution of the identity associated with the operator A. For every measurable subset J ⊆ [0, +∞) we consider the space H J := R(E(J)), namely the range of the projection operator E(J), which is a closed subspace of H. For every µ > 0, we can therefore write H as a direct sum As a consequence, every vector v ∈ H can be written in a unique way in the form +∞) . Here subscripts refer to low and high frequencies with respect to µ. We also point out that In the case where H admits a (finite or countable) orthonormal system {e k } made by eigenvalues of A, and {λ 2 k } is the sequence of corresponding eigenvalues, then H J is just the set of all v ∈ H such that v, e k = 0 for every k ∈ N such that λ 2 k ∈ J. Moreover in this case we have that We are now ready to introduce the class of initial data which is crucial for our decay-error estimates. Definition 2.3 (Assumption on initial data) Let ν > 0 and δ 0 > 1 be two real numbers. We say that a pair of initial conditions • the low frequency component u 0,ℓ,δ 0 ν 2 of u 0 is an eigenvector of A (hence different from zero) corresponding to the eigenvalue ν 2 , • the low frequency component u 1,ℓ,δ 0 ν 2 of u 1 is a multiple (possibly equal to zero) of the corresponding component of u 0 , namely u 1,ℓ,δ 0 ν 2 = βu 0,ℓ,δ 0 ν 2 for some β ∈ R.
In other words, u 0 is the sum of an eigenvector relative to ν 2 and other components corresponding to frequencies greater than or equal to δ 0 ν 2 , while u 1 is the sum of a multiple (possibly equal to zero) of the same eigenvector and other components corresponding to frequencies greater than or equal to δ 0 ν 2 .
Remark 2.4
Let us assume that H admits a (finite or countable) orthonormal system {e k } made by eigenvalues of A, relative to an increasing sequence of positive eigenvalues. Let 0 < λ 2 0 < λ 2 1 be the two smallest eigenvalues. Let us assume that λ 2 0 is simple, and let e 0 be a corresponding eigenvector.
We point out that this assumption is always satisfied in the concrete case where Ω ⊆ R n is a connected bounded open set, H := L 2 (Ω), and Au = −∆u with Dirichlet boundary conditions. The interested reader is referred to Theorem 8.38 of [14].
We can now state the main result of this paper.
(1) If in addition we assume that (2) If in addition we assume that (u 0 , u 1 ) ∈ D(A 2 )×D(A), then there exist ε 1 ∈ (0, ε 0 ) and a constant C such that for every ε ∈ (0, ε 1 ) we have that Remark 2.6 It is possible to show that there is no improvement of decay rates when u 1,ℓ,δ 0 ν 2 is not a multiple of u 0,ℓ,δ 0 ν 2 . The example is similar to Example 2.2, just with λ 0 = λ 1 . In this case a step of the proof of Theorem 3.3 of [6] implies that Here the limit could be ε-dependent, but in any case this prevents |ρ ε (t)| 2 from decaying faster that (1 + t) 1/γ . This shows that also the third condition in Definition 2.3 is needed in order to have an improvement of decay rates.
Heuristics
A toy model for the singular perturbation problem is considering the difference between two solutions of the first order problem with two different initial data. The analogy is reasonable if we accept that the second order equation (1.1) behaves as the first order equation (1.4) when ε is small enough. Then we further simplify the model by taking H := R and A = identity. Thus we have reduced ourselves to considering the difference between two different solutions of a first order ODE.
Despite of the dramatic simplification, the toy model still reveals a rich behavior. Indeed let us consider the following four examples.
(E1) Let us examine equation u ′ + u = 0 (linear and nondegenerate). All solutions decay exponentially, and the difference between two different solutions has the same decay rate of the two solutions alone.
(E2) Let us examine equation u ′ +k(1+t) −1 u = 0 (linear and degenerate). All solutions decay with a polynomial rate, and the difference between two different solutions decays with the same polynomial rate.
Once again solutions and differences between different solutions decay with the same (exponential) rate.
(E4) Let us examine equation u ′ +|u| 2γ u = 0 (nonlinear and degenerate). Now solutions decay as (1 + t) −1/(2γ) , which is consistent with the decay rates in Theorem A and Theorem B. On the contrary, the difference between two solutions with positive data decays as (1 + t) −(2γ+1)/(2γ) . In other words, the decay rate of the difference is faster by a factor (2γ + 1).
These examples seem to suggest that the improvement of decay rates depends both on the nonlinear character and on the degeneracy of the equation. Last example suggests also that the factor (2γ + 1) in the right-hand side of (2.10) is optimal.
Let us consider now the interaction between different Fourier components. For the sake of simplicity we take H, A, λ 2 0 , λ 2 1 , e 0 , and e 1 as in Example 2.2. Then we take the solution u(t) of (1.4) with initial condition u(0) = e 0 , and the solution v(t) of (1.4) with initial condition v(0) = e 0 + e 1 .
It is easy to see that u(t) has a unique component u 0 (t)e 0 , whose coefficient satisfies Once again the solution decays as (1 + t) 1/(2γ) , and an easy computation shows that the nonlinear term is We know from Theorem A that c(t) ∼ (1 + t) −1 , hence v 0 (t) and v 1 (t) decay with a polynomial rate with exponents depending on λ 2 0 and λ 2 1 . In particular, v 1 (t) decays faster than v 0 (t), so that in the limit it is reasonable to assume that c(t) ∼ λ 2γ 0 v 2γ 0 (t). This ansatz uncouples the system, and therefore v 0 (t) becomes the solution of a single equation, the same solved by u 0 (t). This means that it is reasonable to assume Setting c(t) equal to the right-hand side of (2.11), an easy computation shows that which means that there is an improvement of the decay rate equal to λ 2 1 /λ 2 0 . This suggests that the term δ 0 in (2.10) is optimal.
Our heuristic arguments are far from being proofs, even for the toy model of the difference between two solutions of the parabolic problem. Nevertheless, we hope that they can shed some light on the improvement of decay rates, and on the reason why it should depend on some δ defined by (2.10).
Proofs
This section is devoted to the proof of Theorem 2.5. In all proofs we set We also use that the corrector θ ε (t), which is the solution of (1.6), (1.7), is given by the explicit formula In many points we need to split vectors according to the orthogonal sum (2.8). In this case v ℓ,µ and v h,µ denote the components of a certain vector v ∈ H, shortened to v ℓ and v h when µ = δ 0 ν 2 .
Due to our assumptions on initial data, all solutions lie in the space H [ν 2 ,+∞) . Therefore we can always assume, without loss of generality, that the operator is coercive, so that we can apply all the results stated in Theorems A, B, and C.
In all proofs, k 1 , k 2 , . . . are real positive constants, always independent of ε and t. We restart the numeration of constants in each proof.
Preliminaries
We recall some decay estimates for solutions of (1.1), (1.2) which are needed in the sequel. The first one concerns the faster decay of components corresponding to high frequencies. A proof is contained in Theorem 3.1 and Theorem 3.3 of [6].
Then there exist ε 1 ∈ (0, ε 0 ), and a constant M (depending also on µ), such that for every ε ∈ (0, ε 1 ) we have that The second result concerns the decay of second derivatives. The estimate deals with low frequencies, and it follows from Theorem 3.3 and Proposition 4.3 of [6]. We point out that an analogous estimate holds true without restricting to low frequencies provided that initial data are more regular, namely (u 0 , u 1 ) ∈ D(A 2 )×D(A 3/2 ), or with a constant M which depends also on ε.
Then there exist ε 1 ∈ (0, ε 0 ), and a constant M (depending also on µ), such that for every ε ∈ (0, ε 1 ) we have that Now we state and prove two results for ordinary differential equations. The first one is a simple comparison principle, which has already been used in similar forms in [5,6,7,9,10,11,12].
Then we have that z(t) ≤ ψ 2 (t) for every t ≥ 0.
Proof Let us consider the differential equation is equivalent to say that z(t) is a subsolution. On the other hand, due to the monotonicity of ψ(t), it is easy to check that w(t) := ψ 2 (t) is a supersolution of the same equation. Since z(0) = 0 < w(0), the conclusion follows from the standard comparison principle between subsolutions and supersolutions. 2 The second lemma is a comparison result for a more complex differential inequality. The assumptions on the coefficients are exactly those which are satisfied in section 3.3, where this lemma plays a crucial role. Let us assume that there exist constants M 1 , . . . , M 5 such that for every ε ∈ (0, ε 0 ) we have that For every ε ∈ (0, ε 0 ), let z ε : [0, +∞) → [0, +∞) be a function of class C 1 such that z ε (0) = 0, and Then there exist ε 1 ∈ (0, ε 0 ), and a constant M 6 such that for every ε ∈ (0, ε 1 ) we have that z ε (t) ≤ M 6 ε 2 λ 2 (t) ∀t ≥ 0. For the sake of simplicity, when no confusion is possible we omit the dependence on ε, and sometimes also the dependence on t, when writing z ε (t), ψ i (ε, t), Ψ i (ε, t). In any case all constants we introduce are independent of ε and t.
Estimates on the parabolic equation
In this section we collect the estimates on the parabolic equation, not contained in Theorem A, which are needed in the proof of our main result.
The first one is an estimate on second derivatives. In particular, estimate (3.24) is in some sense the parabolic counterpart of (3.5). Here we do not need to restrict to low frequencies because u 0 ∈ D(A 2 ). Then for every δ ∈ (0, 2γ + 1] we have the following conclusions. (1) If u 0 ∈ D(A 3/2 ), then there exists a constant M such that Proof Let us set for simplicity η := δ/γ. Since δ ≤ 2γ + 1, it follows that η ≤ 2 + 1/γ, hence for every t ≥ 0 we have that Basic integral estimates We prove that when u 0 ∈ D(A 3/2 ) we have that and when u 0 ∈ D(A 2 ) we have that To this end, an easy calculation shows that d dt Now we integrate in [0, t], and then we apply the estimate from above in (2.1) with j = 3, and finally estimate (3.25). We obtain that Applying the estimate from below in (2.1) with j = 1, we therefore deduce that which proves (3.26). The proof of (3.27) is analogous (one just needs to add 1/2 to all powers of the operator A).
In this case the solution u(t) of problem (1.4), (1.5) can be written in the form u(t) = u ℓ (t) + u h (t), where u ℓ (t) and u h (t) are the solutions of the linear problems where of course c(t) is given by (3.1). Since u 0,ℓ is an eigenvector of A, it is easy to see that the solution of (3.30) is given by the explicit formula u ℓ (t) = u 0,ℓ e −ν 2 C(t) ∀t ≥ 0.
The estimate from below is trivial. In order to prove the estimate from above, let us consider the well known inequality Setting x := |A 1/2 u ℓ (t)| 2 , and y := |A 1/2 u h (t)| 2 , we obtain that Exploiting (2.1) with j = 1, and (3.37) with j = 1, we obtain that Since δ 0 > 1, integrating in [0, t] we deduce the estimate from above in (3.38).
Proof of key decay-error estimate
This section is the key step in the proof of Theorem 2.5. Here we show that Let c(t), c ε (t), and components of vectors be defined as in the first paragraph of section 3. Let Φ : [0, +∞) → [0, +∞) be the function defined by (3.32). Let us set for simplicity η := δ/γ, and let We claim that z ε (t) satisfies a differential inequality as in Lemma 3.2. If we prove this claim, then from that lemma it follows that On the other hand, the estimate from below in (3.34) implies that From (3.41) and (3.42) we easily conclude (3.40). Thus we can limit ourselves to show that z ε (t) satisfies the assumptions of Lemma 3.2. To this end, we first observe that ρ ε (t) is the solution of the first order equation with initial condition ρ ε (0) = 0. Therefore we have that z ε (0) = 0, and (3.43) The term L 1 is z ε (t) times a coefficient which behaves like (1 + t) −1 , hence whose integral is divergent. Therefore this term alone would prevent (3.41) from being true. Thus the idea is to cancel out L 1 by means of L 2 and one of the terms arising from the expansion of L 3 . In the following paragraphs we carry out this program.
Estimate of L 3 We claim that (3.47) We point out that the first term cancels out the right-hand side of (3.44).
Now we claim that
from which (3.47) follows directly. The proof of (3.51) through (3.53) is the content of the next three paragraphs.
Estimate of L 3,1 From the second order Taylor's expansion of the function σ γ it follows that Setting x := |A 1/2 u(t)| 2 and y := |A 1/2 u ε (t)| 2 , we obtain that Now from (2.1) with j = 1 and (2.3) we have that (note that in (2.1) and (2.3) we need both the estimates from below and the estimates from above because we ignore the sign of γ − 2). From (2.1) with j = 2 and (2.4) we have that The last two terms can be easily estimated exploiting (2.4) and the estimate from below in (3.34). We thus obtain (3.51).
Estimate of L 3,3 First of all we have that
(3.60) Since u 0,ℓ and u 1,ℓ are multiples of an eigenvector of A, it is easy to see that both u ε,ℓ (t) and u ℓ (t) are multiples of the same eigenvector, and the same for ρ ε,ℓ (t). Therefore the vectors A 1/2 ρ ε,ℓ (t) and A 1/2 u ℓ (t) are parallel, hence the square of their scalar product is equal to the square of the product of their norms (this is the point where the last condition in the (ν, δ 0 )-assumption plays a crucial role). It follows that On the other hand, exploiting (2.1) with j = 1 and j = 2, and (3.33), we have that (3.62) Plugging (3.61) and (3.62) into (3.60), and recalling that δ ≤ δ 0 , we obtain (3.53).
Estimate of L 4 Let us fix µ := 8γν 2 . Splitting components corresponding to low and high frequencies with respect to µ, we have that Indeed from Proposition E we have that so that the estimate from above in (3.34) implies that Since we have proved (3.64).
Let us prove the estimate from above in (3.8). Since δ > 1 we have that +∞ 0 1 (1 + s) 1+(δ−1)/(2γ) ds ≤ k 28 , and this settles the integral of the third term in the definition of ψ 1 (ε, t). For the integral of the second term, we exploit that λ(t) ≥ 1, and with the variable change σ = s/ε we obtain that It remains to estimate the integral of the first term. If δ < 2γ + 1 we have that = log(log(e + t)) = log λ(t).
In both cases we have proved (3.8).
In order to prove (3.10), we consider the integral The second integral can be estimated as in (3.66). The first integral is less than a constant if δ < 2γ + 1, and equal to log(e + t) = λ(t) if δ = 2γ + 1. In both cases this proves (3.10).
It remains to prove (3.11), and this is the content of the last paragraph.
Estimates on linear equations
Let us define c ε (t) and c(t) as in (3.1), and let us set Then it is easy to see that ρ ε (t) is the solution of the linear equation with initial data while r ε (t) is the solution of the linear equation with initial data r ε (0) = 0, r ′ ε (0) = 0. (3.81) In this section we forget that g ε (t) is given by (3.77), and that c ε (t) and c(t) are given by (3.1). We just regard ρ ε (t) and r ε (t) as solutions of the corresponding linear equations (which implies also that ρ ε (t) = r ε (t) + θ ε (t), where θ ε (t) given by (3.2)).
Let us assume that ρ ε (t) satisfies the a priori estimate (3.40). Then the following conclusions hold true.
Proof Let us consider the following weighted versions of classical energies Exploiting (3.78) and (3.80), with some computations we obtain that First energy estimate We prove that To this end, from (3.88) we have that (3.91) Let us estimate the four terms. From (3.40) we have that (3.92) From (3.82) we deduce that c ε (t) is bounded. Therefore, using also the explicit expression (3.2) for θ ε (t), from (3.92) we obtain that From (3.40) we obtain also that Let us estimate some of the terms in the right-hand side. Exploiting the fact that c ε (t) is bounded, the explicit formula (3.2) for θ ε (t), and the fact that λ(t) ≥ 1, for the first term we obtain that In the third term of (3.95) we make the variable change σ = s/ε, and we obtain that Moreover, we estimate the fourth term of (3.95) by means of (3.87), and the fifth by means of (3.84). Finally, the estimate from below in (3.82) implies that t 0 (1 + s) 2η−1 |A 1/2 ρ ε (s)| 2 ds ≤ k 16 t 0 c ε (s)(1 + s) 2η |A 1/2 ρ ε (s)| 2 ds.
Conclusion of proof of Theorem 2.5
Let c ε (t) and c(t) be defined by (3.1). Let g ε (t) be defined by (3.77). We already know that (3.40) holds true. Thanks to Proposition 3.5, it is enough to show that assumptions (3.82) through (3.87) are satisfied. Assumption (3.87) is trivial when λ(t) ≡ 1, and follows from a simple integration by parts when λ(t) = log(e + t).
Open problems
The main open problems in the theory of dissipative Kirchhoff equations have been stated in the last section on [13]. In particular, this paper gives a partial answer to the sixth problem presented therein.
Here we state some open questions which are more closely related to the specific degenerate nonlinearity considered in this paper.
• Open problem 1. In the case where δ 0 ≥ 2γ + 1, is the term λ(t) really needed in the estimates of Theorem 2.5?
• Open problem 2. Determine the better decay-error estimates which are true without the (ν, δ 0 )-assumption on initial data. We suspect that nothing more than (2.6) can be true for general data.
• Open problem 3. Is it possible to extend the theory to the case γ ∈ (0, 1)?
• Open problem 4. Is it possible to extend the theory to weak dissipation terms of the form (1 + t) −p u ′ ε (t) with p ≤ 1? | 2011-08-18T14:15:53.000Z | 2008-11-15T00:00:00.000 | {
"year": 2011,
"sha1": "4d9d599edf3d4b2243b27378723c1156997f8367",
"oa_license": null,
"oa_url": "http://arxiv.org/pdf/1108.3758",
"oa_status": "GREEN",
"pdf_src": "Arxiv",
"pdf_hash": "bc8d8e513ef6c7ec3dc5e27b602c4120120b0a6c",
"s2fieldsofstudy": [
"Mathematics"
],
"extfieldsofstudy": [
"Mathematics"
]
} |
201208902 | pes2o/s2orc | v3-fos-license | Nonconsumptive effects of hunting on a nontarget game bird
Abstract Human hunting activity and disturbance can significantly impact prey species through both consumptive and nonconsumptive effects. The nonconsumptive effects of rabbit hunting on Northern Bobwhite (Colinus virginianus; hereafter, bobwhite) are currently unknown. Increased perceived risk of predation by bobwhite during rabbit hunting events may elicit antipredator responses among bobwhite that impact fitness via changes in behavior that ultimately impact population growth. We estimated the nonconsumptive effects of rabbit hunting on bobwhite behavior using telemetry across varying rabbit hunting intensities. Movements were analyzed using Bayesian hierarchical modeling with a before‐after‐control‐impact (BACI) design to determine the effect of rabbit hunting on bobwhite. We observed an overall reduction in bobwhite movement in the presence of rabbit hunting, with a 38% (Posterior Overlap = 0.01) increase in bobwhite step length in the absence of rabbit hunting. We also observed bobwhite maintaining closer proximity to hardwood and escape cover under high rabbit hunting intensity, with a 59% (Posterior Overlap = 0.03) increase in distance from hardwood and a 28% (Posterior Overlap = 0.14) increase in distance from escape cover when rabbit hunting was removed. Synthesis and applications. Heightened antipredator behavior through decreased movement may assist with bobwhite predator avoidance. However, decreased movement and increased use of poor habitats may also have negative effects as a result of reduced foraging time or increased susceptibility to other predators. Future research should attempt to quantify the effect of decreased movement on bobwhite fitness through the evaluation of foraging time and survival in order to continue to improve management efforts for the species.
| INTRODUC TI ON
Understanding predator-prey dynamics and the complexity of community ecology is critical for effective management and wildlife conservation. Anthropogenic disturbances can have profound effects on wildlife by eliciting fear and altering behavior (Gaynor, Hojnowski, Carter, & Brashares, 2018). This fear is often evoked indirectly through auditory and visual anthropogenic cues (e.g., vehicles and construction; Smith et al., 2017). It has been recognized that such human-driven disturbances can have cascading effects across food webs (Smith et al., 2017;Smith, Wang, & Wilmers, 2015).
Within many systems, human hunting activity can exert a greater threat to prey than natural predators (Allendorf & Hard, 2009;Darimont et al., 2009). Additionally, human hunters are unique apex predators as they are bound by regulations and bias that often dictate prey habitat use and spatial patterns (Asmyhr, Willebrand, & Hörnell-Willebrand, 2013;Cromsigt et al., 2013;Lone et al., 2014;Stillfried, Belant, Svoboda, Beyer, & Kramer-Schadt, 2015). Hunting disturbance is known to cause antipredator behavior in prey as a way to mitigate the risk of predation (Lima & Bednekoff, 1999). Prey species attempt to mitigate these risks through behaviors such as altered habitat use and increased vigilance (Creel, Winnie, Maxwell, Hamlin, & Creel, 2005;Clinchy et al., 2016;Embar, Raveh, Burns, & Kotler, 2014;Frid & Dill, 2002;Lima & Dill, 1990). However, antipredator behavior can have fitness costs in terms of reduced foraging effort, greater net energy loss, and increased vulnerability to other predators (Lima & Bednekoff, 1999;Morrison, 1999;Downes, 2001;Eklov & Van Kooten, 2001;Creel, 2018). Though ecological theory addresses both direct and indirect and lethal and nonconsumptive effects of predation, wildlife management often focuses specifically on lethal consumptive effects (Preisser, Orrock, & Schmitz, 2007). However, nonconsumptive effects of hunting can at times be stronger than lethal effects through altered prey behavior (Clinchy et al., 2016;Frid & Dill, 2002;Lima & Bednekoff, 1999). The threat sensitivity hypothesis states that "individuals will trade-off predator avoidance against other activities by altering their avoidance responses in a manner that reflects the magnitude of the predatory threat" (Helfman, 1989). Prey species' perception of predation risk and subsequent decisions are not necessarily a result of direct predator confrontation, but often the result of indirect landscape cues (Schmidt & Kuijper, 2015). With regards to hunting, prey are exposed to numerous indirect cues such as the auditory cues of hunting dogs and firearms and increased landscape disturbance within these areas (Ciuti et al., 2012;Cromsigt et al., 2013). Preys' response to their perception of risk alone may be powerful enough to affect wildlife population dynamics (Zanette, White, Allen, & Clinchy, 2011). Therefore, it is important to have a thorough understanding of nonconsumptive hunting impacts on prey species (Kotler & Holt, 1989).
Nonconsumptive impacts of hunting may be particularly important for understanding the fitness of nontarget game and nongame wildlife. Many game and nongame species are managed in landscapes that often include other game species. As a result, hunting and management activities targeted at one species may have negative nonconsumptive effects on other species. This effect may be amplified if the nontarget prey is also a game species and cannot distinguish whether it is the intended target of predation. Prey response to the threat of predation may be directly correlated with its perception of the encounter (Blumstein, 2003;Edgar, Paul, & Nicol, 2013). While there has been a substantial increase in the study of direct and indirect effects of hunting on prey (Gosselin, Zedrosser, Swenson, & Pelletier, 2014;Lone, Loe, Meisingset, Stamnes, & Mysterud, 2015;McGrath, Terhune, & Martin, 2018;Stillfried et al., 2015), the nonconsumptive effects of hunting on nontarget prey species are understudied. (Brennan, 1991;Hernández, Brennan, DeMaso, Sands, & Wester, 2013;Stoddard, 1931). Broad-scale changes in land use including modern agricultural and forestry practices have resulted in habitat loss that is the primary cause of this decline (Brennan, 1991;Guthery, Peterson, & George, 2000;Williams, Guthery, Applegate, & Peterson, 2004). Bobwhite popularity as a game species, in conjunction with their declines, has made the species a conservation priority and prompted their designation as a flagship species for many upland and grassland bird communities (Brennan & Kuvlesky, 2005;Crosby, Elmore, Leslie, & Will, 2015).
The effect of rabbit hunting on bobwhite antipredator behavior is currently unknown but is a management concern due to the fact that both species coincide in the same habitats and have concurrent hunting seasons. Increased perceived risk of predation by bobwhite during rabbit hunting events may elicit antipredator behavior or alterations to other behavior that, in turn, can affect demography and factors related to hunt quality (e.g., encounter rates). We hypothesized that bobwhite respond to cues during rabbit hunting as a direct threat and mitigate the perceived threat following the predictions of the threat sensitivity hypothesis (Helfman, 1989). We tested this hypothesis by subjecting bobwhites to varying levels of rabbit hunting and predicted that behavior would change across this gradient. We relied on expert knowledge and the literature on bobwhite (McGrath et al., 2018) to make more specific predictions under this theoretical framework to help uncover possible behavioral patterns and strategies. Specifically, we predicted that bobwhites may mitigate risk by reducing movement (i.e., shorter step length and contracted space use) under greater rabbit hunting or increase movement because of more frequent threat encounters. Bobwhite may use Protean behavior which involves more erratic movement patterns in an effort to avoid predation (Jones, Jackson, & Ruxton, 2011), which would be reflected in increased path tortuosity of movement. Moreover, we hypothesized bobwhite would follow the risky space hypothesis (Creel, Winnie, Christianson, & Liley, 2008;Cresswell & Quinn, 2013;Laundré, Hernández, & Altendorf, 2001) by maintaining closer proximity to escape cover (e.g., scrub/ shrubs) and hardwoods where there is less hunting pressure but also poorer quality resources (Lima, 1993;Lohr, Collins, Castelli, & Williams, 2010;Wilhite, 2019).
| Study site
The research was conducted on Di-Lane Wildlife Management Area (WMA), located in Waynesboro, Georgia. The study area encompassed roughly 3,278 ha with ~2,023 ha managed for bobwhite by the Georgia Department of Natural Resources since 1993. Prior to its designation as a public WMA, the property was a private quail plantation and agricultural farm. Habitat management for bobwhite also benefits rabbits, leading to robust populations of both species on the site.
| Experimental design
The study involved a before-after-control-impact (BACI) design (Stewart-Oaten, Murdoch, & Parker, 1986;Underwood, 1994). During the before period of 2016-2017, rabbit hunting opportunities were homogenous across the study site with hunting occurring 5 days per week. This was followed by an after period during 2017-2018 in which rabbit hunting treatments were assigned across the study site in a randomized complete block design (i.e., three levels of rabbit hunting and two spatial blocks). The treatments included 0 days of hunting per week ("No Rabbit Hunting"), 3 days of hunting per week ("Reduced Hunting"), and 5 days of hunting per week ("Reference"), with replicates of each treatment ( Figure 1). The BACI design allows for treatment impacts to be distinguished from background time effects shared by all the replicates and background differences between the treatment and reference sites (Popescu, de Valpine, Tempel, & Peery, 2012).
| Bobwhite trapping and processing
Walk-in funnel traps baited with sorghum were used to capture bobwhite (Stoddard, 1931). Trapping occurred both years from early October to early November, prior to rabbit hunting season.
Traps were uniformly spaced 250-300 m apart across the property within dense cover. All unique individuals captured were weighed, sexed, aged, and given unique number leg bands (National Band & Tag Company) when processed. Individuals captured that weighed ≥130 g were fitted with Very High Frequency (VHF) radio tags from Holohil Systems and American Wildlife Enterprises. During fall and winter, bobwhite congregate in social groups called coveys (DeMaso, Guthery, Spears, & Rice, 1992;Williams, Lutz, & Applegate, 2003).
Caution was taken to not deploy more than 6 radio tags per covey to allow for uniform dispersion of tags across the study site and rabbit hunting treatments. Within a given trapping session, between 150 and 170 radio tags were deployed. Each tag had an estimated battery life of 10-12 months and was equipped with mortality signals. If tagged individuals remained inactive for >12 hr, the signals switched to a rapid mortality beep indicating the individual was deceased.
| Radio telemetry of bobwhite
Bobwhite coveys were monitored to capture fine-scale movement patterns using radio telemetry. Coveys were tracked from approximately 30 min prior to sunrise to approximately 30 min after sunset to capture movement during active periods. Locations were determined via homing telemetry (Amelon, Dalton, Millspaugh, & Wolf, 2009;White & Garrott, 1990) at 30-min intervals. Approximate locations of coveys were taken roughly 20-30 m from the observer to minimize disturbance, with an estimated error rate of 12 m. Global positioning systems (GPS) were used to note observer locations, and compasses were used to note the azimuth to the covey. Intensive telemetry occurred once on all coveys fitted with radio tags during the scope of the study, with individuals being tracked on either a rabbit hunting or bobwhite hunting day throughout the duration of both hunting seasons. Coveys were selected for tracking through random number generation to reduce observer bias.
F I G U R E 1 Rabbit hunting treatments on Di-Lane Wildlife Management Area, located in Waynesboro, GA. Treatment 1 (No Rabbit Hunting) indicates 0 days of rabbit hunting per week, Treatment 2 (Reduced) indicates 3 rabbit hunting days per week, and Treatment 3 (Reference) indicates 5 rabbit hunting days per week. The gridded area specifies the safety zone in which no hunting can occur
| Statistical analysis
Statistical analyses were performed using R (R Core Team, 2017).
Bobwhite movement data from November 2016 to February 2017 were analyzed for the before-time period, and data from November 2017 to February 2018 were analyzed for the after-time period to encompass the full duration of the rabbit hunting seasons. We assumed coveys remained in their respective rabbit hunting treatments for the duration of the treatment year due to the size of the treatments (range: 218.65-383.45 ha) and the decreased dispersal of bobwhite during the overwintering months (Townsend et al., 2003). ). An assessment of map accuracy was conducted by generating 100 equally distributed random points across each land cover type of interest and generating an error matrix compared to ground-truthed values. Map accuracy was determined to be 100% for hardwoods and 77.41% for scrub/shrub vegetation. To determine bobwhite use of hardwood and scrub/shrub, the Euclidean distance from each individual location to the nearest example of that habitat feature was calculated.
| Bayesian hierarchical modeling and BACI ratio
We used a Bayesian hierarchical modeling approach within the jag-sUI package (Kellner, 2018) of R (R Core Team, 2017) to estimate the effects of the treatment and reference groups before and after the implementation of rabbit hunting treatments using the model described below. We assumed normal distributions for the random effects with a mean of 0 and vague gamma-distributed precision terms (1/variance). We used vague normal priors for the fixed effects with a mean of 0 with small precision (0.001). The model was and that all chains converged. We checked chain convergence using the Gelman-Rubin statistic, R-hat, which compared between and within chain variation (Gelman, Carlin, Stern, & Rubin, 2004). R-hat values below 1.1 indicate convergence. Values of all estimated parameters had an R-hat value of 1.1 or below.
To estimate effects of the BACI study, we calculated posterior distributions for BACI ratios to determine the change in metrics in response to treatments while accounting for pretreatment differences. The BACI ratio was calculated using the ratio of pretreatment differences, R t|ref Before = M Treatment before /M Reference before , where M is the posterior distribution for a respective metric and then doing the same with the after-treatment differences, R t|ref After = M Treatment after /M Reference after . We then estimated the posterior distribution of the treatment effect of each trajectory metric as R M BACI = R t|ref After /R t|ref Before . That is, for each MCMC sample, we calculated the mean ratio from the treatments for each trajectory metric after the rabbit hunting treatments were implemented and the mean ratio for the treatments for each trajectory metric before the rabbit hunting treatments were implemented and divided them. We estimated the mean, 2.5 and 97.5 percentiles for the distribution of each R M BACI . BACI ratios deviating from 1 indicate an effect of the treatment implementation, with values >1 indicating a positive relationship and values <1 representing a negative relationship. Percent change is determined by subtracting 1 from the BACI ratio multiplied by 100 for positive effects or 1 minus the BACI ratio multiplied by 100 for negative effects.
For example, a BACI ratio of 1.30 would result in a 30% increase in the tested metric, while a BACI ratio of 0.70 would result in a 30% decrease. Posterior overlap was used to denote the effect of a movement metric, which we here define as the amount of the posterior distribution that overlaps 1 for the BACI ratios.
| Movement summary
Bobwhite coveys were naturally dispersed across the study site both years this, as well as the random selection of the treatment areas, resulted in the varying levels of coveys in each treatment (Table S1). We collected daily movement tracks from all 73 unique tagged Table S2. After the implementation of rabbit hunting treatments, 122 rabbit hunters were reported at Di-Lane from hunter sign-in sheets, with 35% of hunts occurring in the Reduced treatment and 64% occurring in the Reference treatment.
| Hypotheses results
In general, there was support for the threat sensitivity hypothesis. We observed an overall decrease in bobwhite movement in the presence of rabbit hunting indicating partial support for the increased antipredator behavior threat sensitivity prediction (Figures 2 and 3). When rabbit hunting was reduced to 3 hunting days, trajectory distance, or overall daily movement, would decrease 3% when compared to the Reference treatment (Posterior Overlap = 0.16, Table S3, Figure 2). When compared to the Reference, when rabbit hunting was absent, bobwhite increased their trajectory distance by 8% (Posterior Overlap = 0.04, Table S3, Figure 2). Trajectory distance was also increased 11% when rabbit hunting was further reduced from 3 hunting days to 0 days (Posterior Overlap = 0.01, Table S3, Figure 2). Similarly, in the absence of rabbit hunting, bobwhite increased their step length by 38% when compared to the Reference (Posterior Overlap = 0.01, Table S3, Figure 3). Reducing rabbit hunting to 0 days from 3 days increased step length 33% (Posterior Overlap = 0.01, Table S3, Figure 3). Similarly, track straightness decreased 38% when rabbit hunting was reduced (Posterior Overlap = 0.01, Table S3, Figure 4). Track straightness decreased 32% when rabbit hunting decreased from the Reference to 0 days (Posterior Overlap = 0.04, F I G U R E 2 BACI contrasts with shaded regions representing the full posterior distribution for Northern Bobwhite (Colinus virginianus) trajectory distance in relation to rabbit hunting. Points indicate mean values for trajectory distance, with lines representing the 50% and 95% credible interval. BACI contrasts above 1 indicate an increase in trajectory distance while BACI contrasts below 1 indicate a decrease. Reference-Reduced denotes the effect of reducing rabbit hunting from 5 to 3 days. Reduced-No Rabbit Hunting denotes the effect of decreasing rabbit hunting from 3 to 0 days. Reference-No Rabbit Hunting denotes the effect of decreasing rabbit hunting from 5 to 0 days F I G U R E 3 BACI contrasts with shaded regions representing the full posterior distribution for Northern Bobwhite (Colinus virginianus) step length in relation to rabbit hunting. Points indicate mean values for step length, with lines representing the 50% and 95% credible interval. BACI contrasts above 1 indicate an increase in step length while BACI contrasts below 1 indicate a decrease. Reference-Reduced denotes the effect of reducing rabbit hunting from 5 to 3 days. Reduced-No Rabbit Hunting denotes the effect of decreasing rabbit hunting from 3 to 0 days. Reference-No Rabbit Hunting denotes the effect of decreasing rabbit hunting from 5 to 0 days Figure 4). Furthermore, bobwhite would decrease their straight-line track distance 25% when rabbit hunting was reduced to 3 days per week indicating a more tortuous path (Posterior Overlap = 0.01, Table S3, Figure 5).
The risky space hypothesis was supported because bobwhite increased their distance from hardwoods and escape cover (scrub/ shrubs) when rabbit hunting was absent when compared to the Reference treatment. Reducing rabbit hunting from 5 to 3 days yielded a 54% increase in bobwhite distance from hardwoods (Posterior Overlap = 0.01, Table S3, Figure 6). There was a 59% increase in distance from hardwoods when rabbit hunting was absent compared with the Reference (Posterior Overlap = 0.03, Table S3, Figure 6).
There was no difference between the Reduced rabbit hunting and No Rabbit Hunting treatment (Posterior Overlap = 0.51). Relatedly, there was a 22% increase in distance from escape cover when rabbit hunting was reduced from 5 to 3 days (Posterior Overlap = 0.13, Table S3, Figure 7). Similarly, distance from scrub/shrub escape cover also increased when rabbit hunting was removed compared to the Reference, with a distance increase of 28% (Posterior Overlap = 0.14,
| D ISCUSS I ON
We found general support for the threat sensitivity and risky space hypotheses whereas the Protean behavior hypothesis was mostly unsupported. Specifically, bobwhite increased movement, path tortuosity, and distance from escape cover in the absence of rabbit hunting. This increase in movement and distance from escape cover within areas with no rabbit hunting may indicate that bobwhites were able to properly perceive lower risk of predation within these treatments and behave accordingly. The use of the F I G U R E 4 BACI contrasts with shaded regions representing full posterior distribution for Northern Bobwhite (Colinus virginianus) path straightness in relation to rabbit hunting. Points indicate mean values for path straightness, with lines representing the 50% and 95% credible interval. BACI contrasts above 1 indicate an increase in path straightness while BACI contrasts below 1 indicate a decrease. Reference-Reduced denotes the effect of reducing rabbit hunting from 5 to 3 days. Reduced-No Rabbit Hunting denotes the effect of decreasing rabbit hunting from 3 to 0 days. Reference-No Rabbit Hunting denotes the effect of decreasing rabbit hunting from 5 to 0 days F I G U R E 5 BACI contrasts with shaded regions representing the full posterior distribution for Northern Bobwhite (Colinus virginianus) straight-line track distance in relation to rabbit hunting. Points indicate mean values for straight-line track distance, with lines representing the 50% and 95% credible interval. BACI contrasts above 1 indicate an increase in straight-line track distance while BACI contrasts below 1 indicate a decrease. Reference-Reduced denotes the effect of reducing rabbit hunting from 5 to 3 days. Reduced-No Rabbit Hunting denotes the effect of decreasing rabbit hunting from 3 to 0 days. Reference-No Rabbit Hunting denotes the effect of decreasing rabbit hunting from 5 to 0 days F I G U R E 6 BACI contrasts with shaded regions representing the full posterior distribution for Northern Bobwhite (Colinus virginianus) distance from hardwood in relation to rabbit hunting. Points indicate mean values for distance from hardwood, with lines representing the 50% and 95% credible interval. BACI contrasts above 1 indicate an increase in distance from hardwood while BACI contrasts below 1 indicate a decrease. Reference-Reduced denotes the effect of reducing rabbit hunting from 5 to 3 days. Reduced-No Rabbit Hunting denotes the effect of decreasing rabbit hunting from 3 to 0 days. Reference-No Rabbit Hunting denotes the effect of decreasing rabbit hunting from 5 to 0 days BACI design allowed us to reduce the possibility of confounding effects of site and annual variation while overcoming the constraints of implementing large-scale manipulations such as cost and area restrictions.
Increased antipredator behavior by bobwhite was observed within treatments with rabbit hunting pressure through an overall reduction in movement, indicating partial support for the increased antipredator behavior threat sensitivity prediction stemming from the threat-sensitive hypothesis. The observed decline in movement was similar between the Reference and Reduced treatments, indicating similar reactions to the presence of rabbit hunting regardless of the number of hunting days. The increased antipredator behavior observed can be described by a common bobwhite antipredator behavior known as holding, which allows individuals to remain cryptic within their landscape (Stoddard, 1931). The two most well-known antipredator responses of bobwhite are holding and flushing behavior (Stoddard, 1931). The behavior used depends on the individual's perception of the threat. While holding behavior allows individuals to remain camouflaged in their environment, bobwhite tend to flush when they assess a high-risk situation (McGrath et al., 2018).
Flushing is reserved for immediate danger, as it is the most energetically costly and has the largest fitness cost to the individual by decreasing activities such as feeding and increasing visibility to predators (Ydenberg & Dill, 1986).
This evaluation is not necessarily a result of direct predator confrontation, but indirect landscape cues (Schmidt & Kuijper, 2015) such as the auditory cues of hunting dogs (Ciuti et al., 2012;Cromsigt et al., 2013). While both rabbit hunting and quail hunting involve the use of hunting dogs, rabbits are often hunted with hounds (Lowe, 1958), whereas quail are hunted with pointing dogs (McGrath et al., 2018). Many studies have found that loud noise can elicit antipredator behavior, as it is considered a threatening stimulus (Frid & Dill, 2002;Goldstein et al., 2005;McRoberts et al., 2011). In the case of our study, the use of hound dogs, who often howl, for rabbit hunting may provide bobwhite with an auditory cue pertaining to the level of risk on the landscape. This assessment of risk indicates support for the threat sensitivity hypothesis demonstrating that bobwhite are able to perceive an increase in hunting pressure within the respective areas and discern that while rabbit hunting is perceived as a threat, it does not require immediate energetically costly escape tactics.
Bobwhite did not show an increase in erratic movement, or Protean behavior, as a result of rabbit hunting pressure. Decreased path tortuosity within the Reference treatment may be a product of the specific predator present, as predator type determines antipredator escape tactics (Perkins, Boal, Rollins, & Perez, 2014).
Recent literature has demonstrated an increase in bobwhite path tortuosity as a result of evading avian predators when compared to terrestrial threats, such as hunters (Perkins et al., 2014). Moreover, it has been suggested that Protean behavior is energetically costly and may only be utilized if prey are directly targeted by a predator (Jones et al., 2011). These findings reinforce our assessment that bobwhite use other strategies to mediate the risk of rabbit hunting instead of Protean behavior. Increased path tortuosity in the absence of rabbit hunting may be due to an increase in foraging behavior, which also results in tortuous paths, and not Protean behavior. McGrath et al. (2018) found that increased hunting pressure resulted in a decline in bobwhite foraging behavior. The decreased hunting pressure in the absence of rabbit hunting may explain the observed increase in path tortuosity within these areas, allowing bobwhite to decrease antipredator behavior and forage more freely. However, further examination of this subject is needed as current data do not allow us to discern between foraging and particular types of predator avoidance behavior that may be affecting path tortuosity.
Bobwhite were found to increase their distance from escape cover and hardwoods when rabbit hunting pressure decreased, indicating support for the risky space hypothesis. These findings suggest that bobwhite maintained closer proximity to hardwoods and scrub/shrub escape cover as a result of high rabbit hunting pressure. In general, prey has an increased perception of predation risk as their distance from refugia increases and as predator abundance increases (Stankowich & Blumstein, 2005). Perkins et al. (2014) found that bobwhite pursued by hunters tended to use greater cover and that the auditory threat stimulus of gunshots and hunting dogs may elicit this response. Therefore, the presence of rabbit hunters on the landscape may have prompted bobwhite to maintain closer proximity to refuge, even when not being directly pursued. Bobwhite used poorer habitat (e.g., hardwoods) to mediate the perceived higher relative risk on the landscape with the increase in rabbit hunting pressure. Increased use of poor habitat during high rabbit hunting pressure may be a result of a trait-mediated effect (Cresswell, 2008). Trait-mediated effects can have negative effects on individual fitness through reduced F I G U R E 7 BACI contrasts with shaded regions representing the full posterior distribution for Northern Bobwhite (Colinus virginianus) distance from scrub/shrub in relation to rabbit hunting. Points indicate mean values for distance from scrub/ shrub, with lines representing the 50% and 95% credible interval. BACI contrasts above 1 indicate an increase in distance from scrub/shrub while BACI contrasts below 1 indicate a decrease. Reference-Reduced denotes the effect of reducing rabbit hunting from 5 to 3 days. Reduced-No Rabbit Hunting denotes the effect of decreasing rabbit hunting from 3 to 0 days. Reference-No Rabbit Hunting denotes the effect of decreasing rabbit hunting from 5 to 0 days foraging time or forcing individuals from a profitable area due to predation risk (Cresswell, 2008). Being forced from profitable areas to poorer habitats, such as hardwoods, may also increase susceptibility to predation by other species. Kotler (1984) demonstrated that species of desert rodents least vulnerable to predators tended to forage in open areas, whereas the most vulnerable species were restricted to covered areas. Individuals who tend to avoid areas of high-risk exposure to a predator would then spend their lives in energetically poor habitats (Abrahams & Dill, 1989).
In the case of a species with a short life expectancy, such as bobwhite, this is not a suitable option. Therefore, when individuals utilize these poor habitats, they may be more likely to engage in uncertain or risk-sensitive foraging behavior to ensure they receive the necessary nutrients for survival (Abrahams & Dill, 1989).
The increase in distance from refuge observed within the Reduced and No Rabbit Hunting treatments would therefore be an effect of reduced hunting pressure on the landscape, resulting in less restricted movement.
Taken collectively our results indicate that bobwhite adjust their movements to the spatial patterns of rabbit hunters to mediate the perceived predation risk. Bobwhite decreased overall movement in areas in which rabbit hunting occurred regardless of the number of hunting days. Nevertheless, the full extent of the effect of rabbit hunting on bobwhite still needs to be investigated. Heightened antipredator behavior through decreased movement may assist with bobwhite predator avoidance by taking advantage of their cryptic coloration in the landscape (Stoddard, 1931). However, decreased movement and increased use of poor habitats such as hardwoods may also have negative effects as a result of reduced foraging time or increased susceptibility to other predators. Additionally, heightened use of antipredator behavior in avian species has been shown to decrease fitness through a decline in clutch survival and fecundity (Dudeck, Clinchy, Allen, & Zanette, 2018;Zanette et al., 2011). Future research should attempt to quantify the effect of decreased movement on bobwhite fitness through the evaluation of foraging time and survival.
Research should also focus on the physiological effects of rabbit hunting on bobwhite through the examination of stress hormones to help assess the effects of rabbit hunting on bobwhite fitness and subsequent population dynamics.
ACK N OWLED G M ENTS
We would like to thank the Georgia Department of Natural Resources and Warnell School of Forestry and Natural Resources for the financial support over the course of this project (grant number W-GA-F16AF00189). We thank our many technicians: Jamie L. Goethlich, Melissa L. Maleckar, Ravin R. Grove, Collin J. Richter, Joseph D. King, Lacey L. Clarke, and Jake G. Spears.
CO N FLI C T O F I NTE R E S T
None declared.
AUTH O R CO NTR I B UTI O N S
JM, IP, and JM conceived the ideas and designed methodology; JM, NW, and RG collected the data; JM and JM analyzed the data; JM and JM led the writing of the manuscript. All authors contributed critically to the drafts and gave final approval for publication.
DATA AVA I L A B I L I T Y
Data were archived using Dryad, https ://doi.org/10.5061/dryad. b3k21db. | 2019-08-23T06:03:57.073Z | 2019-07-30T00:00:00.000 | {
"year": 2019,
"sha1": "24884927adf5c93f33ab49befb4d64a2dd865e83",
"oa_license": "CCBY",
"oa_url": "https://onlinelibrary.wiley.com/doi/pdfdirect/10.1002/ece3.5479",
"oa_status": "GOLD",
"pdf_src": "PubMedCentral",
"pdf_hash": "77f79199fd6e62b3c8050135bbeaa1c44bbca859",
"s2fieldsofstudy": [
"Biology"
],
"extfieldsofstudy": [
"Biology",
"Medicine"
]
} |
248482016 | pes2o/s2orc | v3-fos-license | Highly Cytotoxic Osmium(II) Compounds and Their Ruthenium(II) Analogues Targeting Ovarian Carcinoma Cell Lines and Evading Cisplatin Resistance Mechanisms
(1) Background: Ruthenium and osmium complexes attract increasing interest as next generation anticancer drugs. Focusing on structure-activity-relationships of this class of compounds, we report on 17 different ruthenium(II) complexes and four promising osmium(II) analogues with cinnamic acid derivatives as O,S bidentate ligands. The aim of this study was to determine the anticancer activity and the ability to evade platin resistance mechanisms for these compounds. (2) Methods: Structural characterizations and stability determinations have been carried out with standard techniques, including NMR spectroscopy and X-ray crystallography. All complexes and single ligands have been tested for cytotoxic activity on two ovarian cancer cell lines (A2780, SKOV3) and their cisplatin-resistant isogenic cell cultures, a lung carcinoma cell line (A549) as well as selected compounds on three non-cancerous cell cultures in vitro. FACS analyses and histone γH2AX staining were carried out for cell cycle distribution and cell death or DNA damage analyses, respectively. (3) Results: IC50 values show promising results, specifically a high cancer selective cytotoxicity and evasion of resistance mechanisms for Ru(II) and Os(II) compounds. Histone γH2AX foci and FACS experiments validated the high cytotoxicity but revealed diminished DNA damage-inducing activity and an absence of cell cycle disturbance thus pointing to another mode of action. (4) Conclusion: Ru(II) and Os(II) compounds with O,S-bidentate ligands show high cytotoxicity without strong effects on DNA damage and cell cycle, and this seems to be the basis to circumvent resistance mechanisms and for the high cancer cell specificity.
Cisplatin and Analogues
The development of metals as anticancer agents began with the coincidental discovery of the biologic activity of cis-[Pt(NH 3 ) 2 Cl 2 ], Cisplatin by Rosenberg in 1965 [1]. Cisplatin was clinically approved in 1978 and targets primarily the DNA leading to DNA adducts, DNA damage, and apoptosis induction [2,3]. Nowadays, platin compounds are used in clinical anticancer treatment against cervical, bladder, head, and neck cancers as single agent and in combination therapy against testicular, ovarian, bladder, and head and neck cancers [4]. Unfortunately, the chemotherapy is limited by side effects, e.g., nephrotoxicity, ototoxicity, neurotoxicity, and innate and acquired resistant mechanism, which limit its [4,38,50,76]. Both the nature of the ligands and the change of the metal (from ruthenium to osmium) results in different anticancer activity, biological activity in general, and may enable the specific targeting of cancer cells or photodynamic therapy and a catalytic activity [14,52,[77][78][79]. Keppler and coworkers investigated some general structure-activity-relationships for osmium(II) and ruthenium(II) complexes; they concluded that the effect of the chosen metal and its anticancer activity is highly ligand-dependent [4]. Ruthenium(II) complexes are more active than their osmium(II) analogues with O,O-chelating ligand systems, whereas N,O/N,N/C,N and S,N osmium(II) compounds show better results [4,[58][59][60]66,67,70,[80][81][82]. As mentioned above, to the best of our knowledge, only the Keppler group analyzed an O,S-chelating system while focusing on different leaving groups and metal centers but did not analyze effects on platinum resistant cells [48,71]. In this work, we analyze different ruthenium(II) complexes and some of their osmium(II) counterparts with O,S-chelating ligands for anticancer properties. Next to investigating the influence of the metal-exchange, we focus on the structure-activity-relationships of different cinnamic acid derivatives as O,S-bidentate ligands. As clinically relevant models, both platinum-sensitive and -resistant epithelial ovarian cancer (EOC) cell lines were chosen for the in vitro comparison of the compounds' cytotoxic effect. While EOC is, in the majority of cases, a platinum-sensitive disease, eventually the majority of patients will relapse and develop a platinum resistance. Platinum resistance is the main limitation for a long-lasting successful therapeutic effect, thus contributing to the low five-year survival rate of approximately 40% [83].
Synthesis
The general structure of the ligand-system and the metal compounds analyzed in this work is given in Figure 1. (A) Overview and substance code of compounds this work is dealing with: β-Hydroxydithiocinnamic acid esters L1-L18 and corresponding Ru complexes Ru1-Ru17 and Os compounds Os3/Os7/Os13 and Os14. (B) General structure of the used ligand system. (C) Structure of analysed complexes with numbers indicating atoms discussed for their NMR signals (Table 1). =C-H 6.97 ppm 6.64 ppm 6.87 ppm * atoms responsible for signals are depicted in Figure 1C.
Cinnamic acid derivatives L1-L18 were synthesized according to published procedures as described in the Supplementary part [51]. For ruthenium(II) and osmium(II) complexes, the corresponding β-Hydroxydithiocinnamic acid ester is deprotonated at the vinyloge acid function with 1 equiv. t-BuOK and afterwards given to a 0.5 equiv. [(η 6 -p-cymene)MCl 2 ] 2 (M = Ru or Os) suspension in THF (Scheme 1). By adding the yellow ester solution to the M(II)-dimer, the color turns dark red and the reaction is stirred over night at room temperature, followed by acidic work up and column chromatography (THF/DCM). =C-H 6.97 ppm 6.64 ppm 6.87 ppm * atoms responsible for signals are depicted in Figure 1C.
Cinnamic acid derivatives L1-L18 were synthesized according to published procedures as described in the Supplementary part [51]. For ruthenium(II) and osmium(II) complexes, the corresponding -Hydroxydithiocinnamic acid ester is deprotonated at the vinyloge acid function with 1 equiv. t-BuOK and afterwards given to a 0.5 equiv.
Characterization
All compounds were characterized by NMR spectroscopy, mass spectrometry, and elemental analysis (see Method section). Results for L13-L18 are in common with those for L1-L12, which were reported earlier (see Supplementary part) [51]. The chemical shifts in 1 H NMR and 13 C{ 1 H} NMR spectra show significant changes after complexation to the metal(II) center for both ligand systems, the O,S-chelating and the arene ligand. Specific changes in the NMR spectra have been already discussed previously for corresponding platinum(II) compounds and are in good agreement for the metal(II) compounds this work is dealing with [51]. Interestingly, the signals of the methine protons are shifted to high-field as a result of their complexation with ruthenium(II)/osmium(II), whereas a low-field shift of the corresponding signals for the platinum(II) complexes were observed, as shown in Figure 2. This is potentially caused by the better donor ability of the cymene ligand. A high-field shift for the 13 C isotope of the -C=S-group was observed previously in the 13 C{ 1 H} NMR spectra of the platinum(II) compounds after complexation and can be confirmed for the ruthenium(II)/osmium(II) complexes as well (see Method section and Table 1). Synthesis for the metal complexes starts with the symmetrical bimetallic complex [(η 6 -p-cymene)MCl2]2 and aromatic signals of the cymene ligand are observed as two doublets, whereas the isopropyl groups resulted in one doublet. Nevertheless, the complexation to the O,S-chelating ligand leads to an unsymmetrical structure and results in chemically non-equivalent aromatic protons and carbons. Thus, four aromatic doublets for the cymene and two doublets for the isopropyl groups in the 1 H NMR spectra, as well as four (instead of two) aromatic carbon signals and two (instead of one) signal for the isopropyl groups in the 13
Characterization
All compounds were characterized by NMR spectroscopy, mass spectrometry, and elemental analysis (see Method section). Results for L13-L18 are in common with those for L1-L12, which were reported earlier (see Supplementary part) [51]. The chemical shifts in 1 H NMR and 13 C{ 1 H} NMR spectra show significant changes after complexation to the metal(II) center for both ligand systems, the O,S-chelating and the arene ligand. Specific changes in the NMR spectra have been already discussed previously for corresponding platinum(II) compounds and are in good agreement for the metal(II) compounds this work is dealing with [51]. Interestingly, the signals of the methine protons are shifted to high-field as a result of their complexation with ruthenium(II)/osmium(II), whereas a low-field shift of the corresponding signals for the platinum(II) complexes were observed, as shown in Figure 2. This is potentially caused by the better donor ability of the cymene ligand. A high-field shift for the 13 C isotope of the -C=S-group was observed previously in the 13 C{ 1 H} NMR spectra of the platinum(II) compounds after complexation and can be confirmed for the ruthenium(II)/osmium(II) complexes as well (see Method section and Table 1). Synthesis for the metal complexes starts with the symmetrical bimetallic complex [(η 6 -pcymene)MCl 2 ] 2 and aromatic signals of the cymene ligand are observed as two doublets, whereas the isopropyl groups resulted in one doublet. Nevertheless, the complexation to the O,S-chelating ligand leads to an unsymmetrical structure and results in chemically non-equivalent aromatic protons and carbons. Thus, four aromatic doublets for the cymene and two doublets for the isopropyl groups in the 1 H NMR spectra, as well as four (instead of two) aromatic carbon signals and two (instead of one) signal for the isopropyl groups in the 13 C{ 1 H} NMR spectra are detectable. For the mass spectra in general, the molecular peak is not observable, only a [M-Cl] + fragment, comparable to literature data [45], and a further fragmentation pathway as observed for the β-Hydroxydithiocinnamic acid derivatives itself. fragment, comparable to literature data [45], and a further fragmentation pathway as observed for the -Hydroxydithiocinnamic acid derivatives itself.
Stability Determination
To investigate the behaviour of the ruthenium(II) complexes Ru1, Ru3, and Ru8 in solution, we analysed kinetic measurements via 1 H NMR spectroscopy (every 1 h, one spectra). NMR signals and behaviour of the ruthenium and osmium compounds is similar, but osmium(II) compounds show a slower ligand exchange mechanism and a higher stability in general [4]. The stability determinations for the osmium(II) compounds using NMR spectroscopy show no structural changes (data for Os3 Supplementary Figure S1).
Stability Determination
To investigate the behaviour of the ruthenium(II) complexes Ru1, Ru3, and Ru8 in solution, we analysed kinetic measurements via 1 H NMR spectroscopy (every 1 h, one spectra). NMR signals and behaviour of the ruthenium and osmium compounds is similar, but osmium(II) compounds show a slower ligand exchange mechanism and a higher stability in general [4]. The stability determinations for the osmium(II) compounds using NMR spectroscopy show no structural changes (data for Os3 Supplementary Figure S1). However, ruthenium(II) compounds exhibit a reduced stability. All 1 H NMR spectra show that the ruthenium(II) molecules are not stable in dmso-d 6 solution. Figure 3 shows the results for Ru1 at 37 • C in dmso-d 6 . The blue spectrum displays the first measurement at t = 0 h and the double-doublets of the cymene ligand changed quickly and already disappeared after 24 h (red spectra). The detailed data showing all of the 1 H NMR spectra for 72 h prove that already after 5 h measurements, the signals for the cymene ligand change to a new signal, resulting in a high-field shift (Supplementary Figure S2). Additional Figure 3 shows that signals of the aromatic region change and the methine proton is disappeared after 24 h (detailed analysis proves a loss after 7 h, Supplementary Figure S2). The same measurements were done also with dmso at room temperature. Similar changes in the spectra occur at room temperature and new species are detectable (exemplified for Ru1 in the Supplementary Figure S3). However, slower speciation processes in comparison to 37 • C measurements occur, which is exemplarily represented by the disappearance of the double-doublets of the cymene ligand after 29 h (rt, Supplementary Figure S3) vs. 5 h (37 • C, Supplementary Figure S2). As reported earlier, dmso molecules are able to bind to the ruthenium(II) center by losing the cymene ligand and changing the structure to an octahedral metal(II) coordination sphere [50]. Thus, an explanation for the new species can be the binding of dmso molecules to the ruthenium(II) center after loss of the cymene ligand representing the new species in the 1 H NMR spectra. To support this hypothesis, the ruthenium(II) complexes were measured under same conditions (rt, 72 h) in CD 2 Cl 2 , and it was shown that the compounds are stable under these conditions in the other solvent (see Supplementary Figure S4). In conclusion, it is shown that the analysed Ru(II) compounds are able to react with dmso at room temperature as well as at 37 • C, but not with dichlormethane. However, ruthenium(II) compounds exhibit a reduced stability. All 1 H NMR spectra show that the ruthenium(II) molecules are not stable in dmso-d6 solution. Figure 3 shows the results for Ru1 at 37 °C in dmso-d6. The blue spectrum displays the first measurement at t = 0 h and the double-doublets of the cymene ligand changed quickly and already disappeared after 24 h (red spectra). The detailed data showing all of the 1 H NMR spectra for 72 h prove that already after 5 h measurements, the signals for the cymene ligand change to a new signal, resulting in a high-field shift (Supplementary Figure S2). Additional Figure 3 shows that signals of the aromatic region change and the methine proton is disappeared after 24 h (detailed analysis proves a loss after 7 h, Supplementary Figure S2). The same measurements were done also with dmso at room temperature. Similar changes in the spectra occur at room temperature and new species are detectable (exemplified for Ru1 in the Supplementary Figure S3). However, slower speciation processes in comparison to 37 °C measurements occur, which is exemplarily represented by the disappearance of the double-doublets of the cymene ligand after 29 h (rt, Supplementary Figure S3) vs. 5 h (37 °C , Supplementary Figure S2). As reported earlier, dmso molecules are able to bind to the ruthenium(II) center by losing the cymene ligand and changing the structure to an octahedral metal(II) coordination sphere [50]. Thus, an explanation for the new species can be the binding of dmso molecules to the ruthenium(II) center after loss of the cymene ligand representing the new species in the 1 H NMR spectra. To support this hypothesis, the ruthenium(II) complexes were measured under same conditions (rt, 72 h) in CD2Cl2, and it was shown that the compounds are stable under these conditions in the other solvent (see Supplementary Figure S4). In conclusion, it is shown that the analysed Ru(II) compounds are able to react with dmso at room temperature as well as at 37 °C, but not with dichlormethane. However, we used freshly prepared stock solutions in dmso for each experiment and diluted these stock solutions within 1 h at RT to the final concentration in cell culture medium (final dmso concentration 0.5%). Therefore, the stability and the potential generation of speciation products in cell culture medium is more relevant but presently unknown. Earlier data show minor changes of the UV-VIS spectra in aqueous solutions pointing to an aquation (ligand exchange chloride to aqua) [50]. Even more important, incubations in protein solution (RNaseA) prove the interaction and binding to proteins [50]. Therefore, it is likely that Ru and eventually Os compounds undergo protein binding and speciation processes in biological systems. Although, the species causative for observed biological effects (see below) is unknown, these effects are attributable to the tested compounds.
Molecular Structures
Ruthenium(II) complexes Ru9, Ru13, and Ru14 as well as L14, L15, L17, and L18 were characterized by means of single crystal X-ray structure determination, whereas the molecular structures of Ru3, L1, L3, L4, L8, and L9 are already known [50,51]. Figure 4 shows the ruthenium(II) complex 14, whereas the molecular structures of Ru9, Ru13, and of the ligands are depicted in the Supplementary part, Figures S5 and S6, and Table S1. Results are in good agreement with the values reported earlier [50]. However, we used freshly prepared stock solutions in dmso for each experiment and diluted these stock solutions within 1 h at RT to the final concentration in cell culture medium (final dmso concentration 0.5%). Therefore, the stability and the potential generation of speciation products in cell culture medium is more relevant but presently unknown. Earlier data show minor changes of the UV-VIS spectra in aqueous solutions pointing to an aquation (ligand exchange chloride to aqua) [50]. Even more important, incubations in protein solution (RNaseA) prove the interaction and binding to proteins [50]. Therefore, it is likely that Ru and eventually Os compounds undergo protein binding and speciation processes in biological systems. Although, the species causative for observed biological effects (see below) is unknown, these effects are attributable to the tested compounds.
Molecular Structures
Ruthenium(II) complexes Ru9, Ru13, and Ru14 as well as L14, L15, L17, and L18 were characterized by means of single crystal X-ray structure determination, whereas the molecular structures of Ru3, L1, L3, L4, L8, and L9 are already known [50,51]. Figure 4 shows the ruthenium(II) complex 14, whereas the molecular structures of Ru9, Ru13, and of the ligands are depicted in the Supplementary part, Figures S5 and S6, and Table S1. Results are in good agreement with the values reported earlier [50]. 1) bond; this is comparable to the already discussed platinum(II) complexes [51]. (1) 86.37 (2)
Biological Behavior
The biological behaviour of all substances was characterized by their cytotoxic activity against a panel of cell lines enabling an understanding of the structure-activity relationship. Cytotoxic activity was determined on ovarian carcinoma cell lines SKOV3 and A2780 as well as their Cisplatin resistant analogues (SKOV3cis and A2780cis) [84,85] and the lung carcinoma cell line A549. Due to a low solubility in water, dmso is used as a solvent for the preparation of a dilution series in cell culture experiments. The toxic influence of dmso was determined earlier and experiments were carried out with 0.5% dmso in cell culture media and this concentration was used as reference sample in each MTT assay (details: Section 3) [51]. Cisplatin was used as a reference substance, and a 4.7 or 3.6 times higher IC50 value was observed for resistant cell lines; see Table 3. Resistance factors (RF) were determined for all substances (for IC50 values and RF of β-Hydroxydithiocinnamic acid esters L1-L18, see Tables S2 and S3, Supplementary part). All investigated ruthenium(II) compounds show lower RF values than Cisplatin on ovarian carcinoma cell lines, ranging from 0.2 to 1.5 (Table 3). Whereas the IC50 values on the non-resistant cell lines are in most cases higher than the IC50 of the reference substance, no increase of IC50 values is observed for the resistant cell lines. Contrary, eight ruthenium complexes show lower IC50 values on SKOV3cis than Cisplatin and four compounds on A2780cis. Thus, it can be concluded that these compounds are able to bypass the Cisplatin resistance mechanism in these cell lines pointing to a different mechanism of action.
The osmium compounds show, in most cases, lower IC50 values than the reference Cisplatin (except for SKOV3 and Os7, 13, and 14, Table 3). To point out, all substances show IC50 values between 0.3-0.4 µM on A2780, whereas Cisplatin has an IC50 value of 1.3 µM. On the resistant analogue of A2780, the activity is more than five times higher for Os3 (0.4 µM) and Os13 (0.8 µM) in comparison to Cisplatin (6.1 µM). Albeit only one compound (Os3) exhibits a lower IC50 value for SKOV3 than Cisplatin, all compounds have a higher activity against SKOV3cis. Remarkably, Os7 shows a 13-times lower IC50 value than Cisplatin (0.6 to 13.5 µM). The most promising candidate, Os3, shows IC50 values between 0.4 µM (A2780) and 2.3 µM (SKOV3cis), generally lower than the range of Cisplatin (1.3 µM A2780-13.5 µM SKOV3cis). Whereas the resistance factors of the ruthenium compounds are in most cases lower than 1, pointing to the specific targeting of resistant cells, the osmium analogues do not behave the same. This confirms earlier published comparison studies showing that osmium analogues of ruthenium complexes exhibit a different biological behavior in vitro (see introduction). Table 4 shows IC50 values for normal primary short-term cell cultures of keratinocytes and fibroblasts as well as the non-cancerous breast epithelial cell line MCF10A. As mentioned before, Cisplatin exhibits numerous side effects by its unselective behaviour and cytotoxic activity against normal cells, which is also reflected by the measured IC50 values against non-cancerous cell cultures. Despite this, both most active compounds, Os3 and Ru14, show high IC50 values for these cells. To conclude, the osmium compounds are in general more active against all five cell lines than Cisplatin and their ruthenium counterparts. This shows the enormous potential for osmium compounds as next generation anticancer drugs. However, the ruthenium compounds are specifically active against Cisplatin-resistant cell lines, meaning they are able to elude the mechanisms of Cisplatin resistance. This indicates the opportunity for ruthenium compounds to be selected for resistant tumors. Additionally, our data showing a higher activity for osmium compared to ruthenium compounds in ovarian and lung cancer cell lines resemble data from Klose et al., identifying tumor type specific activity ratios for isosteric Ru/Os compounds using the NCI-60 cell line panel [86]. Both compound classes do not attack non-cancerous cells resulting in higher cancer specificity compared to Cisplatin. This is confirmed by recent data for breast cancer cell lines showing a high cancer cell selectivity for similar Ru(II) complexes with cinnamic acid derivates [87] and for Ru(II)/Os(II) complexes with N,N-bidentate ligands in various cancer cell models [88]. This higher cancer cell specificity potentially leads to lower side effects during the therapy in vivo. Lower side effects may translate into the treatment with higher doses of the drugs, resulting in earlier and increased effects. Therefore, acquired drug resistance mechanisms arising after several treatments with suboptimal doses may be circumvented by drugs like the osmium compounds due to lower toxic side effects. Altogether, there are possibly two different indications for the ruthenium(II) and osmium(II) complexes. The ruthenium(II) compounds should be further developed for a treatment of Cisplatin resistant tumors, whereas the osmium(II) complexes can be an alternative for the first-line therapy due to higher cytotoxic activity compared to Cisplatin. A further analysis for the different ruthenium(II) compounds to determine structureactivity-relationships shows that five compounds (Ru14, Ru15, Ru2, Ru5, and Ru3) exhibit lower mean IC50 value on Cisplatin resistant cell lines than Cisplatin itself (Supplementary Figure S7E). Interestingly, compounds Ru14, Ru15, and Ru16 are, all together, the most active compounds in comparison to Cisplatin ( Figure 5A). The compound Ru14 shows a lower mean IC50 value than Cisplatin for all cancer cell lines (Supplementary Figure S7A), for all ovarian carcinoma cell lines (Supplementary Figure S7C), for the Cisplatin resistant cell lines (Supplementary Figure S7E), and for all non-resistant cell lines (Supplementary Figure S7B). In conclusion, the determined structure-activity-relationship shows that longer alkyl chains at the aromatic ring lead to higher cytotoxic activity. The most active compound having an ethoxy-group at para-position (Ru14) is followed by Ru15 with an ethoxy-group at ortho-position. Interestingly, compound Ru16 has a butoxy-substituent at meta-position. Thus, it can be concluded that the biological activity is mediated by a longer chain (butoxy) at the meta-position, whereas the orthoand para-positions are more suitable with a shorter chain (ethoxy). To have a further look at the influence of the different ligand systems and substitution patterns, all β-Hydroxydithiocinnamic acid alkyl esters were tested under same conditions as their derived ruthenium(II) complexes ( Figure 5B, Supplementary Table S3). Figure 5B shows the trend of all IC50 values ordered by an increased mean IC50 value (determined for all five cell lines) for the β-Hydroxydithiocinnamic acid alkyl esters. Interestingly, the most active compounds are L17, L14, L18, L16, L13, and L15, showing similar low IC50 values on all cell lines. This confirms the results for the corresponding ruthenium(II) complexes, proving that the longer alkyl chains on the aromatic positions are the most active compounds and that the IC50 values increase by decreasing lipophilicity. All ligands are less cytotoxic than Cisplatin itself and therefore the metal(II) center is necessary for the high cytotoxic activity, what is in clear contrast to the literature for O,S-chelating ruthenium(II) or osmium(II) compounds with thiomaltol ligand [48,71] but confirmed for Ru(II)/Os(II) compounds with N,N-bidentate glycosyl heterocyclic ligands [88]. This is exemplarily shown by the comparison of mean IC50 values for Cisplatin, L14, and Ru14 (Supplementary Figure S8). The ruthenium(II) center strongly decreases the IC50 values in all cases, and therefore the metal is the active part that is supported by the most active ligand system.
The reduced viability under treatment, as measured by the MTT assay, can be a result of cell cycle arrest and/or increased cell death. To further evaluate the anticancer properties of the ruthenium(II) complexes we measured cell cycle distribution and cell death rates after treatment with Ru3 or Ru14. After seeding and attaching, the cells were treated for 48 h with different concentrations of substances. For cell cycle distribution measurements, a recovery phase of 24 h was added after treatment, and cells were fixed and stained with PI for the DNA content. Arresting of cells in specific cell cycle phases gives them time to resolve the DNA damage (G1 arrest) or is an initial step to apoptosis if DNA damage is too severe (G2/M arrest) [89]. As previously shown, Cisplatin (5 µM) efficiently induces cell cycle arrest in G2/M phase in parental A2780 and SKOV3 cells, whereas resistant cells show only a minor G2/M arrest [85]. On the other side, both examined ruthenium complexes show no or only a minor effect on cell cycle distribution ( Figure 6). This is in line with other published ruthenium(II) complexes, which do not all induce cell cycle arrest [89,90]. Therefore, one can suggest that these complexes do not induce high DNA damage levels, leading to cell cycle arrest. For cell death rate analysis, live cells were stained with PI immediately after 48 h treatment. Again, it can be seen that 15 µM Cisplatin efficiently induces cell death in parental ovarian cancer cells [85], where it is 29.9-fold higher for A2780 and 6.3-fold higher for SKOV3 compared to untreated cells. Furthermore, resistant cells show much lower Cisplatin-induced cell death rates (Figure 7). Both complexes, Ru3 and Ru14, have a high capacity to induce cell death in vitro (Figure 7). In A2780 cells, both compounds trigger similar cell death rates in parental and Cisplatin-resistant cells. Cisplatin-resistant SKOV3 are much more sensitive to both ruthenium(II) complexes than the parental counterpart, with a median of 3.3-fold higher sensitivity. Interestingly, Ru3 induced higher cell death rates than Ru14 despite contrary results for IC50 values. For cell death rate analysis, live cells were stained with PI immediately after 4 treatment. Again, it can be seen that 15 µ M Cisplatin efficiently induces cell death in rental ovarian cancer cells [85], where it is 29.9-fold higher for A2780 and 6.3-fold hig for SKOV3 compared to untreated cells. Furthermore, resistant cells show much low Cisplatin-induced cell death rates (Figure 7). Both complexes, Ru3 and Ru14, have a h capacity to induce cell death in vitro (Figure 7). In A2780 cells, both compounds trig similar cell death rates in parental and Cisplatin-resistant cells. Cisplatin-resist SKOV3 are much more sensitive to both ruthenium(II) complexes than the paren counterpart, with a median of 3.3-fold higher sensitivity. Interestingly, Ru3 indu higher cell death rates than Ru14 despite contrary results for IC50 values. Previous studies showed a direct induction of apoptosis by ruthenium(II) comple via ROS production and activation of pro-apoptotic BCL2-family proteins [91,92]. Ru compounds may also inhibit TrxR (thioredoxin reductase), thus resulting in ROS p duction, mitochondrial dysfunction, and apoptosis [93]. ROS production may also lead endoplasmatic reticulum stress-induced apoptosis [36]. In general, many ruthenium complexes with different ligands induce intracellular ROS [94][95][96][97][98][99]. Moreover, the cy toxic activity of Os(II) compounds can be inhibited by vitamin E co-treatment pointing the contribution of ROS [88]. In addition to mitochondrial dysfunction, Ru(II) comple may affect glycolysis [100] or topoisomerase I/II, thus inducing necroptosis [101]. Ru compounds with modified pyrithione ligands were recently described to overco platinum resistance in ovarian cancer cells by inducing cytostatic G1 arrest, TrxR inh tion, and cell membrane damage [102]. Both Ru(II) and Os(II) compounds can also inh proteosynthesis [34,103]. A direct interaction of Ru3 with a model protein (RNaseA) sulted in ligand exchange, binding to histidine residues, and altered coordination sph geometry, pointing to a mode-of-action that involves protein targets [50]. Future stud may identify the specific target proteins enabling molecular docking studies and spec refinement of the organo-metal compound structure. Altogether, presented compoun may use some of these alternative modes of action as well, as we see efficient cell de but no cell cycle arrest induction by Ru3/Ru14. Furthermore, the ruthenium(II) core at Previous studies showed a direct induction of apoptosis by ruthenium(II) complexes via ROS production and activation of pro-apoptotic BCL2-family proteins [91,92]. Ru(II) compounds may also inhibit TrxR (thioredoxin reductase), thus resulting in ROS production, mitochondrial dysfunction, and apoptosis [93]. ROS production may also lead to endoplasmatic reticulum stress-induced apoptosis [36]. In general, many ruthenium(II) complexes with different ligands induce intracellular ROS [94][95][96][97][98][99]. Moreover, the cytotoxic activity of Os(II) compounds can be inhibited by vitamin E co-treatment pointing to the contribution of ROS [88]. In addition to mitochondrial dysfunction, Ru(II) complexes may affect glycolysis [100] or topoisomerase I/II, thus inducing necroptosis [101]. Ru(II) compounds with modified pyrithione ligands were recently described to overcome platinum resistance in ovarian cancer cells by inducing cytostatic G1 arrest, TrxR inhibition, and cell membrane damage [102]. Both Ru(II) and Os(II) compounds can also inhibit proteosynthesis [34,103]. A direct interaction of Ru3 with a model protein (RNaseA) resulted in ligand exchange, binding to histidine residues, and altered coordination sphere geometry, pointing to a mode-of-action that involves protein targets [50]. Future studies may identify the specific target proteins enabling molecular docking studies and specific refinement of the organo-metal compound structure. Altogether, presented compounds may use some of these alternative modes of action as well, as we see efficient cell death but no cell cycle arrest induction by Ru3/Ru14. Furthermore, the ruthenium(II) core atom might be responsible for this effect because of the lack of anticancer behaviour of the ligand L14 (Supplementary Figure S8). To further confirm that Ru compounds use another mechanism of action, DNA damage analyses were conducted for Ru3 and Ru14 (Figure 8). Both Ru compounds (at IC50 concentration) induced less γH2AX-foci as Cisplatin after 24h incubation under the same conditions. This confirms published data pointing to a DNA-independent mode of action for ruthenium compounds [41,71,104]. However, other data show an interaction of ruthenium complexes with DNA [33,43,99,105,106]. These contrary observations may relate to experimental conditions or specific ligands. might be responsible for this effect because of the lack of anticancer behaviour of the ligand L14 (Supplementary Figure S8). To further confirm that Ru compounds use another mechanism of action, DNA damage analyses were conducted for Ru3 and Ru14 (Figure 8). Both Ru compounds (at IC50 concentration) induced less γH2AX-foci as Cisplatin after 24h incubation under the same conditions. This confirms published data pointing to a DNA-independent mode of action for ruthenium compounds [41,71,104]. However, other data show an interaction of ruthenium complexes with DNA [33,43,99,105,106]. These contrary observations may relate to experimental conditions or specific ligands. The presented data suggest that Ru(II) and Os(II) complexes with O,S-chelating -Hydroxydithiocinnamic acid esters are both highly active and specific against cancer cell lines (Os(II) compounds) or Cisplatin resistant cancer cells (Ru(II) compounds). The avoidance of resistance mechanisms seems to be related to another mode of action inducing cell death without high levels of DNA damage or cell cycle arrest. Several limitations must be discussed for the evaluation of these data. Firstly, the biological activity was determined for in-vitro 2D cell culture systems, only. Further analyses in 3D cell cultures or in vivo should clarify the potential for clinical use of the most active compounds (Os3, Ru14). Thereby, the detailed mode-of-action must be identified, although first data point to a potential contribution of protein interactions [50]. Secondly, presented and already published data point to the instability of Ru(II) compounds and the generation of speciation products in dmso and biological systems (Figure 3) [50]. Therefore, it is presently unknown which specific compound directly causes the observed biological effects. However, our experiments show clearly and reproducibly that the tested complexes are the general source of the effects. If future studies can solve these limitations and validate the high cancer cell specific cytotoxicity also against platin resistant tumors, these compounds are likely to improve the treatment of ovarian cancer patients. The presented data suggest that Ru(II) and Os(II) complexes with O,S-chelating β-Hydroxydithiocinnamic acid esters are both highly active and specific against cancer cell lines (Os(II) compounds) or Cisplatin resistant cancer cells (Ru(II) compounds). The avoidance of resistance mechanisms seems to be related to another mode of action inducing cell death without high levels of DNA damage or cell cycle arrest. Several limitations must be discussed for the evaluation of these data. Firstly, the biological activity was determined for in-vitro 2D cell culture systems, only. Further analyses in 3D cell cultures or in vivo should clarify the potential for clinical use of the most active compounds (Os3, Ru14). Thereby, the detailed mode-of-action must be identified, although first data point to a potential contribution of protein interactions [50]. Secondly, presented and already published data point to the instability of Ru(II) compounds and the generation of speciation products in dmso and biological systems (Figure 3) [50]. Therefore, it is presently unknown which specific compound directly causes the observed biological effects. However, our experiments show clearly and reproducibly that the tested complexes are the general source of the effects. If future studies can solve these limitations and validate the high cancer cell specific cytotoxicity also against platin resistant tumors, these compounds are likely to improve the treatment of ovarian cancer patients.
Materials and Techniques
All reactions were performed using standard Schlenk and vacuum-line techniques under nitrogen atmosphere. The NMR spectra were recorded with a Bruker Avance 200 MHz, 400 MHz, or 600 MHz spectrometer. Chemical shifts are given in ppm with reference to SiMe 4 . Mass spectra were recorded with a Finnigan MAT SSQ 710 instrument. Elemental analysis was performed with a Leco CHNS-932 apparatus. Silica gel 60 (0.015-0.040 mm) was used for column chromatography, and TLC was performed using Merck TLC aluminium sheets (Silica gel 60 F 254 ). Chemicals were purchased from Fisher Scientific (Schwerte, Germany), Sigma-Aldrich (Taufkirchen, Germany), or Acros (Nidderau, Germany) and were used without further purification. All solvents were dried and distilled prior to use according to standard methods.
[(η 6 -p-cymene)RuCl 2 ] 2 (0.5 equiv.) was dissolved in 50 mL tetrahydrofurane (THF). The corresponding ligand L1-L12 (1 equiv.) was solved in 25 mL THF and potassiumtert.-butoxylate (t-BuOK, 2 equiv.) was added to that solution and stirred 30 min at rt. The solution of the deprotonated ligand was added dropwise to the suspension of [(η 6 -p-cymene)RuCl 2 ] 2 and stirred at room temperature for 24 h. After adding sulfuric acid (H 2 SO 4 , 20 mL, 2M) to the solution, the mixture was stirred for 30 min at rt and afterwards extracted with dichlormethane (DCM, 3 × 30 mL). The combined organic phases were washed with water (3 × 20 mL), dried over sodium sulfate and after filtration and evaporation of the solvent the crude product was purified with column chromatography.
3.2.1. General Procedure 1: Osmium(II) Complexes with β-Hydroxydithiocinnamic Acid Alkyl Esters, Chlorido, and p-cymene as Ligands (Os1-Os4) [(η 6 -p-cymene)OsCl 2 ] 2 (0.5 equiv.) was dissolved in 50 mL tetrahydrofurane (THF). The corresponding ligand (1 equiv.) was solved in 25 mL THF, and potassium-tert.butoxylate (t-BuOK, 2 equiv.) was added to that solution and stirred 30 min at rt. The solution of the deprotonated ligand was added dropwise to the suspension of [(η 6 -pcymene)RuCl 2 ] 2 and stirred at room temperature for 24 h. After adding sulfuric acid (H 2 SO 4 , 20 mL, 2M) to the solution, the mixture was stirred for 30 min at rt and afterwards extracted with dichlormethane (DCM, 3 × 30 mL); the combined organic phases were washed with water (3 × 20 mL), dried over sodium sulfate and after filtration and evaporation of the solvent the crude product was purified with column chromatography. multiple-scans [108,109]. The structures were solved by direct methods (SHELXS) and refined by full-matrix least squares techniques against Fo 2 (SHELXL-97) [110]. All hydrogen atoms (with exception of the methyl-group at C13 of Ru14 and the methylene-group at C11 of L18 were located by difference Fourier synthesis and refined isotropically. All other hydrogen atoms were included at calculated positions with fixed thermal parameters. Crystallographic data as well as structure solution and refinement details are summarized in Table 4. MERCURY was used for structure representations [111].
Stability Determinations
NMR spectra were measured via NMR spectroscopy on Bruker Avance 400 MHz. Substances were solved in dmso-d 6 or CD 2 Cl 2 and measured directly at 37 • C or room temperature for 72 h. NS = 128 scans, t = 709 s/2891 s break, 72 measurements.
Biological Assays
Ovarian cancer cell lines were cultured under standard conditions (5% CO 2 , 37 • C, 90% humidity) in RPMI medium supplemented with 10% FCS, 100 U/mL penicillin and 100 µg/mL streptomycin (Life Technologies, Dreieich, Germany). Cisplatin (Sigma, Taufkirchen, Germany) was freshly dissolved at 1 mg/mL in 0.9% NaCl solution and diluted appropriately. New ruthenium(II) complexes and ligands were dissolved in dmso. Platinum-resistant A2780 and SKOV3 cells were established by repeated rounds of 3-day incubations with increasing amounts of Cisplatin starting with 0.1 µM. The concentration was doubled after 3 incubations, interrupted by recovery phases with normal medium. Cells that survived the third round of 12.8 µM Cisplatin were defined as resistant cultures. Determinations of IC50 values were carried out using the CellTiter96 non-radioactive proliferation assay (MTT assay, Promega, Mannheim, Germany). After seeding 5000 cells per well in a 96-well plate, cells were allowed to attach for 24 h and were incubated for 48 h with different concentrations of the substances ranging from 0 to 500 µM for Ruthenium and 0 to 1000 µM for ligand tests (0, 1, 10, 50, 100, 500, 1000 µm), for Cisplatin from 0 to 100 µM (0.1, 1, 5, 10, 50, 100 µM). Each measurement was done in triplicate and repeated 3 times. The proportion of viable cells was quantified by the MTT assay and after background subtraction relative values compared to the mean of medium controls were calculated. Non-linear regression analyses applying the Hill slope were run in GraphPad 5.0 software.
To examine cell cycle distribution and cell death rates, 30,000 cells were seeded in 12 well plates. After attaching for 24 h cells were treated with Cisplatin, Ru3 and Ru14 for 48 h at various concentrations for cell cycle and cell death analyses. For cell death analysis, immediately after treatment cells were stained with Propidium Iodid (PI) (1 µg/mL) on ice and the number of dead cells was measured using BD Canto II. For cell cycle distribution, cells recovered for 24 h after treatment. Afterwards, cells were fixed in ice-cold, 50% EtOH for 24 h at −20 • C. For DNA staining, fixed cells were incubated in PBS with 0.05% Triton-X, 0.1 µg/mL RNaseA and 50 µg/mL PI for 1 h at 4 • C in dark. DNA content was measured using BD Canto II.
For the determination of DNA damage induced by the treatment with different substances, histone γH2AX-foci were visualized by immunocytochemical staining. Cells were seeded on coverslips to reach 60-70% confluence after 24 h. After incubation (24 h) with different substances at IC 50 concentrations for the resistant cells, cells were washed 3× with PBS and fixed for 10 min in 4% paraformaldehyde. Cells were again washed 3 times and then permeabilised by incubation with 0.25% Triton-X in PBS for 5min. Primary antibody against γH2AX (clone JBW301, Merck-Millipore, Darmstadt, Germany; diluted 1:2000) was incubated for 1 h at RT, and coverslips were washed 3 times afterwards. Alexa488-labelled secondary anti-mouse antibody (Life Technologies) was used in a 1:1000 dilution in PBS and applied for 1 h at RT. Cells were washed 3 times, counterstained with DAPI, washed again, and embedded in mounting medium (Vectorshield, Vector Laboratories, Burlingame, CA, USA). Slides were stored at 4 • C in darkness until microscopic evaluation was done using a Zeiss LSM 710 laser scanning microscope using a 63× oil-immersion objective. Image analysis was done using ImageJ and the FindFoci PlugIn [112].
Conclusions
In this work, we investigated 18 cinnamic acid derivatives, 17 ruthenium(II) complexes, and 4 osmium(II) complexes, and all of these compounds have been characterized by different methods, including X-ray diffraction analysis. NMR spectra signals have been compared to previously reported platinum(II) complexes and show significant changes in the ligand systems after complexation to metals. Stability determinations for some ruthenium(II) compounds were done with NMR spectroscopy, showing that these compounds are not stable in the solvent dmso, but in different other organic solvents. The biological activity of these complexes have been investigated mainly by IC50 measurements for all substances, as well as by cell cycle arrest, cell death, and DNA damage analyses for two of the ruthenium(II) complexes. Regarding the IC50 values, we can add to the previously reported SARs of ruthenium(II) and osmium(II) complexes by Keppler and coworkers that bearing an O,S-chelating ligand results in lower IC50 values for osmium(II) complexes compared to their ruthenium(II) analogues, but the ruthenium(II) compounds exhibit lower resistance factors [4]. Nevertheless, regarding non-cancerous cell lines, both complexes show a selective activity to cancer cell lines and high IC50 values on non-cancerous cells, pointing to possibly lower toxicity and side effects. The high cancer cell specific cytotoxic activity, also against cisplatin resistant cells combined with the diminished effects on cell cycle arrest and DNA damage point to a different mode of action. This may potentially involve the induction of ROS and mitochondrial dysfunction. Focusing on the structureactivity-relationship of the ruthenium(II) compounds, it is shown that longer alkyl chains at the aromatic ring lead to higher cytotoxic activity of these compounds. For the osmium complexes, most active compound is Os3, with a hydroxy-group at meta-position. Therefore, some of these compounds will be selected for further development, including in vivo experiments. | 2022-05-02T15:04:49.423Z | 2022-04-29T00:00:00.000 | {
"year": 2022,
"sha1": "232789a43a7e0c4061eb9fe8d457a63b8ab43cfe",
"oa_license": "CCBY",
"oa_url": "https://www.mdpi.com/1422-0067/23/9/4976/pdf?version=1652065539",
"oa_status": "GOLD",
"pdf_src": "PubMedCentral",
"pdf_hash": "25d48e9c708acd727470794e1709e47bd1685e41",
"s2fieldsofstudy": [
"Chemistry",
"Biology"
],
"extfieldsofstudy": [
"Medicine"
]
} |
243415543 | pes2o/s2orc | v3-fos-license | The implications of the use of cannabidiol-related products in a safety-sensitive drug testing environment: A medical-legal perspective
Cannabis access laws allow for the use of cannabis in private and the trade, purchase and use of hemp-related products as a complementary medicine and for other benefits. Cannabidiol (CBD) has the treatment potential for several conditions but, with the lack of resources in South Africa to maintain the legislation, products contaminated with delta-9-tetrahydrocannabinol (Δ9-THC) are sold by some suppliers who do not comply with the legislative provisions in terms of the threshold concentrations for Δ9-THC. This dilemma complicates a medical review officer's decision regarding intentional use of Δ9-THC or otherwise, since a CBD user may have purchased the product legally and in good faith. Hemp- and CBD-containing products were analysed by gas chromatography-mass spectrometry and compliance was assessed for CBD and Δ9-THC purity against the legislative thresholds. A strategy based on metabolite ratios is suggested to distinguish between intentional or irresponsible cannabis use and legitimate CBD use.
remained, since SA is a developing country that lacks resources to manage the broad social impact of legalisation. [16] Medical practitioners who function as medical review officers (MROs) in the occupational health setting are confronted with the difficulty of distinguishing between intentional or negligent use of Δ 9 -THC where it is prohibited, as opposed to non-intentional administration using complementary medicine. Delta-9-THC contamination of CBD oils is also critical in other areas where medical practitioners advise on test results: for school drug-testing programmes where CBD oils are sometimes administered to learners by their parents as complementary medicine; parents in custody cases being tested for substance abuse and adjudicated in SA family courts; sports doping tests where such substances are prohibited; and driving under the influence of intoxicating substances.
The risks of incorrect interpretation regarding cannabis testing results have two aspects. First, there is the possibility of crossreactivity of the screening due to non-specific interference from other cannabinoids, leading to false positives, most common in immunoassays. [17] Second, there are difficulties in discriminating the source of Δ 9 -THC in the case of true positives.
The legitimate use of CBD-enriched oil as complementary medicine has gained a tremendous amount of traction recently. Its use as complementary medicine, or as edible and drinkable products, is legal if compliant with the schedules of the Medicines Act. [15,18] There is a danger of compromising safety in the workplace and triggering positive drug tests if the products are contaminated with Δ 9 -THC. [19][20][21][22] The use of CBD-related products may also become the first line of defence for individuals who smoke cannabis and fail threshold drug
IN PRACTICE
tests in the workplace. An uninformed MRO decision may also result in injustice if an individual uses a hemp product not knowing that it is contaminated with Δ 9 -THC.
Relevant legislation
The fundamental human rights related to the legal use of cannabis and its refined products must be balanced against the health and safety of others. [23] The relevant human rights of privacy, freedom, autonomy, freedom of religion and the equal enjoyment of rights and privileges are indicated in the Bill of Rights of the Constitution of the Republic of SA. [24] The legalisation in effect confirmed that cannabis and hemp product users should enjoy equal protection and benefit of the law, including the enjoyment of rights and freedom. [25] The legalisation of the private use of cannabis also did not preclude the health and safety legislation and regulations applicable to workplaces and risk-sensitive environments. [26,27] Screening and confirmation threshold testing, followed by validation by a MRO, are mainly utilised to detect individuals with prohibited substances in their bodies. [28] The schedules of the Medicines Act classify CBD-containing products for human consumption as follows: [15] • All products intended for human ingestion with a Δ 9 -THC purity greater than 0.001 mass percentage are classified as schedule 6. • All CBD-containing products are classified in schedule 4, unless the products are indicated as complementary medicine, with a total mass of less than 600 mg CBD per package, intended to be administered in a regimen of less than 20 mg CBD per dose. A product that complies with this specification is classified in schedule 0.
Any person may possess substances in schedules 0 -2 for medicinal purposes. Schedule 3 -6 substances require a prescription issued by an authorised prescriber for a product registered for use in SA by the South African Health Product Regulatory Authority (SAHPRA). [29] Manufacturers and suppliers must comply with the legal thresholds for Δ 9 -THC and CBD in their products to trade in these formulations legally and avoid liability and possible prosecution.
Composition of cannabis plant extract
Cannabis originates from the plant C. sativa, of which C. indica and C. ruderalis are subspecies. [2,[30][31][32] Cannabis contains cannabinoids, [33] such as cannabidiol (CBD), Δ 9 -tetrahydrocannabinol (Δ 9 -THC), cannabinol (CBN) and tetrahydrocannabivarin (THCV), of which the last three are psychoactive. [2] Most of the psychotropic effects are caused by Δ 9 -THC. [2,322,34,35] In contrast, CBD is considered nonpsychoactive, although it shows positive psychological effects in terms of anxiety and depression [2] and can modulate the psychoactive effects of Δ 9 -THC. [36] C. indica variants or hemp plants [35,37] are known to have higher CBD but lower Δ 9 -THC contents. [32,35] In contrast, sativa variants or marijuana have lower CBD and higher Δ 9 -THC contents, up to 25% (w/w). [32] Hemp is preferred for the manufacture of CBD oils, [38] but all cannabis plants contain Δ 9 -THC to some extent, [39] and the extraction processes used to isolate CBD from plant materials also extract other biologically active compounds such as CBN and Δ 9 -THC. [40] The need for CBD purification is illustrated by studies wherein commercially available CBD products were analysed. These studies found that upwards of 50% of the researched products contained CBD levels that did not correspond with the product labelling. Additionally, 85%, [31] 20% [41] and 100% [42] of the products analysed by each study, respectively, had detectable levels of Δ 9 -THC, with concentrations ranging up to and exceeding the minimum dose of Δ 9 -THC, 2.5 mg/day, above which adverse effects arise.
The present study reports CBD, Δ 9 -THC and CBN levels in a sample of commercially available hemp-related products in SA, analysed in a forensic laboratory with a validated gas chromatography-mass spectrometry (GC-MS) analytical method. The products are assessed for compliance with current legislative threshold specifications for CBD and Δ 9 -THC, to provide information to medical practitioners responsible for determining the risk of an individual's possible cannabis consumption to an organisation.
Experimentally obtained Δ 9 -THC, CBD and CBN concentrations
Products from the local SA market were purchased and analysed for CBD, CBN and Δ 9 -THC by GC-MS isotope dilution, with a linear non-weighted response model. Eight CBD-based products comprising four oils for oral consumption, one 'vape' oil, one tincture, one tea and one chewing gum, obtained from either online shops, cannabis shops, pharmacies or cannabis industry expos, were analysed.
The following non-compliances were observed: • One of the oils with a dark appearance exceeded the legal limit for the Δ 9 -THC content, at 3.809%, or 54.82 mg, which is 22 times higher than the psychoactive dose. [42] This places the product under schedule 6. In addition, the total CBD content for the product was 31.38 mg. • False advertising was a prominent feature, with two of the oils reported as having 'no THC' , but containing 3.8 and 3.2 µg, respectively. • The CBD contents of the chewing gum and the vape oil were significantly lower than the reported values. • CBN was detected in all the samples, with masses ranging up to 0.6 mg. CBN can trigger a positive screening test. [43] • Products purported to contain a total mass of 1 000 mg CBD instead of the legal limit of 600 mg CBD were also available in one of the cannabis shops.
Pharmacodynamics and pharmacokinetics of CBD and Δ 9 -THC
CBD and Δ 9 -THC have similar pharmacokinetics. The shared highly lipophilic character of Δ 9 -THC and CBD results in rapid distribution throughout the body, with both compounds metabolised in the liver by cytochrome P450 enzymes. [44] These compounds are mainly excreted in the faeces as un-metabolised compounds, [44,45] and in the urine as metabolites THC-COOH [46] and CBD-glucuronide. [45] The elimination half-life of these compounds is similar, ranging from 3 to 4 days for THC-COOH [46] and 2 to 5 days for CBD. [46] Therefore, a similar detection window is expected.
Synthesis: Legislation, science and ethics
The cannabis access laws in the form of amendments to the Drugs and Drug Trafficking Act No. 140 of 1992, [47] the Medicines Act [48] and their respective schedules should, in principle, not affect drug test results performed as part of a health and safety programme in workplaces and other areas where drug tests are performed. The national health and safety legislation did not change, and it is still the responsibility of employers to ensure a safe workplace, which includes drug testing. However, Δ 9 -THC-contaminated hemp and CBD products can affect cannabis drug test results in unforeseen ways.
IN PRACTICE
First, CBD product (primarily oils and tinctures) use may trigger positive drug tests since Δ 9 -THC can accumulate in the human body owing to its lipophilic character and extensive half-life, exceeding administrative thresholds or cut-off concentration values. Second, it may become a first-line defence for non-negative screening tests and positively confirmed Δ 9 -THC test results. The integrity of such a defence must be interrogated if a transgression by the employee of workplace policy or contract has indeed occurred, where a delictual liability exists or where criminal sanctions may apply.
The absence of intent must be kept in mind when the MRO assesses the organisation's risk due to the possible use of cannabis by the individual. A strict liability approach is not justified in a workplace drug testing programme, but decisive action must follow for users with no legitimate excuse for intentional or negligent use.
It will be a complex task for the MRO to investigate whether Δ 9 -THC ingestion is due to the ingestion of a contaminated CBD oil product. The MRO may consider analysing the product to verify the Δ 9 -THC purity, since a non-compliant CBD product is already suspicious. Doubt will, however, not be eliminated even if the product does comply with the legislative requirements. Dosage frequency, dose amounts, and the continuous homoeostatic process related to the body's hydration status, which may concentrate drug metabolites in the urine, are all bioaccumulation factors that must be considered.
It will also be problematic for a company to prohibit and enlist the use of hemp-and CBD-containing complementary medicine, purchased legally, in good faith and used correctly according to the prescribed dose regimen in schedule 0. For this reason, legitimate use of CBD products should be considered carefully to exclude the possibility of a confirmed positive test result due to the use of such products that may be contaminated with Δ 9 -THC.
The prohibition of cannabis use and possession in a workplace could justify invoking the Constitutional limitation-of-rights clause within reasonable boundaries. [27] However, in the authors' opinion, it is doubtful that this can be justified for a schedule 0 substance such as CBD when used within all legal bounds.
The issue of contaminated hemp and CBD products may be attributed to the fact that SA does not have adequate resources to maintain the Medicines Act and related regulations' legislative provisions. Also, advertising the products by stating that the product 'contains no THC' is a qualitative statement that should be supported with a definitive value. If the purities are not reported accurately, it amounts to false advertising, which has far-reaching consequences. The authors believe that all cannabis test results must be cautiously treated on a case-by-case basis (ad hoc), even after future mandatory SAHPRA product registration has occurred.
It is relatively easy to imagine the large number of legitimate and illegitimate defences that will be used once roadside testing for cannabis starts in SA.
The way forward
The authors suggest that an experimentally obtained normal reference range of CBD: Δ 9 -THC ratio of drug metabolites in urine or oral fluid be considered, which should be available from a reputable drug testing confirmation laboratory. The CBD:Δ 9 -THC-COOH ratio in urine and possibly also a CBD:Δ 9 -THC ratio for oral fluid can be employed to identify a THC user. A CBD user will have a larger ratio compared with that of a THC user. It will not take long for some cannabis users to understand that a 'normal' CBD:Δ 9 -THC ratio can be skewed by CBD co-administration. Therefore, more research is required to discover unique and selective markers that will enable more accurate discrimination between Δ 9 -THC and CBD users.
Conclusions
The recent changes in the schedules of the Medicines Act related to CBD necessitate proper quality control of CBD products from a compliance drug-testing perspective. The analysis of commercial products containing CBD highlighted quality-control shortcomings. Products commonly did not match the CBD values listed on their packaging, and one of the products was contaminated with Δ 9 -THC, showing levels far above the SA legislative threshold of 0.001 mass percent. CBD users risk administering unknown amounts of cannabinoids, including Δ 9 -THC, and are at risk of positive cannabis drug test results.
It is recommended that medical practitioners consider CBD: Δ 9 -THC metabolite ratios in urine or oral fluid to distinguish between an intentional or irresponsible cannabis user and a legitimate CBD user.
Acknowledgements. None.
Author contributions. The authors contributed equally to the initial concept and preparation of the manuscript.
Conflicts of interest.
None. | 2021-10-15T16:03:41.976Z | 2021-10-05T00:00:00.000 | {
"year": 2021,
"sha1": "4a9428190c022dcbc90bfe860d58d560f7902ae8",
"oa_license": "CCBYNC",
"oa_url": "http://www.samj.org.za/index.php/samj/article/download/13411/9934",
"oa_status": "GOLD",
"pdf_src": "MergedPDFExtraction",
"pdf_hash": "f511f579efafc850fc3bcb7392b8a739db267ad3",
"s2fieldsofstudy": [
"Medicine"
],
"extfieldsofstudy": [
"Medicine"
]
} |
3774479 | pes2o/s2orc | v3-fos-license | Factors associated with different smoking status in European adolescents: results of the SEYLE study
Early onset and long-term smoking are associated with physical and psychological health problems. The aim of the presented analysis was to investigate risk and influencing factors for different smoking status in a big sample of European adolescents. In the context of the “saving and empowering young lives in Europe” (SEYLE) study we surveyed 12,328 adolescents at the age of 13–17 from 11 countries. The survey took place in a school-based context using a questionnaire. Overall 58% reported the onset of ever-smoking under the age of 14 and 30.9% smoke on a daily basis. Multinomial logistic regression model showed significant positive associations between adolescent smoking and internalizing problems (suicidal behavior, direct self-injurious behavior, anxiety), externalizing problems (conduct problems, hyperactivity, substance consumption) and family problems (parental substance consumption, broken home). Our data show that smoking among adolescents is still a major public health problem and adolescents who smoke are at higher risk for mental problems. Further, adolescent smoking is associated with broken home families and parental behaviors. Therefore, early preventive measures are necessary not only for adolescents, but also for their parents.
Introduction
Tobacco smoking among adolescents is a major public health problem and a global concern [1]. Early onset and long-term smoking are associated with physical and psychological health problems [2] and tobacco is still known as one of the most frequently consumed substances in adolescents [3]. Further, many studies reported that adolescent tobacco use is predictive of psychiatric and psychological health problems like depression, substance consumption, as well as personality disorders [4][5][6][7]. Thus, it is necessary to clarify possible risk factors and coherences.
It is universally accepted that the familiar environment and especially parental behavior has an influence on child development and adolescent achievement. Some studies already investigated the relationship of family substance use and the consequences on adolescent legal and drug consumption [8][9][10][11][12][13][14]. Bauman and colleagues [15] already claimed not to underestimate the influence of parental smoking on adolescent smoking behavior and urge a new explanation for the association between parental and adolescent smoking behavior. Although findings across various studies showed only weak and inconsistent associations between parental and adolescent smoking, which is most probably due to methodological issues [16], stronger connections between parental smoking and adolescent smoking have been described recently [17][18][19]. Some studies found that family structure (like intact vs. stepfamilies) is significantly associated with smoking and could be seen as a serious risk factor [20,21].
Further, several studies ascertained strong significant associations between adolescent smoking and substance consumption like alcohol or drug use [22][23][24].
Some previous studies reported a strong association between adolescent substance use and attention-deficit/ hyperactivity disorder (ADHD) or conduct problems [25][26][27][28]. Adolescents suffering from hyperactivity symptoms and ADHD are at increased risk of substance use problems, especially alcohol and nicotine consumption [29,30].
With the implementation of non-suicidal self-injury disorder in the fifth version of the Statistical and Diagnostic Manual of Mental Disorders (DSM-5) the issue around self-injurious behaviors receives an actual impact. In adolescents self-injurious behaviors (SIB) constitute a serious health problem and a major risk factor for future suicidal ideation, suicide attempts and suicide [42][43][44].
By now different opinions exist regarding the association of SIB and substance abuse among adolescents. Some studies report significant correlations between both [45][46][47][48], arguing that similar psychological processes underlie these behaviors [49] or that substance use helps individuals to habituate to SIB [50].
Similarly opposite opinions exist regarding the relationship of adolescent smoking and SIB. Some investigations also established a correlation of smoking and SIB in adolescents [51][52][53]. Brunner and colleagues [54] recently reported an obvious association between adolescent smoking and direct self-injurious behavior (D-SIB). Others, however, ascertained no significant association between smoking and SIB in both genders [52] or at least in boys [55,56].
Those earlier findings were either investigated with smaller sample sizes, adults or children, or they were inconsistent along different studies. Based on this fact and various claims on preventive measures regarding juvenile smoking it is necessary to clarify actual influencing and mediating factors such as family problems (broken home families, parental smoking and substance use), internalizing problems (emotional symptoms, anxiety, previous suicide attempts) and externalizing problems (conduct problems, hyperactivity, alcohol and drug consumption) on the basis of an adequate, representive sample of youths.
The present analysis investigates risk and influencing factors for different smoking status in European adolescents. We hypothesized that the risk for adolescent smoking will be considerably increased in adolescents with internalizing problems (anxiety, emotional symptoms, previous suicide attempts, D-SIB), externalizing problems (conduct problems, hyperactivity, substance use like alcohol or drug consumption) and family problems (parental smoking, family drunkenness, broken homes). We also assumed that the sooner adolescents start smoking and with increasing frequency, the more serious problems emerge.
Participants
The multi-center study "Saving and Empowering Young Lives in Europe" (SEYLE) was initiated to evaluate school-based prevention measures for risk behaviors and 1 3 suicidality in youths. Study design and characteristics of the sample have already been published [57,58]. In total 168 schools, comprising a sample of 12,395 adolescents from 11 countries (i.e., Austria, Estonia, France, Germany, Hungary, Ireland, Israel, Italy, Romania, Slovenia and Spain), with Sweden as the coordination center, were included. In each country eligible schools were randomly selected and ethical permissions were obtained from local ethical committees. Sixty-seven of the 12,395 participating adolescents were excluded, based on missing relevant data and thus a total of 12,328 adolescents [mean age: 14.9 years, range 13-17; number of female/male participants: 6799 (55.2%)/5529 (44.8%)] were included in further analyses.
Questionnaire
Baseline evaluations were conducted between October 2009 and December 2010. The baseline structured selfreport questionnaire was submitted to adolescents aged between 13 and 17. It contained socio-demographic items (e.g., sex, age, religious affiliation, etc.), risk behaviors and mental symptoms. Family setting was assessed by the question: "We would like to know about your family. Please answer this question about your home, where you live permanently or most of the time and put down the people who live with you at your home". Questions from the Global school-based student health survey (GSHS; [59]) were applied to determine risk behaviors, such as alcohol or drug consumption. The following item was used to assess alcohol consumption in youths: "How often do you have a drink containing alcohol? For example, 0.33 l beer or cider; glass of wine or 4 cl of strong alcohol" and drug use was investigated by the question: "During your life, how many times have you ever used drugs". Alcohol consumption of family members was assessed by: "Have you ever seen a family member when they are drunk?". GSHS-Items were also used to identify adolescent and parental smoking behavior. Juvenile smoking behavior was assessed by the questions "Have you ever smoked cigarettes?" and "How many cigarettes did you smoke per day, during the last 6 months?", whereas parental smoking behavior was gathered by the question "Which of your parents or guardians use any form of tobacco?". Emotional symptoms, conduct problems and hyperactivity were investigated by questions of the Strengths and Difficulties Questionnaire (SDQ, [60]). Anxiety was assessed using the Zung Self-Rating Anxiety Scale (SAS; [61]) and suicidality (i.e., previous suicide attempts) was ascertained employing the Paykel Suicide Scale (PSS; [62]).
There are various terms to define self-injurious behaviors such as non-suicidal self-injury (NSSI), deliberate selfharm (DSH) and direct self-injurious behavior (D-SIB). NSSI refers to deliberately inflicted damage to one's body without suicidal intent [63]. The term deliberate self-harm is a more comprehensive term for self-injurious behaviors [64]. DSH is defined as intentional self-injury or self-poisoning irrespective of with or without suicidal intent [65]. DSH also includes indirect damage to one's body such as severe substance abuse or taking overdose [54]. The term DSH so far was used mainly within Europe and Australia, whereas many researchers within Canada and the United States prefer the term NSSI [64].
The present paper focuses on D-SIB, which is defined as self-inflicted damage to an individual's body on purpose, regardless of with or without suicidal intent. It includes direct damage such as self-cutting, -burning, -biting, -hitting, and skin damage by other methods [54]. To investigate adolescents engaging in D-SIB, a 6-item questionnaire was used. This 6-item questionnaire is based on the 9-item Deliberate Self-harm Inventory (DSHI) questionnaire from Bjärehed and Lundh [66], which is a shortened version of the 16-item DSHI by Lundh et al. [67] that originated from the original 17-item DSHI by Gratz [68]. This modified version tends to assess direct self-injurious behavior (D-SIB) to one's body only and self-injurious acts were combined to shorten and simplify the measure. However, it contains the same facets on frequency, severity and duration [54].
Statistical analysis
Statistical analysis was performed using IBM SPSS and GraphPad Prism.
Dichotomous variables were constructed for alcohol consumption (at least twice a week vs. no to low use), drug consumption (at least three times in life vs. no to low use), family drinking (seen family member drunk at least sometimes vs. never seen family drunk), parental smoking (no parent smokes vs. one parent or both parents smoke), family setting (broken homes-one birth parent with or without step-parents vs. both parent households-both birth parents), and previous suicide attempts (lifetime history of suicide attempts vs. no previous suicide attempts).
Group variables were generated for adolescent smoking status (non-smoker-never have smoked cigarettes; nondaily smoker-have ever smoked or do smoke occasionally but do not smoke daily; daily smoker-smoke daily at least one cigarette) and onset of smoking (younger than 10 years; younger than 14 years; 14 years or older).
The relationship of different adolescent smoking status with internalizing, externalizing and family problems was first analyzed using Chi-square test. Group differences were tested by Kruskal-Wallis H test (i.e., nonparametric analysis of variance) followed by post hoc Mann-Whitney U tests for group comparisons. Multinomial logistic regression analysis was used to verify the association between different adolescent smoking status and internalizing, externalizing and family problems controlling for covariate risks. The level of statistical significance was predefined at p < 0.05.
Results
Out of 12,328 participating adolescents, 12.5% reported non-daily smoking and 30.9% smoked daily, with the highest rate in Israel (50.4%) and lowest rate in Ireland (13.4%). 58.0% stated the onset of smoking below 14 years (Table 1). Daily smoking was more common in girls (31.8 vs. 29.8%), whereas early onset (<14) was more common in boys (61.6 vs. 55.3%).
Kruskal-Wallis H test and Chi-square test revealed significant differences between defined adolescent smoking groups and internalizing problems (emotional symptoms, suicidal behavior, direct self-injurious behavior, anxiety), externalizing problems (conduct problems, hyperactivity, substance consumption) and family problems (parental substance consumption, broken home; Table 2).
Significant associations with smoking could be observed for adolescents with direct self-injurious behavior, anxiety, emotional symptoms, conduct problems and hyperactivity. Further, adolescents with previous suicide attempt smoked nearly three times more often than adolescents without previous suicide attempts and adolescents who reported family problems such as parental smoking or living in broken homes reported daily smoking nearly twice as often than adolescents without such family problems. Further, the biggest amount of juvenile daily smokers was assessed in adolescents with alcohol consumption or drug use. Chi-square test revealed significant differences regarding juvenile daily smoking and no or only small differences in the context of non-daily smoking.
Multinomial logistic regression analysis also revealed significant associations of adolescent daily smoking with internalizing problems (emotional symptoms, suicidal behavior, direct self-injurious behavior, anxiety), externalizing problems (conduct problems, hyperactivity, substance consumption) and family problems (parental substance consumption, broken home; Table 3). Significant associations were obtained for the Zung anxiety-score, emotional symptoms and previous suicide attempts. With each increase on the Zung-scale the odds to belong to the daily smoking group than to the non-smoking group rose about 3%. Further, adolescents with previous suicide attempts compared to adolescents with no former suicide attempts had a 1.7 times (OR = 1.69) higher likelihood for belonging to the daily smoking group as to the non-smoking group. Interesting results were found regarding the SDQ-emotional scale.
The likelihood to belong to the non-smokers rather than to daily smokers increased about 10% with each rise on the SDQ-emotional scale.
In addition, multinomial logistic regression revealed significant associations of adolescent daily smoking with conduct problems (OR = 1.22), hyperactivity (OR = 1.15), alcohol consumption (OR = 4.61) and drug use (OR = 19.29). With increasing conduct problems and hyperactivity scores the likelihood for belonging to the daily smoking group rose about 20% and 14%, respectively. Furthermore, adolescents who drink alcohol at least twice a week compared to adolescents who drink no or less alcohol had a 4.6 times higher probability to belong to the daily smoking group than to the non-smoking group. Besides, adolescents who used drugs at least three times in their life compared to adolescents who did not or few times even had a 19.3 times higher likelihood to belong to the daily smokers as to the non-smokers. Family problems were also significantly associated with adolescent daily smoking. Multinomial logistic regression analysis revealed significant associations of parental smoking (OR = 1.66), family member drunkenness (OR = 1.79) and living in broken homes (OR = 1.43).
Multinomial logistic regression analysis also revealed smaller but still significant associations of adolescent nondaily smoking with internalizing problems (emotional symptoms, suicidal behavior, direct self-injurious behavior, anxiety), externalizing problems (conduct problems, hyperactivity, substance consumption) and family problems (parental substance consumption, broken home; Table 3). A negative association was also found regarding the SDQemotional scale. No significant associations were found between adolescent non-daily smoking and anxiety, as well as previous suicide attempts.
Adolescent smoking and direct self-injurious behavior
Another association with adolescent daily and non-daily smoking was found for D-SIB.
Chi-square test revealed a significant difference between defined adolescent smoking groups and D-SIB (χ 2 = 640.208; df = 2; p < 0.001). In the non-smoking group, 7.9% reported D-SIB, whereas in the daily smoking group 26.5% of the adolescents showed D-SIB. Further, 56.4% of adolescents with D-SIB and only 26.5% of adolescents without D-SIB reported daily smoking (p < 0.001).
Further group comparisons revealed statistically significant differences between different adolescent smoking status and D-SIB (p < 0.001). The largest differences emerged between the non-smoking group and the daily smoking group (effect sizes; DSHI, r = 0.29). Smaller but still significant differences were found between non-smokers and non-daily smokers (DSHI, r = 0.12) and between non-daily Table 1 Prevalences for different adolescent smoking status (non-smoker, non-daily smoker, daily smoker), internalizing problems (anxiety, emotional symptoms, previous suicide attempts, D-SIB), externalizing problems (conduct problems, hyperactivity, substance use like alcohol or drug consumption) and family problems (parental smoking, family drunkenness, broken homes) smokers and daily smokers (DSHI, r = 0.14). Non-daily and daily smokers obtained significantly higher mean scores on the DSHI compared to adolescent non-smokers. Multinomial logistic regression analysis revealed that with each increase on the DSHI the odds of belonging to the adolescent daily or non-daily smoking group than to the non-smoking group rose about 36.5 or 22.8% (p < 0.001) for both genders, all age groups and countries. Even after controlling for influencing factors these findings remained significant (Table 3). With increasing D-SIB scores the likelihood of belonging to the daily or non-daily smoking group rather than to the non-smoking group was now about 19 or 15% and still significant (p < 0.001).
Age of onset
The sooner adolescents started smoking, the more they smoked at a later point in time and the higher were their mean scores for smoking frequencies. Adolescents who started smoking below 10 years reported 7 cigarettes per day on average, whereas adolescents that reported the onset of smoking under the age of 14 smoked on average 6 cigarettes per day. Adolescents who started smoking only after 14 years reported smoking four cigarettes per day (Fig. 1). Further, the earlier adolescents started smoking the higher the scores they reached on the DSHI score. Group comparisons revealed statistically significant differences between different onset of smoking groups and D-SIB (p < 0.001).
Discussion
We ascertained high prevalence rates for non-daily and daily smoking in a large sample of European adolescents. Also the WHO [3] and OECD [1] indicated high prevalences regarding these topics, therefore corroborating our results. Accordingly, smoking among youths is a serious health problem worldwide.
Adolescent smoking and internalizing problems
As a very high percentage of youths reported the onset of smoking under 14 years, our data revealed significant differences between different onset of smoking groups and DSHI scores. Many studies state that the early onset of regular tobacco use is predictive of psychiatric and psychological health problems like depression, substance consumption as well as personality disorders [4][5][6][7]. Thus, our findings suggest that the early onset of smoking is associated with D-SIB. Our data further verify a coherence of adolescent smoking behavior (especially daily smoking) and D-SIB. Klonsky and Muehlenkamp [49] also argue that self-injurious behavior and substance abuse have to be strongly associated, because both include causing physiological harm to the body. In addition, prior studies found similar correlations between smoking and self-injurious behavior in youths [45][46][47][48]69], arguing that similar psychological processes underlie these behaviors [49] or that substance use helps individuals to habituate to SIB [50]. Further, our data show a relationship between adolescent smoking and psychological issues like anxiety, emotional symptoms and previous suicide attempts. Several studies found similar associations corroborating, for example the relationship between adolescent smoking and anxiety. Patton et al. [39] already stated that subjects reporting high levels of anxiety were twice as likely to be smokers and that anxiety predicted the initiation of experimental smoking [39]. Further, Johnson et al. [36] suggested that early cigarette smoking may increase the risk of certain anxiety disorders during late adolescence. Recently, Cavazos-Rehg et al. [32] reported that smoking cessation is associated with risk reduction of anxiety even among smokers who have had a pre-existing disorder. This is in contrast to the hypothesis that smoking might function as a sort of compensation or self-medication and suggests the assumption of juvenile smoking as a risk factor for anxiety disorders.
Regarding emotional symptoms we found interesting different results. Whereas Chi-square test revealed positive associations between emotional symptoms and adolescents smoking, multinomial logistic regression analysis showed negative associations between these two topics. These findings in turn support the hypothesis that smoking might function as a sort of self-medication. Also Audrain-McGovern and colleagues [70] provide evidence for selfmedication processes in the relationship between adolescent smoking and depression.
The association of smoking behavior and suicidality has already been demonstrated in several studies [31, 33-35, 37, 38, 40, 41]. Our findings partially confirm these previous results for a large sample of European adolescents.
To summarize, our data show a relationship between adolescent smoking and psychological issues like anxiety, emotional symptoms, previous suicide attempts and D-SIB.
Adolescent smoking and externalizing problems
The present paper further suggests that hyperactivity as well as conduct problems increase the risk for smoking in adolescents. Previous studies have documented that adolescents with attention-deficit/hyperactivity disorder (ADHD) or conduct problems are at increased risk of substance use problems [25][26][27][28]. Chang et al. [29] found that especially hyperactivity and impulsivity predict early onset of tobacco and alcohol use in youths. Recently, Roberts et al. [30] also stated that specifically hyperactive symptoms were associated with alcohol and nicotine use in young adults, underlining that increased levels of impulsivity are thought to contribute to their increased levels of risk.
Our results further show that there exists a relationship between adolescent smoking and several other risk factors. The strongest associations with juvenile smoking were observed between other substance consumptions like drug and alcohol use. Several studies ascertained strong significant associations between these topics as well and therefore corroborate our results [22][23][24].
Thatcher and Clarke [71] proposed that psychological dysregulation can be seen as a predictive phenotype for substance use disorders in adolescents. It is characterized by cognitive, behavioral and emotional difficulties in childhood, connecting heritable predispositions and early environmental influences to later substance use disorders. During adolescence, brain circuits including those for motivation, reward and decision-making are not yet fully developed, thereby making this group more susceptible to addiction [72].
Adolescent smoking and family problems
We further found that family behavior and issues like parental smoking, family drunkenness and living in broken homes influences adolescent smoking behavior. Previous studies confirm the influence of family factors on adolescent smoking behavior. Especially the relationship of parental smoking or tobacco use and adolescent smoking is reported to be very high [17,73]. Bauman et al. [15] already verified that lifetime parental smoking was as strongly correlated with adolescent smoking as peer smoking. Farkas et al. [74] found that parental smoking cessation keeps adolescents off smoking and that the earlier parents quit smoking, the less likely their children are to become smokers. In addition to that, Almutairi [75] showed that parents had been perceived to be the first source for smoking. Further, Nosa et al. [19] found that exposure to smoking at home seems to be an important risk factor for ever-smoking. Compared to prior findings we found strong effects regarding this topic. Adolescents with smoking parents had a higher likelihood for belonging to the daily smoking group. Furthermore, Griesbach et al. [20] found that family structure (like intact vs. stepfamilies) is significantly associated with smoking among 15-year-olds in seven European countries. Recent findings of Du et al. [21] in multicultural students of Hawaii also show that family structure could be a serious risk factor for smoking among young people. These results demonstrate how important and necessary it is to raise awareness in parents and legal guardians of the damaging influence of their behavior on the smoking behavior of their children to ideally cause lifestyle changes and to make their homes smoke-free.
Limitations and strengths
It is important to mention that our data do not imply causality, meaning that they do not give an explanation about cause and effect. This limitation of the study is due to the cross-sectional design, where a direct link between determinants and outcome is missing. Concerning this matter further research will be needed. For example, does smoking itself represent a form of self-injurious behavior or does it rather function as a compensation of existing problems?
Another limitation of this analysis would be the missing cross-cultural comparison. Our study comprised adolescents from 11 different countries from all across Europe. Of course there exist cultural differences between the majority of countries, which might also influence adolescent smoking behavior. Future analysis of differences across countries might therefore help to develop country and culturespecific treatment programs. Nevertheless, controlling for country in our regression analysis did not attenuate the effect of the influencing factors we found.
The major strength of this study is its large sample size together with the random selection of schools across 11 representative study sites in Europe. A huge body of literature corroborates our results, most of which are studies from North America. Therefore, to our knowledge, this study is the first investigating factors associated with adolescent smoking in Europe on such a large scale.
Conclusion
To summarize, the fact is that we have identified very high prevalences and early onset of smoking in European adolescents and that smoking and internalizing problems (emotional symptoms, suicidal behavior, direct self-injurious behavior, anxiety), externalizing problems (conduct problems, hyperactivity, substance consumption) as well as family problems (parental substance consumption, broken home) are consistent and highly associated in an adequate, representative sample of European youths. Further, our data suggest that smoking intensity, which is modulated by both early onset of smoking and family problems, predicts the intensity of externalizing and internalizing problems. Thus, reducing the intensity of smoking from daily to non-daily or preferably no smoking may reduce the intensity of the associated symptoms and might have major clinical and public health consequences.
Therefore, early preventive measures-not only for adolescents but also for their parents-are necessary and essential and should not be neglected. The recently evaluated prevention program "Youth Aware of Mental Health" (YAM) in which a decrease of mental problems, especially suicidal ideations and suicide attempts in high schools was observed [76], could also be developed for young people to prevent early smoking and its problematic outcomes. However, further research will be needed to develop new strategies regarding adolescent smoking and to prevent poor outcomes regarding D-SIB and associated psychological issues in adolescents. | 2017-11-04T17:06:40.222Z | 2017-04-06T00:00:00.000 | {
"year": 2017,
"sha1": "2d6fb2c1442e071f1203954269b90fa2cf64e90c",
"oa_license": "CCBY",
"oa_url": "https://diglib.uibk.ac.at/ulbtirolfodok/content/titleinfo/4569326/full.pdf",
"oa_status": "GREEN",
"pdf_src": "PubMedCentral",
"pdf_hash": "00cd1c3673d12ca4dd8c9455646211f20ba902e2",
"s2fieldsofstudy": [
"Medicine",
"Sociology"
],
"extfieldsofstudy": [
"Medicine"
]
} |
4693881 | pes2o/s2orc | v3-fos-license | Quantitative TaqMan® real-time PCR assays for gene expression normalisation in feline tissues
Background Gene expression analysis is an important tool in contemporary research, with real-time PCR as the method of choice for quantifying transcription levels. Co-analysis of suitable reference genes is crucial for accurate expression normalisation. Reference gene expression may vary, e.g., among species or tissues; thus, candidate genes must be tested prior to use in expression studies. The domestic cat is an important study subject in both medical research and veterinary medicine. The aim of the present study was to develop TaqMan® real-time PCR assays for eight potential reference genes and to test their applicability for feline samples, including blood, lymphoid, endocrine, and gastrointestinal tissues from healthy cats, and neoplastic tissues from FeLV-infected cats. Results RNA extraction from tissues was optimised for minimal genomic DNA (gDNA) contamination without use of a DNase treatment. Real-time PCR assays were established and optimised for v-abl Abelson murine leukaemia viral oncogene homolog (ABL), β-actin (ACTB), β-2-microglobulin (B2M), β-glucuronidase (GUSB), hydroxymethyl-bilane synthase (HMBS), hypoxanthine phosphoribosyltransferase (HPRT), ribosomal protein S7 (RPS7), and tryptophan 5-monooxygenase activation protein, zeta polypeptide (YWHAZ). The presence of pseudogenes was confirmed for four of the eight investigated genes (ACTB, HPRT, RPS7, and YWHAZ). The assays were tested together with previously developed TaqMan® assays for feline glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and the universal 18S rRNA gene. Significant differences were found among the expression levels of the ten candidate reference genes, with a ~106-fold expression difference between the most abundant (18S rRNA) and the least abundant genes (ABL, GUSB, and HMBS). The expression stability determined by the geNorm and NormFinder programs differed significantly. Using the ANOVA-based NormFinder program, RPS7 was the most stable gene in the tissues studied, followed by ACTB and ABL; B2M, HPRT, and the 18S rRNA genes were the least stable ones. Conclusion The reference gene expression stability varied considerably among the feline tissues investigated. No tested gene was optimal for normalisation in all tissues. For the majority of the tissues, two to three reference genes were necessary for accurate normalisation. The present study yields essential information on the correct choice of feline reference genes depending on the tissues analysed.
Background
The domestic cat is an important study subject not only in veterinary medicine but also in medical research. It plays an essential role as a laboratory model for human infectious, hereditary and endocrine diseases and allows the study of topics such as host-pathogen interactions, defence mechanisms, and development of prophylactic or therapeutic regimens. Of importance in this context is not only the feline immunodeficiency virus, the single naturally occurring animal model for HIV-AIDS pathogenesis [1,2] and the feline leukaemia virus (FeLV), an important model for retrovirus and tumour research [3,4], but also other infectious agents, some related to fatal human diseases [1,5]. Furthermore, in the genome of the cat, various mutations have been characterised that are associated with genetic diseases, and 280 phenes have been reported, 136 of which could potentially serve as models for human hereditary diseases http://omia.angis.org.au/. Models under investigation include the glycogen storage disease type IV reported in the Norwegian Forest cat, the only reported animal model for this pathology [6], or the obesity-associated form of diabetes mellitus in the domestic cat that is similar to the type 2 diabetes mellitus in humans [7]. For the latter, the domestic cat presents a valuable model for understanding the molecular mechanisms linking obesity to the development of insulin resistance, hypertension, and atherosclerosis [7]. The potential of the cat as an animal model and the similarities in genome organisation between humans and felids [8,9] provide the basis for a wide range of gene expression studies.
Quantitative real-time PCR assays are a method of choice for reliable and fast quantification of transcription levels in gene expression studies, and they are used frequently in many areas of modern research. Real-time PCR provides quantification of input templates over a broad linear range, low sample consumption, rapid throughput of large sample numbers, and low risk of contamination [10][11][12]. Accurate normalisation is of fundamental importance to obtaining sound results. Normalisation is usually achieved by simultaneous amplification of reference genes along with the target gene. Several publications emphasise the need for more than one reference gene for exact analysis of transcription levels [13][14][15]. When selecting reference genes, several critical points should be considered, including a stable, experimentally-independent expression pattern of the candidate gene, the absence of processed pseudogenes, an adequate level of expression, and a lack of potential co-regulation among target and reference genes [16][17][18].
For the domestic cat a number of potential reference genes have been studied using pair-wise correlation analysis (geNorm) [19] and real-time PCR systems based mainly on SYBR Green chemistry [13,[20][21][22]. The SYBR Green principle has the advantage that it is less costly; however, TaqMan ® systems usually have a higher specificity and lead to less non-specific product formation than SYBR Green assays. No systematic study using TaqMan ® realtime PCR assays for potential feline reference genes is available. In addition, no comparisons of pair-wise analysis with ANOVA-based methods (NormFinder) [23] have been published, and most assays for feline reference genes were conducted using pathological samples. Data on tissues from healthy cats are largely missing.
Thus, the purpose of the present study was to i) develop and optimise TaqMan ® real-time PCR assays for potential feline reference genes and ii) evaluate the suitability of these assays for normalisation in the blood and other tissues from clinically healthy cats and neoplastic tissues. The earlier tissues were chosen to cover those frequently included in studies investigating infectious diseases and immunological, endocrine, metabolic, and inflammatory disorders. The neoplastic tissues originated from FeLVinfected cats; the expression of reference genes may differ in neoplastic tissues [16,24]. For stability comparison of the potential reference genes, two programs were used: the ANOVA-based NormFinder and the geNorm.
Sample collection
All domestic cats included in this study had been in experimental studies officially approved by the veterinary office of the appropriate Swiss Canton. They were kept in groups under optimal ethological conditions. Clinically healthy cats were available from negative control groups, and they were sacrificed for reasons unrelated to this study. Tissue samples were collected upon necropsy from 15 clinically healthy cats (ten neutered males and five intact females). The tissues were histologically examined and verified to be free of pathological alterations. They originated from lymphatic tissues including bone marrow (n = 11), mesenteric lymph node (n = 10), and spleen (n = 10); from the endocrine tissues of the adrenal gland (n = 11), pancreas (n = 13), thyroid (n = 10), and parathyroid (n = 7); from the gastrointestinal tissues of the parotid gland (n = 9), duodenum (n = 10), and ileum (n = 10); and from the brain (n = 13), myocardium (n = 10), kidney (n = 14), and liver (n = 9). The cats ranged in age from 1.25 to 13 years (median age 3.8 years). In addition, EDTA-anticoagulated whole blood samples were collected from 11 specific pathogen-free (SPF) cats (five males at the age of 0.5 years, five neutered males at 6 years, and two spayed females at 14 years). Upon necropsy, neoplastic tissues (n = 12, including tissue from liver (1), spleen (2), kidney (2), mesenteric lymph node (3), ileum (1), and thymus (3) were collected from six FeLV-infected cats (three neutered males, one neutered and two intact females; ages of 3 to 13 years). Five of the cats had been diagnosed with malignant lymphoma; one had leucosis. All tissues were snap-frozen upon collection and stored at -80°C until extraction of nucleic acids.
Nucleic acid extractions
Tissues (30-35 mg, in duplicate) were homogenised prior to RNA extraction in 350 μl of RLT buffer (Qiagen, Hombrechtikon, Switzerland) containing 3.5 μl β-mercaptoethanol, together with a 5 mm Ø steel bead (Schieritz & Hauenstein, Arlesheim, Switzerland) in a Mixer Mill MM 300 (Retsch, Haan, Germany). Samples were then processed using the RNeasy Mini Kit (Qiagen) following the manufacturer's recommendations. In a preliminary experiment using selected samples (n = 10), the effect of a digestion step on the RNA binding silica gel membrane of the spin column, performed according to the manufacturer's instructions with RNase-free DNase, was assessed (on-column DNase treatment). In addition, for bone marrow, lymph node, spleen, and thyroid samples, the RNeasy Plus Mini Kit (Qiagen) with genomic DNA (gDNA) Eliminator spin columns and RLT plus buffer was applied according to the manufacturer's recommendations. The presence of contaminating gDNA was assessed using GAPDH quantitative reverse transcriptase (RT-) PCR with a minus-reverse transcription control with Reverse Transcriptase qPCR Mastermix (Eurogentec, Seraing, Belgium). RNA was extracted from 1 ml of blood within 60 minutes of collection using the QIAamp RNA Blood Mini Kit (Qiagen) and stored at -80°C until further use. gDNA was extracted from tissues using the DNeasy Blood & Tissue Kit (Qiagen). For all RNA and DNA extractions, negative controls consisting of 100 μl of phosphate buffered saline were prepared with each batch to monitor for crosscontamination.
First-strand cDNA synthesis
The RNA yield and the ratio of absorbance at 260 nm to 280 nm (A 260 /A 280 ratio) were measured using the Nano-Drop ND-1000 Spectrophotometer (NanoDrop Technologies, Witec, Littau, Switzerland). Samples containing < 10 ng/μl of RNA were excluded from the study. Firststrand cDNA was synthesised in quadruplicate using the High Capacity cDNA Reverse Transcription Kit (Applied Biosystems, Rotkreuz, Switzerland) and random primers according to the manufacturer's instructions. The amount of input RNA in each reaction was calculated to be 2 μg. The cDNA GAPDH copy number/RNA GAPDH copy number ratio was calculated as a measure of the efficiency of the cDNA synthesis; this ratio was used to normalise the reference gene copy numbers as assessed by quantitative real-time PCR.
Development of real-time PCR assays for feline reference genes
Using Primer Express™ software (versions 2 and 3, Applied Biosystems), primers and TaqMan ® probes were designed for eight potential reference genes: ABL, ACTB, B2M, GUSB, HMBS, HPRT, RPS7, and YWHAZ (essential gene-specific data are given in Table 1). The sequences and information on gene organisation were retrieved from Ensembl http://www.ensembl.org/index.html, GenBank http://www.ncbi.nlm.nih.gov and the Genome Annotation Resource Fields (GARFIELD) http:// lgd.abcc.ncifcrf.gov [25]. All systems were designed so that the predicted amplicons would span exon-exon boundaries ( Table 2). The eight primer pairs (Microsynth, Balgach, Switzerland) were tested for amplification of the appropriate amplicon length using 5 μl of cDNA in a total volume of 25 μl per reaction on a Rotor-Gene 6000 realtime rotary analyser (Corbett, Mortlake, Australia) using In order to test for potential amplification of pseudogenes or of gDNA, the eight primer pairs were also assayed with gDNA under the same conditions. Moreover, the possible presence of pseudogenes for the eight assays was assessed using the Ensembl Genome Browser. In all PCR assays, water was used as a negative control.
Optimisation of quantitative real-time PCR assays
After the primers had been tested for correct amplification of the estimated amplicon length, the eight newly designed real-time TaqMan ® PCR systems were optimised using cDNA and a 3 × 3 primer matrix with 50, 300, and 900 nM end concentrations. Each of the nine conditions was run in quadruplicate under the conditions described above. Moreover, using the best primer concentration, five different probe (Microsynth) end concentrations (50,100,150,200, and 250 nM) were tested for optimal performance. The optimised assays were tested together with a feline GAPDH TaqMan ® real-time PCR assay developed previously in our laboratory [26] and a universal 18S rRNA gene (Applied Biosystems).
Production of DNA standards for absolute quantification
cDNA synthesised from feline tissue samples was used to generate standard templates for absolute quantification of ABL, ACTB, B2M, GUSB, HMBS, RPS7, and YWHAZ. The corresponding sequences were amplified using primers enclosing the TaqMan ® real-time PCR sequences under conditions described (Table 3) [27]. The gel purified amplification products (Gen Elute PCR Clean-Up Kit, Sigma-Aldrich, Buchs, Switzerland) were subjected to a 3' A-tailing reaction (Sigma) and ligated into the TOPO TA cloning vector pCRII (Invitrogen, Basel, Switzerland), selected by Ampicillin resistance, followed by sequencing (Microsynth). Plasmids were linearised by restriction digestion with BamHI (Promega, Wallisellen, Switzerland), SpeI (New England BioLabs, Berverly, MA, USA), or KpnI (Roche, Rotkreuz, Switzerland) and then gel purified. The copy numbers were calculated based on spectrophotometric analysis (NanoDrop ND-1000). Carrier salmon sperm DNA (Invitrogen) at a concentration of 30 μg/ml was used for the tenfold serial dilutions of the standard templates, and aliquots of the dilutions were stored at -20°C until use. For the GAPDH assay, the DNA standard described previously [26] was used. For the HPRT and 18S rRNA assays, cDNA from kidney tissue of a clinically healthy cat was diluted tenfold in carrier salmon sperm DNA and in nuclease free water, respectively, to produce an arbitrary standard. The copy numbers of the latter samples were estimated by matching the resulting threshold cycle (Ct) values with those of the feline GAPDH standard.
Efficiency, sensitivity, linear range and precision of the real-time PCR assays
The efficiency of the newly designed assays was calculated as described [28] using the following equation: E = (10 (-1/ slope) )-1. The sensitivity of the seven new systems (for which DNA standards had been produced) was deter-mined by an endpoint dilution experiment: ten replicates of the dilutions containing 10 2 , 10 1 , and 10 0 standard template copies per reaction, respectively, were tested. The sensitivity of the assay is given by the dilution in which at least seven of 10 replicates are still positive [29]. The linear range of amplification and the precision of all newly developed TaqMan ® real-time PCR assays were determined using tenfold serial dilutions of the plasmid or arbitrary standards. For the precision analysis, the dilutions were chosen according to the ranges of Ct values that were characteristic for the expression levels of the particular reference genes in the tissues. Intra-run (n = 10) and between-run (n = 5) analytical performances of the PCR measurements were determined using these control materials.
Data evaluation and statistics
For stability comparison of candidate reference genes, the Microsoft Excel Add-in NormFinder [23] was applied.
Comparisons were made with calculations performed using the geNorm version 3.4 [19]. The NormFinder uses an ANOVA-based model [23], while the geNorm calculates the stability using a pairwise comparison model [19].
In addition, the geNorm ranks candidate reference genes according to the average expression stability, M [19]. Genes with the lowest M values have the most stable expression; a cut-off of 1.5 was proposed, above which the variation is assumed to be too high for accurate normalisation [19]. Moreover, the optimal number of reference genes required for accurate normalisation was estimated using the geNorm. To this end, the pairwise variation V n/ n+1 between sequential normalisation factors containing an increasing number of reference genes was calculated. If V n/n+1 < 0.15, the recommended number of reference genes is given by n; the inclusion of an additional reference gene is not required [19]. Statistical analyses were performed with GraphPad Prism for Windows, Version
RNA extractions and gDNA contamination
RNA extractions from blood using the QIAamp Blood Mini Kit and from tissues using the RNeasy Mini Kit yielded RNA with a low level of contaminating gDNA (< 1%), with the exceptions of bone marrow, lymph node, spleen, and thyroid. When RNA extraction was performed for these four tissues using the RNeasy Plus Mini Kit, contaminating gDNA levels were < 1%. These additionally processed RNA samples were used for analysis of these four tissues. No on-column DNase treatment was used in the main experiment because the loss of RNA due to DNase treatment was > 85%, as determined in a preliminary experiment (data not shown). RNA purity was estimated from A 260 /A 280 ratio; this ratio ranged from 1.7 to 2.1.
Evaluation of the primer pairs
When the primers were tested in a conventional PCR with cDNA, all assays yielded PCR products of the predicted size ( Table 2). The primers were then assessed using the same procedure and gDNA to test for the amplification of gDNA and the possible presence of pseudogenes. Bands of the size of the cDNA were found for ACTB, HPRT, RPS7, and YWHAZ, indicating the presence of processed pseudogenes for these genes, but not for ABL, B2M, HMBS, and GUSB (Table 2). This was consistent with the results retrieved from Ensembl. In addition, for ACTB, GUSB, and HMBS, PCR products presumably of the size of the gDNA, including the introns, were detected ( Table 2).
Evaluation and optimisation of the newly developed realtime PCR assays
Primer and probe concentrations for the eight newly designed TaqMan ® real-time PCR assays were optimised using cDNA (for final concentrations see Table 4). When the real-time TaqMan ® PCR assays were tested using gDNA instead of cDNA, specific amplification was found for ACTB, HPRT, RPS7, and YWHAZ, confirming the presence of pseudogenes (Table 2). In addition, amplification was detected for GUSB (Table 2).
Efficiency, sensitivity, linear range and precision of the real-time PCR assays
The amplification efficiencies of the eight newly designed assays and the feline GAPDH real-time PCR were ≥96%.
The lower detection limit of the assays for ABL, ACTB, B2M, GUSB, RPS7, and YWHAZ was equal to one copy of target standard plasmid per reaction in an endpoint dilution experiment (7 to 10 out of 10 reactions positive). For HMBS the lower limit of detection was < 100 copies per reaction (10 out of 10 reactions positive). For all newly developed TaqMan ® real-time PCR assays, we observed linearity of the assay over a ≥10 8 -fold range. The coefficients of variation ranged from 0.44% (B2M) to 1.18% (YWHAZ) for the intra-run precision analysis and from 0.49% (ACTB) to 2.15% (HMBS) for the between-run analysis.
Expression levels of candidate reference genes
Transcription of the ten candidate reference genes was detectable above background in all tissue and blood samples from all cats tested. The potential reference genes were classified into three groups according to their transcription levels (all healthy tissues and blood samples were included in the analysis; Figure 1a). The difference in median expression levels was 10 6 between the most abundant and least abundant transcripts: 18S rRNA showed a high transcription level (median copy number/reactioñ 10 9 ); ACTB, GAPDH, B2M, HPRT, and RPS7 were found to have intermediate transcription levels (median copy number/reaction 2.6 × 10 4 to 9.3 × 10 4 ), and ABL, GUSB, and HMBS had low transcription levels (median copy number/reaction 0.5 × 10 3 to 1.9 × 10 3 ; Figure 1a). YWHAZ had a transcription level between the intermediate and low levels (median copy number/reaction 6.9 × 10 3 ). Individual candidate reference genes had different expression levels across all studied tissues; the transcription levels differed significantly among all different reference genes when all tissues were included in the analysis (pF < 0.0001; pD < 0.05), with the exceptions of ACTB, GAPDH and B2M; GUSB and HMBS; and HPRT and RPS7 Expression levels of candidate reference genes in different tissues Figure 1 Expression levels of candidate reference genes in different tissues. a) All healthy tissues combined, b) adrenal gland, c) bone marrow, d) myocardium, e) brain, and f) blood samples. Values are given as copy numbers per PCR. For the 18S rRNA gene and HPRT the copy numbers were calculated using an arbitrary standard (see M&M). Data are shown as box plots. Boxes extend from the 25th to the 75th percentile; a horizontal line represents the median, and the error bars extend down to the smallest and up to the largest value. Expression levels were analyzed for statistical differences using the Friedman test (pF-values as indicated in the figure). Significant differences between two particular genes were analyzed by the Wilcoxon test for paired samples (asterisks and circles, respectively, mark statistically significant differences between two genes; pW ≤ 0.0039). Copies/reaction a.
( Figure 1a). The latter three groups of reference genes did not have significantly different transcription levels (pD > 0.05; Figure 1a). A reference gene transcription level pattern similar to that seen in all tissues combined was found when individual tissues were examined (for a representative example, see Figure 1b), with the following particular exceptions. In the bone marrow samples HMBS transcription was significantly higher than GUSB transcription (pW = 0.0010; Figure 1c), in the myocardium and brain samples GAPDH transcription was significantly higher than B2M transcription (pW = 0.0020; Figure 1d and 1e), in the blood B2M and ACTB transcription levels were higher than GAPDH transcription (pW = 0.0039; Figure 1f), and in the liver YWHAZ was significantly lower than RPS7 and HPRT (pW = 0.0039, data not shown). When expression levels of the individual potential reference genes were analysed among different tissues significant differences were found. The most prominent were the following: GAPDH was significantly higher in the myocardium, brain, and blood samples than in most of the other tested tissues (pKW < 0.0001; pD < 0.001 for 11 of the other tested tissues; Figure 2a); ABL was significantly lower in the bone marrow samples than in the majority of the other tested tissues (pKW < 0.0001; pD < 0.001 for eight of the other tested tissues; Figure 2b), B2M was higher in the blood samples (pKW < 0.0001; pD < 0.001 for twelve of the other tested tissues; Figure 2c), and YWHAZ was higher in the blood and brain samples than
a.
A d r e n a l g l a n d P a n c r e a s P a r a t h y r o i d
b.
A d r e n a l g l a n d P a n c r e a s P a r a t h y r o i d
c.
A d r e n a l g l a n d P a n c r e a s P a r a t h y r o i d in the majority of the other tissue samples (pKW < 0.0001; pD < 0.001 for 11 and 12, respectively, of the other tested tissues; Figure 2d). No particular differences in expression levels were observed when the neoplastic tissues were compared to the healthy tissues (pW > 0.05 for all genes tested; data not shown).
Expression stability of candidate reference genes in different tissues
The stability of reference gene expression was estimated based on the calculations of the geNorm and NormFinder software, and the rank order given by the two programs differed significantly (Table 5; for details see also Additional Files 1 and 2). However, partial agreement was found between the NormFinder results and the results according to the M values of the geNorm program (Table 5); for three tissues (bone marrow, duodenum, and kidney) the rank order was identical. The two genes that ranked best were identical with both methods (manual M value ranking and NormFinder) for the majority of the tissues except for lymph node, parotid gland, liver, and blood samples. All further stability analyses were made using the NormFinder results.
The stability of the genes varied considerably depending on the tissues tested. When tissues were analysed individually, no single gene was found among the three bestranked genes in all of the tested tissues. In 11 of the 14 healthy tissues, RPS7 ranked among the three most stable genes, followed by ACTB in eight and ABL in six out of 14 tissues (Table 5). Less stable were GUSB and YWHAZ; they were among the most stable genes in five of the 14 tissues used. GAPDH was among the three most stable genes in the NormFinder ranking in four out of 14 tissues. HMBS, B2M, HPRT, and 18S rRNA were the least stable, HMBS with two, B2M and HPRT with one, and 18S rRNA with no rankings among the most stable genes in the 14 tissues tested. When all 14 healthy tissues were included at once in the analysis (Table 5), RPS7, GUSB, and YWHAZ ranked as the best three reference genes, while ABL and ACTB ranked fourth and fifth. Exceptions were found in certain healthy tissues; RPS7 was less stable in the ileum, lymph node, and thyroid samples. Moreover, ACTB ranked last in the pancreas samples (Table 5). In the blood samples, GAPDH and B2M ranked better than in other tissues, but RPS7 ranked only second to last (Table 5). Remarkably, in the neoplastic tissues the two most stable genes were identical to those in all the healthy tissues (RPS7 and ACTB). ABL, GUSB, and YWHAZ were less stable than in the healthy tissues ( Table 5).
Number of reference genes for optimal normalization
The optimal number of reference genes for normalisation was calculated using the geNorm program. The number of recommended reference genes for optimal normalisation varied considerably depending on the tissue being tested. For brain, myocardium, lymph node, and adrenal gland the pairwise variation V 2/3 was ≤ the proposed cut-off of 0.15 (Additional File 2); therefore, two reference genes should be sufficient for accurate normalisation in those tissues. Similarly, for parathyroid, parotid gland, liver and kidney, three; for thyroid, four; for spleen and neoplastic tissues, five; and for blood, seven reference genes were necessary for accurate normalisation according to the geNorm program (Additional File 2). For four tissues the pairwise variation V always exceeded the cut-off of 0.15. The recommended number of reference genes for these four tissues (lowest V value) was: five for the pancreas, six for ileum and bone marrow, and seven for the duodenum (Additional File 2). For all tissues combined, the optimal number of reference genes that would have been necessary for normalisation exceeded ten using the reference genes tested in this study. For the three tissue groups, endocrine, lymphatic, and gastrointestinal tissues, the V value always exceeded the cut-off of 0.15; the recommended number of reference genes for these tissue groups (lowest V value) was six (Additional File 2).
Normalisation
When tissues were analysed individually, a normalisation factor was calculated for all tissues with the exception of the pancreas. For the latter, all M values calculated by geNorm exceeded the proposed cut-off value of 1.5, and no normalisation factors based on the geometric mean of multiple reference genes could be computed (Additional File 2). When tissue groups were analysed, a normalisation factor was calculated for the endocrine and gastrointestinal tissues but not for the lymphatic group. No normalisation factor was calculated for all tissues combined.
Discussion
The present study is the first to develop and evaluate realtime TaqMan ® assays for the quantification of a series of potential feline reference genes. The assays were applied using feline peripheral blood and tissue samples from healthy cats. The latter were chosen based on their applicability in research areas such as the investigation of immune functions or metabolism, characterisation of infections, inflammatory reactions, or excretion patterns of pathogens. In this regard, the selected tissue categories also met the claim of suitability regarding their potential use in animal models for several human diseases. The expression stability of potential reference genes was analysed and compared for the feline species for the first time using pair-wise and ANOVA-based analyses; the two methods yielded discrepant results concerning the gene expression stability.
We selected ten commonly used mammalian reference genes featuring a broad range of cellular functions (Table 1). Some other potential candidate genes, such as Cyclo- philin A, the TATA box binding protein or the transferring receptor CD71, were not included because they are regulated by or known to interact with retroviruses [30][31][32][33]; the latter are a major research interest of our group.
Processed pseudogenes are known to hamper data interpretation in mRNA transcription analysis [36,37]. We therefore included potential reference genes that are known to lack pseudogenes in humans, i.e., ABL, B2M, GUSB, and HMBS [36,38]. We confirmed that these four genes also lack pseudogenes in the cat (at least for the sequences included in our assays). For the other four newly developed assays (ACTB, HPRT, RPS7, and YWHAZ), we demonstrated the presence of processed pseudogenes. For feline GAPDH the presence of one copy of a pseudogene had been reported earlier [39]. To minimise co-amplification of pseudogenes, gDNA contamination of the assayed RNA was reduced to a minimum by choosing the optimal RNA extraction method for each tissue. The inclusion of a DNase digestion step was omitted because it significantly reduced the RNA yield. The absence of a DNase treatment and thus the presence of sufficient amounts of RNA may explain why the PCR signals were clearly above background for all samples, in contrast to other reports in which HPRT, ABL, or HMBS could not (reliably) be detected [17,40].
We designed the eight new assays to span an exon-exon junction in order to reduce the possible interference of gDNA -including conventional but not processed pseudogenes. The assays for those four potential reference genes lacking processed pseudogenes did not amplify gDNA, with the exception of GUSB. For the latter, the amplicon size from gDNA was only 532 bp due to a short intron and the primer design software had positioned the exon-exon junction only three nucleotides before the 3' end of the probe ( Table 2).
The expression level of an ideal reference gene should not undergo tissue-specific and experimentally-dependent variation and should be similar to that of the target genes [16][17][18][19]41,42]. The RNA transcription levels of the ten selected candidate reference genes in the blood samples and 15 tissues differed considerably, but most of them were within a range that could be used for proper normalisation of many target genes. Only the 18S rRNA gene expression level was impracticably high. Significant differences were found when the expression levels of the potential reference genes were analysed in different tissues. GAPDH expression was higher in brain, myocardium, and blood samples; this is consistent with results reported for human tissues [19,43]. B2M expression was significantly higher in blood. This has also been demonstrated for human leukocytes [19].
The expression stability of the tested reference genes was calculated using the NormFinder and the geNorm software. The geNorm uses a pairwise comparison model that provides a combination of two genes whose expressions are most correlated in the tested sample [19], while the ANOVA-based model of the NormFinder program selects the highest ranked gene based on the highest expression stability due to minimal estimated intra-and intergroup variation [23]. Using the geNorm, co-regulated genes may become highly ranked independent of their expression stabilities [23]. In contrast, the ANOVA-based approach is not significantly affected by co-regulation of candidate reference genes [23]. Therefore, the differences found between the reference gene rankings automatically produced by the two programs may indicate that at least some of the examined genes were co-regulated. A better agreement was found between the automatic NormFinder ranking and the ranking performed manually using the geNorm M values. To our knowledge, the differences observed between the automatically calculated geNorm gene ranking and the ranking using the M values of the geNorm program has not been reported previously.
No general best reference gene has been found for all species. Moreover and in agreement with findings from other species [17,41,42,53], in the cat no single gene was suitable for accurate normalisation in all investigated tissues. This highlights the need for proper validation of reference genes in the respective tissues preceding any experimental set-up. It has been demonstrated that normalisation of data sets with different reference genes, such as GAPDH and ACTB, may influence the outcome of the study; the expression profiles of target genes were markedly influenced and statistically significant differences between study groups were present or absent depending on the choice of the reference genes [54][55][56].
For the majority of the examined feline healthy tissues RPS7, ACTB, and ABL were among the most stably expressed genes. When all healthy tissues were combined for analysis, the three most stable genes were RPS7, GUSB, and YWHAZ. These results are only partially in agreement with those of a recent validation of feline reference genes using SYBR Green real-time PCR assays and pairwise analysis [13]. In the latter study, RPS7 was also found as the most stable gene in six tissues under investigation; YWHAZ ranked fourth (after two ribosomal protein genes not included in the present study), and GUSB was found among the least stable genes; ACTB and ABL were not examined [13]. Three feline tissues were included in both the previous [13] and the present study. While for the kidney a partial agreement was found in the gene ranking (RPS7 and YWHAZ were among the stable ones; HPRT and GAPDH were rather unstable), the results for liver and myocardium differed significantly. The observed discrepancy may be (apart from the differences in the applied PCR and mathematical methods) due to the tissues under investigation. We based our evaluation primarily on tissues from healthy cats. In contrast, the study from Penning and co-workers [13] used mainly pathological tissues. Pathological processes can alter the expression levels of reference genes [55,57], therefore it is of importance to evaluate potential reference genes in healthy tissues. Generally, healthy tissues are included in most study design as internal controls. Thus, with the present study, we provide baseline data on reference genes for many future studies investigating feline tissues. Furthermore, the number of samples studied may have influenced the evaluation: in the previous study, for the majority of the tissues, a small number of identical samples (i.e. kidney: n = 2 healthy tissue; n = 3 tumours; n = 3 chronic kidney failure) was available [13].
Although in the present study B2M in general was not a very stable reference gene in the tissues, it ranked second to fourth in stability in the blood samples. This would confirm a finding in human blood cells, where B2M seemed a good choice for normalisation in leukocytes, while it was one of the least stable genes in tissues [19]. For the neoplastic tissues, only a limited number of samples from FeLV-infected cats with malignant lymphoma and leucosis were available. The gene ranking order but not the expression levels of the reference genes in the neoplastic tissues differed significantly from those in the healthy tissues. Nonetheless, the two most stable genes in the neoplastic tissues (RPS7 and ACTB) were identical to those in the investigated healthy tissues. Thus, these two genes could serve as a good first choice to be tested in neoplastic tissues in future studies.
Using the geNorm program, we calculated that two to three reference genes were required for accurate normalisation in most tissues. According to Vandesompele and co-workers, using the three best reference genes is a valid normalisation strategy in most cases; it results in much more accurate and reliable normalisation than the use of only one reference gene does [19]. The use of a limited number of reference genes is also supported by the finding that no significant effect on the relative quantity of the target gene expression was demonstrated when using the combination of the two best genes compared to using five of the six most stable genes [34].
From our results, we recommend consulting the literature for appropriate reference genes according to the tissues and species under investigation. If healthy tissues are included in the study design (e.g. baseline values, negative controls) the reference genes should be evaluated in healthy individuals. If the corresponding data is not available, preliminary experiments for the identification of optimal reference genes are necessary. If this is impossible, we recommend using RPS7 as the primary choice for a reference gene in feline tissues (with tissue-specific limitations documented in the present study); results should be confirmed using other reference genes, such as ACTB, ABL, GUSB, and/or YWHAZ.
Conclusions
In this study, we investigated the most reliable feline reference genes for normalisation of gene expression data in blood samples and 15 different tissues, including endocrine, lymphatic, and gastrointestinal tissues, using realtime TaqMan ® PCR assays and pair-wise and ANOVAbased analyses. Our results indicated that stability of the reference genes varies among the different tissues, and no gene was found among the most stable ones for all the tissues under investigation. Moreover, significant differences were found using either pair-wise or ANOVA-based analysis approaches. The three most stable genes were RPS7, ACTB, and ABL, while B2M, HPRT, and the 18S rRNA gene were the least stable ones. The frequently used GAPDH had an acceptable stability in a few tissues. These data emphasise the need for proper validation of candidate reference genes in the respective tissues and species in healthy individuals preceding the initiation of any experimental gene expression study.
Authors' contributions
YK performed and analysed the research and drafted the manuscript. AKH designed the assays, performed statistical analyses, prepared the figures, and co-drafted the manuscript. VC and MLM participated in the assay design and data analysis and revised the manuscript. BZ performed molecular assays. PO performed the necropsies, the macroscopic and histological analysis, and the sample collections and revised the manuscript. BR was responsible for the SPF cats, housing, animal care, and the blood collec-tions and revised the manuscript. CER contributed to the study design (endocrine tissues) and provided some feline tissues. HL contributed to the study design. RHL conceived and supervised the study, participated in the data analyses, and edited the manuscript. All authors read and approved the final manuscript. | 2018-05-08T18:16:30.225Z | 0001-01-01T00:00:00.000 | {
"year": 2009,
"sha1": "09062e21cd6a3b2402016bdfa835339110f8b2e1",
"oa_license": "CCBY",
"oa_url": "https://bmcmolbiol.biomedcentral.com/track/pdf/10.1186/1471-2199-10-106",
"oa_status": "HYBRID",
"pdf_src": "PubMedCentral",
"pdf_hash": "b820c4bfbba3fe55e50877bec7a2441b20e6dfc9",
"s2fieldsofstudy": [
"Biology"
],
"extfieldsofstudy": [
"Medicine",
"Biology"
]
} |
226221852 | pes2o/s2orc | v3-fos-license | Quasiperiodic criticality and spin-triplet superconductivity in superconductor-antiferromagnet moire patterns
Quasiperiodicity has long been known to be a potential platform to explore exotic phenomena, realizing an intricate middle point between ordered solids and disordered matter. In particular, quasiperiodic structures are promising playgrounds to engineer critical wavefunctions, a powerful starting point to engineer exotic correlated states. Here we show that systems hosting a quasiperiodic modulation of antiferromagnetism and spin-singlet superconductivity, as realized by atomic chains in twisted van der Waals materials, host a localization-delocalization transition as a function of the coupling strength. Associated with this transition, we demonstrate the emergence of a robust quasiperiodic critical point for arbitrary incommensurate potentials, that appears for generic relative weights of the spin-singlet superconductivity and antiferromagnetism. We show that the inclusion of residual electronic interactions leads to an emergent spin-triplet superconducting state, that gets dramatically enhanced at the vicinity of the quasiperiodic critical point. Our results put forward quasiperiodicity as a powerful knob to engineer robust superconducting states, providing an alternative pathway towards artificially designed unconventional superconductors.
I. INTRODUCTION
Unconventional superconductivity [1,2] encompasses one of the most exotic states found in quantum materials. In particular, recent interest in topological superconductors hosting Majorana states has been boosted by their potential for topological quantum computing. [3,4] However, unconventional superconductivity and in particular spin-triplet superconductivity remains a highly elusive state in natural compounds. [5][6][7][8] Whereas several compounds have been identified as a potential candidates for spin-triplet superconductivity, [9][10][11][12][13][14] finding generic mechanisms for its engineering remain still an open challenge. [15,16] A highly successful strategy for engineering spin-triplet superconductors consists of focusing on materials with potential coexisting magnetic and superconducting orders. [15,16] This procedure has been heavily exploited for the engineering of Majorana bound states with a variety of platforms [17][18][19][20][21][22][23][24]. Most of these schemes have relied on engineering periodic structures with competing orders, while its study in non-periodic systems has remained relatively unexplored. [25,26] In stark contrast, the study of conventional superconductivity in disordered, [27][28][29] and quasiperiodic systems [30][31][32][33] has a long history, in particular, demonstrating potential critical advantages of non-periodicity for engineering robust superconducting states.
Quasiperiodic patterns display a never-repeating arrangement of elements [34,35], yet hosting long-range order. Due to the lack of conventional periodicity, standard band-structure arguments no longer hold, and their electronic structure exhibits a notably rich behavior [36], such as the presence of confined states [37][38][39], fractal dimensions [40][41][42], pseudogap in the density of states [43][44][45][46], and unconventional conduction properties [47][48][49][50]. More importantly, the incommensurate structure of quasicrystals has prominent effects on the electron eigen- (a) Engineered antiferromagnetic atomic chain on top of a superconducting twisted graphene bilayer. The twisted system shows a spatially modulated superconducting state (yellow), that coexists with the spatially modulated antiferromagnetism of the chain. Panel (b) shows the modulation of the superconducting and antiferromagnetic order parameters along the chain direction, associated to the Hamiltonian of Eq. 1.
states. Incommensurate potentials give rise to electronic wave functions extended, localized or critical [51][52][53][54], and ultimately can host topological states of matter fully associated to the quasiperiodicity. [55,56] Here we demonstrate that quasiperiodic patterns arising from a combination of spin-singlet superconductivity and antiferromagnetism provide a powerful platform to engineer spin-triplet superconductivity. In particular, we show that this antiferromagnetic-superconductor pattern hosts a localization-delocalization phase transi-tion, with an associated quasiperiodic critical point with multifractal wavefunctions. We further show that upon inclusion of residual interactions, a spin-triplet superconducting state emerges, that gets dramatically enhanced at the proximity of the localization-delocalization critical point. Our results show that magnetic-superconducting quasiperiodic patterns, as those found in atomic chains in twisted van der Waals materials, provide a new mechanism to engineer unconventional superconducting states by exploiting quasiperiodic criticality. Our manuscript is organized as follows. First, in Sec. II we introduce a realization of our model, and we show the emergence of a critical point in quasiperiodic superconductorantiferromagnet patterns, separating the extended and localized regime. In Sec. III we show that interactions give rise to a spin-triplet superconducting state, and analyze its dependence with respect to the details of the quasiperiodic modulation. In Sec. IV, we demonstrate the robustness of the interaction induced spin-triplet superconducting state with respect to perturbations in the quasiperiodic Hamiltonian. Finally, in Sec V we summarize our conclusions.
II. ANTIFERROMAGNET-SUPERCONDUCTOR QUASIPERIODIC CRITICALITY
The system that we will study combines a spatially modulated antiferromagnetism and superconductivity, as shown in Fig. 1ab. This type of spatially modulated parameters appears in generic twisted twodimensional materials that combine superconductivity and magnetism. [24] Here we will focus on a specific case in which the system is purely one dimensional. This situation can be realized by taking a twisted graphene multilayer in a superconducting state, [57][58][59] whose superfluid density follows the modulation of the moire pattern, and depositing an array of ad-atoms on top it [60][61][62][63][64][65][66][67] ( Fig. 1a) The ad-atoms will have a long-range antiferromagnetic order stemming from the graphene RKKY interaction, [68] leading to a one-dimensional antiferromagnetic state. [69][70][71] Both electronic orders will have a modulation following the moire pattern, effectively realizing to a one dimensional model with modulated antiferromagnetism and superconductivity. [72][73][74][75] We describe this system combining a modulated superconducting and antiferromagnetic exchange by the following effective Hamiltonian [76][77][78][79]: where c † i,s (c i,s ) denotes the fermionic creation (annihilation) operator for site n and spin s, and σ z is the spin Pauli matrix. The first term denotes the kinetic energy of the system, the second term denotes the spatially modulated antiferromagnetism, and the third term corresponds to the modulated superconductivity. The parameters ∆ and m are responsible for the strength of the modulation corresponding to the antiferromagnetism and superconductivity, respectively, and Ω is the wavelength of the modulation. The model of Eq. 1 assumes that the superconducting state will be stronger when the magnetism is weaker (Fig. 1b), as often happens for spin-singlet superconductivity. For convenience, we will parameterize the superconducting and antiferromagnetic strength as m = λ sin α and ∆ = λ cos α, so that the net strength of the quasiperiodic modulation can be defined by the parameter λ = √ m 2 + ∆ 2 . For irrational values of Ω, the model of Eq. 1 lacks translational symmetry and thus does not accept a description in terms of Bloch states. As a result, the eigenstates of this Hamiltonian are not guaranteed to be extended states, as the Hamiltonian is inherently non-periodic. Whereas random disorder creates localization at arbitrarily small coupling constants in one-dimension, [80] quasiperiodic patterns are known to give rise to a localization transition at finite coupling constant. [52,81] In particular, it is worth noting that for ∆ = 0, our model is mathematically equivalent to the Aubry-Andre-Harper model. [52] Therefore, in the limit of ∆ = 0, the previous model will have a localization transition at m = 2t, so that for m < 2t all the states will be extended and for m > 2t all the states will be localized. As we show below, the generalized model of Eq . 1 with ∆ = 0 shares many of the characteristics of the AAH model, in particular a critical transition at finite coupling constant.
We now address the localization-delocalization transition in the previous model. In order to determine the extended and localized nature of the states, we compute the inverse participation ratio (IPR) of each eigenstate Ψ n as where i runs over all the components of each eigenstate. For localized states whose wavefunction spans a certain number of sites L, the value of the IPR is a finite nonzero number. In stark contrast, for extended states, the value of the IPR scales as 1/L, becoming zero in the thermodynamic limit.
Let us now explore the model of Eq. 1, and in particular, analyze how the localization of the states evolve as we increase the strength of the quasiperiodic modulation λ. We show in Fig.2a the evolution of the IPR for the different eigenstates, as a function of the modulation strength λ for α = π/3, which corresponds to taking ∆/m ≈ 0.57. In particular, as shown in Fig.2abc all the states remain extended up to λ = 2t is reached, at which point all become localized. Note that the single in-gap modes that remain all the time localized correspond to topological edge states. [55,56] This can be systematically studied by looking at the average IPR of the states as a function of ∆ and m. This is shown in Fig. 2d, where it can be seen that a boundary with the functional form ∆ 2 + m 2 = λ 2 = 4t 2 separates the localized region from the extended region. At the previous phase boundary separating extended from localized states, wavefunctions with critical behavior emerge. The different nature of the extended, localized, and critical states can be easily observed by plotting individual wavefunctions. In particular, we show in Fig. 2efg the wavefunction closest to charge neutrality for an extended (Fig. 2e), critical (Fig. FIG. 3. Interaction-induced spin-triplet superconducting order as a function of the position (a), and spatial profiles of the spin-singlet superconductivity and antiferromagnetism (b). It is observed that the spin-triplet component is maximal in the regions where the spin-singlet superconductivity and antiferromagnetism coexist.
2f) and localized (Fig. 2g) regime. Whereas the extended wavefunctions span over the whole system (Fig. 2e), localized wavefunctions are strongly localized in a few lattice sites (Fig. 2g). The critical wavefunction of Fig. 2f are characterized by multifractal revivals. [82][83][84][85][86][87][88] This will become especially important in the next section, as the multifractal behavior of the states will substantially increase the impact of interactions in the system.
III. INTERACTION-DRIVEN SPIN-TRIPLET SUPERCONDUCTIVITY
Let us now move on to consider the impact of interactions in the previous quasiperiodic system. In particular, we will show that the inclusion of interactions will lead to spin-triplet superconductivity, where the criticality driven by the quasiperiodic pattern can be used as a knob to enhance superconducting order parameter close to the critical point. Local interactions of the form H int ∼ n c † n,↑ c n,↑ c † n,↓ c n,↓ are already accounted for in the stagger antiferromagnet and superconducting terms of the Hamiltonian. Therefore, we will now consider the effect of residual non-local interactions, in particular nearest neighbor density-density interactions. For that sake, we will now include an interaction term in our Hamiltonian of the form where V controls the strength of the nearest-neighbor attractive interaction. We solve the previous interacting term using a mean-field approximation . We will focus in this last term, whose contribution to the mean-field Hamiltonian is of the form where by definition of spin-triplet ∆ s,s n,n+1 = −∆ s ,s n+1,n . As the spin-triplet component of the interaction induced superconducting state has several degrees of freedom, it is convenient to define an spatially dependent d-vector d n,n+1 that parameterizes the spin-triplet superconducting order as where σ are the spin-Pauli matrices. As a first step, it is interesting to look at the real-space distribution of the unconventional superconducting state. In particular we show in Fig. 3 the real-space distribution of the spin-triplet state defined as We observe that the spin-triplet density follows the quasiperiodic pattern, and that it becomes zero in regions only having antiferromagnetism or s-wave superconductivity (Fig. 3ab). Interestingly, we find that such spintriplet component is maximal right in the region where the s-wave superconductivity and antiferromagnetism coexist in the same footing (Fig. 3ab), highlighting the key interplay of magnetism and superconductivity for driving as spin-triplet superconducting state. Moreover, it is interesting to examine the specific type of spin-triplet state that the interactions promote. In particular, we find that the d n,n+1 is always locked to the same direction of the antiferromagnetism, which in terms of the superconducting order parameters is associated to an interaction induced spin-triplet ∆ ↑↓ n,n+1 order parameter for antiferromagnetism in the z-axis.
We now move on to examine the impact of the quasiperiodic criticality on the induced spin-triplet state. As anticipated above, the critical behavior of the wavefunctions is known to provide an effective mechanism for enhancing electronic instabilities. [89][90][91][92][93] To verify this, we now compute the selfconsistent spin-triplet order parameter of Eq. 6 as a function of the modulation strength λ, as shown in Fig. 4a. It is observed that, as the modulation strength increases, the induced spin triplet parameter grows, becoming maximal around the critical point and decreasing as the system goes deeper into the localized regime. The enhancement associated to the critical point can also be verified by computing the induced spintriplet order parameter as a function of the interaction strength V , as shown in Fig. 4b. It is seen that for all coupling constants, the spin-triplet state is stronger close to the critical point λ = 2t, than deep into the extended (λ = t) or localized (λ = 5t) regime.
We now move on to examine the impact of the two quasiperiodic modulations as parameterized by α. As we showed above, the extended-localized transition takes place for λ = 2t, and independently on the value of α. This means that a critical point appears independently on the relative strengths between ∆ and m, and it only depends on λ = √ ∆ 2 + m 2 . In stark contrast, the emergence of a spin-triplet component due to interactions turns out to be highly dependent on α, as shown in Fig. 4c. In particular, we observe that when the system is purely antiferromagnetic or purely superconducting (α = 0, π/2) the spin-triplet component generated is exactly zero. In comparison, the spin-triplet state is maximal for α = π/4, that corresponds to having an equal weight on the singlet superconducting and antiferromag- netic order parameters. This observation emphasizes the importance of the coexistence of antiferromagnetism and superconductivity for the emergence for the interaction induced spin-triplet state.
We finally consider the impact of the spatial modulation frequency Ω in the interaction induced spin-triplet state. As shown in Fig. 4d we observe that the enhancement at the critical point happens for generic values of the modulation frequency. In the limit of small Ω, the system is essentially formed by patches of superconductor and antiferromagnet, [76][77][78][79] having a typical length on the order l ∼ 1/Ω, and thus the interaction between the superconducting and antiferromagnetic state happens in a limited part of the system. In comparison, for Ω ≈ π/2 there is a quick oscialltion between the two orders, promoting a dense coexistence of antiferromagnetism and spin-singlet superconductivity in the system. We observe that the interaction induced spin-triplet component is especially strong in this regime (Fig. 4d), reflecting the key interplay between spin-singlet superconductivity and antiferromagnetism for driving the unconventional superconducting state.
IV. ROBUSTNESS TO PERTURBATIONS
In this section, we address the robustness of our phenomenology with respect to perturbations. In particular, we will focus on the impact of next to nearest-neighbor hopping and Anderson disorder. The next to nearestneighbor hopping breaks the bipartite nature of the lattice, whereas the Anderson disorder would drive the system to a localized state for all λ. In particular, we obtain that the critically enhanced spin-triplet superconducting state also happens with those additional perturbations, as elaborated below. Second neighbor perturbations are expected to appear in a realization of the previous model and break the original bipartite nature of the system. The previous term is included in our Hamiltonian by means of a perturbation of the form The results with this additional perturbation are shown in Fig. 5a, where we took t = 0.2t. It is observed that the enhancement of the interaction-induced spin-triplet state happens in the presence of this additional perturbation. It is worth to note that in the presence of second neighbor hoppings, the localization-delocalization transition becomes state dependent, and it will no longer happen at λ = 2t. Nevertheless, it is observed that the qualitative behavior remains analogous to the idealized case with t = 0. This is especially important for potential realizations of our model in twisted two-dimensional materials and cold atoms setups, as generically these systems present small additional contributions to the Hamiltonian such as a second neighbor hopping. Next, we consider the impact of random disorder in the system, included as an onsite Anderson perturbation.
where n is a random number between [−0.1, 0.1]t. First, it is interesting to note that the inclusion of an arbitrarily small amount of disorder would drive the extended states to a localized regime. As a result, in the presence of disorder the localization-delocalization transition as a function of λ is completely destroyed, as the state becomes localized for all λ. The disorder strength λ will define a minimal localization length for the system. As λ is ramped up, the system will go from a localized regime dominated by the disorder, to a regime in which the localization is dominated by the quasiperiodic potential. Although the critical point is washed out, the enhancement of the superconducting state will still be visible at this quasiperiodic-disorder localization crossover. This is shown in Fig. 5b, where it is seen that the spin-triplet enhancement close to the former critical point is still visible. This phenomenology shows that even in experimental setups that host small imperfections, the enhancement of an unconventional spin-triplet superconducting state can be observed.
V. CONCLUSION
To summarize, we have demonstrated that antiferromagnet-superconductor moire patterns show a critical point associated with a localization-delocalization transition. We showed that the quasiperiodic criticality happens for arbitrary ratios between the superconducting and antiferromagnetic order parameters, and that the critical point is universally located in a curve defined by the two order parameters. Upon inclusion of residual electronic interactions, we demonstrated the emergence of an unconventional spin-triplet state, whose d-vector is locked along the antiferromagnetic spin direction. We showed that the emergence of this unconventional superconducting state is finely related to the interplay between antiferromagnetism and superconductivity, having a spatially inhomogeneous superconducting order maximal when the two parent orders coexist. We finally showed that this phenomenology happens for generic quasiperiodic modulation frequencies and survives the presence of perturbations to the Hamiltonian. Ultimately, the phenomenology presented can be realized in twisted graphene superlattices with atomically engineered impurities, and generically on moire patterns between two-dimensional antiferromagnets and superconductors. Our results put forward antiferromagnetic-superconducting quasiperiodicity as a powerful knob to engineer robust superconducting states, providing a new route towards the design of artificial unconventional superconductors. | 2020-11-02T02:00:42.660Z | 2020-10-30T00:00:00.000 | {
"year": 2020,
"sha1": "432cde8b1fcb00d445fc405bba78190e5db7bcff",
"oa_license": "CCBY",
"oa_url": "http://link.aps.org/pdf/10.1103/PhysRevResearch.3.013262",
"oa_status": "GOLD",
"pdf_src": "Arxiv",
"pdf_hash": "432cde8b1fcb00d445fc405bba78190e5db7bcff",
"s2fieldsofstudy": [
"Physics"
],
"extfieldsofstudy": [
"Physics"
]
} |
56254253 | pes2o/s2orc | v3-fos-license | OUTCOME OF NEONATES WITH THROMBOCYTOPENIA
OBJECTIVE: To determine etiology, onset, clinical features and outcome of neonates with thrombocytopenia. METHODS: 140 neonates having bleeding or having platelet count (<1.5lakhs/μl) were selected from those admitted to NICU’S attached to MR Medical College, Gulbarga. Initial platelet count was done on admission and counts were repeated 12 hours after any therapeutic intervention. OBSERVATION AND RESULTS: Severe thrombocytopenia (<50000/μl) was present in 8.5%, moderate (50, 000-1, 00, 000/μl) in 17%. Majority (45.33%) were preterm and the major cause was sepsis in 51.3%.Mucosal bleed was the most common presentation. Mortality was 37% in severe and 3.9% in moderate thrombocytopenia group. CONCLUSION: Significant association is observed with maternal PIH, Late onset sepsis, NEC and sepsis with DIC .Prematurity, IUGR, Birth asphyxia were common associated morbidities. Severe thrombocytopenia in sick neonates, in NICU, is a poor prognostic indicator.
INTRODUCTION: Thrombocytopenia (platelet count (<1.5lakhs/µl) is one of the most common haematological problems in NICU with 18-35% of neonates developing this problem. 1 The overall prevalence of thrombocytopenia in neonates ranges from 1 to 5% [2][3][4][5][6] and is reported to be much higher in neonates admitted to neonatal intensive care units, ranging from 22 to 35%. [2][3][4][5][6] More common among extremely low birth weight, preterm babies or sick neonates. In contrast, only 2% of the neonates are thrombocytopenic at birth with Severe Thrombocytopenia (platelet count <50, 000/µl) occurring in less than 3/1000 term infants. 7 In the past decade there have been a lot of research article pouring in regarding the etiology, clinical profile and management of this entity, Neonatal Thrombocytopenia in the NICU's. 7,8 The influence of thrombocytopenia on the outcome of neonate is a subject that has not been studied in detail in the past. Neither have articles assessed the value of neonatal thrombocytopenia as a prognostic indicator in sick neonates. After a detailed search of the indexed medical literature, it was found that there have been only few articles on this topic from India. 9,10 One article is a study of the association between maternal PIH and neonatal thrombocytopenia while the others are case reports and case series reports. The paucity of studies from India and the increasing prevalence of this condition in our NICU, instigated us to determine the etiology, incidence, onset, clinical profile and immediate outcome of the neonates admitted to NICU's attached to M. R. Medical College, Gulbarga.
METHODS: 140 of 550 consecutive neonates admitted to NICU's attached to M. R. Medical College, Gulbarga during the period of one and half years irrespective of their underlying morbidity were taken up for the study. At admission, the parents or guardians were informed about the study. A detailed history inclusive of maternal history and obstetric history with a focus on history suggestive of a bleeding and its type in the new born or the mother was obtained. A history of PIH, gestational diabetes mellitus, premature rupture of membrane, Rh isoimmunisation in the mother, any drug consumption was taken.
Every neonate was examined in detail for purpuric /petechial rashes, mucosal bleeding etc. All neonates at admission underwent a gastric lavage to look for any altered blood in the aspirate. Maternal blood was differentiated from neonatal blood using the Apt-Downey test. All neonates underwent blood investigations like complete blood count, peripheral smear study, blood culture, sepsis work up (absolute neutrophil count, total WBC count, micro ESR, C reactive protein).
Relevant data was entered in a proforma and analyzed. Statistical analysis was done by chi square test, continuous variables were analyzed using unpaired two tailed student t test or by one way analysis of variance (ANOVA).
RESULTS:
Of 550 consecutive neonatal admissions, subjects were divided into 3 groups based on their platelet counts: Prevalence of thrombocytopenia on the whole was 25.45% of which severe thrombocytopenia (<50, 000/µl) was 8.5%, mild to moderate thrombocytopenia (>50, 000/ µl) was 17.01%.The mean platelet count for all the groups were 1.603Lakhs/ µl. Among the various predisposing factors, maternal PIH was significantly associated with thrombocytopenia (p value <0.001) According to the age at presentation, severe thrombocytopenia was seen after 72 hours of life (p value 0.012) and it was significant. Septicaemia, as proven by blood culture, was significantly associated with thrombocytopenia. Prevalence of septicaemia was 60% in the severely thrombocytopenic group.34.40% and 25.12% in the mild to moderate and no thrombocytopenic group respectively. Mucosal bleeding was significantly associated with thrombocytopenia; prevalence was 65.95% in the severely thrombocytopenic group. The proportion of mortality was high in the severely thrombocytopenic group, but the proportion of babies with a not satisfactory immediate outcome was higher in mild to moderate thrombocytopenia and no thrombocytopenia group. 10 . It is evident from the table that in our study there is a slight higher prevalence of neonatal thrombocytopenia. This higher prevalence is probably due to higher proportion of septicemic neonates in our NICU admissions, 31.3%, while it was lower in the other studies, for e.g. in the study conducted by Castle et al the prevalence of septicemia was just 7.5%. 8 The proportion of severe thrombocytopenia among the neonatal thrombocytopenias, 33.57% in our study, is also on the higher side. This is once again probably a reflection of a higher contribution of septicemia to neonatal thrombocytopenia in our NICU than other etiologies. Septicemia is reported to result in severe thrombocytopenia rather than its milder form in various studies. 11
Studies on neonatal thrombocytopenia in NICUs
Etiological Profile: The etiological profile on the whole was similar to other NICU studies from India, with septicemia and perinatal asphyxia accounting for the majority of the admissions. 11 Septicemia accounted for most of the cases in both the severe and mild to moderate thrombocytopenia group. Perinatal asphyxia accounted for most of the admissions in the no thrombocytopenia group. Septicemia with DIC accounted for 28% of the cases of severe thrombocytopenia.
Prevalence of septicaemia
Castle et al 8 10% Hale oren et al 9 5.4% Our study 28.17% Predisposing Factors: Maternal PIH was significantly associated with neonatal thrombocytopenia (P<0.00l) and the odds ratio of the association was calculated to be 4.8 (with a C.I of 2.4-9.3). This finding is in agreement with studies conducted by Burrows et a1. 12
Prevalence of maternal PIH
Burrows et al 12 68.1% Our study 72.17% But maternal PIH is associated with mild to moderate thrombocytopenia rather than severe thrombocytopenia in other studies while in our study it was associated with severe thrombocytopenia. This could once again be explained by the frequent exposure of these neonates to infection, due to the relatively high prevalence of septicemia in our study that leads to a precipitous fall in platelet count.
It was shown that 55.31% of the severely thrombocytopenic neonates presented after 72 hours of life while only 25% and 21.7 % of neonates did so in the other two groups. This finding once again reiterates the well documented association that majority of the severely thrombocytopenic neonates present after 72 hours and the common etiology, in these neonates, are acquired ones such as septicemia and NEC. 13 Immediate Outcome: Mortality rate was very high, 37%, among the severely thrombocytopenic neonates while it was only 3.72% and 3.92% respectively in the mild to moderate and no thrombocytopenia groups. The proportion of a "Non-satisfactory" outcome was more (80.39%) in the mild to moderate thrombocytopenia group while it was 30 and 52.77% in the severely thrombocytopenia and no thrombocytopenia group. Hence a poor immediate outcome was associated with thrombocytopenia. This association might be due to the higher degree of severity of the underlying illness or due to an increased susceptibility of the neonates to complications, in the severely thrombocytopenic group. | 2019-03-13T13:28:42.843Z | 2014-04-24T00:00:00.000 | {
"year": 2014,
"sha1": "de2b084ee523a4bfcbe28fecde7f426019c77062",
"oa_license": null,
"oa_url": "https://doi.org/10.14260/jemds/2014/2471",
"oa_status": "GOLD",
"pdf_src": "MergedPDFExtraction",
"pdf_hash": "d74b83e3d970f561d0069521d416f5fe825cf13c",
"s2fieldsofstudy": [
"Medicine"
],
"extfieldsofstudy": [
"Medicine"
]
} |
31179802 | pes2o/s2orc | v3-fos-license | The Estimation of the Quenching Effects After Carburising Using an Empirical Way Based on Jominy Test Results
graphical and analytical solutions to solve the information transfer from the Jominy test samples to real parts are shown. The essay regarding the analytical solutions for the information transfer from the Jominy test samples to real parts includes detailed information and exemplifications concerning the essence and using the Maynier-Carsi and Eckstein methods in order to determine the quenching constituents proportions corresponding to the different carbon concentrations in carburized layers, respectively the hardness profiles of the carburized and quenched layers. In the final of the chapter, taking into account the steel chemical composition, the geometrical characteristics of the carburized product, the quenching media characteristics, the heat and time parameters of the carburising and the correlations between these values and the Jominy test result, an algorithm to develop a software for the estimation of the quenching effects after carburising, based on the information provided by Jominy test, is proposed. are determined by the presence of residual austenite and its presence implication on the hardness in the superficial layer; the obtained algorithm allows the very easily determination of the information regarding the effects of the carburising and quenching process on layer characteristics, starting from information provided by Jominy test. Metallurgical Engineering is the science and technology of producing, processing and giving proper shape to metals and alloys and other Engineering Materials having desired properties through economically viable process. Metallurgical Engineering has played a crucial role in the development of human civilization beginning with bronze-age some 3000 years ago when tools and weapons were mostly produced from the metals and alloys. This science has matured over millennia and still plays crucial role by supplying materials having suitable properties. As the title, "Recent Researches in Metallurgical Engineering, From Extraction to Forming" implies, this text blends new theories with practices covering a broad field that deals with all sorts of metal-related areas including mineral processing, extractive metallurgy, heat treatment and casting. jominy-test-
The particularities of quenching process after carburising
The aim of the quenching process after carburizing is to transform the "austenite" with high and variable carbon content of the carburized layer in quenching "martensite", respectively the core austenite in non martensitic constituents (bainite, quenching troostite, and ferriteperlite mixture). This goal is achieved by transferring the parts from carburizing furnace into a cooling bath containing a liquid cooling (quenching) medium. The transfer can be directly made from the carburizing temperature (direct quenching), or after a previous precooling of parts from the carburizing temperature to a lower quenching temperature (direct quenching with pre-cooling). In both ways, the austenitic grain size is the same (depending on the chosen carburising temperature and time), but the thermal stresses are different, being higher in the case of direct quenching and lower in the case of direct quenching with pre-cooling, due to higher thermal gradient achieved in the first cooling variant. Consequently, the risks of deformation or cracking of the parts are lower in the pre-cooling quenching, this variant being most commonly used in the industrial practice.
On the other hand, the result of quenching is influenced by three factors: one internal, intrinsic hardenability of steel (determined by its chemical composition -carbon content, alloying elements type and percentage) and two external (technological)thickness of the parts, expressed by an equivalent diameter D ech (the actual diameter in the case of cylindrical parts, or the diameter calculated using empirical relations for the parts with non cylindrical shapes) and cooling capacity of quenching media, expressed by relative cooling intensity -H (in rapport with a standard cooling media -still or low agitation industrial water at 20°C). In Fig. 1 an empirical diagram of transformation of non cylindrical sections (prisms, plates) in circular sections with the equivalent diameter D ech is shown; in Table 1, the indicative values of the relative cooling intensity of water and quenching oils -H are given depending on their degree of agitation related to the parts that will be quenched. If the parts have hexagonal section, it shall be considered that the cylindrical equivalent section has the D ech equal to the "key open" of hexagon. Lately, in the industrial practice, the so-called synthetic quenching media with cooling capacity that can be adjusted in wide limits have also been used, from the values specific to mineral oils to those specific to water, by varying the chemical composition, temperature and degree of agitation.
The degree of agitation of quenching media can be adjusted by the power and /or frequency of propellers or pumps type agitators, mounted in the quenching bath integrated in the carburizing installation (batch furnaces).
The external factors (D ech , H) determine cooling law of the parts, respectively cooling curves of the points from surface or internal section of the parts; the internal factor (steel hardenability) determines quenching result, expressed by structure obtained from transformation of continuously cooled austenite from austenitizing temperature to final cooling temperature of assembly -parts-quenching medium.
To foresee or verify the structural result of quenching, the overlapping of the real cooling curves (determined by external factors D ech and H) over the cooling transformation diagram of austenite of chosen steel at continuous cooling can be made, which is a graphical expression of intrinsic hardenability of steel.
The diagram of austenite transformation at continuous cooling allows steel to achieve both a quantitative assessment of the quenching structure, the estimation sizes being the proportions of martensitic and non martensitic constituents and to estimate the hardness of quenching structure.
Use of Jominy frontal quenching sample for estimation of quenching process results
The estimation of the steel quenching effects represents an extremely complex stage due to large number of variables that influence this operation, respectively: steel chemical composition, austenitizing temperature in view of quenching, the parts thickness and the quenching severity of the quenching media. The problem can be solved in an empirical way using the frontal quenched test sample, designed and standardized by W. E. Jominy and A. L. Boegehold and named Jominy sample (Jominy test). The simple geometry of sample and the way of performing the Jominy test covers a large range of cooling laws, their developing in terms of coordinates T-t being dependent on the distance from the front quenched part to the end of the Jominy sample. Using these curves a series of kinetic parameters of the cooling process can be obtained: cooling time and temporary (instantaneous) cooling speeds or cooling speeds appropriate for different thermal intervals. From its discovery (1938) until present, the Jominy test has been the object of numerous determinations and interpretations, evidenced especially by means of drawing of the cooling curves, of the points placed at certain distances from the frontal quenched end. The European norm, ISO/TC17/SC7N334E, Annex B1, specify the aspect of the Jominy samples cooling curves in the surface points placed at the distances dj=2.5; 5; 10; 15; 25; 50 and 80 mm from the frontal quenched end (Fig.2 [1]). This representation has the advantage that can be applied to each steel and for each austenitizing temperature T A in terms of quenching, in the common limits T A =830~900°C, because has on the ordinate axis the relative temperature θ=T/T A , respectively the ratio between the current temperature T (in a point placed at the Analyzing the cooling curves from Fig. 2, a difference between those taken from the ISO standard and those from G. Murry work, is observed. To clarify these discrepancies and to adopt an unique and argued assessment of the way of setting out of the cooling curves in the case of Jominy samples, we can start to analyze the modality in which the heat transfers from Jominy sample to the ambient, during cooling of the sample from the austenitizing temperature T A to the ambient temperature T amb took place. In principle, the heat flow in a point P of the Jominy sample, placed at distance x from the frontal quenched end and at the distance r from the axis of the sample, at time t after the start of cooling is given by the differential equation: this can be solved in the following univocity conditions: a. initial condition: T (x,0) =T A ; b. boundary conditions of first order: T (0,t) =T water jet (on the water cooled surface) T (x,t) =T amb (along the cylinder generator) c. boundary conditions of second order defined by the specific heat flux through the frontal cooled surface and through the external cylindrical surface cooled in air, which are proportional with the negative temperature gradients: The heat loss during sample cooling takes place by means of three mechanisms: conduction -at the contact interface between cooling water and direct cooled surface, the heat loss value being a function of time: convection -of the ambient air, the heat loss value being a function of the T (x,t) -T amb difference where is the convection heat transfer coefficient: radiation -from the cylindrical surface: where is the radiation constant, ; is the emissivity coefficient of the sample surface; 8 24 J 5.67. 10 ms K The solving of the differential equation (1) lead to a solution representing the general form of the cooling curves equations of points placed at the distance x from the frontal quenched end: where the parameter c has speed dimensions (m/s), and the rapport c/x is a constant on which the temporary (instantaneous) cooling speed has a linear dependency: To simplify the analysis, without introducing further errors, can be admitted that T amb~0 and noting T (x,t) =T S (the current surface temperature) and T S /T A =θ (the relative surface temperature), the final solution can be written as: The solution (6) makes the direct connection between the relative temperature θ and time t, values that represent the coordinates in which are drawn the cooling curves of the points (planes) placed at the distance x from the frontal quench end of the Jominy sample.
In the work [2] the using of the relation (4) is exemplified in the case of Jominy samples austenitized at T A =1050 o C, for which the cooling curves of the points placed at the distances x=1, 10, 20 and 40 mm from the frontal quench end (Fig.4) were drawn and on which the ordinate referring to the relative temperature θ=T/1050 and also the ISO and Murry cooling curves for distances x = 1.5 mm (Murry), 9mm (Murry),10 mm (Murry), 20 mm (ISO and Murry) and 40 mm (Murry) were also drawn.
Using data taken from continuous curves presented in Fig.4 and replacing the notation x which represents the distance from the frontal quenched end of Jominy sample with E, the value of parameter c (from eq. 6) has found as c = 0.28, so that eq.(6) of cooling curves will get a particular form (7) and the inverse function, t = f (θ) will have the expression (eq.8) On the other hand, from Fig. 4 it can be seen that between the aspect of the actual cooling curves experimentally determined by Murry and ISO and that obtained by calculation, using the relations (7) and (8), there are differences which are substantially and simultaneously amplifying with the decreasing of the relative temperature θ. In conclusion, we can say that the theoretical analysis presented above is incomplete in that it fails to consider some effects of interaction between the types of heat loss during cooling of the Jominy samples.
Taking into account the higher matching of the Murry curves to theoretical curves, were mathematically processed the data provided by Murry curves and was noted that these are best described also by an exponential function, having the general form t=aE b , and the particular form as: where the parameter a depends on θ also by means of an exponential relation: In conclusion, the relations describing the dependencies tf E (,) -eq.(8) and ftE (, ) eq. (7), will have the following particularly forms: Once the Jominy sample kinetic parameters are known for a sample made from a certain charge of steel, they can be attributed to a specific part (with an equivalent diameter, D ech known) made from the same charge of steel, taking into account that both the part and also the Jominy test sample to be processed in the same technological conditions (same austenitizing temperature T A and time t A ) and same quenching media (with the same relative cooling intensity, H). This condition will be accomplished in the case where the Jominy sample is "embedded" in the heat treatment charge, composed of identical parts and follows the same processing sequence, in the same heating and cooling equipment. The correlation between the Jominy test sample and real part with the equivalent diameter of D ech is usually graphically provided: a first chart was built by Jatczak [3] (for parts with equivalent discrete diameters of 12.5 mm, 19 mm, 25 mm, 38 mm, 50 mm and 100 mm). Jatczak diagram provides a graphical solution for the function dj = f (Dech, H, r), 0 ≤ r ≤ where R is the coordinate position of the point on the part cylindrical section, that has the same cooling law (curve) with that of points situated in the plane placed at the distance dj from the frontal quenched end of the Jominy sample.
For parts subject to carburizing, the correlation diagram -cylindrical part -Jominy sample will become the curve from the Jatczak diagram, referring to part surface S (as the layer thickness δ is very small compared to the equivalent radius of the part). In this case, the correlation function has the form dj = f (D, H) or D = f (dj, H), where D is the diameter of www.intechopen.com cylindrical part with length L ≥ 3D (or equivalent diameter of the parts with other forms of the section) and dj is the distance from the frontal quenched end of Jominy sample.
In Fig.6,the correlation diagram-Jominy sample -superficial layer of cylindrical parts drawn by U.Wyss [4] based on the method of Grossmann and numerous literature data is reproduced.
-For a cooling intensity of H=0.45, corresponding to intensive agitated oil: -For a cooling intensity of H=0.60, corresponding to strong agitated oil: -For a cooling intensity of H=1.00 , corresponding to low agitated water: The values of cooling intensity, H, shown above, are suitable for quenching oil used at normal temperatures (50 ~ 80 °C) and with different degrees of agitation (of oil and/or parts) in a relatively wide range, starting from absence of agitation (H=0.25) to strong agitation (H=0.6), or a shower or pressure jet oil (H = 1.00), this last situation being equivalent for low agitated cooling water at 20 ° C, .
With these observations, the graphical relation between the carburized layer of part with diameter D and points from the Jominy sample, carburized in the same conditions, in accordance with the scheme shown in Fig.7 is achieved, where both the superficial layer of part and also the Jominy sample are represented at very high magnification in rapport with actual size (the dimension of the carburized layer δ~0.5~2mm and the equivalent distance in the Jominy sample E ~ 2 ~ 20mm).
The graphical solving of correlation between real parts -Jominy sample
From the graphical representation shown in the Fig. 7 results that the A', B' and C' points of the carburized layer of the Jominy test sample, equivalent to A,B and C points of the part carburized layer, are located on the intersections of the horizontal plane placed at the E distance from the frontal cooling plane of the Jominy test piece with the vertical planes placed at the O, δ ef and δ tot from the Jominy test sample generator, characterized through the C s , C ef and C m constant carbon contents.
Based on the above considerations, a graphical solution of the issue regarding the correlation between the Jominy test sample and a part with D diameter, both carburized in identical conditions (same carbon profile and same hardness profile in the carburized layer) has developed by U. Wyss. Using of Wyss graphics solution requires the knowledge of the equivalent diameter of the part (D), the cooling intensity of quenching media (H), the carbon profile of carburized layer and the hardenability curves, respectively the hardness = f (%C) curves at various depths 0 ≤ δ ≤ δ tot in the case hardened layer of the Jominy test sample.
www.intechopen.com The information sources used by Wyss in developing of the scheme shown in Fig. 8 were the following: the D=f(dj) dependence for H=0.35, has been taken from Fig. 6; -the carbon profile curve has been experimentally plotting by means of sequential corrections of the case hardened layer of a part with dimensions of Φ35 x105 mm; Fig. 8. Deduction of the hardness profile in the carburizing layer for parts(d) and for the Jominy test sample from Jominy hardenability curves (c) for a given carbon profile of carburizing layer (b), after carburising at C s = 0.8% C of parts made of a case hardening steel with composition (0.16% C, 1% Mn, 1% Cr) and diameter D = 35 mm and subsequent quenching in oil with cooling intensity H = 0.35 (according to Wyss [4]).
the hardenability curves has been experimentally plotting by means of measuring of the HRC hardness on planes parallel with the Jominy test piece generator, corrected at the depths of the case hardened layer at which the carbon content is that mentioned on curve (0.60%C; 0.52%C; 0.45%C; 0.35%C; 0.29%C and 0.16%C).
The algorithm of using of the graphical solution is shown by arrows in Fig. 8 and involves the following steps: in the diagram (b) the horizontals lines corresponding to the hardenability curves from the diagram(c) will be plotted and the points of intersection with the carbon profile curve will be determined; from these points, vertical lines extending in the space of the diagram (d) are drawn and cross the horizontals plotted in an earlier stage (from the intersection points of the vertical E = 10 mm (diagram (a)) with the hardenability curves drawn for different carbon concentrations in the case hardened layer (diagram (c)); the intersection points belong to the hardness profile curve available for the existing space diagram (d). The point D in the diagram (d) in which the hardness profile curve crosses the horizontal corresponding to HRC ef hardness has the abscise corresponding to the effective depth δ ef (in the example discussed, for HRC ef = 52.5 results δ ef =1.38 mm); -from the crossing point D, the vertical line which will meet the carbon profile curve at point B of which horizontal corresponds to the actual carbon content, C ef (for example the analysis made for δef = 1.38 mm results C ef = 0.4% C).
Essay regarding the analytical solving of the real parts-Jominy sample correlation
The above graphical solution can be transformed into an analytical solution if the equations of the following curves are available: a for 0.6≤H≤1,00; 2≤E≤12(mm) and10≤D≤100 (mm): The parts that will be case hardened by instillation and pyrolysis of organic liquids are usually thin pieces with D ech ≤ 50mm, which are cooled in mineral oil with cooling intensity of H ~ 0.25 ~ 0.60. To this category of products it can be applied the above mentioned relations no. (18-19) thus achieving results very close to those achieved using Wyss relations (Fig.9).
where C δ represents the carbon content measured at the δ depth in rapport with the surface at the case hardening end; C m represents the carbon content of the non -case hardened core and C s is the surface carbon content at the end of case hardening.
where t k represents the carburising time, h=K/D, represents the relative coefficient of mass transfer, K-is the global coefficient of mass transfer in the case hardening medium; D -the diffusion coefficient of carbon in austenite.
c. For the Jominy hardenability curbes (c) have been deduced by E. Just [6] several regression equations having the general form: (25-26).The three relations produce results significantly closer each to another and also very close to those provided by the graphical dependencies for a series of German steels presented in the work [7]. Therefore, it was adopted for explaining of the hardenability curve the relation no. (27) In this purpose, the data from the Table 2 were used and exponential, logharitmic and logistic functions were explored, their graphics having the ordinate at origin different from zero and positive (is known the fact that the technical iron can be quenched to a structure of "massive" acicular ferrite, close to the martensite with low carbon and which, according to relation ( and their principle graphics being shown in Fig. 10. The formulas no. (34) and (35) give results very close each to another and also close to the experimental data referring to steels with carbon content in the limits 0.1~0.8%.
Furthermore the Johnson function can be used with satisfactory results also for the calculation of the hardness of the quenched layers in which the martensite proportion decreases to 50%.
The general calculation relation and the auxiliary relations are shown in Fig.11.
Whereas in many literature works the hardness is expressed in Vickers units, is necessary also a Rockwell-Vickers equivalence relation. In this purpose, mathematical tables and graphics equivalence HRC-HV -were processed and the following relation have been obtained, depending on the load used to determine the Vickers hardness: for loads F≤1Kgf (9,8N) a. Concerning the data provided by the graphical dependencies and relations from Fig.11 it must be specified that these are referring to the "ideal case" in which cooling of the austenite subject to quenching is achieved below M f temperature, who, like the M s temperature decreases with the increasing of steel carbon content (austenite) and becomes negative at higher carbon concentrations than 0.6%. In this case, if the austenite is cooled to room temperature or even above, in structure will remain a significant proportion of residual austenite, which decreases the hardness below the level indicated by the curves in Fig.11.
Typically, the proportion of residual austenite is calculated by Koistinen-Marburger relation: Returning to the analytical solution of the correlation between Jominy sample and real parts, which should finally allow to draw the hardness curve of the carburized and quenched www.intechopen.com layer, is noted that this solution is materialized in a mathematical model of post carburising quenching, whose solving algorithm is based on knowing of the initial data referring to the following independent variables: a. chemical composition of steel, respectively the alloying factor; . the geometry of parts subject to carburising, respectively the equivalent diameter D ech ; c. the cooling intensity of the quenching medium, respectively the Grossmann (H) relative cooling intensity; d. the requested effective hardness (HRC ef ); e. the requested effective case depth(δ ef ).
Starting from this initial data, the algorithm for determining of the hardness curve of carburized and quenched layer will require the following sequence of steps: Step I taking into account the geometry of parts subject to processing, the equivalent diameter D ech is calculated with one of the equivalence relations mentioned in Fig.1.
Step II taking into account D ech and h, is calculated the equivalent distance E from the frontal quenched end of the Jominy sample by means of the relation (19) for several German case hardening steels (Fig.12) with the average chemical composition (according to DIN-tab.3), without mentioning of the calculation formula or the effective values of the alloying factors S. Fig. 12. Correlation between the effective carbon content and the Jominy equivalent distance for different German steels with the average standard chemical composition [4].
In addition, U. Wyss suggested the using of a linear relation to calculate the actual carbon content as it follows: In which the slope f c is a hardenability factor of steel, having the values written on the corresponding lines, drawn in Fig.12.
In connection with this approach of the problem, it is observed that the use of relation (46) is possible only for certain intervals corresponding to equivalent distance E, intervals on which the value obtained for C ef52.5 is not lower than 0.33% and is not different more than ±0.03% in rapport with curve (parable) replaced by the line corresponding to a given steel. These limits for E are mentioned also in the table placed on the right of Fig. 12.
In the work has been calculated the effective carbon content with formula (45), both for the German steels and also for the Romanian steels having an average chemical composition according to DIN and STAS (Table 3), using for the calculation of the alloying factor the relation S=21Mn+22Cr+7Ni+33Mo. The variation of the effective carbon content with the Jominy equivalent distance for the specified steels is shown in Fig. 13.
The analysis of the curves from Fig.13 led to following conclusions: a. for the steels with close values of the alloying factor, S, the curves are close positioned or even overlapped (case of 20MoNi35, 16MnCr5, 18MnCr10 and 21TiMnCr12 steels, having S=44.3 ~45.25 and of the steels 15CrNi6 şi 21MoMnCr12, having S~55.55); b. the ordinates at origin and the rate of curves are strongly decreasing with the increasing of the value of the alloying factor, S. As a result, the curves can be replaced with straight lines with different slope, but also with ordinates having different origins, both being dependent of alloying factor S. Putting the condition that the replacing lines do not lead to deviations higher than ±0,03%C, the generalized equation of these lines was: Table 4. The values of parameters ef C 0 , c f and the intervals for E in which is applied the linearizing relation (47) for the German and Romanian steels with medium standardized chemical composition.
In fact, the effective carbon content can has lower values than those ensuring a certain minimum proportion of quenching martensite. If in Fig.11 will be drawn the horizontal corresponding to the effective hardness of 52.5 HRC can be found that this value is assured by the following combinations of effective carbon contents and respectively martensite percentages in the hardening structure: On the interval 80≤M≤100%, the effective carbon content has a lineraly variation with the martensite proportion, according to relation: Setting the condition that for the effective case depth the actual proportion of martensite to be within the required range (to provide appropriate mechanical characteristics of the carburized and quenched layer), can be noted that the maximum amount of the effective carbon content should not exceed 0, 56% (value which is close to the carbon surface content of about 0.8%, with a drastic reduction of the carburizing depth, particularly in steels with low hardenability, respectively with an alloying factor S ≤ 30).On the other hand, the value of the C ef52,5 will not decrease more below 0.34%C because even the quenching structure for the effective depth is fully martensitic, its hardness decreases significantly below the set value (52.5HRC). This is the reason for why U.Wyss adopted for the 17CrNiMo6 steel the www.intechopen.com amount C ef =0.33%, although in the carburized layer of steel, the information offered by relation (45) and Fig.13 shown that the hardness of 52.5HRC can be obtained even for the content of 0.17%C of core (at E=13 mm). In a subsequent paper [7], Weissohn and Roempler suggest as minimum value for the carbon content with the concentration of 0.28% (for which HRC 100M~4 9.4, according to Fig.11). Adopting a value below that of the 0.34% could be justified for alloyed steel intended for carburising, due to the fact that alloyed martensite has a hardness higher with 1~2HRC than that of unalloyed steels.
In conclusion, for the calculation of the effective carbon content ( ef HRC C ,52.5 ) can be used the linearizing relations (47-49),with supplementary restrictions: ≤0.56% (respectively 100≥M≥80%) Step IV Using C ef and δ ef , can be calculated the carburising time (t k ) at isothermal carburising with a single cycle or the active carburising time (t k ) ,respectively the diffusion time (t D ), at carburising in two steps. The performing of this calculation supposes the knowing of thermal regime (t k, t D ), the chemical regime(C potK, C pot D ), the corresponding evaluation of the global mass transfer in the carburising medium (K); and the diffusion coefficient of carbon in austenite (D).
Step V is based on knowing of the carbon profile and of the cooling law (curve) of the layer and has as final purpose the drawing of the layer hardness profile. The carbon profile can be determined after the step IV, and the cooling curve of layer can be drawn using the relation (11).
The most direct method for determining and drawing of the hardness profile consist in overlapping of the cooling curve of the case hardened layer on the transformation diagrams of continuous cooling of austenite (CCT diagrams) "of different steels" from the layer, steels with carbon content that decreases from surface to core. The method is illustrated in Fig.14, for the case where the diagrams for the austenite transformation, corresponding for three steels with different carbon content that will be carburized, are known: a. with carbon content of core, C m ; b. with effective carbon content, C ef ; c. with carbon content of surface, Cs Because the temperatures corresponding to Ms point and the transformation ranges for the three diagrams are placed differently in the plane T-t (at a lower position and to the right, as the carbon content increases), the intersections of these with the cooling curve led to different structures (decreasing of the proportions of bainite and increasing of the proportions of martensite), respectively with different hardnesses (HRC m < HRC ef < HRC 100M For an accurate drawing of the hardness profile, is necessary to know a minimum number of 5-6 austenite transformation diagrams, corresponding to different carbon contents for a steel having in its chemical composition, all the other elements that are permanently accompanying and alloying elements with the same contents. But, this kind of technical "archive" is not currently available, even in the richest databases for the usual casehardening steels. To overcome these difficulties can be used a hardness calculation method based on knowledge of chemical composition and of a kinetic parameter characteristic to cooling law of the case hardened layer. This parameter can be the cooling time at passing through a certain temperature enclosed between the austenitizing temperature (T A ) and ambient temperature (T amb ). Most of the kinetic parameters of this type are the times of passing through temperatures of 700°C, 500°C and 300°C respectively t 700 ,t 500 şi t 300 , highlighted also on CCT diagrams in Fig. 14 ). The advantage of using of these kinetic parameters is that can be built structural diagrams in coordinates T-lgt or T-lgv, in which the cooling curves are replaced by verticals lgt or lgv and on basis of these, also structural diagrams in coordinates % structural constituents -lgt (lgv).
Method Maynier-Carsi
In the works [8] and [9] is used a calculation method derived from the analysis of 251 diagrams of transformation of austenite at continuous cooling, method in where the kinetic factor taken in consideration is the instantaneous cooling speed at passing of the cooling curve through the temperature of 700°C,respectively: In the calculation, the authors have introduced also the austenitizing parameter: parameter (of the austenitic grain size), and Ei represent the proportion of carbon, adding elements and alloying elements.
The particular forms of the regression relations are given in The two relations offer results in accordance with data of Fig. 15, for 1.5≤E≤30mm.
Admitting that the austenitizing temperature, for the data shown in Fig.15 is T A =850°C (which is not specified in the work [9], but is usually used in other works in the field), the absolute instantaneous cooling speed v 700 can be replaced with the relative speed v 0,825TA , which will be calculated with the relation: , value which is very close to that given by the relation(54), this is confirming also the validity of the relations (11) and (12).
Taking into account the critical speeds (calculated with the relations from Table 5) and the carbon profile curve of the carburized layer allow the overlapping of the structural diagrams at different carbon concentrations and taking into account the speed v 700 (v 0,825TA ) of layer, allow the positioning of the vertical of this speed in the space of the structural diagram and the deriving of the proportions of the quenching constituents for each carbon content (respectively for each depth) of carburized layer.
The Eckstein method
In paper [10], another drawing method of the hardness profile curve is provided, which is considered to be described as a complex exponential function as it follows: where S H is the surface hardness, respectively the martensite hardness which has the superficial carbon content C s , E is the equivalent Jominy distance in the depth of the case hardened layer, and b and c are the coefficients dependent on the steel chemical composition.
For the calculation of the Jominy equivalent distance, the author provides the formula: Due to fact that in both relations, the independent variable is the cooling time A T t 0.59 , become necessary to find a modality for calculation of this time depending on part diameter , D, on cooling intensity of the quenching medium, H and on the carburising depth δ. In this purpose, were used the data provided by the graphical dependencies from work [10], referring to the experimental determination of the time A t /5 in accordance with the carburising depth δ, which are combined with the data provided by Fig. 16 and 6 In order to properly use the relation (58), the b and c coefficients also have to be known; for these coefficients no information is available in the technical literature, including the work [10].
In conclusion, the effects of post carburising quenching process can be quantified by the calculation algorithm required by Maynier-Carsi, but corrections have to be applied; these corrections are determined by the presence of residual austenite and its presence implication on the hardness in the superficial layer; the obtained algorithm allows the very easily determination of the information regarding the effects of the carburising and quenching process on layer characteristics, starting from information provided by Jominy test. | 2017-09-16T14:55:55.547Z | 2012-03-23T00:00:00.000 | {
"year": 2012,
"sha1": "ecfa8e35aa320496c6f61027c760f9467bd625e3",
"oa_license": "CCBY",
"oa_url": "https://www.intechopen.com/citation-pdf-url/32752",
"oa_status": "HYBRID",
"pdf_src": "ScienceParsePlus",
"pdf_hash": "5792a1aaaa60ff19510db3ac52ebe21aa598eb72",
"s2fieldsofstudy": [
"Engineering",
"Materials Science"
],
"extfieldsofstudy": [
"Materials Science"
]
} |
238228919 | pes2o/s2orc | v3-fos-license | Neurodegeneration, memory loss, and dementia: the impact of biological clocks and circadian rhythm
Introduction: Dementia and cognitive loss impact a significant proportion of the global population and present almost insurmountable challenges for treatment since they stem from multifactorial etiologies. Innovative avenues for treatment are highly warranted. Methods and results: Novel work with biological clock genes that oversee circadian rhythm may meet this critical need by focusing upon the pathways of the mechanistic target of rapamycin (mTOR), the silent mating type information regulation 2 homolog 1 (Saccharomyces cerevisiae) (SIRT1), mammalian forkhead transcription factors (FoxOs), the growth factor erythropoietin (EPO), and the wingless Wnt pathway. These pathways are complex in nature, intimately associated with autophagy that can maintain circadian rhythm, and have an intricate relationship that can lead to beneficial outcomes that may offer neuroprotection, metabolic homeostasis, and prevention of cognitive loss. However, biological clocks and alterations in circadian rhythm also have the potential to lead to devastating effects involving tumorigenesis in conjunction with pathways involving Wnt that oversee angiogenesis and stem cell proliferation. Conclusions: Current work with biological clocks and circadian rhythm pathways provide exciting possibilities for the treating dementia and cognitive loss, but also provide powerful arguments to further comprehend the intimate and complex relationship among these pathways to fully potentiate desired clinical outcomes.
Introduction
Neurodegenerative disorders pose a significant challenge for diagnosis, preventing disease progression, and providing treatment. Cognitive loss in relation to Alzheimer's disease (AD) is an excellent example since diseases that include AD are the result of multiple underlying mechanisms [1][2][3][4][5][6] (Table 1). For example, many pathways may lead to memory loss and involve neuronal and vascular cell injury related to metabotropic receptors, lipid dysfunction, cellular metabolic dysfunction with diabetes mellitus (DM), astrocytic cell injury, β-amyloid (Aβ), heavy metal disease, loss of access to bright light, tau, mitochondrial damage, oxidative stress, acetylcholine loss, and excitotoxicity [1,3,.
In addition, cognitive disorders raise significant financial concerns [1,[31][32][33][34]. Greater than 800 billion United States dollars (USD) per year are required to treat dementia equaling approximately 2 percent of the global Gross Domestic Product. Social and medical services by the year 2030 may possibly equal 2 trillion USD per year in the United States. Currently, greater than 5 million patients have AD and it is estimated that 4 million receive care at a yearly cost of 3.8 billion USD. Furthermore, the market revenue to provide treatments for AD may not be fully appreciated, but at minimum it may be greater than 11 billion USD. Many new social and medical services will be necessary to meet this challenge such that 60 million additional care workers will be needed [35][36][37]. These projections do not consider that all cases of dementia may not have been identified and diagnosed at this time [38,39].
Cognitive loss impacts a large spectrum of the population. Dementia in the United States affects greater than 5 million people [4]. Many of these cases, 60 percent, are diagnosed as AD [4,6,17,[40][41][42][43]. Case of AD that are familial in origin comprise under 2% of all cases [4]. In familial AD that affects 200 families worldwide, mutations in the presenilin 1 or 2 genes occurs and an autosomal dominant mutated amyloid precursor protein (APP) gene exists. In these familial AD patients, illness can present prior to 55 years of age [44][45][46]. Familial AD can be the result of mutations in chromosome 21 leading to changes in APP, mutations in chromosome 14 causing changes in presenilin 1, and mutations in chromosomes 1, 14, and 21 such that mutations in chromosome 1 lead to changes in presenilin However, it is the sporadic version of AD that leads to illness in patients over age 65 and represents the cases of AD in ten percent of the population in the world. The ϵ4 allele of the apolipoprotein E (APOE) gene represents an additional risk of developing AD in the sporadic group.
Biological clocks and circadian rhythm pathways for dementia treatment
Current attempts to treat dementia such as with cholinesterase inhibitors may lead to a decrease in the presenting symptoms but ultimately do not block the progression of the disease, such as in AD [27,45,47,48]. Other treatments for cognitive loss can focus on metabolic disorders, such as diabetes mellitus (DM) [1,20,27,41,49,50], and on vascular disease [19,45,[51][52][53]]. Yet, there exist other risks for developing vascular cognitive loss that can affect the efficacy of treatments such as tobacco use, alcohol consumption, hypertension, and a low level of education [20,39,[54][55][56][57]. With reference to metabolic disease, tight glucose control in the serum in combination with early diagnosis of DM may assist to limit the progression of the disease, but complications from DM can still ensue [6,[58][59][60][61][62][63][64][65][66][67][68][69]. Given the need for novel strategies directed against memory loss and dementia, exciting new avenues of development are now focusing upon biological clock mechanisms and include the pathways of the mechanistic target of rapamycin (mTOR), its associated pathways of mTOR Complex 1 (mTORC1), mTOR Complex 2 (mTORC2), the silent mating type information regulation 2 homolog 1 (Saccharomyces cerevisiae) (SIRT1), mammalian forkhead transcription factors (FoxOs), the growth factor erythropoietin (EPO), and the wingless pathway of Wnt pathway (Fig. 1).
With neurodegeneration and aging studies, experimental studies with Parkinson's disease (PD) using 6-hydroxydopamine (6-OHDA) during chronic treatment with levodopa show depressed levels of BMAL1 and RORα, indicating that memory loss in PD patients also may be a result of medication that alters circadian rhythm clock genes [106]. Cognitive impairment with memory loss and neuronal injury may occur as a result of sleep fragmentation during extended space flight which alters circadian rhythm [108,109]. Changes in the DNA methylation of biological clock genes may foster memory loss and changes in behavior since rhythmic methylation of BMAL1 has been shown in the brains of individuals with AD [70]. In experimental studies AD using mice, significant alterations have been observed in RNA clock gene expression that may suggest a dysfunction in the clock pathways during cognitive loss [110].
Circadian rhythm disruption and the wingless wnt pathway
Lifespan can be affected by biological clock genes. Lifespan in Drosophila melanogaster is decreased through three arrhythmic mutants involving ClkAR, cyc0 and tim0. In addition, mutations in ClkAR with increasing age can result in dysfunction with ambulation. Through the promotion of Clk function, the locomotor deficits in Drosophila were reversed. This loss of function appears linked to the absence of dopaminergic neurons instead of insults from oxidative stress [75]. Other studies in Drosophila also suggest negative effects with alterations in circadian rhythm [6,80,84] (Table 1). For example, TIMELESS, a mammalian homolog of Drosophila circadian rhythm gene, can lead to cell death and has increased expression in nasopharyngeal carcinoma. During increased TIMELESS expression, cell growth pathways are fostered that involve the wingless pathway of Wnt/βcatenin and resistance against chemotherapy to lead to cell apoptosis, such as with cisplatin, is increased [89]. Wnt proteins are cysteine-rich glycosylated proteins that can affect development of neurons, immune system function, tissue fibrosis, angiogenesis, stem cell development, and cancer [111][112][113][114]. Yet, detrimental effects with Wnt pathways can result to promote increased vascular growth of tumors [111,115,116] and tumorigenesis [40, [117][118][119][120][121]. As a result, these mechanisms may work in conjunction with TIMELESS. There also is evidence for sleep fragmentation and disruption of biological clock genes with shift work to indicate that these lighting and international travel are other examples that can lead to circadian rhythm disturbance [79]. Sleep deprivation affects circadian rhythm and can prevent the clearance of Aβ, α-synuclein, and tau that are tied to the progression of nervous system disorders that include AD and PD [34,109]. Some work suggests that female healthcare workers with extended night shift work may be at enhanced risk for breast cancer [122]. The circadian gene hClock during increased expression also can lead to cancer and colorectal cancer metastatic disease through promotion genes that activate angiogenesis [123].
Previous studies also suggest in experimental studies with AD that a baseline cyclic circadian rhythm that controls autophagy is necessary to reduce Aβ deposition and prevent memory loss [129,148]. Alterations in environmental homeostasis [82,129,149] can alter circadian rhythm that results in loss of cognitive ability [2,19,49,50,84,150]. Sleep fragmentation also can produce changes in hippocampal autophagy proteins and decrease memory function [4,127,[151][152][153][154]. Cellular protection is dependent on the activation of autophagy with circadian clock proteins during insults with stroke, since loss in the function of the PER1 circadian clock protein can increase cerebral ischemia [128].
In regard to the mTOR pathway, mTOR is a 289-kDa serine/threonine protein kinase and is vital during nervous system disease and memory loss [2,19,20,25,49,[155][156][157]. mTOR is also known as the mammalian target of rapamycin and the FK506-binding protein 12-rapamycin complex-associated protein 1 [19,85,158,159]. mTOR is the main component of the protein complexes mTOR Complex 1 (mTORC1) and mTOR Complex 2 (mTORC2) [160][161][162]. mTORC1 and mTORC2 are then divided into additional components [2,107,[163][164][165]. mTORC1 is composed of Raptor, Deptor (DEP domaincontaining mTOR interacting protein), the proline rich Akt substrate 40 kDa (PRAS40), and mammalian lethal with Sec13 protein 8, termed mLST8 (mLST8) [20,40,166]. mTORC1 activity is controlled through a number of pathways that includes PRAS40 by blocking the association of p70 ribosomal S6 kinase (p70S6K) and the eukaryotic initiation factor 4E (eIF4E)-binding protein 1 (4EBP1) with Raptor [167,168]. Rapamycin is an agent that can inhibit mTOR activity [164,[169][170][171][172]. Rapamycin blocks the activity of mTORC1 through its association with immunophilin FK-506-binding protein 12 (FKBP12) that attaches to the FKBP12 -rapamycin-binding domain (FRB) at the carboxy (C) -terminal of mTOR to impede the FRB domain of mTORC1 [4]. mTORC2 is composed of Rictor, Deptor, mLST8, the mammalian stress-activated protein kinase interacting protein (mSIN1), and the protein observed with Rictor-1 (Protor-1) [167,173,174]. mTORC2 oversees remodeling of the cytoskeleton through PKCα and the migration of cells through the Rac guanine nucleotide exchange factors P-Rex1 and P-Rex2 and through Rho signaling [175]. Cognitive decline can be associated with the loss of mTOR activity and altered circadian rhythm during extended space flight [108]. Ischemia in the brain that leads to stroke may be altered by alteration in circadian rhythm genes and fluctuations in the activity of mTOR [124,128]. Other studies suggest that the absence of period2 (PER2), a mammalian circadian clock protein, can increase mTOR activity and chemotherapy drug resistance [125]. mTOR also maintains a relationship with the silent mating type information regulation 2 homolog 1 (Saccharomyces cerevisiae) (SIRT1). SIRT1 maintains an inverse relationship with mTOR [19,[176][177][178][179][180]. SIRT1 can also affect pathways of autophagy [49, 65,163,178,[181][182][183][184][185][186]. SIRT1 activity can lead to the expansion of neurites and promote the survival of neurons during conditions that limit nutrients that involves mTOR inhibition [187]. SIRT1 can foster growth of tumors during autophagy induction that requires the blockade of mTOR, indicating that autophagy and SIRT1 can be targeted to control tumorigenesis [183]. SIRT1 is necessary to foster autophagy and mTOR inhibition during oxidative stress to preserve mitochondrial function in embryonic stem cells [188]. During periods of elevated serum glucose, SIRT1 can block mTOR to offer vascular cell protection [189]. SIRT1 with the blockade of mTOR activity can increase photoreceptor cell survival [177] and limit cell senescence [190]. It is also important to note that some pathways that lead to nerve cell injury require a relationship between mTOR and SIRT1 that is symbiotic. During the loss of dopaminergic neuronal cells, it has been observed that a balance in activities of SIRT1, mTOR, and forkhead transcription factors are required to promote neuronal cell survival [191]. It also has been demonstrated that SIRT1 and mTOR absence during obesity can suppress core circadian components CLOCK and BMAL1 and lead to loss of metabolic cellular homeostasis. The agent metformin, an inhibitor of mTOR activity [4,65,72], can prevent such processes during obesity in experimental mouse models and can reverse the loss of SIRT1 function during inhibition of the circadian components CLOCK and BMAL1 [192].
Through SIRT1 pathways, the coenzyme ß-nicotinamide adenine dinucleotide (NAD + ) has an important function with clock genes that is linked to mTOR [20,66,72,192,205]. Control of circadian rhythm by SIRT1 and melatonin can impact glucose tolerance in cells [102]. Dementia onset can be dependent upon melatonin, a pineal hormone that controls circadian rhythm [81,88,95], as well as mTOR through autophagy induction [90,206]. During the process of aging, circadian rhythm cycles involving melatonin can affect infection with coronavirus disease of 2019 (COVID-19) [94], cellular metabolism [90,103], mitochondrial dysfunction [81], oxidative stress [207,208], and inflammatory mediators [206,209]. In addition, SIRT1 can affect biological clock rhythm through stem cell function [210] and inflammation during obesity [91] and neurodegeneration [209]. Cellular NAD + pools fluctuate with circadian rhythmicity and with aging [72]. SIRT1 in connection with CLOCK:BMAL1 can control the circadian expression of nicotinamide phosphoribosyltransferase (NAMPT) that is required for NAD + production. SIRT1 also through the NAMPT promoter can promote the circadian synthesis of its own coenzyme [211]. Yet, NAD + cellular pools can become depleted during impairment of mitochondrial function to result in cell injury with cellular NAD + pools oscillating with free nicotinamide levels and promoting cell injury, metabolic dysfunction, and loss of cognitive function [205]. SIRT1 regulation of biological clock genes also can affect cognitive function though growth factors, such as EPO [161,197,[212][213][214]. The EPO gene is present on chromosome 7 and represents a single copy in a 5.4 kb region of the genomic DNA [215,216]. The gene encodes for a polypeptide chain protein that has 193 amino acids [64,217]. EPO later undergoes the removal of a carboxy-terminal arginine 166 in the mature human and recombinant human EPO (rhEPO). A protein with a molecular weight of 30.4 kDa and 165 amino acids is generated as the mature protein [218][219][220][221]. EPO expression occurs in the brain, uterus, and liver [64,161,164,215,216,222,223], but the principal site for the production and secretion of EPO is the peritubular interstitial cells of the kidney [216,217,[224][225][226][227]. It is important to note that expression of EPO is overseen by oxygen tension changes and not by the concentration of red blood cells [64,228,229].
In relation to SIRT1, EPO prevents metabolic dysfunction by modulating adipose energy homeostasis in adipocytes through the combined activation of peroxisome proliferatoractivated receptor-α (PPAR-α) and SIRT1 [213] (Table 1). EPO promotes vascular cell protection in the brain through SIRT1 nuclear subcellular trafficking and blocks mitochondrial depolarization, cytochrome c release, BCL2 associated agonist of cell death (Bad) activity, and caspase activation [212]. EPO can increase human cardiomyocyte survival through SIRT1 activation during chemotherapy toxicity [197] and prevent brain neuronal cell loss through the up-regulation of SIRT1 [214]. EPO can block memory loss during AD [5,43], control metabolic pathways [230,231], and block mitochondrial dysfunction [197,216,222,[232][233][234]. However, control of biological clock gene pathways appear to be necessary for EPO and SIRT1 to offer cellular protection. Some studies indicate that during hypoxia specific clock genes, that include BMAL1 and PER2, are required for the production of EPO [235].
Mammalian forkhead transcription factors (FoxOs)
Mammalian FOXO proteins of the O class are transcription factors and play a significant role in the nervous system. FoxO family members include FOXO1, FOXO3, FOXO4, and FOXO6 [67,164,[245][246][247] (Table 1). FoxO proteins bind to deoxyribonucleic acid (DNA) through the FoxO-recognized element in the C-terminal basic region of the forkhead DNA binding domain. With the binding to DNA by FoxOs, target gene expression is blocked or promoted through fourteen protein-DNA contacts with the primary recognition site located at α-helix H3. Phosphorylation or acetylation of FoxOs can change the binding of the C-terminal basic region to DNA to inhibit FoxO transcriptional activity [48,152,199,248]. FoxOs are intimately connected circadian rhythm since they are linked to SIRT1 [4,34,48,85,[248][249][250][251][252]. For example, insulin-phosphatidylinositol 3-kinase (PI3K) signaling that occurs in the liver is overseen by FoxO3 control of circadian rhythmicity through modulation of Clock. Loss of FoxO3 impairs the circadian amplitude and rhythmicity [253]. Autophagy induction also is dependent on mammalian FOXO proteins of the O class [12,164,202,254,255]. FoxO1 transcription factors [256] oversee the myelination of nerves that requires oligodendrocyte progenitor cells and determine the progression of disorders that include multiple sclerosis [257]. Additional studies indicate that epigenetic changes in DNA methylation and genetic variations of FoxO3a and FoxO1 also can affect demyelinating disorders [258]. Yet, it is important to state that a fine balance in FoxO activity is necessary to lead to the protection of cells since activation of FoxOs with autophagy can be beneficial. Sequestering and clearance of detrimental intracellular accumulations by FoxOs and autophagy can lead to increased survival of neurons [246,259,260].
In regard to SIRT1, blockade of the activity of FoxOs by SIRT1 can promote cell survival [19,67,[249][250][251]. However, FoxOs can attach to the SIRT1 promoter region to further change forkhead transcription [181]. This mechanism permits FoxOs to use auto-feedback mechanisms to regulate the activity of SIRT1. FoxO proteins, including FoxO1, can oversee SIRT1 transcription and increase the expression of SIRT1 [261]. These studies suggest an intimate relationship between SIRT1 and FoxOs. Interestingly, SIRT1 and FoxOs can synergistically increase cell survival. SIRT1 and FoxO3a can work in unison to block memory loss and Aβ brain toxicity, mitochondrial dysfunction, and oxidative stress [5,152,262,263].
Future perspectives
Neurodegenerative disorders that involve cognitive loss and dementia impact a significant proportion of the world's population and lead to a large financial burden for all nations. Adding to these concerns is the knowledge that cognitive disorders present almost insurmountable challenges for treatment since they are multifactorial in origin and can result from multiple pathways that involve Aβ, tau, metabotropic receptors, excitotoxicity, lipid dysfunction, mitochondrial damage, astrocyte injury, loss of access to bright light, heavy metal disease, acetylcholine loss, oxidative stress, and metabolic dysfunction that involves DM. Novel new therapeutic strategies are desperately warranted. New investigations may meet this need with work that highlights biological clock genes that oversee circadian rhythm and involve the pathways of mTOR, SIRT1, FoxOs, EPO, and the Wnt/β-catenin pathway (Fig. 1). These pathways are complex in nature and intimately tied to autophagy induction that can sequester intracellular accumulations and potentially reduce cognitive loss under some conditions. Dysfunctional changes in biological clock genes and circadian rhythm can result in motor deficits, memory impairment, and the progression of dementia. Even chronic treatment regimens that occur during PD can alter circadian rhythm function and foster dementia. The pathways of autophagy may be one mechanism to oversee circadian rhythm homeostasis that can become lost during conditions of chronic sleep fragmentation.
The pathways that impact circadian rhythm have an intricate relationship that can lead to both beneficial as well as detrimental clinical effects. For example, blockade of mTOR activity can change circadian rhythm, affect memory function, and increase neuronal cell injury such as during stroke. SIRT1 can oversee the production of NAD + pools that have been tied to circadian rhythmicity and if these cellular pools become depleted, cell injury and metabolic dysfunction can ensue with cognitive loss. Furthermore, without circadian rhythm control, the protective capability of EPO and SIRT1 may become absent and lead to mitochondrial dysfunction and the loss of cognition. In regard to FoxOs, SIRT1 and FoxOs may be required to work in unison to limit cognitive loss, mitochondrial dysfunction, and oxidative stress. Yet, it is important to remember that Wnt pathways that function in conjunction with circadian clock gene pathways, such as TIMELESS, may promote new angiogenesis and tumorigenesis. In addition, other circadian genes that include hClock also may promote metastatic colorectal cancer through the promotion of angiogenesis-related gene activity and vascular cell growth.
These observations serve to form a strong foundation for the further investigation of biological clock genes and circadian rhythm in regards to their significant role in neurodegenerative disorders such as dementia. The circadian pathways involving mTOR, SIRT1, FoxOs, EPO, and the Wnt can offer considerable potential for the understanding and treatment of memory loss and neurodegnerative disorders. Yet, it is the intimate and complex relationship among these pathways that is most intriguing and potentially offers the greatest insight to harness this knowledge for the innovative treatment of dementia.
Acknowledgment
We appreciate the reviewers for their opinions and suggestions.
Funding
This research was supported by the following grants to Kenneth Maiese: American Diabetes Association, American Heart Association, NIH NIEHS, NIH NIA, NIH NINDS, NS053956, and NIH ARRA.
AD
Alzheimer's disease The circadian biological clock gene pathways are intricately related but complex in nature. The pathways of the mechanistic target of rapamycin (mTOR), the silent mating type information regulation 2 homolog 1 (Saccharomyces cerevisiae) (SIRT1), mammalian forkhead transcription factors (FoxOs), the growth factor erythropoietin (EPO), and the wingless Wnt/β-catenin pathway can lead to beneficial outcomes and employ autophagy induction that may provide cellular protection, metabolic homeostasis, and prevent dementia and cognitive loss. Yet, biological clocks and alterations in circadian rhythm, such as during sleep disruption and fragmentation, also have the potential to lead to devastating effects involving tumorigenesis in conjunction with pathways involving Wnt that oversee angiogenesis and stem cell proliferation. Circadian rhythm disruption can result from shift work, exposure to artificial lighting, and from sleep fragmentation. A fine balance in the oversight of circadian biological clock gene pathways is required to foster safe and efficacious clinical outcomes for the treatment of dementia and cognitive loss.
Neurodegeneration and Dementia: Circadian Rhythm Biological Clock Gene Pathways
• Cognitive loss in relation to Alzheimer's disease is an excellent example of complex disorders that are multi-factorial in origin and may involve several mechanisms as etiologies that include cellular injury from β-amyloid, tau, metabotropic receptors, excitotoxicity, lipid dysfunction, mitochondrial damage, loss of access to bright light, acetylcholine loss, oxidative stress, and metabolic dysfunction with diabetes mellitus.
• Current strategies to treat cognitive loss are limited and do not adequately address disease onset and progression. Innovative work with biological clock genes that oversee circadian rhythm can offer new strategies for the treatment of dementia that employ the pathways of the mechanistic target of rapamycin (mTOR), the silent mating type information regulation 2 homolog 1 (Saccharomyces cerevisiae) (SIRT1), mammalian forkhead transcription factors (FoxOs), the growth factor erythropoietin (EPO), and the wingless Wnt/β-catenin pathway.
• Autophagy in combination with biological clock gene pathways are dependent upon mTOR. Studies suggest that a basal circadian rhythm that modulates autophagy and mTOR pathways involving mTOR Complex 1 (mTORC1) and mTOR Complex 2 (mTORC2) may be necessary to prevent cognitive decline and cellular toxicity with amyloid deposition. mTOR also holds an inverse relationship with SIRT1 and these pathways may be necessary to support core circadian components CLOCK and BMAL1 and prevent cellular metabolic dysfunction.
• SIRT1,a histone deacetylase, regulates ß-nicotinamide adenine dinucleotide (NAD + ) cellular NAD + pools that fluctuate with circadian rhythmicity and can impact cell function, metabolism, and loss of cognitive function. Oversight with SIRT1 of circadian rhythm pathways may be required for growth factor EPO cellular production and protection.
• FoxOs that can control circadian rhythmicity, such as through the modulation of Clock, can also bind to SIRT1 promoter regions to function through autofeedback mechanisms to regulate SIRT1 activity. SIRT1 and FoxOs can work in unison to block cognitive loss and prevent amyloid toxicity, mitochondrial dysfunction, and oxidative stress injury.
• Wnt proteins are cysteine-rich glycosylated proteins that can affect neuronal development, immunity, tissue fibrosis, angiogenesis, stem cell proliferation, and cancer. Wnt pathways that function in conjunction with circadian clock gene pathways, such as TIMELESS, may promote new angiogenesis and tumorigenesis. Furthermore, disruption of circadian rhythms with sleep fragmentation may increase the risk for developing cancer and other circadian genes that include hClock also may promote the metastasis of colorectal cancer through the enhanced expression of angiogenesis-related genes. | 2021-10-01T06:16:50.996Z | 2021-09-30T00:00:00.000 | {
"year": 2021,
"sha1": "9bfdd646f37aeb10fc9e1454cdecb4c64c7106df",
"oa_license": "CCBY",
"oa_url": "https://www.fbscience.com/Landmark/articles/pdf/Landmark4971.pdf",
"oa_status": "HYBRID",
"pdf_src": "PubMedCentral",
"pdf_hash": "3217f2944b1b8ebb54e8a480952c3f746c003f80",
"s2fieldsofstudy": [
"Medicine",
"Biology"
],
"extfieldsofstudy": [
"Medicine"
]
} |
235259969 | pes2o/s2orc | v3-fos-license | The impact of work-place social capital in hospitals on patient-reported quality of care: a cohort study of 5205 employees and 23,872 patients in Denmark
Background Decision-makers increasingly consider patient-reported outcomes as important measures of care quality. Studies on the importance of work-place social capital–a collective work-place resource–for the experience of care quality are lacking. We determined the association between the level of work-place social capital and patient-reported quality of care in 148 hospital sections in the Capital Region of Denmark. Methods This cross-sectional study combined section-level social capital from 5205 health care professionals and 23,872 patient responses about care quality. Work-place social capital encompassed three dimensions: trust, justice and collaboration. Patient-reported quality of care was measured as: overall satisfaction, patient involvement, and medical errors. Linear regression analysis and generalized linear models assessed the mean differences in patient reported experience outcomes and the risk of belonging to the lowest tertile of care quality. Results A higher level of work-place social capital (corresponding to the interquartile range) was associated with higher patient-reported satisfaction and inpatient and acute care patient involvement. The risk of a section belonging to the lowest tertile of patient involvement was lower in sections with higher social capital providing inpatient (RR = 0.39, 0.19–0.81 per IQR increase) and acute care (RR = 0.53, 0.31–0.89). Patient-reported errors were fewer in acute care sections with higher social capital (RR = 0.65, 0.43 to 0.99). The risk of being in the lowest tertile of patient-reported satisfaction was supported for acute care sections (RR = 0.47, 0.28–0.79). Conclusions Although we found small absolute differences in the association between patient-reported experience measures and social capital, even a small upward shift in the distribution of social capital in the hospital sector would, at the population level, have a large positive impact on patients’ care experience. Supplementary Information The online version contains supplementary material available at 10.1186/s12913-021-06498-x.
Introduction
Hospitals must increase efficiency and quality of care to meet growing patient demands and expectations [1]. To these ends, hospitals restructure how they deliver health care [2] by introducing management systems such as lean [3] or value-based health care [4], merging departments or hospitals, [5] or by implementing hospital-wide IT enterprise systems [6]. However, restructuring how hospitals operate at the frontline [7,8] may reduce health care professionals' psychosocial work environment and well-being [9] known to influence organisational performance [10]. To maintain a good psychosocial work environment and wellbeing under circumstances of change and uncertainty, research within and outside the health care domain finds that having a high level of social capital is important [11,12]. Social capital is an inter-personal phenomenon [13] consisting of trust, justice, and collaboration components that inheres in the structure of relations between actors and among actors [14]. It may help to foster work-place participation, reciprocity, and interpersonal trust [11] that in turn allow employees to collaborate about solving complex organisational tasks [15] for mutual benefit and with higher performance [16].
Delivering efficient and safe health care is a complex task requiring interdisciplinary collaboration, to which end health care researchers have studied social capital [17] and the related measure of relational coordination [18]. This literature finds that higher levels of social capital improve care coordination [19], patient care [20,21] and safety [22], and reduces the length of stay [23]. For health care professionals, social capital improves job satisfaction [24][25][26], work engagement [24,27], knowledge sharing [22] and innovation adoption [28], and reduces burnout [21,29], long-term sickness absence [30], and work-home conflict [31]. At the hospital level, social capital is associated with better quality management systems [32] and clinical risk management [33]. Nevertheless, research studying the effect of social capital on patient-reported experience measures remain scarce [34] even though they represent vital measures of health care performance [35,36] because the patient is uniquely positioned to assess the experienced care quality [37]. Despite social capital being an inter-personal phenomenon, previous studies also typically investigate individual-level rather than group-level social capital, thereby limiting our understanding of the extent to which the aggregate, collective level drives care and patient-reported outcomes.
Given these knowledge gaps, this article aims to determine the relationship between work-place social capital and patient-reported quality of care. For the purpose of this article, we operationalise patient-reported care quality as patient's overall satisfaction with their acute, in-or outpatient care, level of perceived involvement in decision processes, and occurrence of patient-reported medical errors, thereby studying three central parameters of health care performance. We hypothesise that a higher level of social capital in a hospital section is associated with higher patient satisfaction, greater involvement of patients, and fewer medical errors. To test this hypothesis, we use survey data from 5205 employees and 23, 872 patients from 148 hospital sections spanning 16 health care institutions serving 1.8 million citizens in the Capital Region of Denmark.
Study population
The Well-being in HospitAL Employees cohort To capture the psychosocial work environment of health care workers, we identified eligible hospital sections by using data from the Well-being in HospitAL Employees (WHALE) cohort [38]. The WHALE cohort initiated in 2011 and followed up with new waves in 2014 and 2017 contains survey information from hospital employees within the Capital Region of Denmark about their physical and psychosocial work environment and detailed information on the organisational structure (including organisation, department and section). In 2014, 37,720 health care employees working at 1089 hospital sections within the Capital Region of Denmark were invited to the survey. Of that number, 84% participated by filling in a self-reported questionnaire. These data allowed for, first, conducting the analysis of social capital at an aggregated level and, second, linking the aggregated measure with patient-reported quality at the same organisational level.
The national survey of patient experiences
The Unit for Evaluation and User-involvement (UEU) in Denmark conducts yearly surveys of patients' experiences of the care received across inpatient, outpatient, and acute sections in all Danish hospitals (see [39] for a detailed elaboration). In 2014, our sample year, the UEU invited a random selection of patients seen in the different hospital sections between August 1 and October 31. Up to 400 patients were included from each hospital section: in case fewer were seen within the inclusion period, the UEU invited all attending patients. Data were collected from all hospital sections except physiotherapy, occupational therapy, anaesthesiology, radiology, diagnostic imaging, biochemistry, physiology, and nuclear medicine because they mostly provided services to other sections that had the primary patient responsibility.
Data selection
Data was derived largely from administrative databases reducing issues of missing information. Since only aggregated measures were used, single missing values for individual employees or patients did not exclude these cases from the data. However, information at the aggregated level was considered for analysis only if > 50% of participants at a given section provided valid information. Due to differences in the hierarchal administrative organisation of sections in WHALE and the national survey of patient experiences (NSPE), not all sections from the latter data set were identifiable in the former. We identified 69 out of 100 sections providing acute care, 51 out of 79 sections of inpatient care, and 89 out of 193 sections providing outpatient care in both data sets. To preserve anonymity for smaller sections or sections with only few patient reports, we excluded sections with < 3 employees (3 sections) or < 10 patient reports of quality of care (19 sections). In addition, one section was excluded due to insufficient data on social capital or covariates. This resulted in a total study sample of 148 unique hospital sections, 34 of which had an overlap in the care they provided: 44 provided inpatient care, 60 acute care, 78 sections provided outpatient care, 33 sections provided both acute and inpatient care, and one section provided both acute and outpatient care. For the purpose of our analysis, we used the combined total of 182 hospital sections, thus including the 34 sections proving care in two contexts in both of those study samples. The identifiability and overlap of sections in the WHALE and NSPE cohorts are illustrated in Fig. 1. Across nine hospitals in the Capital Region of Denmark, we included a total of 5205 employee responses about social capital measures and 23,872 patient responses about their perceived quality of care. Patients reporting from hospital sections that could not be identified in WHALE differed in some regards (Additional file 1: Appendix 1). Patients in the unidentifiable hospital sections were more likely to be women and < 60 years of age and they reported receiving a slightly higher average quality of care, more outpatient care and less acute care, and the acute and outpatient care they received were more likely to exceed 24 h.
Social capital
Social capital was measured by eight items [40] covering the three dimensions of trust, justice, and collaboration between employees at the same hierarchical level and with their manager(s) at higher hierarchical levels [38]. Examples of items of the three dimensions are, respectively: 'Do you trust the information that comes from the management?', 'Is the work distributed fairly?', and 'Do you and your colleagues take responsibility for creating a nice atmosphere and tone of communication?' The full list of items is available in Additional file 1: Appendix 2. The response categories were on 5-and 7-point Likert scales ranging from 'not at all' to 'to a very large extent', which we converted into a scale ranging from 0 to 100 to be able to aggregate answers. We measured individual-level social capital as the mean of the converted item responses with higher scores representing higher social capital. Individual-level social capital was recorded as missing if the employee responded to fewer than four of the eight items. To measure social capital at the section level, we aggregated the mean of individuallevel social capital within each hospital section. Sectionlevel social capital was recorded as missing if data were available for < 50% of employees. Similar operationalisations of aggregated social capital have previously been applied within the WHALE cohort, and we have estimated the Cronbach's alpha coefficient of the social capital scale to be 0.85 for the 2014 survey.
Patient-reported quality of care
We used distinct surveys covering the same three dimensions of quality of care that were collected from the three groups (inpatient, outpatient, and acute care) as part of the NSPE [33]. Overall satisfaction was measured by three items concerning patients' satisfaction with treatment, care, and the experience as a whole. Patient involvement was measured by five items. Three items concerned the extent to which the health care staff asked the patient about their experiences with the disease, talked to the patient about disease self-management, and considered the patient's needs when planning the discharge (not including outpatient care). Two items concerned the extent to which the patient and next of kin had the opportunity to take part in shared decisionmaking. Response categories for overall satisfaction and patient involvement were on a 5-point Likert scale from 'to a very large extent' to 'not at all'. Overall satisfaction and patient involvement were operationalised as the mean level of patients' responses (range 1-5) with higher values reflecting a more positive evaluation. Medical errors were measured by patients' reporting of the occurrence of errors during their stay (yes/no). The included scales of patient-reported quality of care have previously Fig. 1 The identifiability of hospital sections in the Well-being among HospitAL Employees cohort and the National Survey of Patient Experiences in the Capital Region of Denmark been found reliable using item response theory models within the Danish patient population [41].
The average quality of care was aggregated within hospital sections based on the mean values of patients' replies to the items measuring satisfaction and patient involvement. A patient observation was included in the analysis if they had provided a minimum of two and three responses (two for outpatient cares) to the included satisfaction and involvement dimensions. An aggregated measure of section-level quality was included if > 50% of patients provided valid responses regarding a given dimension. Medical errors were measured as the average reported occurrence of errors among patients attending each hospital section.
Covariates
Both patient and hospital section characteristics were included as covariates. Information on the patient composition within each hospital section included the distribution of age, gender, and length-of-stay that were measured as aggregated measures of patients' responses. Using data obtained from administrative databases at the Capital Region of Denmark, we included relevant hospital section characteristics: affiliated hospital, size, complexity, proportion of employees on long-term sickleave, and use of part-time workers (see below for an elaboration of how these were operationalised).
Statistics
Descriptive analyses were carried out to assess the mean level of patient satisfaction, patient involvement and occurrence of medical errors at the section-level according to hospital section characteristics of both patient and employee compositions.
To determine the relation between section-level social capital and patient-reported quality of care, we used an ordinary least square linear regression analysis to assess the mean differences in level of patient satisfaction, patient involvement, and occurrence of medical errors. To determine the relation between section-level social capital and the risk of being in the lowest tertile of patientreported quality of care, we used generalized linear models with a Poisson distribution and log link rather than a logistic model because of the high prevalence of the outcome. This also enabled the direct estimation of risk ratios. Confidence intervals were estimated by using robust error variances. The lowest tertile was selected as the low patient-reported quality of care group due to the distribution of the outcomes, where a lower threshold would have resulted in unstable analyses due to few observations.
The full distribution of section-level social capital in hospital sections providing acute, in-and outpatient care, respectively, is displayed in Fig. 2. All associations between section-level social capital and patient-reported quality of care were assessed using the interquartile range (IQR) of social capital in each of the three types of care sections. We used the IQR as a metric of change because it allowed us to capture the impact of shifting a section from the lowest (25% cut-off point) to the highest quartile (75% cut-off point) of social capital. For the three types of sections the IQR was six, six and ten for, respectively, inpatient, acute and outpatient care, meaning that the effects estimated correspond to an improvement in section social capital of this magnitude.
The IQR was similarly used in the adjusted analyses where each IQR-unit increase in social capital was analysed to determine the association with the three outcome measures. The analyses were adjusted for potential confounding from section-level information on both patient and employee compositions. Potential patient composition confounders comprised: gender (proportion of female patients), age (proportion ≥ 60 years of age), and length-of-stay (proportion receiving care for < 24 h, not including outpatient care). Potential employee composition confounders comprised: hospital, hospital section size (number of active employees, i.e., not absent due to parental leave or education), complexity (number of employee-defined smaller work-units, i.e., employees referring to the same manager (described elsewhere: 9, 30), proportion of part-time workers (< 37 h/week), and proportion of employees on long-term sick-leave (exceeding 29 days within one year before assessment). We analysed the three patient groups (inpatient, outpatient, and acute) separately because of the different ways in which care is delivered across these functions [42].
Sensitivity analyses were performed to determine if effects could be ascribed to any of the three components of social capital (trust, justice or collaboration) by analysing each aspect separately.
Results
Hospital section and patient characteristics Table 1 shows mean levels of patient-reported satisfaction, involvement, and the occurrence of medical errors according to patient and hospital section characteristics.
The mean level of patient-reported quality of care was generally lower for patients receiving acute care than those receiving inpatient or outpatient care. The level of reported patient involvement was lower for patients staying for more than 24 h or who were 60 years of age or older. Older patients, however, were generally more satisfied with their care and less likely to report medical errors. Women and men reported similar mean levels of satisfaction, involvement and occurrence of errors.
Regarding hospital section characteristic, those providing acute care had lower patient-reported satisfaction, involvement and higher medical errors than those providing inpatient or outpatient care. Patient-reported quality of care was slightly higher for hospital sections within the highest quartile of social capital, although the pattern was not consistent across low, medium, and high levels of social capital. Slightly lower levels of patientreported quality of care were also found for hospital sections with higher rates of long-term sickness absence among employees, a larger number of employees, and higher section complexity (i.e. number of work-units). The use of part-time workers resulted in similar levels of patient-reported quality of care.
Patient satisfaction
Generally, the majority of patients reported a high degree of satisfaction with their care. A larger variation in the mean level of satisfaction was seen among hospital sections providing acute care compared to inpatient and outpatient care (Fig. 3). The positive correlation between section-level social capital and mean level of satisfaction among inpatient and acute care seen in Fig. 3 was also found in the adjusted analyses (inpatient: 0.09 (0.00, 0.19), acute: 0.08 (0.01, 0.15) presented in Table 2. However, the association between section-level social capital and risk of a hospital section being in the lowest tertile of patient-reported satisfaction was supported only for acute care, with approximately half the risk associated with an IQR increase in social capital (RR = 0.47; 95% CI: 0.28 to 0.79) ( Table 3).
Patient involvement
The mean level of reported patient involvement was between 3 (to some extent) and 4 (to a high extent), with the level being slightly lower for acute care. As Fig. 3 shows, there was a positive correlation between section-level social capital and the mean level of patient involvement in hospital sections providing acute or inpatient care. Yet, in the adjusted analyses the association was apparent only for inpatient care, where each 6-unit increase in social capital was associated with a 0.15 (0.03, 0.27) higher mean level of patient involvement ( Table 2). The risk of a hospital section belonging to the lowest tertile of mean patient involvement was lower in sections with high social capital among both inpatient (RR = 0.39, 95% CI: 0.19 to 0.81) and acute care (RR = 0.53, 95% CI: 0.31 to 0.89) ( Table 3).
Medical errors
The mean occurrence of medical errors reported by patients varied between the three types of care, with a higher occurrence and variation in acute than inpatient and outpatient care, with reported proportions of 13, 9, and 5%, respectively (Fig. 3). The weak negative correlation between section-level social capital and the mean occurrence of medical errors seen in Fig. 3 for acute and inpatient care was not supported in the adjusted analyses ( Table 2). The risk of being in the tertile of hospital sections with the highest occurrence of patient-reported errors was lower in sections with higher levels of social capital caring for acute patients (RR = 0.65, 95% CI: 0.43 to 0.99) while there was no association for inpatient and outpatient care (Table 3).
Sensitivity analyses
The adjusted analysis of the correlations between the three components of social capital (trust, justice, and collaboration) with each of the three measures of quality of care (Additional file 1: Appendix 3a-c) show patterns similar to the main analysis based on the combined measure of section-level social capital, but with a larger variance. In terms of absolute differences, the adjusted effect estimates point in the same direction as the main analysis, but the greater variance results in wider confidence intervals (Additional file 1: Appendix 4).
The association in the main analysis between section-level social capital and the risk of being in the lowest tertile of patient-reported quality was partly supported when analysing the three components separately (Additional file 1: Appendix 5). A statistically significant association was found between sectionlevel trust and patient satisfaction and between section-level justice and patient involvement for inpatient care. For acute care a statistically significant association was found between section-level trust and patient satisfaction, between section-level justice and patient satisfaction and patient involvement, and between section-level collaboration and patient involvement. No associations were found for outpatient care regarding the separate dimensions.
Discussion
The study investigated the association between sectionlevel social capital and the level of patient-reported quality of care among 148 Danish hospital sections spanning acute, inpatient, and outpatient care delivery. Our research extends the existing literature on work-place social capital by analysing the effect of social capital on patient-reported outcomes, an outcome measure increasingly seen as a vital indicator of health care performance. We found that a higher level of section-level social capital in hospitals was associated with higher patient-reported satisfaction and involvement for inpatient and acute care but not for outpatient care. Although the absolute differences found in our study are small, elevated to the entire population of health care professionals and patients, even a small upward shift in the distribution of social capital in the hospital sector would improve the care delivered and experienced by patients.
Studying the association with patient-reported outcomes at the section-level renders it possible to study the interpersonal phenomenon of social capital at the relevant group level where health care professionals together deliver health care and jointly contribute to the patient experience. By measuring social capital at the aggregated group level, we add to the current literature on social capital and performance outcomes that do not adequately consider the former as an interpersonal phenomenon. Moreover, the current evidence base relies predominantly on reporting by the health care professionals and not the receiver of the care. Compared to previous studies, our study more fully adjusts for two sets of covariates known to influence how hospitals operate and how health care professionals deliver care.
Analyses were adjusted for, first, the level of organisational complexity (operationalised as the section size and number of work units in a section) and, second, the type of work environment (operationalised as the proportion of employees with prior long-term sick-leave and use of part-time workers). Similar to our study, earlier studies also find that workplace social capital is an organisational resource that leads to better care outcomes such as length-of-stay [18,23], care quality [20,21], and patient safety [22] and a better work environment as measured by job satisfaction [24][25][26], work engagement [24,27], knowledge sharing [22], burnout [21,29], and longterm sickness absence [30].
The present analysis benefits from relying on a large data set of systematically collected register data. Data on workplace social capital came from a large and representative sample of hospital sections in the Capital Region of Denmark with a response rate of 84%, from which we included 148 hospital sections totalling 5205 employee responses. The detailed information on the composition of hospital sections and patient-level information enabled comprehensive adjustment for potential confounding at the level of both patients and employees. The analyses relied on 23,872 patient reports on validated measures of patient satisfaction, patient involvement, and medical errors regarding outcome measures. Relying on independently collected answers on the sides of the exposure (workplace social capital) and outcome variables (patient-reported quality of care) safeguards against common method bias [43]. For example, research shows that health care professionals' willingness to report medical errors depends on the level of psychological safety [44].
Nevertheless, relying on cross-sectional data precludes longitudinal assessment of the relationship between [20,21,29], primarily surveys one occupation-specific sample (nurses). This may limit the generalisability of the empirical evidence to other health care occupations working in hospitals, most notably the medical profession, which occupies a unique position due to its high level of professional autonomy [45]. However, all participants have reported on their collective work environment, including relations to all professions, including doctors. Further, since the survey is anonymous, it is unlikely that nurses would have reported a high level of social capital if their collaboration with doctors was strained. The study would have benefitted from including covariates such as staff-topatient ratios and the type of medical speciality of each hospital section because they likely influence the psychosocial work environment and the quality of care health care professionals deliver to patients. Another limitation is the response rate (61% for inpatient, 45% for acute, and 56% for outpatient care) among the invited random sample of patients asked to report their patient experiences. These response rates could result in selection bias and influence the analysis if the responding and nonresponding patients experienced different levels of care quality. Participation was generally lower for acute care, patients with a length-of-stay less than 24 h, and patients younger than 60 years. Relatedly, if the work context and environment differ between included and excluded types of hospital sections, the results may not apply to those types of sections excluded in this study (for example, where patient contact typically is a sub-part of the care pathway as is the case for diagnostic imaging, biochemistry, and nuclear medicine). Our findings inform hospital management and policymakers, who increasingly understand patient quality as delivering patient-centred and coordinated care, in the following ways: moving hospital sections from having a section-level social capital belonging to the lowest 25% to the highest 25% (i.e., the IQR) would most likely result in an increase in patient-reported quality of care in most settings. Although there is a low variance in the distribution of section-level social capital, implementation of supportive collaborative practices would render it possible to start shifting the distribution and raise the level of social capital among a larger share of health care professionals. From the literature, relevant workenvironment approaches include employing participatory workshops [46], joint collaborative committees [47], psychological safety [44], transformational leadership [48,49], and supportive organisation designs [50]. That social capital in hospitals is associated with better quality management systems [32] and clinical risk management [33] amplifies why hospital management should pursue developing this inter-personal resource. Because workplace social capital is an organisational resource for employee well-being and the perceived quality of care for patients, our findings highlight the merits of investing health care managers' and clinicians' time in efforts to promote social capital among employees. This opens up for using work-place social capital to achieve a high level of care quality in hospitals that restructure and rationalise care delivery to meet calls for more and better health care.
Conclusion
This study extends the existing literature on workplace social capital by analysing the effect of social capital across 148 Danish hospital sections spanning acute, inpatient, and outpatient care on patient-reported quality of care. Although we found small absolute differences in the association between social capital and overall patient satisfaction, patient involvement and patient-reported medical errors, even a small upward shift in the distribution of social capital in the hospital sector would at the population level have a large positive impact on patients' care experience.
Additional file 1: Appendix 1. Individual level patient characteristics among patients receiving care at NSPE hospital sections according to merge status with WHALE Appendix 2. Items covering section social capital and patient-reported quality of care. Appendix 3a. The correlation between section trust and patient-reported quality of care. Appendix 3b. The correlation between section justice and patient-reported quality of care. Appendix 3c. The correlation between section collaboration and patient-reported quality of care. Appendix 4. Dimensions of section social capital and mean differences in patient-reported quality of care in 148 hospital sections. Appendix 5. Dimensions of section social capital and risk ratios (95% CI) of being in the lowest tertile of patientreported quality of care in 148 hospital sections. publicly available. Data are, however, available from the authors upon reasonable request and with permission of the Capital Region of Denmark.
Declarations
Ethics approval and consent to participate The study was approved by the Danish Data Protection Agency through the joint notification of The Faculty of Health and Medical Sciences at The University of Copenhagen (record no 514-0531/20-3000). All data was handled according to GDPR guidelines. Survey-based studies do not require ethical approval by the Danish National Committee on Health Research Ethics according to Danish Law (Section 14, part 2) because 'Notification of questionnaire surveys and medical database research projects to the system of research ethics committee system is only required if the project involves human biological material' (https://en.nvk.dk/rules-and-guidelines/act-onresearch-ethics-review-of-health-research-projects).
Consent for publication
Not applicable. | 2021-06-01T13:48:02.991Z | 2021-05-31T00:00:00.000 | {
"year": 2021,
"sha1": "e4f71114297cf5decae3be453e537a08776a09e1",
"oa_license": "CCBY",
"oa_url": "https://bmchealthservres.biomedcentral.com/track/pdf/10.1186/s12913-021-06498-x",
"oa_status": "GOLD",
"pdf_src": "PubMedCentral",
"pdf_hash": "0303e5578eda00172e6d47df08dab3025c695e88",
"s2fieldsofstudy": [
"Sociology",
"Medicine"
],
"extfieldsofstudy": [
"Medicine"
]
} |
256221680 | pes2o/s2orc | v3-fos-license | Practical Implementation of the Comprehensive Geriatric Assessment to Optimise Care for Older Adults with Cancer
Whilst cancer remains a very serious health problem at any stage, cancer combined with increasing age creates an even more challenging situation for health care providers [...].
Introduction
Whilst cancer remains a very serious health problem at any stage, cancer combined with increasing age creates an even more challenging situation for health care providers. Age is the principal non-modifiable risk factor for all cancer types. There is a rising incidence of cancer among older adults worldwide. In Belgium, the highest incidence rate is situated in the 80-84 age group [1]. In 2020, 60% of women and 70% of men in Belgium were aged 65 years or older when diagnosed, an increase of 6% for both sexes compared to 2010 [1]. Older adults with cancer can be a vulnerable group of individuals, as they present with unique characteristics that have naturally developed as a consequence of aging, such as altered physiology, functional impairment, and comorbidities [2].
In geriatric oncology, there is an awareness of the heterogeneity of the population of older adults with cancer and the spectrum of impairments that may be experienced. Decision making should be based on the patient's functional age rather than the chronological age, as chronological age alone is often a poor indicator of the physiological and functional status of older adults [3]. Some older adults will present as independent individuals, whereas others may be at a moderate risk of health deterioration or even have a high risk of functional decline or mortality. These different profiles, ranging from fit over vulnerable to frail, can be determined through a geriatric assessment, and more extensively through a comprehensive geriatric assessment (CGA). The CGA is the gold standard in geriatric oncology to identify patients at a high risk of adverse outcomes and optimize cancer and overall management [4,5]. This multidisciplinary, in-depth evaluation helps physicians to assess the objective health of the older adult while evaluating vulnerabilities in different age-related domains such as functional status, physical performance, cognition, nutrition, social status, emotional status, comorbidity, and polypharmacy. The appropriate screening and identification of vulnerable or frail older adults is an important step to administer a safe and effective cancer treatment. Evidence-based data based on the geriatric profile are essential to determine potential risks and benefits older adults with cancer could encounter during and after cancer treatment. The use of geriatric screening tools such as the CGA helps physicians to develop a coordinated plan for cancer treatment and to guide interventions tailored to the individual. The integration of general geriatric principles focusing on the patient rather than the tumour represents the first step towards individualized treatment decisions, and consequently, the avoidance of over-and undertreating older adults with cancer.
In a recent report on Clinical Cancer Advances, the American Society of Clinical Oncology (ASCO) stressed the importance of an equal opportunity to participate in, be recognized for, and benefit from research for all populations [6]. Amongst others, the optimisation of care for older adults with cancer was mentioned as one of the research priorities in order to accelerate progress against cancer, with the following focus areas: (1) the use of practice-based data to better understand the efficacy and toxicities of cancer treatments; (2) the development of standardized methods to characterize physiologic aging, such as geriatric assessment; and (3) testing the role of geriatric assessment-guided management in improving outcomes using personalized care, in order to minimise the undertreatment of fit patients and the overtreatment of vulnerable or frail patients.
The CGA as a Tool to Assist in the Management of Cancer-Related Toxicity
The first challenge highlighted by the ASCO is the use of practice-based data to better understand the efficacy and toxicities of cancer treatments in older adults with cancer. Indeed, the underrepresentation of (vulnerable) older adults with cancer in phase II/III clinical trials continues to pose a major problem. This forces physicians to rely on their own clinical expertise, rather than the extrapolation of evidence-based data to assess the benefits and risks of a particular therapy for an (vulnerable) older adult with cancer. The outcome of a case-control study by our research group supports that the majority of older adults with cancer included in clinical trials have a fit profile (4). Those with a vulnerable profile, who represent the majority of older adults with cancer in real practice, are underrepresented in clinical trials [7].
This calls for research to perform subpopulation studies or to include older adults with cancer in later stages of clinical trials. Another tool to address this challenge is the use of artificial intelligence (AI). Platforms using AI and natural language processing represent a potential tool to obtain valuable insights about the dynamics of patients' reserves and cancer treatments in a plethora of cancer types, generating a snapshot of the real-world patient population.
At present, our research group is preparing a research project on the effectiveness and toxicity of combination therapies for older adults coping with stage IV kidney cancer. In this post-marketing observational study, a trial-specific sample size calculation will be applied to recruit separate cohorts of fit and vulnerable older adults with cancer for each immunotherapy combination regimen. Fleming's Two-Stage Design with an admissible adaptive design will be utilised as a formal stopping rule for a high rate of adverse events leading to trial discontinuation (as compared to the data provided in the respective landmark trials). This could lead to the avoidance of regimens that are associated with too much toxicity in vulnerable (as compared to fit) older adults with stage IV kidney cancer (in collaboration with Dr S. Kim [8]). The serial performance of a geriatric assessment will help us to identify which older patients will truly benefit from immunotherapy combinations and at what risk (discerning fit versus vulnerable older patients). With this project, we aim to contribute to the body of knowledge on how to appropriately treat this patient population, responding to the ASCO's call for achieving equity in cancer research and hereby representing all the different individuals.
The use of AI to provide practice-based data to better understand the efficacy and toxicities of cancer treatments represents another tool to assess excess toxicity and the maintenance of beneficence in real life populations. In that regard, the general hospital Groeninge collaborates on a multicentric project of LynxCare ® (Leuven, Belgium) on the Real-World Evidence Observational Study of Cancer Patients Treated with Immune Checkpoint Inhibitors [9]. These tools and their outcomes may contribute to greater equity in cancer research and, therefore, represent all the different individuals that are being treated. For our research group, these methods of AI will also be exploited to determine the outcomes of the geriatric assessment and the according management contributing to the optimisation of the CGA as such.
CGA in Routine Practice: The Kortrijk Older Adult Oncology Care Model
The second challenge set by the ASCO refers to the development of standardized methods to characterize physiologic aging. Indeed, a patient's chronological age is often a poor indicator of the physiological and functional status of older adults [3]. There is, thus, a need for a more reliable tool to assess one's objective health and to perform clinically justified decision making regarding the treatment plan of an older adult with cancer.
Potential solutions include the identification of biomarkers of aging and clinical pharmacology in older adults. A widely used tool already available for the characterization of physiologic aging is the use of a geriatric assessment such as the CGA. Although the importance of the CGA is promoted by international organisations, including the National Comprehensive Cancer Network (NCCN), its implementation may lag behind, as it involves a dedicated team accomplishing the geriatric assessments, which is embedded as routine practice in clinical settings.
With the support of the National Cancer Plan in Belgium, the "oncogeriatric clinic" was founded at the OECI-designated Kortrijk Cancer Centre in 2010. This clinic is staffed by both scientific research associates responsible for the research aspect and oncopsychologists responsible for daily practice, hereafter referred to as trained healthcare workers (THCWs). Throughout the years, we have optimized the workflow of the oncogeriatric team at the Kortrijk Cancer Centre, which resulted in the "Kortrijk older adult oncology care model" (Figure 1). The initial step in routine practice is the administration of the Geriatric 8 (G-8) questionnaire. The G-8 screening tool identifies those older adults with cancer who would benefit from a CGA [10,11], and indicates quality-adjusted survival [12]. This two-step approach is performed by the team of oncopsychologists of the oncogeriatric clinic who are trained to administer the geriatric assessments (THCW-led pathway). If there are no concerns about the patient's ability to tolerate cancer-specific treatment, the patient will be screened with the G-8 screening tool and sequentially receive standard cancer treatment when G-8 negative. However, if there are concerns and the patient effectively scores 14 or less on the G-8, the patient undergoes a short CGA (sCGA) performed by the THCW (Table 1). In the case of obvious concerns, such as a high-risk surgery, older adults with cancer can be referred to the geriatrician-led pathway (Figure 1). The geriatrician-led pathway involves an in-depth evaluation of multiple domains of the CGA, including extended CGA (eCGA), at the geriatric day clinic by a multidisciplinary team, which is supervised by the geriatrician. This is often combined with scheduled pre-operative or staging examinations at the geriatric day clinic to mitigate time toxicity for the patients.
Comorbidities
Assessment of the number of co-existing morbidities and rating accordingly Charlson Comorbidity Index (CCI) [20] Polypharmacy Assessment of the number of medications Not applicable
Testing the Role of Geriatric Assessment-Guided Management
To continue the challenge of the development of standardized methods, this focus area goes hand in hand with the according management of the defined vulnerabilities. The integration of the CGA in oncology has provided several advantages, promoting its use in routine cancer care. They include, but are not limited to, guidance in treatment decision, the prediction of chemotherapy toxicity, and the prediction and improvement of survival and quality of life [12,21,22]. Nevertheless, it should be noted that conducting a CGA alone does not add any value to the patient's treatment plan without informing consequent tailored interventions. The latter was also stressed in the third focus area by the ASCO: it is a challenge to test the role of geriatric assessment-guided management in improving outcomes using personalized care, in order to minimise the undertreatment of fit patients and the overtreatment of vulnerable or frail patients. Besides toxicity management, the CGA is also essential to meet the quality standard stipulated by the International Psycho-Oncology Society (IPOS), stressing the importance of the integration of a psychosocial domain into routine care to provide quality cancer care [23].
According to the ASCO, it remains a challenge to provide supportive care interventions and care delivery interventions to improve outcomes using personalized care. Potential solutions entail a decent workflow on specific vulnerabilities uncovered by performing a CGA, in order to provide each older adult with cancer with the cancer care that
Testing the Role of Geriatric Assessment-Guided Management
To continue the challenge of the development of standardized methods, this focus area goes hand in hand with the according management of the defined vulnerabilities. The integration of the CGA in oncology has provided several advantages, promoting its use in routine cancer care. They include, but are not limited to, guidance in treatment decision, the prediction of chemotherapy toxicity, and the prediction and improvement of survival and quality of life [12,21,22]. Nevertheless, it should be noted that conducting a CGA alone does not add any value to the patient's treatment plan without informing consequent tailored interventions. The latter was also stressed in the third focus area by the ASCO: it is a challenge to test the role of geriatric assessment-guided management in improving outcomes using personalized care, in order to minimise the undertreatment of fit patients and the overtreatment of vulnerable or frail patients. Besides toxicity management, the CGA is also essential to meet the quality standard stipulated by the International Psycho-Oncology Society (IPOS), stressing the importance of the integration of a psychosocial domain into routine care to provide quality cancer care [23].
According to the ASCO, it remains a challenge to provide supportive care interventions and care delivery interventions to improve outcomes using personalized care. Potential solutions entail a decent workflow on specific vulnerabilities uncovered by performing a CGA, in order to provide each older adult with cancer with the cancer care that is required without overshooting the vulnerabilities, but also without undertreating those older adults with a fit profile.
The CGA can adequately identify several vulnerabilities that could require an appropriate referral. For example, a referral to a dietitian may be made in the case of unintentional weight loss in the last 6 months, or offering access to complementary therapies such as Emotional Freedom Techniques if the patient would present with subjective cognitive problems [24]. When standard treatment is not an option for an older adult with cancer, it might be feasible to receive cancer treatment when taking special precautions, such as dose modification or opting for a less toxic regimen. For older adults who appear to have a frail profile or non-modifiable abnormalities, the geriatrician or oncologist will examine if alternate treatment options are available. If not, these patients will receive supportive or palliative care. Further examples of modifiable abnormalities that require the appropriate scheduling of one or more interventions performed by the multidisciplinary geriatric oncology team are mentioned in Figure 1.
Examples to support the importance of the geriatric assessment-guided management may be delivered by our research group via the earlier mentioned example of the cohort study of fit and vulnerable older adults with stage IV kidney cancer, which will be set up for four immunotherapy combination regimens. The performance of serial CGA throughout the treatment period will be used to test the role of geriatric assessment-guided management to minimise the undertreatment of fit patients and the overtreatment of vulnerable or frail patients.
Previously, our research group has performed extensive research on several psychosocial domains included in the CGA [23]. One of the current challenges we address in the population of older adults with cancer is the assessment of depressive symptoms. Depression has been identified in approximately 28% of older adults with cancer and its prevalence can have a significant impact on the patient's ability to receive life-sustaining treatment for their cancer [25]. Within the CGA, the emotional status is assessed by the 15-item version of the Geriatric Depression Scale (GDS-15). Yet, since the GDS is only a screening tool, the GDS-15 is often considered too time-consuming in clinical practice. Moreover, it contains a number of inappropriate questions that may also lead to an inaccurate assessment of depression in older adults with cancer. This domain thus requires optimisation where the GDS-15 might be replaced by a shorter screening tool. A pilot study by our research group identified the Patient Health Questionnaire two-item version (PHQ-2) as a valid screening tool to accurately assess depressive symptoms in older adults with cancer (manuscript by Tack L et al. in preparation). This pilot study is an example of the continued development of the CGA into a more effective and tailored aspect of geriatric cancer care.
Implementation of the CGA in Daily Oncology Practice
The integration of the geriatric assessments in routine cancer care would represent a major added value. Several international oncology societies have published guidelines on the different domains included in the CGA and the performance of the CGA by a multidisciplinary geriatric oncology team, including geriatric oncology nurses, dietitians, psychologists, oncologists, geriatricians, and other health care professionals [26][27][28]. However, in practice, given high workloads in healthcare, we doubt the universal recommendation of a full multidisciplinary team to perform the CGA in all patients at every transition (e.g., change in line of treatment). Given the obvious benefits of geriatric cancer care, psychologists and other members of the multidisciplinary oncogeriatric team should receive adequate education to undertake CGAs and to challenge barriers related to culture, language, misinformation, and cancer stigma.
The increasing population of older adults with cancer comes along with a high socioeconomic burden, which calls for an effective use of the available geriatric assessment tools and healthcare professionals. A Belgian national consortium led by researchers of the KU Leuven identified 70% of the older adults with cancer screened by the G-8 as vulnerable (G-8 ≤ 14) [29]. To limit the use of resources and staff, we apply a two-step approach at the Kortrijk Cancer Centre (Figure 1). In the first step, our oncopsychologists are the THCWs who conduct the G-8 and sCGA and simultaneously introduce the availability of psychological support to the patients. The two-step approach allows the first step to be performed by other healthcare professionals, such as an advanced practice nurse, an occupational therapist, a physical therapist, or a nurse at the geriatric or oncology department, when provided with adequate training to become a THCW. This two-step approach facilitates the implementation of geriatric assessments, increases coverage, and makes it feasible for implementation in routine practice. In the future, tele-sCGA [30] and the validation of shorter screening tools (a research focus of our research group [16,[31][32][33]) to be applied in the sCGA could further facilitate implementation in oncology practice.
To continue with the efficiency of performing a CGA, we trust a centred approach with a THCW identifying vulnerabilities as the first step. This does not neglect the importance of a multidisciplinary geriatric oncology team: in our view, they are complementary to the role of the THCW. In the outlined two-step approach, the responsibility for the management of the identified vulnerabilities represents the second step, which should be assigned to this team. Therefore, it is certainly necessary to have a multidisciplinary geriatric oncology team in place to start addressing the outcome of both the sCGA and eCGA, and accordingly schedule the necessary interventions ( Figure 1). They also bear the responsibility of adding the principles of geriatrics into the daily practice of geriatric oncology, which requires training specific to their discipline with focus on the older adult with cancer. This last notice was recently highlighted by SIOG's patient advocate Ms. Beverly Canin, who stated "research training should include how to engage with patients not just as subjects or peripherally but as collaborators and partners".
In conclusion, we propose the implementation of the CGA as a two-step approach performed with older adults who appear vulnerable on the G-8. There is a call from international organisations to standardize cancer care, including the performance of geriatric assessments. We believe there is great potential in the performance of the screening and CGA by a THCW, complemented by a multidisciplinary oncogeriatric team available to manage the identified vulnerabilities. This requires upskilling of the healthcare professionals involved, which in turn will raise awareness and, thus, also improve the quality of cancer services for older adults. | 2023-01-25T16:17:39.875Z | 2023-01-23T00:00:00.000 | {
"year": 2023,
"sha1": "95d95895424cd8f6b0f373e72940e334d2979897",
"oa_license": "CCBY",
"oa_url": "https://www.mdpi.com/2308-3417/8/1/18/pdf?version=1674465663",
"oa_status": "GOLD",
"pdf_src": "PubMedCentral",
"pdf_hash": "f39e9d7c563e1c7d58f83ad4914265aefb418942",
"s2fieldsofstudy": [
"Medicine"
],
"extfieldsofstudy": [
"Medicine"
]
} |
11505239 | pes2o/s2orc | v3-fos-license | Magnitude of Morning Surge in Blood Pressure Is Associated with Sympathetic but Not Cardiac Baroreflex Sensitivity
The ability of the arterial baroreflex to regulate blood pressure may influence the magnitude of the morning surge in blood pressure (MSBP). The aim was to investigate the relationships between sympathetic and cardiac baroreflex sensitivity (BRS) and the morning surge. Twenty-four hour ambulatory blood pressure was recorded in 14 young individuals. The morning surge was defined via the pre-awakening method, which is calculated as the difference between mean blood pressure values 2 h before and 2 h after rising from sleep. The mean systolic morning surge, diastolic morning surge, and morning surge in mean arterial pressures were 15 ± 2, 13 ± 1, and 11 ± 1 mmHg, respectively. During the laboratory protocol, continuous measurements of blood pressure, heart rate, and muscle sympathetic nerve activity (MSNA) were made over a 10-min period of rest. Sympathetic BRS was quantified by plotting MSNA burst incidence against diastolic pressure (sympathetic BRSinc), and by plotting total MSNA against diastolic pressure (sympathetic BRStotal). Cardiac BRS was quantified using the sequence method. The mean values for sympathetic BRSinc, sympathetic BRStotal and cardiac BRS were −1.26 ± 0.26 bursts/100 hb/mmHg, −1.60 ± 0.37 AU/beat/mmHg, and 13.1 ± 1.5 ms/mmHg respectively. Significant relationships were identified between sympathetic BRSinc and the diastolic morning surge (r = 0.62, p = 0.02) and the morning surge in mean arterial pressure (r = 0.57, p = 0.03). Low sympathetic BRS was associated with a larger morning surge in mean arterial and diastolic blood pressure. Trends for relationships were identified between sympathetic BRStotal and the diastolic morning surge (r = 0.52, p = 0.066) and the morning surge in mean arterial pressure (r = 0.48, p = 0.095) but these did not reach significance. There were no significant relationships between cardiac BRS and the morning surge. These findings indicate that the ability of the baroreflex to buffer increases in blood pressure via reflexive changes in MSNA may play a role in determining the magnitude of the MSBP.
INTRODUCTION
In humans, blood pressure (BP) fluctuates according to a circadian rhythm, whereby it decreases during sleep and increases in the morning (Muller et al., 1989). In line with this circadian rhythm, the onset of adverse cardiovascular and cerebrovascular events such as myocardial infarction, sudden cardiac death, and stroke occur more frequently in the hours of the morning (Willich et al., 1987;Muller et al., 1989;Argentino et al., 1990;Elliot, 1998). The increase in blood pressure observed in the first 2-3 h after waking from nocturnal sleep is known as the morning surge in blood pressure (MSBP; Atkinson et al., 2014). Importantly, the MSBP has been shown to have prognostic value, as individuals with an accentuated MSBP are more likely to experience deleterious cardiovascular and cerebrovascular events (Kario et al., 2003;Li et al., 2010;Pierdomenico et al., 2014). In addition to an unfavorable prognosis, an accentuated MSBP is associated with left ventricular hypertrophy (Kaneda et al., 2005;Yano et al., 2009), greater internal carotid intima-media thickness (Yano et al., 2009), an unfavorable atherosclerotic plaque phenotype (Marfella et al., 2007) and a prothrombotic environment (Kario et al., 2011).
Previous research indicates that the sympathetic nervous system has a significant influence on the MSBP. For example, the MSBP is reduced in response to α1-adrenergic antagonist therapy (Kario et al., 2004) and centrally acting sympatholytic agents (Hashimoto et al., 2003). Furthermore, individuals with an exaggerated MSBP have higher levels of urinary catecholamine excretion (Marfella et al., 2005) and an MSBP with a larger αadrenergic component is more intimately associated with silent cerebral infarcts (Kario et al., 2004). Investigating the relationship between muscle sympathetic nerve activity (MSNA) and the MSBP directly, it has been found that the resting levels of MSNA do not correlate to a greater MSBP (Hering et al., 2001). Whilst these findings suggest that baseline sympathetic nerve activity does not influence the magnitude of the MSBP, the mechanisms involved in blood pressure regulation are dynamic and so resting MSNA is not the only influence that the sympathetic nervous system can have on the MSBP.
The arterial baroreflex buffers spontaneous fluctutations in blood pressure through its actions on the heart and vasomotor drive to resistance vessels in, for example, skeletal muscle (Ackermann, 2004;Wehrwein and Joyner, 2013). The ability to alter heart rate or MSNA in response to a given change in arterial pressure is known as baroreflex sensitivity (BRS; Wehrwein and Joyner, 2013). BRS is clinically relevant: it plays a role in a number of diseases and is associated with a poorer prognostic outcome. For example, studies have shown that BRS is reduced in aging, diabetes, obesity, coronary artery disease, hypertension, heart failure and obstructive sleep apnoea (Skrapari et al., 2006;Monahan, 2007). An impaired sensitivity of the vascular sympathetic baroreflex (baroreflex modulation of MSNA) or cardiac baroreflex (baroreflex modulation of heart rate) could prevent the buffering of blood pressure increases that occur upon awakening, contributing to an exaggerated MSBP. Two previous studies have investigated aspects of this relationship. Okada et al. (2013) discovered that sympathetic BRS is reduced in elderly, hypertensive individuals with an exaggerated systolic MSBP, defined using the "sleep-trough" method in which morning blood pressures are compared to the trough in blood pressure during sleep (Xie et al., 2015). This method is influenced by dipping status; that is, the extent to which blood pressure decreases during sleep (Yano and Kario, 2012). It may be argued that other measures of the MSBP that are centered around the waking event may be a more realistic measure of the MSBP. Lambert et al. (2014) used a measure of morning blood pressure, known as BP Power , to examine the relationships between BRS, the MSBP and sympathoexcitation during a cold pressor test. The BP Power approach is unique as it incorporates the rate of rise of morning BP in addition to the amplitude of the MSBP (Head et al., 2010). Lambert et al. (2014) did not find a relationship between sympathetic BRS and BP Power or rate of rise, although changes in MSNA burst amplitude during the cold pressor test were positively associated with these measures of the morning surge. Despite its advantages, this novel approach to assessing the MSBP is not yet widely used thus limiting the scope for comparisons with previous studies of different populations. Furthermore, the sample of participants included individuals being treated for hypertension and a wide age range.
The major issue with research surrounding the MSBP is that it can be quantified using various methods (Stergiou et al., 2008;Atkinson et al., 2014), and the different methods used by the aforementioned studies impairs their comparability. Perhaps the most widely used method for quantifying the MSBP is the preawakening method. For this method, morning blood pressures are typically compared to those in the 2 or 3-h period prior to waking (Xie et al., 2015) and thus the measure is centered around waking. The aim of this paper is to investigate the relationship between BRS and the MSBP using both the pre-awakening and sleep-trough methods for quantifying the morning surge. In addition, sympathetic BRS will be quantified using both "burst incidence" (threshold technique) and "total MSNA" methods in order to account for baroreflex-driven changes in MSNA burst amplitude. It is hypothesized that individuals with reduced sympathetic BRS have larger pre-awakening morning surges in blood pressure.
Participants and Ethics
This study was conducted under the approval of the Human Research Ethics Committee of Western Sydney University and satisfied the Declaration of Helsinki. Fourteen healthy young volunteers (12 male, 2 female; age 19-27 years) participated in this study. All participants provided written informed consent prior to taking part. Participants who had known cardiovascular, neuromuscular, or respiratory conditions, as well as those who smoked or took regular medication were excluded. The changes in hormone levels associated with the menstrual cycle have been proven to influence MSNA and sympathetic BRS (Minson et al., 2000). Accordingly, females participated in all experimental protocols during the early follicular (low-hormone) phase of their menstrual cycle.
Ambulatory Protocol
Ambulatory blood pressure was recorded using an ambulatory blood pressure monitor (Spacelabs 90217A, Spacelabs Healthcare, Snoqualmie WA, USA) for 24 h, to ensure that recordings were taken during periods of both sleep and wake. The monitors were programmed to take blood pressure readings every 20 min during wakefulness and every 30 min during sleep over the 24 h. Before the recording, participants indicated the times that they normally slept and the monitor was set accordingly to minimize disturbance to sleep. During the period of recording, participants were required to complete an activity diary indicating activities that were undertaken during the day as well as their sleep and wake times. Actigraphy was completed simultaneously using an activity monitor (Actigraph wGT3X-BT, Actigraph, Pensacola, FL) worn on the waist to confirm the time of waking. Participants were instructed to undertake their usual activities but to refrain from strenuous exercise and any activities that were not normally a part of their daily routine. On completion of the recording, participants were asked to indicate the quality of their sleep.
Laboratory Protocol
At a time separate from the period of ambulatory blood pressure recordings, participants attended the Human Autonomic Laboratory at the School of Medicine, WSU (Campbelltown, NSW, Australia). All experiments took place in the morning, beginning at 0800h. The following variables were recorded non-invasively: heart rate (ECG), blood pressure (Finometer Pro, Finapres Medical Systems, Amsterdam, The Netherlands), and respiration (Pneumotrace, UFI, Morro Bay CA, USA). MSNA was recorded from fascicles of the common peroneal nerve supplying the pretibial flexors via tungsten microelectrodes (FHC, Bowdoinham, ME, USA) inserted percutaneously at the level of the fibular head. Multi-unit neural activity was amplified (gain 20,000, bandpass 0.3-5.0 kHz) using an isolated amplifier (NeuroAmp EX, ADInstruments, Sydney, Australia) and stored on computer (10 kHz sampling) using a computer-based data acquisition and analysis system (PowerLab 16SP hardware and LabChart 6 Pro software; ADInstruments, Sydney, Australia). Mean voltage of the nerve signal was computed via the root mean square (RMS) feature in LabChart, using a moving average of 200 ms. Participants were seated in a semi reclined posture in a comfortable chair with the legs supported in the extended position. The variables described above were then continuously measured over a 10-min period.
Ambulatory Blood Pressure Characteristics and MSBP
For each participant, mean values of systolic BP, diastolic BP, MAP, and heart rate were calculated for each time period of the day (across the entire 24 h, during times of wakefulness and during times of sleep). Two widely recognized definitions of the MSBP were quantified for each participant: (i) the pre-awakening morning surge and (ii) the sleep-trough morning surge (Xie et al., 2015). The pre-awakening morning surge was determined via the difference between the average BP recordings 2 h after rising from sleep and the average BP recordings over 2 h before rising from sleep (Figure 1). The sleep-trough morning surge was determined as the difference between the average BP over 2 h after rising from sleep and the average of three consecutive BP recordings, centered on the lowest reading during sleep (Figure 1). Both definitions of MSBP were quantified using systolic BP, diastolic BP, and MAP. Using systolic blood pressure as the example, interval A represents the three systolic blood pressure values centered around the lowest systolic blood pressure obtained during sleep, interval B represents the 2 h prior to rising from sleep and interval C represents the 2 h after rising from sleep. Pre-awakening morning surge in blood pressure is determined via the difference between the mean of the systolic blood pressures captured during interval C and the mean of the systolic blood pressures captured during interval B. Sleep-trough morning surge in blood pressure is determined via the difference between the mean of the systolic blood pressures captured during interval C and the mean of the three systolic blood pressures represented by interval A.
Laboratory Data Analysis
All laboratory data analysis was performed by one investigator to minimize any subjective differences in defining sympathetic bursts. Beat-to-beat values were extracted from LabChart (ADI Instruments, Sydney, Australia) for systolic BP, diastolic BP, R-R interval, and MSNA. The experimental records for one participant are shown in Figure 2. MSNA bursts were detected and measured from the RMS nerve signal using a custom-written analysis program (LabView, National Instruments, USA). For each participant, the number of MSNA bursts per 100 heart beats (MSNA burst incidence) and number of MSNA bursts per minute (MSNA burst frequency) were calculated. Sympathetic BRS and cardiac BRS analyses were performed using the spontaneous techniques described below.
Sympathetic Baroreflex Sensitivity: Burst Incidence Method
Sympathetic BRS was quantified by plotting burst incidence against diastolic blood pressure, as described by Kienbaum et al. (2001). Prior to the determination of sympathetic BRS, the nerve trace was shifted to account for the sympathetic baroreflex conduction delay and this was adjusted for each participant to account for inter-individual differences in burst latency. The mean shift applied was 1.31 ± 0.03 s. To help remove nonbaroreflex stimuli, the diastolic pressure values of each cardiac cycle were assigned to 3 mmHg bins for each participant (Ebert and Cowley, 1992;Taylor et al., 2011). The corresponding MSNA burst incidence (bursts/100 HB) was then calculated for each bin. Sympathetic BRS was quantified by plotting MSNA burst incidence against the mean diastolic BP for each bin. A weighting procedure was applied according to the number of cardiac cycles for each diastolic BP bin, as the highest and lowest diastolic pressures contain fewer cardiac cycles (Kienbaum et al., 2001). The acceptance level for the linear regression analyses was set at r > 0.5 (Rudas et al., 1999;Hart et al., 2011b;Okada et al., 2013). These baroreflex values will be referred to as sympathetic BRS inc for the purpose of differentiating it from other methods of obtaining sympathetic BRS.
Sympathetic Baroreflex Sensitivity: Total MSNA Method
All sympathetic bursts obtained during the MSNA recording were normalized to the largest burst, with the largest being assigned a total integrated nerve activity value of 1000. The 0 nerve activity value was determined from the mean voltage during a >5 s period of neural silence between sympathetic bursts. Diastolic blood pressures for each cardiac cycle were assigned to 3 mmHg bins. Total MSNA was determined for each bin using the segregated signal averaging approach described by Halliwill (2000). In this approach, 2-s windows synchronized with the R wave were used to determine the MSNA for the cardiac cycles within each 3 mmHg diastolic pressure bin. MSNA windows were time shifted so as to account for the latency between R waves and sympathetic bursts and the time shift varied from individual to individual. The mean time shift was 1.33 ± 0.04 s. The total integrated nerve activity was determined using the area under the signal-averaged curve for each bin and expressed as arbitrary units (AU) per beat. Linear regression was performed by plotting total MSNA against diastolic BP with the acceptance level set at r > 0.5 FIGURE 2 | Laboratory data obtained from a 21 year old male. The baroreflex coordinates an increase in both MSNA burst incidence and heart rate in response to a transient decrease in blood pressure (A). Conversely, rising systolic and diastolic blood pressure causes a fall in heart rate and inhibition of MSNA bursts (B). MSNA burst incidence increases again in response to a drop in diastolic blood pressure (C).
Frontiers in Neuroscience | www.frontiersin.org (Rudas et al., 1999;Hart et al., 2011b;Okada et al., 2013). Data points were weighted according to the number of cardiac cycles per diastolic BP bin. These sympathetic BRS values will be referred to as "sympathetic BRS total " to differentiate these values from the sympathetic BRS values obtained via the burst incidence method.
Cardiac Baroreflex Sensitivity: Sequence Method
The sequence method was used to determine cardiac BRS. In this method, up and down sequences are identified. "Up sequences" consist of three or more consecutive cardiac cycles for which there is a rise in systolic BP coinciding with lengthening of the R-R interval. "Down sequences" consist of three or more cardiac cycles for which there is a fall in systolic BP coinciding with shortening of the R-R interval. The threshold for changes in systolic BP was set at 1 mmHg and the threshold for changes in R-R interval was set at 6 ms (Parati et al., 1988). Sequences containing changes smaller than these thresholds were not used in the assessment of cardiac BRS. To account for baroreflex delays, systolic BP values were matched to either the concurrent heartbeat for R-R intervals of 800 ms or a 1 beat delay for shorter heart periods, typically 500-800 ms (Eckberg and Eckberg, 1982). Cardiac BRS was quantified by plotting R-R interval against systolic BP for each sequence (r ≥ 0.8 acceptance level) and taking the average for up and down sequences combined (Monahan et al., 2001;Okada et al., 2013). Values of cardiac BRS were accepted when the number of sequences was ≥3 for both up and down sequences.
Statistical Analysis
In order to test the hypothesis that the morning surge in BP is negatively correlated to BRS, separate linear regression analyses were performed to determine the relationships between MSBP and BRS variables. The analyses were performed for systolic, diastolic and MAP components of MSBP, relating each to sympathetic BRS and cardiac BRS. Linear regression analyses were also performed to examine the relationships between BRS variables and secondary ambulatory blood pressure characteristics (mean 24 h BP, BP whilst awake, and BP whilst asleep). All statistical analyses were performed using Prism 6 (GraphPad, USA) and the alpha level was set at 0.05.
Participant Characteristics
Recordings of MSNA were successfully obtained in all 14 participants. The mean age of these participants was 23 ± 1 years and mean body mass index was 24.3 ± 0.8 kg/m 2 . Resting MSNA burst incidence and MSNA burst frequency were 44 ± 5 bursts/100 HB and 30 ± 3 bursts/min, respectively. Neither MSNA burst frequency nor MSNA burst incidence was related to the measures of the MSBP (P > 0.05).
Ambulatory Blood Pressure Characteristics and MSBP
At least two thirds of the blood pressure readings were successful for all participants, thus meeting the minimum requirements for ambulatory blood pressure recordings (O'Brien et al., 2005). None of the participants reported severely disrupted sleep. The mean number of blood pressure recordings over the 24 h period, during sleep and during wake were 61 ± 1 (range 51-67), 16 ± 1 (range 12-22), and 44 ± 1 (range 37-50), respectively. The average time of going to sleep was 23:43 h (range 22:22-00:44 h) and the average time of rising was 07:28 h (range 05:10-10:12 h). The mean hours of sleep was 8.2 ± 2.2 h. Mean values for systolic BP, diastolic BP, MAP, and heart rate over 24 h and during sleep and times of wakefulness are displayed in Table 1. All cardiovascular variables were significantly lower during sleep than wakefulness (p < 0.05). The mean MSBP values are displayed in Table 2. Linear regression analyses revealed no significant relationships between BRS (sympathetic BRS inc , sympathetic BRS total , cardiac BRS) and blood pressure averaged over the 24-h recording period, wakefulness or sleep (p > 0.05).
Sympathetic Baroreflex Sensitivity: Total MSNA Method
Thirteen participants exhibited significant sympathetic BRS total slopes (r > 0.5). The participant that did not was excluded from analyses involving sympathetic BRS total . The mean sympathetic BRS total was −1.60 ± 0.37 AU/beat/mmHg. The BRS total value for the same participant was identified as an outlier (−5.4 AU/beat/mmHg).
Relationship between Baroreflex Sensitivity and MSBP
Sympathetic BRS inc and "Pre-awakening" MSBP Linear regression analysis did not reveal a significant relationship between sympathetic BRS inc and pre-awakening systolic MSBP (r = 0.33, p = 0.25). However, a significant negative relationship was identified between sympathetic BRS inc and pre-awakening diastolic MSBP (r = 0.62, p = 0.02) and between sympathetic BRS inc and pre-awakening morning surge in MAP (r = 0.57, p = 0.03). Since sympathetic BRS values are negative, a positive r-value indicates that low sympathetic BRS is associated with a larger MSBP. With the outlier removed the findings were consistent albeit with steeper linear regression slopes. The relationship remained significant between sympathetic BRS inc and pre-awakening diastolic MSBP (r = 0.72, p = 0.006; Figure 3B) and between sympathetic BRS inc and pre-awakening morning surge in MAP (r = 0.74, p = 0.004; Figure 3C). There was still no significant relationship between sympathetic BRS inc and pre-awakening systolic MSBP (r = 0.38, p = 0.20; Figure 3A).
Sympathetic BRS total and "Pre-awakening" MSBP Linear regression did not reveal a significant relationship between sympathetic BRS total and pre-awakening systolic MSBP (r = 0.36, p = 0.23). There was a relationship between sympathetic BRS total and pre-awakening diastolic MSBP but this did not reach statistical significance (r = 0.52, p = 0.066). There was also a trend for a relationship between sympathetic BRS total and pre-awakening morning surge in MAP (r = 0.48, p = 0.095). This trend was significant with removal of the outlier noted above (r = 0.61, p = 0.03; Figure 4C). With the removal of the outlier the relationship between sympathetic BRS total and preawakening morning surge in DBP neared significance (r = 0.56, p = 0.059; Figure 4B). There was still no significant relationship between sympathetic BRS total and pre-awakening systolic MSBP (r = 0.43, p = 0.16; Figure 4A).
Sympathetic BRS total and "Sleep-Trough" MSBP
Linear regression analyses revealed no significant relationships between sympathetic BRS total and sleep-trough systolic MSBP (r 0.18). Removal of the outlier revealed a trend for relationship between sympathetic BRS total and sleep-trough morning systolic MSBP that did not reach significance (r = 0.52, p = 0.08). With the outlier removed, there were significant relationships between sympathetic BRS total and sleep-trough diastolic MSBP (r = 0.67, p = 0.02) and sleep-trough morning surge in MAP (r = 0.66, p = 0.02).
DISCUSSION
The aim of this study was to determine the relationship between BRS and the MSBP in healthy, young individuals. Our major finding is that the magnitude of the pre-awakening morning surge in diastolic and mean arterial pressure is greater in individuals who have a lower sympathetic but not cardiac BRS. This supports the hypothesis that the ability of the sympathetic baroreflex to buffer increases in blood pressure may play a role in determining the magnitude of the morning surge.
Sympathetic Baroreflex Sensitivity and the MSBP
In this study, the magnitudes of the pre-awakening morning surge in diastolic and mean arterial pressure were exaggerated in individuals with lower sympathetic BRS inc . By contrast, no significant relationships were found between pre-awakening systolic MSBP and sympathetic BRS. Similar trends were found between sleep-trough MSBP variables and sympathetic BRS but none reached significance, except with the removal of an outlier. It should be noted, however, that the outlier in the present data set (>1.5 interquartile ranges below the first quartile) still represents a typical sympathetic BRS value for a healthy young as reported by our research group and others (Hart et al., 2011b). The "sleep-trough" method is more likely to be influenced by dipping status, as it is centered around the lowest BP reading obtained during sleep. "Dipping status" is also a risk factor for cardiovascular and cerebrovascular disease (Kario et al., 2001;Yano and Kario, 2012), which may confound the prognostic significance of the MSBP when using the sleeptrough method. The pre-awakening MSBP is centered around the waking event and this could explain why pre-awakening MSBP was more intimately related to sympathetic BRS. However, Okada et al. (2013) examined the sleep-trough systolic MSBP and found that it is associated with sympathetic BRS inc, at least in elderly, hypertensive individuals. This relationship was not present in normotensive individuals, although the morning surge in diastolic and MAP were not examined.
In the current study, pre-awakening diastolic MSBP and morning surge in MAP were significantly correlated to sympathetic BRS, whilst systolic MSBP was not. The occurrence of bursts of MSNA is driven by physiological fluctuations in diastolic BP (Sundlof and Wallin, 1987) and, as a result, diastolic BP is more intimately related to sympathetic baroreflex function. Considering this, it seems logical that the diastolic MSBP, as opposed to the systolic MSBP, is correlated with sympathetic BRS. Previous research concerning the risks associated with the MSBP tends to have involved the use of systolic BP to quantify the morning surge (Xie et al., 2015). As a result, the elevated cardiovascular risk associated with the systolic MSBP has been well established. Although the prognostic significance of the diastolic MSBP is less studied, research suggests that an exaggerated diastolic MSBP may be associated with increased risk of cardiovascular events. In a large study by Li et al. (2010), involving 5645 participants, an increased hazard ratio was identified for all-cause mortality with both an exaggerated pre-awakening systolic and diastolic MSBP. An exaggerated pre-awakening diastolic MSBP (≥21.6 mmHg), was associated with a hazard ratio of 1.26 (CI 1.01-1.56) for all cause mortality and 1.40 (CI 1.00-1.96) for fatal and nonfatal cardiac events. For all cardiovascular and cerebrovascular events, a hazard ratio of 1.21 (CI 0.95-1.54) was determined, although this did not reach statistical significance. Nevertheless, these findings suggest that the pre-awakening diastolic MSBP may still have some predictive value. Further research should be conducted to confirm the prognostic significance of diastolic MSBP.
Cardiac BRS and the MSBP
We have previously identified a significant but modest relationship between cardiac and sympathetic BRS in young individuals . The findings suggested that cardiac BRS predicts only around 10% of the variability in sympathetic BRS and therefore one cannot reliably predict the other. In support of the current study, it also suggests that a relationship may exist between the morning surge and cardiac or sympathetic BRS but not necessarily both. In contrast to sympathetic BRS, which is determined using diastolic BP, cardiac BRS is quantified using systolic BP (Parati et al., 1988). Therefore, similar to the relationship between sympathetic BRS and diastolic MSBP, it may be speculated that cardiac BRS is associated with an exaggerated systolic MSBP. However, we did not find a relationship between cardiac BRS and either the systolic or the diastolic component of the MSBP. Cardiac BRS may be unrelated to the MSBP due to the fact that it represents a marker of reflex vagal modulation. Our findings support the studies by Okada et al. (2013) and Lambert et al. (2014) in which no significant relationship was reported between cardiac BRS and either the morning surge in systolic pressure or MAP, respectively. Our previous work indicates that when males and females are assessed separately the relationship between cardiac and sympathetic BRS is significant only for females. In light of this, the study of larger groups of males and females is warranted to determine whether the present findings are influenced by gender. Since the control of blood pressure via sympathetic vasoconstriction appears to be more dominant in males than females (Hart et al., 2011a(Hart et al., , 2014, it may be postulated that the sympathetic baroreflex has a greater influence on the morning surge in males than in females.
Methodological Considerations
A major issue with the MSBP is that there has been no consensus regarding the precise definition of the MSBP and as a consequence, researchers have utilised a number of different methods to quantify the morning surge (Stergiou et al., 2008;Atkinson et al., 2014). For the reasons described earlier, the preawakening definition may be considered a more realistic measure of the MSBP. However, some participants had morning blood pressure increases that spanned more than 2 h after rising and this is a limitation of the method used. Nonetheless, the risk associated with the pre-awakening morning surge has been more extensively studied (Xie et al., 2015) than definitions that include BP readings over a longer time period after rising, which is why these definitions were not considered in the present study. The BP Power is a promising method as it provides a comprehensive evaluation of an individual's MSBP. It has also been found that the rate of rise of the MSBP is greater in hypertensives (Head et al., 2006) and is an independent risk factor for stroke and myocardial infarction (Luo et al., 2013). Further study of this method in the context of sympathoexcitation and circadian variation may allow the mechanisms involved in the MSBP to be unraveled. Nevertheless, the fact that we report significant correlations between the MSBP and sympathetic BRS using the pre-awakening method obviate the use of the BP Power method in the current study.
To our knowledge, only two studies have previously examined the relationship between sympathetic BRS and the MSBP (Okada et al., 2013;Lambert et al., 2014) with both studies using the burst incidence method to quantify sympathetic BRS. The burst incidence method, otherwise known as the threshold technique, involves plotting MSNA burst incidence against diastolic BP and is associated with high success rates for significant baroreflex slopes (Kienbaum et al., 2001). It is important to recognize that the burst incidence method does not incorporate changes in MSNA burst amplitude. The present study is the first to examine the relationship between sympathetic BRS total and the MSBP, thus allowing the incidence and the amplitude of MSNA bursts to be taken into account. The total MSNA method (Halliwill, 2000) involves plotting total MSNA against diastolic blood pressure in order to quantify sympathetic BRS and incorporates both burst strength and burst incidence. Whilst we were unable to obtain a significant BRS total slope in one participant, the success rate (13/14, 93%) remained comparable with that of the burst incidence method (14/14, 100%). It is interesting to note that when an outlier was removed, sympathetic BRS inc and sympathetic BRS total were both correlated with the MSBP. This may be expected because the two methods are not mutually exclusive. However, it tends to suggest that the ability of the baroreflex to modulate the occurrence of bursts (burst incidence) may be the more influential factor in determining the magnitude of the MSBP, as the addition of burst strength into the analyses had little effect on the relationships between sympathetic BRS and MSBP variables. This supports the concept that changes in MSNA burst incidence are driven primarily by the baroreflex whereas the modulation of MSNA burst amplitude may involve the influence of other modulators, such as chemoreceptors (Malpas et al., 1996).
This study provides evidence to elucidate the influence of the sympathetic nervous system on the MSBP. Although the study of healthy, young individuals limits the ability to generalize the results to clinical populations, it is known that hypertension is associated with both exaggerated MSBP (Kario et al., 2003) and diminished cardiac BRS (Grassi et al., 1998). Further examination of sympathetic baroreflex function and its relation to the pre-awakening MSBP in this population is warranted. However, the current findings suggest there is potential for the use of appropriately timed antihypertensive therapy, specifically targeting the sympathetic nervous system, that may be useful in reducing the cardiovascular morbidity and mortality in individuals with an exaggerated MSBP.
Limitations
We note that the present study is limited by a relatively small sample size and it is possible that investigation of a larger population may have allowed further relationships to be identified. However, the study was sufficiently powered to detect a significant relationship between the diastolic and mean arterial components of the pre-awakening MSBP and sympathetic BRS. This is a novel finding in healthy, young populations. Although the baroreflex response to rising pressures may be considered most relevant to the MSBP, we did not perform separate BRS analyses with respect to rising and falling pressures. The sequence method lends itself to quantifying cardiac BRS in response to rising and falling pressures, but the assessment of sympathetic BRS does not involve the detection of sequences and therefore does not allow for such differentiation. Hart et al. (2011b) have developed a sequence technique for sympathetic BRS, but this comes with its own limitations as it involves sequences of only two consecutive cardiac cycles and does not provide significant slopes in all individuals. For these reasons separate analyses were not performed for rising and falling pressures for sympathetic BRS, and rising and falling cardiac BRS values were pooled for consistency. The technique of ambulatory blood pressure monitoring is also associated with some limitations. Given the differing schedules of participants during the day, the precise times of wake and sleep for each participant were unable to be controlled. However, individuals who took part in shift work or who had abberant sleep patterns were excluded from the study. It is possible that BRS changes under different conditions and throughout the day. Whilst all recordings were made at rest, our findings are based upon measurements taken at only one time point during the day. However, we have previously shown that sympathetic BRS is not significantly different between the morning and afternoon . The ambulatory and laboratory protocols were not performed on the same morning and it is acknowledged that this is a limitation and may have influenced the findings.
CONCLUSION
We have uncovered significant relationships between sympathetic BRS and both the diastolic and mean arterial components of the pre-awakening MSBP in healthy, young individuals; the higher the MSBP, the lower the sympathetic BRS. This finding suggests that the ability of the baroreflex to buffer increases in blood pressure may have an influence on the magnitude of the MSBP.
AUTHOR CONTRIBUTIONS
Experiments were performed in the School of Medicine (Western Sydney University). All authors were involved in the design of the experiments and/or data acquisition and analysis of the data, as well as the writing or editing of this manuscript. All authors approved the final version of the manuscript and agree to be accountable for all aspects of the work. | 2017-05-04T18:15:33.094Z | 2016-09-08T00:00:00.000 | {
"year": 2016,
"sha1": "f2d53cb6867de7b46da0a2a20a3db5661fe2c5c0",
"oa_license": "CCBY",
"oa_url": "https://www.frontiersin.org/articles/10.3389/fnins.2016.00412/pdf",
"oa_status": "GOLD",
"pdf_src": "PubMedCentral",
"pdf_hash": "f2d53cb6867de7b46da0a2a20a3db5661fe2c5c0",
"s2fieldsofstudy": [
"Medicine",
"Biology"
],
"extfieldsofstudy": [
"Medicine"
]
} |
247892390 | pes2o/s2orc | v3-fos-license | Siltuximab-Related Favorable Clinical Outcome for a Patient Suffering from Idiopathic Multicentric Castleman Disease
RationalCastleman disease is a rare lymphoproliferative disorder that can be subdivided into unicentric and multicentric forms, the latter of which causes a spectrum of serious medical conditions. Here, we present a case of idiopathic multicentric Castleman disease in the eighth decade of life. Patient Concerns. First hospitalized due to unexplained progressive anemia, the patient was readmitted to the hospital 18 months later with suspected lymphoma. Clinical examination revealed a progressive lymphadenopathy. Diagnoses. Histopathologic lymph node features, anemia, elevated CRP and IL6 levels, splenomegaly, and hypoalbuminemia indicated multicentric Castleman (MCD) disease. Interventions. The patient was treated intravenously with a dose of 11 mg/kg siltuximab every 3 weeks. Outcomes. Timely correct diagnosis through the stringent use of consensus diagnostic criteria and sufficient siltuximab therapy has considerably promoted favorable clinical outcomes in a patient suffering from MCD.
Introduction
Castleman disease (CD) is a rare lymphoproliferative disorder first reported by pathologist Benjamin Castleman in 1954 [1]. CD is subdivided into unicentric CD (UCD), involving just one enlarged lymph node, and multicentric CD (MCD), affecting multiple lymph nodes [2]. While UCD is considered a benign condition, MCD is characterized by a variety of clinical and laboratory abnormalities with a poor prognosis in otherwise untreated patients [3]. MCD can be caused by human herpesvirus-8 (HHV-8; HHV-8-associated MCD), with the majority of cases consisting of human immunodeficiency virus (HIV) infected or immunocompromised patients [4]. Little is known about the etiology of HHV-8-negative MCD, referred to as idiopathic MCD (iMCD). iMCD accounts for up to 50% of all MCD cases and can lead to cytokine stormmediated multiple organ system dysfunction, apart from other severe clinical manifestations [4]. e diagnosis of iMCD can be challenging because it is both a rare disorder and a mimicker of other clinical conditions such as malignant lymphoma, cancer, or autoimmune diseases [5]. Recently, Fajgenbaum et al. established evidence-based consensus diagnostic criteria for HHV-8negative/iMDC, which are subdivided in major, minor, and exclusion criteria [4]. Major criteria concise enlarged lymph nodes and histopathologic features consistent with the iMCD spectrum (i.e., regressed/atrophic/atretic/hyperplastic germinal centers (GC), follicular dendritic cell (FDC) prominence, and vascularity). Minor criteria consist of 6 laboratory and 5 clinical features (elevated ESR or CRP, anemia, thrombocytopenia/cytosis, renal dysfunction or proteinuria, polyclonal hypergammaglobulinemia, hypoalbuminemia and constitutional symptoms, large spleen and/or liver, fluid accumulation, eruptive cherry angiomata or violaceous papules, lymphocytic interstitial pneumonitis, respectively) of which at least 2 must match the patient's symptoms (with ≥1 laboratory criterion). iMCD can be further subclassified into iMCD-thrombocytopenia, ascites, reticulin fibrosis, renal dysfunction, organomegaly (iMCD-TAFRO), or iMCD, not otherwise specified (iMCD-NOS) [2]. Considering exclusion criteria, it is crucial to rule out malignant, infectious, and autoimmune conditions that can mimic iMCD [4].
While therapeutic strategies for UCD are more or less complete surgical removal, the multicentric form of the disease involves many more different approaches. Considering the guidance from van Rhee et al. [6], the consensus treatment algorithm for iMCD patients, regardless of clinical severity, starts with administering anti-IL6directed treatment [6]. Up to now, siltuximab, an anti-IL6 chimeric monoclonal antibody, is the only approved drug for the treatment of HHV-8-negative iMDC in Europe and the USA (US Food and Drug Administration, 2014; European Medicines Agency, 2016) [7,8]. For people who do not respond to siltuximab, a broad spectrum of other treatment options exist, like tocilizumab or rituximabbased therapeutic approaches, to name a few; however, it is currently difficult to clearly determine their effectiveness [3].
Report of a Case
We report on a 78-year-old female patient diagnosed with HIV-negative, HHV-8-negative iMCD in 2021, with a history of nicotine abuse and comorbidities such as hyperthyroidism, coronary sclerosis, and bilateral knee gonarthrosis, leading to right total knee replacement in November 2020. As permanent therapeutic intervention, the patient received yronajod (175 µg qd), pantroprazol (40 mg qd), Torasemid (5 mg qd), and Oxycodone (20 mg qd). During 2021, she received anti-SARS-CoV-2 vaccination (1st dose in April; 2nd dose in July).
As of November 2019, the patient was hospitalized due to progressive anemia (haemoglobin 10 g/dl, MCV 82 fl), persistent elevated levels of CRP (8-11 mg/dl), and weight loss (20 kg). Initially suspected as lymphoma, this diagnosis could not be clearly confirmed. CT scan of the thorax and abdomen revealed highly suspicious paraaortic and interaortocaval lymph nodes (largest paraaortic node: 3.3 × 2.6 × 1.1 cm). While subsequent examinations in March 2020 and March 2021 showed essentially steady lymph node progression (axillary lymph nodes from 6 to 8 mm; largest para-aortal node: 2.5 × 2.2 × .0 cm), these findings were considered not indicative for lymphoma. e general condition of the patient appeared to be mainly stable since November 2019; Eastern Cooperative Oncology Group (ECOG) performance status has been assessed of 1 to 2.
However, as of June 2021, the patient was referred to our hospital by her general practitioner still with lymphoma as an initial tentative diagnosis. As well, she suffered from rheumatism-like musculoskeletal pain, mainly in the upper extremities.
Biopsy of a left-sided suprainguinal lymph node revealed atrophic germinal centers, positivity for CD21 in follicular dendritic cells, and a partial loss of proliferating B cells with abundant surrounding T-cells ( Figure 1). Plasma cells were increased and appeared widely IgG-positive, exhibiting a polyclonal pattern for kappa and lambda light chains without enhanced expression of IgG4.
Both progressive lymphadenopathy in combination with histopathologic lymph node features are consistent with the iMCD spectrum (plasma cell subtype) and are referred to as major criteria.
Since anemia, elevated CRP and IL6 levels, splenomegaly, and hypoalbuminemia meet 5 out of 11 minor consensus criteria, iMCD in the patient could be confirmed. e lack of thrombocytopenia, ascites, reticulin fibrosis, and renal dysfunction could clearly rule out iMCD-TAFRO, therefore we claimed the criteria are met for the existence of a less aggressive subtype iMCD-NOS.
Considering the consensus treatment algorithm for iMCD-NOS, the patient started therapy with siltuximab in August 2021 at a dose of 11 mg/kg intravenously every 3 weeks [6].
After 5 cycles of siltuximab, she presented in a general good condition with just a slight increase of lower limb edema. e therapy was well tolerated and no serious adverse side events could have been observed. Moreover, the patient experienced a remarkable decrease of rheumatismlike musculoskeletal pain. Haemoglobin as well as CRP and ferritin levels normalized (12.5 g/dl, normal 11.5-15.4 g/dl; 0.06 mg/dl, normal <0.50 mg/dl; 120.0 ng/ml, normal 13.0-150.0 ng/ml). TSH values decreased to 0.186 mU/l (normal <0.270-4.200 mU/l) while and FT4 values increased to 2.13 ng/dl (normal 0.90-1.70 ng/dl), both indicating hyperthyroidism which is an already known comorbidity in the patient.
CT scan of the thorax and abdomen revealed no essential changes in size of the spleen as well as of para-aortal and interaortocaval lymph nodes.
Discussion
More than half a century after Benjamin Castleman's initial description of a case, later termed as CD, a variety of lymphoproliferative disorders constitute the spectrum of CD. However, CD is a very rare and potentially serious lymphoproliferative disorder. International efforts have been made to define diagnostic criteria allowing to differentiate between particular forms of CD and to rule out mimickers of CD [4]. 2 Case Reports in Hematology e patient who attended our hospital was in her eighth decade of life and had a history of nicotine abuse and comorbidities. Her clinical presentation with features like progressive anemia and persistent elevated levels of CRP did not automatically trigger the suspicion of CD since CD is a rare condition with an estimated 10-year period prevalence of 2.5 cases per million in North America, which may also be applicable to Germany [9]. After ruling out lymphoma as an initial tentative diagnosis, we examined the possibility for CD. Because of the wide range of potential clinical outcomes, a correct diagnosis of not only CD but also the subtype is essential for treatment strategy [10]. Further, thorough differential diagnostic examination following the scheme of Fajgenbaum et al. [4] could reveal the MCD-subtype HHV-8-negative iMCD-NOS in our patient and rule out lymphoma as the initial tentative diagnosis.
Anti-IL6-directed therapy is recommended for the treatment of HHV-8-negative iMDC [7,8]. Since siltuximab is an anti-IL6 chimeric monoclonal antibody, we decided to treat the patient with this therapeutic agent following established guidelines [6]. erapeutic outcome after 5 cycles of siltuximab administration (3 weeks each cycle) can be considered very promising. e patient reported toleration of the treatment and decrease of rheumatism-like musculoskeletal pain. IL6 plays a role in rheumatoid arthritis and it is a common observation that inhibiting IL6 ameliorates the symptoms of both rheumatoid arthritis and iMCD [11,12]. erefore, a positive effect on rheumatism-like musculoskeletal pain of our patient was not unexpected.
Blood values improved generally, in particular haemoglobin as well as CRP and ferritin levels normalized. CT scan of the thorax and abdomen exhibited stable disease. However, a slight increase of lower limb edema could have been observed as well as a slight deterioration of indicators for hyperthyroidism. Overall, siltuximab appears to be a safe and effective treatment of HHV-8-negative iMDC in our patient.
Conclusions
Improving iMCD patient outcomes is essentially dependent on timely correct diagnosis and sufficient therapy of the disease. e consistent use of international, evidence-based consensus diagnostic criteria described by Fajgenbaum et al. [4] as well as consensus treatment strategies [3] helped us rule out lymphoma as an initial tentative diagnosis and to minimize unfavourable outcomes for an iMCD-NOS patient.
e application of anti-IL6-directed therapy with siltuximab appeared as a safe and effective treatment strategy. | 2022-04-03T16:33:12.357Z | 2022-03-30T00:00:00.000 | {
"year": 2022,
"sha1": "f622501837ff14aa8d6d567277c858860f448212",
"oa_license": "CCBY",
"oa_url": "https://downloads.hindawi.com/journals/crihem/2022/1840589.pdf",
"oa_status": "GOLD",
"pdf_src": "PubMedCentral",
"pdf_hash": "6e2a6e748212182771a9c356879826e1772ea5a3",
"s2fieldsofstudy": [
"Medicine"
],
"extfieldsofstudy": [
"Medicine"
]
} |
46927368 | pes2o/s2orc | v3-fos-license | The educational impact of Mini-Clinical Evaluation Exercise (Mini-CEX) and Direct Observation of Procedural Skills (DOPS) and its association with implementation: A systematic review and meta-analysis
Introduction Mini Clinical Evaluation Exercise (Mini-CEX) and Direct Observation of Procedural Skills (DOPS) are used as formative assessments worldwide. Since an up-to-date comprehensive synthesis of the educational impact of Mini-CEX and DOPS is lacking, we performed a systematic review. Moreover, as the educational impact might be influenced by characteristics of the setting in which Mini-CEX and DOPS take place or their implementation status, we additionally investigated these potential influences. Methods We searched Scopus, Web of Science, and Ovid, including All Ovid Journals, Embase, ERIC, Ovid MEDLINE(R), and PsycINFO, for original research articles investigating the educational impact of Mini-CEX and DOPS on undergraduate and postgraduate trainees from all health professions, published in English or German from 1995 to 2016. Educational impact was operationalized and classified using Barr’s adaptation of Kirkpatrick’s four-level model. Where applicable, outcomes were pooled in meta-analyses, separately for Mini-CEX and DOPS. To examine potential influences, we used Fisher’s exact test for count data. Results We identified 26 articles demonstrating heterogeneous effects of Mini-CEX and DOPS on learners’ reactions (Kirkpatrick Level 1) and positive effects of Mini-CEX and DOPS on trainees’ performance (Kirkpatrick Level 2b; Mini-CEX: standardized mean difference (SMD) = 0.26, p = 0.014; DOPS: SMD = 3.33, p<0.001). No studies were found on higher Kirkpatrick levels. Regarding potential influences, we found two implementation characteristics, “quality” and “participant responsiveness”, to be associated with the educational impact. Conclusions Despite the limited evidence, the meta-analyses demonstrated positive effects of Mini-CEX and DOPS on trainee performance. Additionally, we revealed implementation characteristics to be associated with the educational impact. Hence, we assume that considering implementation characteristics could increase the educational impact of Mini-CEX and DOPS.
used in other studies evaluating the educational impact of Mini-CEX/DOPS [14]. By applying Barr's adaptation of Kirkpatrick's four-level model, the educational impact can be analyzed on different levels: Level 1 describes learners' reactions, Level 2a includes modification of attitudes/perceptions, Level 2b addresses the acquisition of knowledge/skills, Level 3 analyzes change in behavior, Level 4a is concerned with change in organizational practice, and Level 4b comprises benefits to patients/clients [15]. Whereas for multisource feedback, Miller and Archer [14] found evidence demonstrating positive effects on trainees' performance (Level 2b), this was not the case for Mini-CEX and DOPS. With regard to Mini-CEX and DOPS, all articles investigated postgraduate medical trainees' perceptions of the tools (Level 1) rather than outcomes such as change in behavior or benefits for patients. As the available evidence on the educational impact of Mini-CEX and DOPS was scarce in 2010, and was limited to postgraduate medical trainees and outcomes on Kirkpatrick Level 1 [14], it would be interesting to ascertain whether more evidence has been gathered in the interim, especially on higher Kirkpatrick levels, including evidence on undergraduate medical trainees and trainees in other health professions.
Mini-CEX and DOPS should be used in the workplace during daily clinical work. However, it has been shown that the assessments are perceived as additional workload [16]. Hence, implementation of Mini-CEX and DOPS is challenging, which could affect their educational impact. In another context, Durlak and DuPre [17] demonstrated that the extent to which a program is implemented successfully significantly influences the outcomes of that program. Within implementation, Durlak and DuPre [17] distinguish eight characteristics: 1) fidelity (the extent to which the innovation corresponds to the originally intended program), 2) dosage (how much of the original program has been delivered), 3) quality (how well different program components have been conducted), 4) participant responsiveness (the degree to which the program stimulates the interest or holds the attention of participants), 5) program differentiation or program uniqueness (the extent to which a program's theory and practices can be distinguished from other programs), 6) monitoring of control (which involves describing the nature and amount of services received by members of the control groups), 7) program reach (the rate of involvement and representativeness of program participants) and 8) adaptation (which refers to changes made in the original program during implementation). As all these characteristics might be relevant for the educational impact of Mini-CEX and DOPS, it would be interesting to consider characteristics of the setting and implementation status as potential influences.
Thus, we aim to answer the following research questions: 1. "What evidence is there that Mini-CEX and DOPS have an educational impact on undergraduate and postgraduate trainees in the health professions?" 2. "Are characteristics of the setting in which Mini-CEX and DOPS take place, or the implementation status of Mini-CEX/DOPS associated with the educational impact of the tools?" Demonstrating the educational impact of Mini-CEX and DOPS in a systematic review would be helpful for educators in terms of justifying the use of the tools in health professions training. Knowing which characteristics of the setting and of the implementation status influence the educational impact of Mini-CEX and DOPS would advance our understanding of when and how to use Mini-CEX and DOPS in order to increase their educational benefits.
Methods
To guide our work, we used the recommendations of Cook and West [18] and followed their seven key steps, from defining a focused question to analyzing and synthesizing the results.
For the first step, we defined the focused research questions as stated at the end of the introduction using the PICO statement. As population, we defined undergraduate and postgraduate trainees in the health professions; as intervention, we defined Mini-CEX and DOPS; as outcome, we defined educational impact operationalized using Barr's adaptation of Kirkpatrick's four-level model. We did not define any comparison. To answer the first research question and to summarize all of the existing literature, we chose a systematic review as the most suitable strategy (step II). To answer our second research question, we extracted the relevant data from the studies included and analyzed whether there are associations of the educational impact with characteristics of the setting and with characteristics of the implementation status. The study protocol (http://dx.doi.org/10.17504/protocols.io.nmpdc5n) and data (https://doi. org/10.6084/m9.figshare.6275288.v1) are available online.The authors did not receive any specific funding for this review. A PRISMA checklist was completed and adhered to (S1 Table).
Literature search (data sources and search terms)
In the third step, we assembled a team including a librarian. In order to identify relevant articles as comprehensively as possible, the search strategy was developed and discussed in close collaboration with our librarian. Together, we decided to use the following electronic databases: Scopus, Web of Science (all databases), and Ovid, including All Ovid Journals, Embase, ERIC, Ovid MEDLINE(R), and PsycINFO (step IV). Search terms were "mini-CEX" OR "mini clinical evaluation exercise" OR "direct observation of procedural skills" OR "work-based assessment" OR "workplace-based assessment" OR "supervised learning event" OR "supervised learning events". We analyzed all articles published between the first description of Mini-CEX in 1995/DOPS in 2003 and December 2016 (date of access: 02.12.2016). Hits from the different databases were exported to an EndNote library and duplicates were removed.
Article selection
To identify articles relevant to our research question, we defined the following selection criteria (step V): 1. Article type: Original research articles were included; other article types such as reviews, conference abstracts, letters or editorials were excluded. Two authors independently scanned the articles for eligibility. Differences in article selection were discussed until consensus was reached. Articles were selected in two rounds: First, titles and abstracts were scanned, and second, remaining articles were selected based on full texts. In the first round, 63 out of 1478 articles were not in consensus and had to be discussed based on the full text. Based on the full text, inclusion/exclusion was clear and all articles were classified unanimously. To visualize the review process, we used the four-phase flow diagram as recommended in the PRISMA statement [19].
Data extraction
From the articles included in our review, the authors extracted the following key information (step VI): setting (institution, Mini-CEX/DOPS, purpose of Mini-CEX/DOPS, feedback recipient, mandatory Mini-CEX/DOPS, number of Mini-CEX/DOPS, assessment sheet, feedback provider, similar tools), study design (aim of study, method, intervention, control, sample), implementation status (fidelity, dosage, quality, participant responsiveness, program differentiation, monitoring of control, program reach, adaptation) [17], and reported outcome.
Data analysis
As a final step, the authors analyzed the data regarding the quality of the studies, educational impact, and the potential influences on the educational impact (step VII). Effects of Mini-CEX and DOPS were analyzed separately. Articles describing the educational impact of both Mini-CEX and DOPS were separated into individual studies.
Study quality. The methodological quality of the studies was assessed using the MERSQI (Medical Education Research Study Quality Instrument) [20]. The MERSQI consists of ten items and takes into account study design (single-group cross-sectional or single-group posttest only: 1 point; single-group pretest and posttest: 1.5 points; nonrandomized, two groups: 2 points; randomized controlled trial: 3 points), sampling (Institutions: one institution: 0.5 points; two institutions: 1 point; three or more institutions: 1.5 points. Response rate: < 50% or not reported: 0.5 points; 50%-74%: 1 point; ! 75%: 1.5 points), type of data (assessment by study participant: 1 point; objective: 3 points), validity evidence for evaluation instrument scores (content validity: 1 point; validity evidence on internal structure: 1 point; relationships to other variables: 1 point), data analysis (Sophistication: descriptive analysis only: 1 point; beyond descriptive analysis: 2 points. Appropriateness: data analysis appropriate for study design and type of data: 1 point), and outcomes (satisfaction, attitudes, perceptions, opinions, general facts: 1 point; knowledge, skills: 1.5 points; behaviors: 2 points; patient/health care outcome: 3 points). The MERSQI demonstrated high interrater reliability (0.72-0.98) and a strong association with the 3-year citation rate and journal impact factors [20]. The minimum MERSQI score is 5 and the maximum is 18.
Educational impact. Reported outcomes were classified according to Barr's adaptation of Kirkpatrick's four-level model [15]. The model consists of level 1 (learner's reaction), level 2a (modification of attitudes/perceptions), level 2b (acquisition of knowledge/skills), level 3 (change in behavior), level 4a (change in organizational practice), and level 4b (benefits to patients/clients) [15]. We used this framework in a strict manner and classified self-assessed improvements in performance into Kirkpatrick level 1 (learner's reaction).
For meta-analysis, effect sizes were calculated using Hedges' g (standardized mean differences) for each comparison [21]. If information reported in the articles was insufficient, we requested means and standard deviations from the corresponding authors via e-mail or ResearchGate. To quantify heterogeneity across studies, we used the I 2 -and tau 2 -statistics [22]. A random effects model was used to pool weighted effect sizes.
Potential influences. As potential influences on the educational impact of Mini-CEX/ DOPS, we considered characteristics of the setting and of the implementation status [17]. Within the setting, we considered the purpose for which Mini-CEX/DOPS was used, whether the use of Mini-CEX/DOPS was mandatory, and what the assessment sheet looked like.
Within the implementation status, we considered fidelity, dosage, quality, participant responsiveness, program differentiation, monitoring of control, program reach, and adaptation. To perform the analyses, implementation characteristics were translated to the context of Mini-CEX and DOPS and manifestations were defined (high, medium, low; or yes or no). Since there were no pre-existing definitions of manifestations of implementation characteristics, we had to define these ourselves. Thus, the definitions were partly influenced by the data reported in the studies, e.g. to decide on the level of program differentiation (the extent to which Mini-CEX/DOPS can be distinguished from other programs such as in-training evaluation reports, case based discussion, or multisource feedback), we first examined which other programs were described in the articles. Based on this information, we discussed their similarity with Mini-CEX/DOPS and defined three manifestations of program differentiation, as described in S2 Table. We discussed this operationalization until the whole research team agreed on its appropriateness. According to these categories, two authors classified the single characteristics' manifestations for each study.
To analyze whether there is an association of the educational impact with characteristics of the setting and with characteristics of the implementation status of Mini-CEX/DOPS, we used Fisher's exact test for count data [23]. For statistical analyses, we used R [24] and the R package "meta" [25]. Statistical significance was defined by a 2-sided α < 0.05.
We assume a negligible risk of bias across and within studies. One major bias across studies is the publication bias favoring the publication of studies demonstrating positive results. With regard to Mini-CEX and DOPS, however, a relevant proportion of studies show negative results [26][27][28][29]. We thus assume negligible publication bias in this review. With regard to the study design, there might be bias within studies. For example, some studies were sequential cohort studies, with limited randomization. In the meta-analyses, however only studies with compatible study designs and compatible bias within the studies were pooled. Thus, we did not further adjust for potential bias within studies.
Search results
Our search identified 2397 records. After duplicates were removed, 1478 records remained. By screening titles and abstracts, 1367 records were removed. The remaining 111 records were assessed based on their full text. Of these, 85 articles were excluded, and 26 articles met all eligibility criteria and were included in our review (Fig 1).
Two studies reported high educational impact (high satisfaction with DOPS; DOPS was perceived as useful), three studies reported medium educational impact (moderate satisfaction with DOPS; DOPS was perceived as rather useful or not useful), and two studies reported low educational impact (DOPS was not perceived as useful). For detailed information on the studies, please refer to S3 Table. Outcomes on Kirkpatrick level 2a (modification of attitudes and perceptions). We found no studies reporting an educational impact of Mini-CEX or DOPS on Kirkpatrick level 2a.
Outcomes on Kirkpatrick level 2b (acquisition of knowledge and skills). Two articles, encompassing three studies, reported effects of Mini-CEX on Kirkpatrick level 2b [33,38]. These studies aimed to investigate the effect of Mini-CEX on trainees' performance. Study designs were sequential cohort studies. The study of Kim, Willett [33] compared mandatory formative Mini-CEX to no or voluntary Mini-CEX, measuring undergraduate medical trainees' performance in a summative end-of-year objective structured clinical examination. The other two studies by Suhoyo, Schönrock-Adema [38] compared mandatory formative and summative Mini-CEX to the existing assessment program and measured undergraduate medical trainees' performance in an objective structured long examination record.
Educational impact was high in two studies and medium in one study. Effect sizes ranged from 0.10 to 0.48 and were pooled in a meta-analysis, which revealed a significant standardized mean difference (SMD) of 0.26 (95% CI, 0.05-0.47; p = 0.014) (Fig 2). Effect sizes of the single studies were not heterogeneous (I 2 = 60%, tau 2 = 0.0206, p = 0.08).
Two articles reported on the educational impact of DOPS on Kirkpatrick level 2b [9,44]. These studies were both randomized controlled trials. Hengameh, Afsaneh [44] evaluated the effect of formative DOPS on undergraduate nursing trainees' performance and compared it to the routine method, which included a subjective judgment of trainees' general skills. As outcome measures, they used validated and reliable assessment checklists for two procedures: intravenous catheterization and changing dressing. Roghieh, Fateme [9] investigated the effect of mandatory formative DOPS on undergraduate nursing trainees' performance. In their study, DOPS was performed in addition to the routine assessment program to which it was compared. Outcomes were measured using two checklists, one for trainees' skill levels in arterial blood sampling and one for trainees' skill levels in endotracheal suctioning.
Educational impact was high in both studies. As both studies reported two outcome measurements each, two effect sizes were calculated for each study. Effect sizes ranged from 2.53 to 6.02. Pooling effect sizes in a meta-analysis revealed an SMD of 3.33 (95% CI, 2.31-4.35; p<0.001) (Fig 2). Effect sizes of the single studies were significantly heterogeneous (I 2 = 82%, tau 2 = 0.8528, p<0.01).
DOPS. Analyzing the studies that investigated DOPS, no significant links between the setting or the implementation status and the educational impact were found. There was no significant link between the educational impact and the mandatory use of DOPS (n = 6, p = 1), participant responsiveness (n = 6, p = 0.2), or program differentiation (n = 7, p = 0.114). With regard to the purpose of DOPS, the assessment sheet, fidelity, dosage I and II, quality, monitoring of control, program reach, and adaptation, the reported data were insufficient to calculate Fisher's exact test for count data.
Discussion
In this systematic review, we summarized the available evidence on the educational impact of Mini-CEX and DOPS from 1995 to 2016 and analyzed associations of the educational impact with characteristics of the setting and characteristics of the implementation status. We identified 26 articles, 17 on Mini-CEX, six on DOPS, and three on both tools. The majority of the articles investigated effects of Mini-CEX/DOPS on learners' reactions (Kirkpatrick level 1), showing mixed results. Four articles reported effects on trainee performance (Kirkpatrick level 2b) and were synthesized into two meta-analyses: one on DOPS and one on Mini-CEX. Both meta-analyses revealed a positive effect of Mini-CEX or DOPS on trainee performance. We found two characteristics of implementation status to be associated with the educational impact: quality and participants' responsiveness.
In the meta-analyses the effect of DOPS was about ten times higher than that of Mini-CEX [9,33,38,44]. This might be due to the different study designs: Whereas the studies on DOPS focused on specific procedures and aligned intervention and outcome measurement, the studies on Mini-CEX used a more general approach and did not match intervention and outcome measurement as closely. In the studies on DOPS, trainees received feedback on their performance in predefined procedures, and afterwards, performance in exactly these procedures was assessed and compared to the control. In the studies on Mini-CEX, the tool could be used for any situation within trainees' internships, and the outcome measurements were more general, using the regular end-of-term assessments. Hence, in the studies on Mini-CEX, a large proportion of the effect could have remained invisible.
Since the educational impact in the meta-analysis on DOPS as well as the reported outcomes on Kirkpatrick level 1 on Mini-CEX and DOPS varied, we examined whether the setting in which the tools take place and their implementation status affect their educational impact. In fact, we found two associations between the implementation status of Mini-CEX and its educational impact: quality and participant responsiveness. The correlation between the quality of implementation and the educational impact indicates that low quality is associated with low educational impact and high quality is associated with high educational impact. In other words, the proper conducting of Mini-CEX, including a realistic reflection of trainee performance during direct observation, followed by a constructive feedback conversation, seems to be a prerequisite for its educational impact [49][50][51]. This might sound logical, but indicates the importance of implementation [17,52]. The positive association between participant responsiveness and the educational impact of Mini-CEX most likely stems from their strongly overlapping definitions. Since no study on Kirkpatrick level 2b commented on participant responsiveness, only studies on Kirkpatrick level 1 were entered into the analysis. Thus, the definitions of the educational impact on Kirkpatrick level 1 and participant responsiveness are very similar. Both are concerned with satisfaction and perceived helpfulness of the tools. The only difference is that the educational impact focuses on trainees' perspectives, and participant responsiveness considers the perspectives both of trainees and supervisors. Therefore, the question of whether the positive association between participant responsiveness and educational impact also holds true for higher Kirkpatrick levels remains open and should be considered in future studies. Besides the two positive associations with participant responsiveness and quality, no other characteristics of the setting and the implementation status showed significant associations with the educational impact of Mini-CEX. With regard to DOPS, no significant associations were found at all. This might be due to the limited data base, as on average, only three out of eight characteristics of the implementation status were reported.
As hypothesized, our study demonstrates that proper implementation is positively associated with the educational impact of Mini-CEX. This finding has several implications for practitioners and researchers in health professions education. For practitioners, our results suggest that it is essential to assure proper implementation and integration of the assessments into daily routine in order for them to have an educational impact. For researchers, it is crucial to take implementation characteristics into account when evaluating the educational impact of Mini-CEX and DOPS. For example, a negative result, such as no effect of Mini-CEX/DOPS on trainees' performance, might be explained by improper implementation of the tools. Addressing implementation characteristics, such as fidelity, dosage, quality, participant responsiveness, program differentiation, monitoring of control, program reach, and adaptation, when evaluating the tools can also help in improving the educational impact in the future. To facilitate further research on factors influencing the educational impact, rigorous reporting is necessary. Above and beyond the usual reporting standards [53,54], the implementation framework of Durlak and DuPre [17], as applied in this systematic review, could be useful to improve the reporting of educational interventions and to guide implementation processes. Since the available evidence was limited to Kirkpatrick levels 1 and 2b, further research should also consider effects of Mini-CEX and DOPS on higher Kirkpatrick levels, such as changes in behavior (Kirkpatrick Level 3), organizational practice (Kirkpatrick Level 4a), or benefits to patients (Kirkpatrick Level 4b). The evidence on Kirkpatrick level 2b was limited to undergraduate trainees. Therefore, it would be interesting to examine whether the positive effects of Mini-CEX and DOPS also hold true regarding postgraduate trainees.
Strengths
To our knowledge, we are the first to report meta-analyses on the educational impact of Mini-CEX and DOPS. By applying a sensitive and broad search strategy, including undergraduate and postgraduate medical trainees as well as trainees from health professions other than human medicine, we assume that we detected all relevant articles on the educational impact of Mini-CEX and DOPS. To analyze potential influences on the educational impact of Mini-CEX and DOPS, we applied a framework of implementation characteristics according to Durlak and DuPre [17].
Limitations
Despite our comprehensive search strategy, we found only four articles which reported effects of Mini-CEX/DOPS on Kirkpatrick level 2. Whether the positive effects on undergraduate trainees also hold true in postgraduate settings needs to be investigated. Although we analyzed eleven potential influences on the educational impact of Mini-CEX and DOPS, we found only two to be significantly associated with the educational impact of Mini-CEX. Due to the limited data basis, we might have underestimated the number of associations of the educational impact of Mini-CEX and DOPS with characteristics of the setting and of the implementation status.
Conclusions
Despite the limited evidence regarding the educational impact of Mini-CEX and DOPS as well as the limited reports on implementation status, we demonstrated positive effects of Mini-CEX and DOPS on trainee performance in meta-analyses and identified two implementation characteristics to be associated with the educational impact of Mini-CEX. The positive results of the meta-analyses might help educators to justify and strengthen the use of these tools in health professions training. The finding that two characteristics of the implementation status are associated with the educational impact puts further emphasis on the proper implementation of Mini-CEX and DOPS in order to increase their educational impact.
Supporting information S1 Table. | 2018-06-21T13:03:54.504Z | 2018-06-04T00:00:00.000 | {
"year": 2018,
"sha1": "61d566c26a65dc3c3d2bc9de553e4c2a8d6fcf86",
"oa_license": "CCBY",
"oa_url": "https://doi.org/10.1371/journal.pone.0198009",
"oa_status": "GOLD",
"pdf_src": "Adhoc",
"pdf_hash": "2a0133af6b837d25ae94e01134a2ce4e94dd0afe",
"s2fieldsofstudy": [
"Psychology"
],
"extfieldsofstudy": [
"Medicine",
"Psychology"
]
} |
14344029 | pes2o/s2orc | v3-fos-license | HIV-1 Reverse Transcriptase Still Remains a New Drug Target: Structure, Function, Classical Inhibitors, and New Inhibitors with Innovative Mechanisms of Actions
During the retrotranscription process, characteristic of all retroviruses, the viral ssRNA genome is converted into integration-competent dsDNA. This process is accomplished by the virus-coded reverse transcriptase (RT) protein, which is a primary target in the current treatments for HIV-1 infection. In particular, in the approved therapeutic regimens two classes of drugs target RT, namely, nucleoside RT inhibitors (NRTIs) and nonnucleoside RT inhibitors (NNRTIs). Both classes inhibit the RT-associated polymerase activity: the NRTIs compete with the natural dNTP substrate and act as chain terminators, while the NNRTIs bind to an allosteric pocket and inhibit polymerization noncompetitively. In addition to these two classes, other RT inhibitors (RTIs) that target RT by distinct mechanisms have been identified and are currently under development. These include translocation-defective RTIs, delayed chain terminators RTIs, lethal mutagenesis RTIs, dinucleotide tetraphosphates, nucleotide-competing RTIs, pyrophosphate analogs, RT-associated RNase H function inhibitors, and dual activities inhibitors. This paper describes the HIV-1 RT function and molecular structure, illustrates the currently approved RTIs, and focuses on the mechanisms of action of the newer classes of RTIs.
Introduction
Since the human immunodeficiency virus (HIV) has been established to be the etiological agent of the acquired immunodeficiency syndrome (AIDS) [1,2], an originally unpredicted number of drugs have been approved for the treatment of the HIV-infected patients [3]. This success in effective drugs identification, certainly unique in the treatment of viral infections, together with the use of such armamentarium in different combination therapeutic regimens, has transformed a highly lethal syndrome into a chronic disease [4]. The management of this disease, however, is still complex and worrisome due to problems such as monitoring of therapy efficacy, chronic administration drug toxicity, poor tolerability, drug resistance development, or therapy adjustment after treatment failures [4]. For all these reasons, the search for new inhibitors, possibly acting with molecular mechanisms different from the ones of the already approved drugs or anyway showing different patterns of drug resistance and, possibly, with diverse drug-associated chronic toxicity, is still a worldwide health care issue.
The success in HIV infection therapy is certainly related to the fact that the HIV life cycle has been intensely dissected; several of its steps have been validated as drug targets, and, subsequently, a number of viral inhibitors have been identified and developed against many of them [3,4]. Among the HIV proteins which have been deeply characterized as major drug targets is the reverse transcriptase (RT), the virus coded enzyme that converts the ssRNA viral genome into the dsDNA provirus which is consequently imported into the cell host nucleus and integrated into the host chromosome by another virus-coded protein, integrase (IN). The present paper focuses on the RT function within the virus cycle, its molecular structure, the mechanism of action of the Step 4: DNA synthesis proceeds, and the RNase H function cleaves the RNA strand of the RNA:DNA at numerous points leaving intact two specific sequences (cPPT, 3 PPT) resistant to the RNase H cleavage.
Step 6: RNase H hydrolysis of the PPT segments and the junction of the tRNA:DNA hybrid, freeing the PBS sequence of the (+)strand DNA.
Step 8: strand transfer of the PBS sequence of the (+)strand DNA that anneals to the PBS on the (−)strand DNA. DNA synthesis then continues with strand displacement synthesis.
Step 9: the product is a linear dsDNA with long terminal repeats (LTRs) at both ends. currently approved RT inhibitors (RTIs), and the newer classes of RTIs and their modes of action.
Retrotranscription Process
After the HIV particle fuses with the host cell surface, the viral particle content is released within the host cell cytoplasm where the viral ssRNA genome serves as template to obtain a proviral dsDNA that is integrated into the host genome, becoming a source of mRNAs coding for viral proteins and ssRNA genomes that, together, will form the new viral particles. The conversion of the viral ssRNA genome into integration-competent dsDNA, termed retrotranscription (Figure 1), is characteristic of all retroviruses, and its accomplishment requires viral as well as cellular elements, among which the most important is the virus-coded RT protein.
Each HIV particle contains two copies of (+)ssRNA genome sequence of 9,7 kb [5] coding for structural and nonstructural proteins and having, in the 5 -and 3 -ends, two identical sequences. Near the 5 -end of the viral genome there is an 18-nucleotides-long segment, termed primer binding site (PBS), which is complementary to the 3end 18 nucleotides of the human tRNALys3. When the cellular tRNA is hybridized to the PBS, it serves as an RNA primer, and the RT-associated DNA polymerase function can initiate the first (−)strand DNA synthesis using the viral RNA genome as a template (Figure 1). After tRNA elongation until the ssRNA 5 -end, there is a first (−)strand strong-stop DNA. In fact, the (−)strand DNA synthesis generates an RNA:DNA hybrid that is a substrate for the RT-associated ribonuclease H (RNase H) function which selectively degrades the RNA strand of the RNA:DNA hybrid [6], leaving the nascent (−)strand DNA free to hybridize with the complementary sequence at the 3 -end of one of the two viral genomic ssRNAs. A strand transfer, therefore, occurs from the R region at the 5 -end of the genome to the equivalent R region at the 3 -end (see Figure 1). After this step, termed (−)strand transfer, (−)strand synthesis can continue along the viral RNA starting from its 3 -end. Whilst DNA synthesis proceeds, the RNase H function cleaves the RNA strand of the RNA:DNA at numerous points. Although most of the RNase H cleavages do not appear to be sequence specific, there are two specific purine-rich sequences, known as the polypurine tracts (PPTs), that are resistant to the RNase H cleavage and remain annealed with the nascent (−)strand DNA. These two well-defined sites are located in the central part of the HIV-1 genome. In particular, the 3end PPT defines the 5 -end of the viral coding (+)strand DNA synthesis since this PPT serves as primer [7,8]. The (+)strand DNA synthesis continues to the 5 -end of the (−)strand DNA and uses also the 18-nucleotides PBS sequence of the tRNA as a template. Importantly, the 19th base from the 3 -end of tRNALys3 is a methyl A, and the presence of this modified base blocks the RT, generating a (+)strand strong-stop DNA. Subsequently, the RNase H function cleaves the RNA segment of the tRNA:DNA hybrid, freeing the PBS sequence of the (+)strand DNA and allowing it to anneal to the complementary site near the 3 -end of the extended (−)strand DNA [9]. Then, a bidirectional synthesis occurs to complete a viral dsDNA that has a 90-nucleotides single-stranded flap at the center. This unusual situation is probably solved by host mechanisms, and one candidate for flap removal is the flap endonuclease-1 (FEN-1) [8]. Finally, a specific cleavage removes the PPT primers and exposes the integration sequence to facilitate the insertion of the viral dsDNA into the host chromosome.
RT Structure and Functions
As a major target for anti-HIV therapy, RT has been the subject of extensive research through crystal structure determinations, biochemical assays, and single-molecule analyses. RT derives from a virus-coded polyprotein that is processed by the viral protease to give rise to two related subunits of different length, the p66 and the p51, that share a common amino terminus and combine in a stable asymmetric heterodimer [10]. Analysis of the crystal structure of RT reveals that p66 is composed of two spatially distinct domains, polymerase and RNase H domains ( Figure 2). The polymerase domain shows a characteristic highly conserved structure that resembles a right hand, consisting of fingers (residues 1-85 and 118-155), palm (residues 86-117 and 156-237), and thumb (residues 238-318) subdomains. The p66 subunit also comprises the connection subdomain (residues 319-426) and RNase H domain (residues 427-560) [11,12]. The p51 subunit lacks the RNase H domain and has the same four subdomains of the p66 polymerase domain whose relative positions, however, are different. For this reason, the p51 subunit folds differently from p66; it does not have enzymatic activities while it serves to anchor the proper folding of the p66 subunit that performs all the catalytic functions. RT is primarily responsible for several distinct activities that are all indispensable for the retrotranscription process: RNA-and DNA-dependent DNA synthesis, RNase H activity, strand transfer, and strand displacement synthesis [13]. The presence of all these functions in a single protein is facilitated by the highly dynamic RT nature which allows RT to spontaneously slide over long distances of RNA:DNA and DNA:DNA duplexes, to easily target the primer terminus for DNA polymerization, to rapidly access multiple sites, and, hence, to make up for its low processivity [13]. RT sliding does not require energy from nucleotide hydrolysis, and it is supposed to be a thermally driven diffusion process [13]. Noteworthy, it has been recently shown that RT can bind to the nucleic acid substrates in two different orientations, termed "RNase H cleavage competent orientation" and "polymerase competent orientation," and that each of them allows to catalyze one of the two RT-associated enzymatic activities [14]. These two binding modes are in a dynamic equilibrium, and it has been demonstrated that RT can spontaneously and rapidly switch between these orientations without dissociating from the substrate. This flipping can be influenced by the presence of small molecules as nucleotides that stabilize the polymerase competent orientation or inhibitors that, conversely, destabilize it [8]. Together, shuttling and switching give rise to a very complex series of conformational changes that increase enormously the replication efficiency, combining DNA polymerization and RNA cleavage. The DNA synthesis, catalyzed by both RT-associated RNA-and DNAdependent DNA polymerase activities (RDDP and DDDP, resp.), occurs with a mechanism that is similar to other DNA polymerases [15]. The polymerase active site is located in the middle of the palm, fingers, and thumb subdomains. In particular, the palm subdomain is very important for positioning of the primer terminus in the correct orientation for nucleophilic attack on an incoming dNTP [16]. Three aspartic acids residues (D110, D185, and D186) located in the palm subdomain of p66 bind the divalent ion cofactor (Mg 2+ ) through their catalytic carboxylates group, and are essential for catalysis ( Figure 2) [17]. DNA synthesis requires that RT binds to the template:primer on the priming binding site; this interaction is stabilized by a change of the conformation of the p66 thumb (from close to open). Then, the dNTP binds at the nucleotide binding site to form an RT:DNA:dNTP ternary complex [18]. Afterwards, a conformational change of the fingers traps the dNTP, precisely aligning the α-phosphate of the dNTP and the 3 -OH of the primer inside of polymerase active site (this is actually the rate limiting step). Under these conditions, the enzyme catalyzes the formation of a phosphodiester bond between the primer 3 -OH and the dNMP with the release of a pyrophosphate. Then, the pyrophosphate is free to go out of the catalytic site. Finally, translocation of the elongated DNA primer frees the nucleotide-binding site for the next incoming dNTP or, alternatively, RT can dissociate from the complex. Compared to cellular DNA polymerases, RT exhibits a very low processivity, typically dissociating from the substrate after synthesizing only a few to a few hundred nucleotides. This may contribute to the fidelity of RT and results in the accumulation of mutations during reverse transcription.
Importantly, during its DNA polymerase activity RT can run up against several template secondary structures. Particularly, the RNA template can form stable RNA:RNA interactions that can occlude the polymerization site and/or displace the primer terminus. In this case, RT has been shown to realize a strand displacement synthesis, in which the sliding movement can contribute to the reannealing of the primer, displacing the RNA [17].
DNA-Directed RNA Cleavage.
RT is able to degrade selectively the RNA portion of an RNA:DNA hybrid and to remove the priming tRNA and PPT. This RNase H function is essential for virus replication since RNase H-deficient viruses are noninfectious [19]. The RNase H domain is located at C-terminus of the p66 subunit, 60Å far from polymerase active site ( Figure 2) equivalent to 17 nucleotides of a DNA:DNA hybrid and/or 18 nucleotides of a RNA:DNA hybrid [20]. The RNase H active site contains a highly conserved, essential, DDE motif comprising the carboxylates residues D443, E478, D498, and D549, that can coordinate two divalent Mg 2+ cations, consistently with the proposed phosphoryl transfer geometry [21]. Mutations in any of the D443, D498, and E478 residues abolish enzyme activity [22,23]. The RNase H domain can catalyze a phosphoryl transfer through nucleophilic substitution reactions on phosphate ester. This action occurs through the deprotonation of a water molecule, with the production of a nucleophilic hydroxide group that attacks the scissile phosphate group on the RNA previously activated by coordination with the Mg 2+ cofactor [24]. The reason for the RNase H cleavage specificity for the RNA portion of the RNA:DNA hybrid mainly relies on its particular minor groove width and its interaction with the "primer grip" (an extensive network of contacts between the hybrid phosphate backbone and several residues far ∼4-9 bp from the RNase H active site) [16]. The RNA:DNA hybrid has a minor groove width of ∼9-10Å, that is intermediate between the A-and B-form of other double-stranded nucleic acids (dsNA). The HIV-1 RNase H hydrolyzes much less efficiently hybrids with lower widths, such as the PPTs that show a width of 7Å probably due to the presence of A-tracts [17,25]. This fact allows the PPT recognition as RNA primers for DNA synthesis and may also represent a further specific viral target.
The RNase H catalysis can occur in a polymerasedependent or polymerase-independent mode, and it is possible to distinguish three different cleavage types: "DNA 3end-directed cleavage," "RNA 5 -end-directed cleavage," and "internal cleavage" [26]. The former acts during (−)strand DNA synthesis, when the RNase H active site cleaves the RNA in a position based on the binding of the polymerase active site to the 3 -end of the new (−)DNA [27]. The second one acts when RT binds to a recessed RNA 5 -end annealed to a longer DNA strand, and the RNase H function cleaves the RNA strand 13-19 nucleotides away from its 5 -end. The internal cleavage occurs since the RNA cleavage is slower than DNA synthesis, and, given that a viral particle contains 50-100 RTs molecules and only two copies of (+)RNA, all the nonpolymerizing RTs can bind to the hybrid and degrade the RNA segment by a polymerase-independent mode [16].
Strand Transfer.
The strand transfer is a critical step during the reverse transcription process in which two complementary ssNAs have to anneal to allow the pursuance of DNA synthesis ( Figure 1). In both (−) and (+)strand transfers the ssNA develops secondary structures: the R region consists of a strong-structured motif TAR hairpin and a poly(A) hairpin [28]. Also the PBS sequence at the 3 -end of the (−)strand DNA can form a stable hairpin structure. Therefore, RT is helped in performing this step by the presence of the viral-coded nucleocapsid (NC) protein [29,30]. The strand transfer process, together with the RT fidelity and the presence of other host factors such as APOBEC [31], helps to explain the high rate of recombination events to allow HIV to evolve rapidly and develop resistance to drugs.
Pyrophosphorolysis.
As most DNA polymerases, RT can catalyze the reversal of the dNTP incorporation that is termed pyrophosphorolysis. RT has the ability to carry out this reverse reaction using a pyrophosphate (PPi) molecule or an NTP, such as ATP, as the acceptor substrate [32][33][34] giving rise to a dinucleotide tetraphosphate (formed by the excised dNMP and the acceptor ATP substrate) and a free 3 -OH end as reaction products. This RT function is particularly important, as discussed later, in some drug resistance mechanisms.
Current RTIs: Structure, Mode of Action, and Resistance
The approved combination treatments used for HIV-1 include two classes of RTIs that target the viral enzyme with two different mechanism of action. The first class comprises compounds known as nucleoside/nucleotide RT inhibitors (NRTIs/NtRTIs), while the second class comprises compounds known as nonnucleoside RT inhibitors (NNRTIs).
Nucleoside RT Inhibitors.
There are currently eight NRTIs clinically available, structurally resembling both pyrimidine and purine analogues [3]. Pyrimidine nucleoside analogues include thymidine analogues such as 3 -azido-2 ,3 -dideoxythymidine (zidovudine, AZT), and 2 ,3 -didehydro-2 ,3 -dideoxythymidine (stavudine, d4T) and cytosine analogues such as (−)-2 ,3 -dideoxy-3 -thiacytidine (lamivudine, 3TC), 2 ,3 -dideoxycytidine (zalcitabine, ddC) which, however, is no longer recommended due to peripheral neuropathy [35], (−)-2 ,3 -dideoxy-5-fluoro-3 -thiacytidine (emtricitabine, FTC), and [(−)-2 -deoxy-3 -oxa-4 -thiacytidine) (dOTC). Purine nucleoside analogues include (IS-4R)-4-[2-amino-6(cyclopropylamino)-9H-purin-9yl]-2-cyclopentane-I-methanol (abacavir, ABC) and 2 ,3 -dideoxyinosine (didanosine, ddI) as guanosine and adenine analogues, respectively ( Figure 3) [3]. These agents, in order to inhibit reverse transcription, have to be phosphorylated by cellular kinases to their triphosphate derivatives. All NRTIs follow the same mechanism of RT inhibition: once activated to their triphosphate form, they are incorporated by RT into the growing primer ( Figure 4), competing with the natural dNTPs and terminating DNA synthesis due to their lack of the 3 -hydroxyl group ( Figure 5). Therefore, once incorporated into dsDNA they prevent the incorporation of the incoming nucleotide. Importantly, while HIV-1 RT uses these NRTIs as substrates, the cellular DNA polymerases do not recognize them with the same affinity. Under selective drug pressure, drug resistant viral mutants can gain a competitive advantage over wt virus and become the dominant quasispecies. HIV-1 resistance to NRTIs usually involves two general mechanisms: NRTI discrimination, that reduces the NRTI incorporation rate, and NRTI excision that unblocks NRTI-terminated primers. A simple example of discrimination is steric hindrance in which there is a selective alteration of the NRTI binding and/or incorporation rate such as in the case of the M184V mutation and 3TC [36,37], where the valine substitution makes steric contacts with the sulfur of the oxathiolane ring of 3TC triphosphate, preventing its proper positioning for catalysis [38]. Even though the discrimination mechanism is less obvious for other NRTIs, in which structurally poorer compounds (e.g., the ones just lacking the 3 -OH group) should be differentially recognized, mutations in the nucleoside-binding site such as K65R, T69D, L74V, V75T, located in the β3-β4 loop of the p66 fingers subdomain, have : Amino acid residues involved in RTI binding. RT two subunits are in green (p66) and in gray (p51). The catalytic residues of the polymerase active site and the RNase H active site are colored in yellow. NRTIs and NtRTIs interact with residues close to the polymerase active site (blue). NNRTIs bind in a hydrophobic pocket next to the polymerase active site (magenta). RHRTIs such as DKAs, N-hydroxyimides, N-hydroxy quinazolinediones and naphthyridine derivatives bind in the RNase H active site (in yellow on the right). Vinylogous ureas bind to a hydrophobic pocket at the interface between the RNase H domain and the p51 subunit (cyan). Hydrazone derivatives have been proposed to bind two different sites (red). One located between the polymerase active site and the NNRTI-binding pocket (sharing a few residues with it) and the second one located between the RNase H and the connection domain. Anthraquinone derivatives have been proposed to bind to the first hydrazone pocket next to the NNRTI-binding site.
been reported to allow a better RT discrimination between NRTI triphosphates and natural dNTPs, since they are involved in the RT interaction with the incoming dNTP [39,40]. Differently, M41L, D67N, D70R, L210W, T215F/Y, and K219Q mutations, located around the dNTP-binding pocket and also termed thymidine analogs mutations (TAMs), increase NRTI excision. In particular, D67N and K70R are the most important in the excision of 3 -end NRTIterminated DNA while T215F/Y may increase the RT affinity for the excision substrate ATP so that the NRTI excision is reasonably efficient at ATP physiological concentrations [32,40,41]. Other TAMs such as M41L and L210W may stabilize the 215F/Y interaction with the dNTP-binding pocket [42], whereas the K219Q mutation may increase the RT processivity to compensate the higher rate of 3nucleotide removal [32,34]. Recently, mutations in the connection and RNase H domains have also been shown to confer NRTI resistance [43][44][45][46][47]. In particular, connection mutations such as E312Q, G335C/D, N348I, A360I/V, V365I, and A376S have been shown to increase AZT resistance up to 500-fold in the context of TAMs by reducing RNase H activity [43]. This RNase H-dependent mechanism of NRTI resistance has been proposed to be due to an increase in NRTI excision determined by a reduction of RNase H activity [44]. In contrast, the connection mutation G333D, in the context of TAMs and M184V mutation, increases discrimination against 3TC-MP incorporation [48], suggesting an RNase Hindependent mechanism of NRTI resistance probably due to long-range interactions and conformational changes in the connection domain [49].
Nucleotide RT Inhibitors.
NtRTIs, such as (R)-9-(2phosphonylmethoxypropyl)-adenine (tenofovir, PMPA) ( Figure 6), are compounds that already have a phosphonate group resistant to hydrolysis [3]. Therefore, they only need two phosphorylation steps to be converted to their active diphosphate derivatives, abbreviating the intracellular activation pathway and allowing a more rapid and complete conversion to the active agent [50,51]. Similarly to NRTIs, NtRTIs are phosphorylated to the corresponding diphosphates by cellular enzymes and serve as alternative substrates (competitive inhibitors); once incorporated into the growing viral DNA, they act as obligatory chain terminators [50]. NtRTIs such as tenofovir are taken as prodrugs to facilitate penetration of target cell membranes. Subsequently, endogenous chemolytic enzymes release the original nucleoside monophosphate analogue that exerts its action [51].
Crystallography, molecular modeling and docking studies have revealed that first generation NNRTIs assume a butterfly-like conformation [53][54][55][56][57]. The stabilization of the NNRTI binding in the allosteric site is accomplished through (i) stacking interactions between the NNRTIs aromatic rings and the side chains of Y181, Y188, W229, and Y318 residues in the RT lipophilic pocket; (ii) electrostatic forces (particularly significant for K101, K103, and E138 residues); (iii) van der Waals interactions with L100, V106, V179, Y181, G190, W229, L234, and Y318 residues; (iv) hydrogen bonds between NNRTI and the main chain (carbonyl/amino) peptide bonds of RT [53,54,58,59]. Larger first-generation inhibitors, such as delavirdine, extend towards the flexible loop containing the P236 residue, while maintaining stacking interactions with the tyrosine residues 181 and 188 and hydrogen bonding with K103 [60]. Stacking interactions are less important in the case of efavirenz binding, while hydrogen bonds between the inhibitor and the protein backbone of K101 and K103 residues are critical [61]. First-generation NNRTIs, such as nevirapine and delavirdine, easily select resistant RTs that contain single amino acid mutations such as Y181C, K103N, and Y188C [62,63], that change their key hydrophobic interactions at the NNRTI binding site. Second-generation NNRTIs, such as efavirenz and dapivirine, usually require two or more mutations in the HIV-1 RT before significantly decreasing their antiviral potency. In general, two or more HIV-1 RT mutations are clustered in the NNRTI pocket, suggesting a direct stereochemical mode of reduction of NNRTI binding, even though other mechanisms may also be present such as the one shown by V108I mutation that induces resistance by perturbing the Y181 and Y188 residues [61] or the one proposed for K103N mutation that should stabilize the apo-RT conformation and, hence, create an energy barrier to NNRTIs binding, reducing their potency [61]. Interestingly, NRTI-resistant mutant virus strains keep full sensitivity to the inhibitory effects of NNRTIs, and vice versa. Recently, however, mutations in the connection and RNase H domains such as N384I, T369I, and E399D have been shown to confer resistance to both NRTIs and NNRTIs probably by altering the template:primer positioning [44,47,64].
New Nucleoside RT Inhibitors
The NRTIs therapeutic use is limited by several factors [65]. Firstly, drug-drug interactions with other NRTIs used in combination treatments such as the one observed between AZT and D4T, that share the same phosphorylation pathway and show a less than additive effect when used in combination [66], or between ddI and tenofovir which determine an increase in single drugs toxicity [65]. Secondly, drug-drug interactions with other molecules such as the one observed when ABC or tenofovir is administered with some protease inhibitors [65,67], or when ABC is administered with ethanol [68]. Thirdly, several adverse events such as mitochondrial toxicity (linked to myopathy, cardiomyopathy, anemia, lipoatrophy), drug hypersensitivity reactions, and renal dysfunctions have been associated with NRTI treatment [65]. Fourthly, as described above, the selection of NRTIresistant strains, which is still the main limitation in view of the need for life-long antiviral treatments. Particularly, it has been reported that almost 50% of the viremic patients actually harbor M184V RT mutant strains and that 6-16% of the patients have been infected with viruses resistant to at least one drug and, hence, have a poorer response to therapy and a lower barrier to select further drug-resistant strains [65,69]. Given this scenario, the new NRTIs which are currently under investigation are sought to have a favorable resistance profile, reduced adverse effects, and/or a novel mechanism of action.
Nucleoside RT Inhibitors in Development Acting as Chain
Terminators. (−)-2 -deoxy-3 -oxa-4 -thiocytidine (Apricitabine, ATC) ( Figure 8) is a (−)enantiomer deoxycytidine analog with a favorable resistance profile. In fact, ATC shows only a 2-fold potency reduction on TAM strains, with or without the M184V mutation, and on K65R mutant strain, while it shows a 10-fold potency reduction on Q151M mutant strains [70][71][72]. ATC has a favorable toxic profile with little effects on mitochondrial DNA levels [73], while it shows negative drug-drug interactions when administered with 3TC or FTC [74]. Overall, ATC seems to be a good candidate in NRTI-experienced patients including individuals who have experienced virological failure on 3TC and FTC containing regimens or harboring M184V mutant strains. In fact, ATC has successfully completed the primary endpoint of a phase IIb trial in drug-resistant HIV patients with the M184V mutation. L-β-2 ,3 -didehydro-2 ,3 -dideoxy-5-fluorocytidine (Elvucitabine, L-d4TC) (Figure 8) is an L-cytidine analog under investigation in phase I/II clinical trials that is more potent than 3TC and that shows no mitochondrial toxicity [75] and an interesting protecting effect on the mitochondrial toxicity due to other NRTIs [76]. L-d4TC resistance profile shows that it selects for M14V RT mutants [77] and has a 10-fold potency reduction on K65R mutant strains [78].
(±)-β-2 ,3 -dideoxy-3 -thia-5-fluorocytosine (Racivir, RCV) ( Figure 8) is a racemic mixture of (+) and (−)FTC currently under evaluation in phase II/III clinical trials as part of a combination therapy. While both molecules inhibit RT [83], (−)FTC is better phosphorylated than (+)FTC in cells [84], and, therefore, it shows a higher potency in virus inhibition [85]. The RCV resistance profile is interesting; in fact, (−)FTC selects for M184V-resistant strains, while (+)FTC selects for T215Y-resistant strains [86]. Since the simultaneous selection of these two amino acid mutations is incompatible, such racemic mixture orthogonal resistance profile determines a delay in the onset of the drug resistance selection [87]. The long-term mitochondrial toxicity, however, is still to be fully assessed since (+)FTC triphosphate is only 36-fold selective for RT versus DNA polymerase γ [88].
Nucleoside RT Inhibitors with Innovative Mode of Action.
The RT inhibition by NRTIs can also be achieved by mechanisms different from the classical chain termination due to the lack of a 3 -hydroxyl group. In particular, new classes of inhibitors with new modes of action are the translocationdefective RT inhibitors (TDRTI), the delayed chain terminators RT inhibitors (DCTRTI), the lethal mutagenesis RT inhibitors (LMRTI), and the dinucleotide tetraphosphates (N p4 Ns).
Translocation-Defective RT Inhibitors. TDRTIs are
NRTIs with modifications of the sugar moiety that block the RT translocation after the NRTI incorporation. 4 -ethynyl-2-fluoro-2 -deoxyadenosine (EFdA) (Figure 9) is the most potent derivative of a series of 4 -substituted nucleoside analogs which, differently from the other NRTIs, have a 3hydroxyl group [91]. EFdA is able to inhibit many drugresistant strains several orders of magnitude more potently than the other approved NRTIs. For instance, it inhibits the M184V mutant strain with an EC 50 value of 8 nM, while some other drug-resistant strains are even hypersensitive to EFdA [92]. Importantly, RT can use EFdA triphosphate (EFdA-TP) as substrate but, despite the presence of the 3 -hydroxyl group, the incorporated EFdA monophosphate (EFdA-MP) blocks further DNA synthesis since the enzyme is not able to efficiently translocate on a RNA:DNA or a DNA:DNA hybrid containing a 3 -terminal EFdA-MP [93] ( Figure 10). In fact, on the one hand, the North (C2exo/C3 -endo) EFdA sugar ring conformation (which is the proper 3 -terminus position for in-line nucleophilic attack on the α-phosphate of the incoming dNTP) has been shown to be required for efficient binding at the primer-binding and RT polymerase active sites suggesting that, once incorporated into the DNA, the EFdA 3 -hydroxyl group is not likely to prevent by itself additional nucleotides incorporation, and, thus, it does not contribute to the mechanism of chain termination [94]. On the other hand, molecular modeling studies suggested that the 4 -ethynyl of EFdA may fit into a hydrophobic pocket defined by residues A114, Y215, F160, M184 and the aliphatic D185 chain [93]. Hence Figure 11: Mechanism of action of DCTRTIs. The RT is represented as a pale green circle with the priming binding site in cyan (P) and the nucleotide binding site in white (N). The RNA template is shown in blue and the (−)strand DNA in purple. DCTRTI triphosphate (strong green) is incorporated into the growing DNA chain (1). After further nucleotides addition, its presence blocks DNA elongation, probably through steric hindrance interference (yellow) between the RNA:DNA hybrid and the RT nucleic-acid-binding cleft (2). In addition, their incorporation can also block the synthesis of the (+)strand DNA affecting the base pairing (3).
complementary nucleotide is prevented [93]. Notably, in spite of the fact that the diminished translocation makes the 3 -EFdA-MP terminus DNA an excellent substrate for NRTI excision, the net excision process has been reported to be not very efficient, apparently because once the nucleotide is excised through pyrophosphorolysis to form EFdA-TP, the latter is rapidly reincorporated [93]. Moreover, it has been recently reported that EFdA is a poor substrate for DNA polymerase γ (it is incorporated 4,300-fold less than dATP), suggesting minimal mitochondrial toxicity [95].
Delayed Chain Terminators RT Inhibitors.
DCTRTIs are NRTIs that allow further incorporation of dNTPs into the growing DNA chain since they have a 3 -hydroxyl group. However, after further nucleotide addition, their presence blocks DNA elongation, probably through steric hindrance interference between the RNA:DNA hybrid and the RT nucleic acid binding cleft, close to the polymerase active site ( Figure 11). They can also block the synthesis of the (+)strand DNA affecting the base pairing. 2 ,3 -dideoxy-3 C-hydroxymethyl cytidine (PPI-801) ( Figure 9) has been reported to allow the incorporation of one additional dNTP prior to mediating chain termination [65]. Interestingly, the incorporated PPI-801 is not accessible for nucleotide excision, and, therefore, this class of compounds is proposed to be attractive because it should be active also on NRTI-resistant strains with enhanced 3 -end nucleotide excision.
8-isopropyl-amino-2 -deoxyadenosine (8iPrNdA) (Figure 9) is a recently reported molecule belonging to a series of nucleoside analogs with a natural deoxyribose moiety and modifications at position 8 of the adenine base [96]. These modifications may induce a steric clash with helix αH in the thumb domain of the p66 subunit, causing delayed chain termination. In fact, once incorporated into the elongated DNA, 8iPrNdA stops the further DNA synthesis after the incorporation of three additional dNTPs [96]. Even though the potency and selectivity of 8iPrNdA are not very high, it is an interesting example of an NRTI with modifications on the adenine base and not on the sugar moiety.
Lethal Mutagenesis RT Inhibitors.
LMRTIs are NRTIs that allow further incorporation of dNTPs into the growing DNA chain. However, their incorporation causes a significant increase of nucleotide mismatches that determines a high mutation rate that eventually leads to viral replication suppression. 5-hydroxydeoxycytidine (5-OH-dC) ( Figure 9) is a deoxycytidine analog that can efficiently base pair with both guanosine and adenosine nucleotides [97]. Viral growth in the presence of 5-OH-dC determines a 2.5-fold increase in G to A substitutions and a decline in viral infectivity over serial passages [97]. The fact that a relatively small increase in the HIV mutation frequency has a large effect on viral lethality substantiates the concept that the HIV mutation frequency is close to the error threshold for the viability of the quasispecies and that NRTIs that may significantly increase mutation frequency can act almost analogously to the cellular cytidine deaminase APOBEC3G [97]. 5-aza-5,6-dihydro-2 -deoxycytidine (KP-1212) ( Figure 9) is a deoxycytidine analog with a modified base and a natural sugar moiety that can also base pair with both guanosine and adenosine nucleotides [98]. The virus grown in the presence of KP-1212 accumulates a number of mutations that, eventually, stops its replication [98]. KP-1212 has been reported to interact also with DNA polymerase γ [99], suggesting a possible mitochondrial toxicity that, however, has not been observed in cells [98].
Dinucleotide Tetraphosphates.
As described above, nucleotides excision is a major mechanism of NRTI resistance. During this mechanism RT catalyzes the pyrophosphorolysis of, for instance, a 3 -AZT-MP terminated DNA. In fact, in the presence of the PPi donor ATP, RT catalyzes the excision reaction which results in the production of a dinucleoside tetraphosphate (i.i. A p4 AZT) freeing the 3end for further DNA elongation. Notably, X-ray crystal studies have shown that the AMP part of the A p4 AZT dinucleotide ( Figure 9) binds differently to wt and drugresistant mutant RTs [100]. These observations demonstrate that (i) RT can catalyze the reverse reaction and (ii) drug resistance mutations create a high-affinity ATP-binding site and open the possibility of designing drugs that can inhibit the enzyme mimicking the N p4 N excision product that may be particularly active on NRTI-resistant strains. Up to now, a few N p4 Ns have been synthesized that are able to inhibit wt and AZT-resistant RTs in the low micromolar range [101]. Notably, while the tetraphosphate linker, that avoids the intracellular phosphorylation step, is a potential advantage of these molecules, it is also an obstacle for their stability and cellular permeability. More studies dedicated to a further exploration of the ATP-binding site may lead to potent and innovative drugs.
New Nonnucleoside RT Inhibitors
The NNRTIs therapeutic use is limited mainly by the selection of NNRTI resistant virus, even though drug hypersensitivity and/or serious central nervous system dysfunctions are also toxicity issues for some NNRTIs. For this reason, there is still an active focus on the development of new NNRTIs, especially for compounds with high potency against K103N, Y181C, and Y188V mutant viruses. Besides the fact that more than 30 different conformational classes of NNRTIs have been reported to date [102,103], the recent development of new NNRTIs has been focused on the identification of molecules that retain high conformation flexibility and positional adaptability in order to adjust the inhibitor conformation to the NNRTI-binding pocket, whose shape is different according to the presence of the diverse amino acid residues involved in NNRTI resistance. In fact, while first-generation NNRTIs, such as nevirapine, delavirdine, or efavirenz, bind to RT in "two-wing" (or "butterfly-like") conformation, the most recently developed NNRTIs show a "U" (or "horseshoe") conformation which gives an increased plasticity to these derivatives [104,105]. Success stories of such an approach are the latest approved NNRTIs, etravirine and rilpivirine (Figure 7), and another compound under clinical investigation in phase I/II clinical trials, dapivirine ( Figure 12) [104,105].
Another complementary strategy used to improve the NNRTIs performance is to design derivatives that make strong interactions with highly conserved amino acid residues in the NNRTI-binding pocket such as F227, W229, L234, and Y318 [105,106]. In fact, these first three residues are part of the primer grip region that maintains the primer terminus in an appropriate orientation for the nucleophilic attack on the incoming dNTP. Specifically, the W229 residue is the prime candidate residue for drug design, and, in fact, among others, the above-mentioned rilpivirine has been reported to make strong interactions with the indole ring of W229.
Another reported interesting NNRTI is 3-(4-(2-methyl-1H-imidazo[4,5-c]pyridin-1-yl)benzyl)benzo[d]thiazol-2(3H) -one (CP94707) (Figure 12) that inhibits, even though not very potently, wt and mutant Y181C and Y188C RTs at the same concentrations and shows only a 2-fold reduction in potency of inhibition on K103N RT [107]. CP94707 makes little contact with Y181 and Y188 residues, while it makes aromatic ring stacking interactions with W229 amino acid [107]. In addition, CP94707 binding to RT results in rearrangement of the distally positioned Y115 side chain, 15Å away, to a conformation that is incompatible with binding of dNTPs. Y115, in fact, can act as a gatekeeper residue that discriminates between deoxynucleotides and ribonucleotides. Therefore, it has been proposed that CP94707 may have a nonconventional mode of action [108].
Nucleotide Competing RT Inhibitors
A series of indolopyridones, therefore belonging to the NN-RTIs, have been shown to inhibit RT interacting differently Molecular Biology International 13 from the classic NNRTIs. In particular, 5-methyl-1-(4nitrophenyl)-2-oxo-2,5-dihydro-1H pyrido [3,2-b]indole-3carbonitrile (INDOPY-1) (Figure 13) (i) inhibits also HIV-2 RT [111], while the other NNRTIs are inactive against this enzyme; (ii) it is active against K103N, Y181C, and Y188C mutant RTs as potently as on wt RT, while it is 3.6fold less active against the K103N/L100I double-mutant RT [112]; (iii) it is active on TAM viruses, while it is 3-to 8fold less effective on M184V or Y115F mutant viruses, it is more than 100-fold less potent on the M184V/Y115F doublemutant virus, and it is slightly more effective on K65R mutant virus [111][112][113]. In addition, the INDOPY-1 analog 1-(4-nitrophenyl)-2-oxo-2,5-dihydro-1H-pyrido [3,2-b] indole-3-carbonitrile (VRX329747) (Figure 13) selected HIV-1 RT mutated at the amino acid residues M41L, A62V, S68N, G112S, V118I, and M184V, which are all located around the incoming nucleotide-binding site [112]. Further, binding and biochemical studies revealed that (i) the M184V mutation reduces the affinity to INDOPY-1, while the Y115F mutation facilitates the dNTP binding, and their combined effects enhance the ability of the enzyme to discriminate against the inhibitor [113]; (ii) RT complexed with INDOPY-1 is trapped in the posttranslocational state [113]; (iii) the INDOPY-1 has preference with respect to substrate primer identity since its binding to RT is higher on a DNA:DNA versus a RNA:DNA primer:template [114]; (iv) when assayed by steady-state kinetic analysis with homopolymeric template primers, INDOPY-1 inhibits RT-catalyzed DNA polymerization with a competitive [111] or mixed-type [112] mode with respect to dNTPs. Overall, these observations suggest that the binding site of the indolopyridones and nucleotide substrates can at least partially overlap and they are therefore proposed as Nucleotide competing RT inhibitors (NcRTIs).
4-dimethylamino-6-vinylpyrimidines (DAVPs) is another class of compounds that have been reported to compete with the incoming dNTP and therefore can be considered NcRTI [115,116]. However, differently from INDOPY-1, DAVP1 ( Figure 13) is 4000-and 5000-fold less potent on mutant K103N and Y181C RTs, respectively [115], and binds also to unligated RT (while INDOPY-1 binds only to the RT:template:primer complex) [116]. X-ray crystal studies have confirmed that DAVP1 binds to an RT site that is distinct from the NNRTI-binding pocket, and it is close to the RT polymerase catalytic site [117]. This site is located in a hinge region, at the interface between the p66 thumb and p66 palm subdomains, that comprises the amino acid residues M230 and G231 (participating to the primer grip region and helping in the correct positioning of the 3 -OH end of the DNA primer), G262, K263 and W266 (involved in the template primer recognition), M184 and D186 (the first is involved in DNA synthesis fidelity, while the second is part of the catalytically essential YXDD motif) [117]. Hence, the DAVP1 binding site is located in a region critical for the correct positioning of the 3 -OH primer for the in-line nucleophilic attack by the incoming dNTP and the subsequent chemical bond formation with its α-phosphate. Notably, the X-ray study also revealed that in the RT/DAVP-1 complex the RT conformation is analogous to the "closed" conformation observed in unliganded RTs (with the p66 thumb subdomain folded into the DNA-binding cleft) and differs from that observed in RT/NNRTI complexes that has a hyperextended "open" conformation [117]. However, considering the proposed binding site, the reason for the loss of DAVP1 activity against K103N and Y181C mutant RTs remains unclear. While it has been hypothesized that DAVP1, owing to its small size, could travel between the NNRTI and nucleoside-binding pockets [117], more studies are needed to understand the DAVP1 mode of action.
PPi Analogs Inhibitors
Foscarnet (phosphonoformate, PFA) ( Figure 13) is a PPi analogue that targets the DNA polymerase of herpes viruses as well as the RT of retroviruses [118]. Foscarnet is used intravenously to treat opportunistic viral infections, particularly CMV retinitis in patients with AIDS, but its pharmacokinetic profile is complicated by nephrotoxicity [119]. When assayed against HIV-1 RT, it competitively blocks pyrophosphorolysis and PPi exchange reactions, suggesting that foscarnet and PPi share overlapping binding sites [120]. It has been shown that foscarnet traps the RT pretranslocated complex preventing the binding of the next nucleotide, and, thus, the pretranslocated complex has been proposed as a target for drug discovery [121]. In vivo and in vitro foscarnet-resistant HIV-1 variants have been shown to carry mutations in the RT gene at several positions, including W88G/S, E89K/G, L92I, A114S, S156A, Q161L, and H208Y [122][123][124][125]. Notably, most of the mutations that reduce the susceptibility to PFA also confer hypersensitivity to AZT and it has been suggested that foscarnet analogs may inhibit the phosphorolytic rescue of NRTI-terminated primers and be used to prevent the excision-based mode of NRTI resistance [126].
RNase H Inhibitors
Despite the fact that the RT-associated RNase H function is essential for the reverse transcription process as well as the RT-associated DNA polymerase function, no effective RNase H RTIs (RHRTIs) have been developed yet. In the last few years, however, a few classes of RHRTI that are specifically targeted to the RNase H active site ( Figure 4) have been identified [19,127]. Most of them are able to chelate the divalent magnesium ion within the RNase H active site, but they also exert a high cellular toxicity, possibly due to an unspecific metal chelation, since the RNase H active site is an open pocket and offers, at least so far, little elements for selective small-molecule optimization. Figure 14) were designed to coordinate the two metal ions in the active site of RNase H and showed no interactions with the polymerase metal-binding site [128]. However, so far they have not been further developed.
Dual RHRTI and IN
Other derivatives that have also been developed as DRH-INIs are N-hydroxyimide. The prototype of these inhibitors was the 2-hydroxyisoquinoline-1,3(2H,4H)-diones (NHI) ( Figure 15) [137,138] that was shown, by crystal structures with the isolated RNase H domain, to bind to RT in a strictly metal dependent manner, confirming the metalion-mediated mode of action. More recently, other Nhydroxyimide derivatives were synthesized such as DRH-INIs [139,140]. Interestingly, the methyl 2-Hydroxy-1,3-dioxo-1,2,3,4-tetrahydroisoquinoline-4-carboxylate analog (CNHI) (Figure 15) has also been shown to inhibit the replication of the double-mutant G140S/Q148H, which is the most resistant strain to the INI raltegravir [140], indicating that it is possible to design compounds with the same scaffold that may (i) inhibit both RNase H and IN and (ii) inhibit specifically one of the two enzymes. Further studies will be needed to dissect the specifics of the two active sites. ion [141]. These two derivatives were further developed into more potent analogs that, however, were devoid of antiviral activity in cell culture [142]. Molecular modeling studies showed that they bind to an hydrophobic pocket comprising residues V276, C280, K281, K275, R277, and R284 of the p51 thumb and residues G541 and H539 of the RNase H domain (Figure 4) [142]. Further studies are certainly warranted since this new pocket is highly attractive for RHRTIs development.
Dual RNase H and Polymerase Inhibitors
An interesting class of RHRTIs is the hydrazone derivatives, whose first reported analog was N-(4-tert-butylbenzoyl)-2-hydroxy-1-naphthaldehyde hydrazone (BBNH) ( Figure 17). derivative, (E)-3,4-dihydroxy-N -((2-hydroxynaphthalen-1yl)methylene)benzohydrazide (DHBNH) (Figure 17), has been reported to bind near the polymerase active site in a pocket different from the NNRTI-binding site and also >50Å away from the RNase H active site (Figure 4) [146]. Hence, it was hypothesized that DHBNH may either perturb the trajectory of the template primer, so that RNase H cannot operate on its substrate, or that it may also bind to a second site, in or near the RNase H domain, that was not seen in the crystal. More recently, molecular docking studies on a series of hydrazone analogs proposed that they bind to a pocket that includes residues Y405, W406, Q500, and Y501 of p66 subunit, and, hence, they form hydrophobic interactions with RT and with base pairs in the groove of the RNA:DNA substrate [147]. In fact, residues D499 and A502, adjacent to Q500, which were perturbed by the hydrazone derivatives presence [147], are part of the primer grip of the RNase H domain and play a role in aligning the DNA:RNA substrate with the active site. Therefore, the hydrazones binding to Q500 may disrupt the primer grip's role in the activity of RNase H. A second class of DNNRTI is a series of emodin [148] and alizarine anthraquinone derivatives [149,150] such as 1-acetoxy-9,10-dioxo-9,10-dihydroanthracen-2-yl 4bromobenzoate (KNA-53) (Figure 17), that inhibits both RT-associated functions of wt and K103N RTs and only the RNase H function of Y181C RT. Mode of action studies and molecular dynamic simulation led to proposing that the anthraquinone derivatives bind to the site adjacent to the NNRTI pocket, which was originally reported [146] for the hydrazones derivatives ( Figure 4) [149]. Accordingly, it has been suggested that the anthraquinone inhibition of the RNase H function may be due to a change in the RNA:DNA hybrid RT accommodation, induced by their binding, which results in a possible variation in the nucleic acid trajectory toward the RNase H catalytic site [149].
It is important to note that questions have been raised regarding the use of combinations between RHRTIs and NRTIs. In fact, RHRTIs have been proposed to lead to an increase in NRTIs resistance by mimicking the RNase Hdependent mechanism of NRTI resistance of some connection domain mutations [43]. Recently, however, studies on the effects of some RHRTIs on the HIV-1 susceptibility to AZT and 3TC have shown that none of the tested RHRTIs decreased NRTI susceptibility, while only one DNNRTI decreased AZT susceptibility by 5-fold [154]. More studies are needed to fully understand the interplay between RNase H inhibition and NRTIs susceptibility as well as its clinical relevance.
Other Potential Targets in RT
The increase in knowledge regarding HIV life cycle and specifically the function of the HIV RT and its essential interactions with other proteins will reveal potential drug targets. Even though no inhibitors have been identified yet, to the best of our knowledge, the DNA synthesis initiation (with an RNA:RNA primer), the PPT hydrolysis, the strand transfer, and pyrophosphorolysis RT functions are all potential aspects of the RT activities that may be targeted by small molecules. In addition, RT makes contact with other viral proteins such as NC and IN. These binding surfaces might be potential targets since their disruption may alter viral protein efficiency. Furthermore, some cellular factors have been described to interact with RT (and with the RT:IN complex) during reverse transcription and may have a role in its function [158]. Therefore, a better understanding of these interactions may offer other new target sites. Finally, intracellular immunity approaches may also involve proteins that affect RT functions and may thus offer additional target possibilities [31]. In conclusion, although RT has been the very first targeted HIV protein and is probably the most explored one, it still presents uninvestigated (or under investigation) functions and aspects that still make it a new fascinating target for innovative drug development. | 2018-04-03T04:37:22.191Z | 2012-06-20T00:00:00.000 | {
"year": 2012,
"sha1": "48243faee41be2e7fd7743026cdf44b42b876a88",
"oa_license": "CCBY",
"oa_url": "http://downloads.hindawi.com/archive/2012/586401.pdf",
"oa_status": "GOLD",
"pdf_src": "PubMedCentral",
"pdf_hash": "48243faee41be2e7fd7743026cdf44b42b876a88",
"s2fieldsofstudy": [
"Biology"
],
"extfieldsofstudy": [
"Biology",
"Medicine"
]
} |
9844046 | pes2o/s2orc | v3-fos-license | Functional heterogeneity of small ubiquitin-related protein modifiers SUMO-1 versus SUMO-2/3.
Post-translational modification marked by the covalent attachment of the ubiquitin-like protein SUMO-1/SMT3C has been implicated in a wide variety of cellular processes. Recently, two cDNAs encoding proteins related to SUMO-1 have been identified in human and mouse. The functions and regulation of these proteins, known as SUMO-2/SMT3A and SUMO-3/SMT3B, remain largely uncharacterized. We describe herein quantitative and qualitative distinctions between SUMO-1 and SUMO-2/3 in vertebrate cells. Much of this was accomplished through the application of an antibody that recognizes SUMO-2 and -3, but not SUMO-1. This antibody detected multiple SUMO-2/3-modified proteins and revealed that, together, SUMO-2 and -3 constitute a greater percentage of total cellular protein modification than does SUMO-1. Intriguingly, we found that there was a large pool of free, non-conjugated SUMO-2/3 and that the conjugation of SUMO-2/3 to high molecular mass proteins was induced when the cells were subjected to protein-damaging stimuli such as acute temperature fluctuation. In addition, we demonstrated that SUMO-2/3 conjugated poorly, if at all, to a major SUMO-1 substrate, the Ran GTPase-activating protein RanGAP1. Together, these results support the concept of important distinctions between the SUMO-2/3 and SUMO-1 conjugation pathways and suggest a role for SUMO-2/3 in the cellular responses to environmental stress.
SUMO-1 is a highly conserved, small ubiquitin-related modifier that has been shown to be covalently conjugated to a variety of cellular proteins (1)(2)(3)(4). Like ubiquitin, SUMO-1 is believed to form an isopeptide bond between the carboxyl terminus of SUMO-1 and a lysine side chain(s) of the target protein (2,3,5). The conjugation of SUMO-1 to cellular proteins has been implicated in multiple vital cellular processes, including nuclear transport, cell cycle control, oncogenesis, inflammation, and the response to virus infection (6 -8). It has been proposed that SUMO-1 conjugation may function antagonistically to ubiquitin conjugation (9) and/or that SUMO-1 conjugation may regulate the target protein's interaction with other cellular components (5,10,11).
Among known SUMO-1 substrates in vertebrate species, the Ran GTPase-activating protein RanGAP1 is the most abundant and best characterized. It is a highly conserved protein that enhances GTP hydrolysis on Ran, a Ras-related small nuclear GTP-binding protein required for nucleocytoplasmic trafficking (12,13). It has been demonstrated both in vitro and in vivo that a single lysine residue at position 526 in the C terminus of mouse RanGAP1 is modified by SUMO-1 (5,10). A large fraction of SUMO-modified RanGAP1 appears to be tightly associated with the nuclear envelope via interaction with RanBP2/Nup358, a component of cytoplasmic filaments in the nuclear pore complex (NPC) 1 (1,4,14,15). Unmodified RanGAP1 is present in the cytoplasm, suggesting that the conjugation of a ubiquitin-like moiety at the C-terminal domain may target RanGAP1 to the NPC (5,10).
Two cDNAs coding for proteins related to SUMO-1 have been isolated from human and mouse cDNA libraries and are referred to as SUMO-2/SMT3A and SUMO-3/SMT3B (6,16,17). Both transcripts are expressed in a wide range of tissues and cell types, suggesting that their gene products play a role in some fundamental cellular processes. A comparison of the amino acid sequences of SUMO-1, -2, and -3 has revealed that SUMO-1 shares 48% identity with SUMO-2 and 46% identity with SUMO-3. Since SUMO-2 and -3 share 95% identity, it is reasonable to group SUMO-2 and -3 into a subfamily distinct from SUMO-1. It has been previously reported that both SUMO-2 and -3 could be transferred to other proteins in a pattern similar to that of SUMO-1 when SUMO-2/3 was transiently overexpressed in mammalian culture cells (18,19). This result suggested that SUMO-2/3 might function in a capacity similar to that of SUMO-1. Very little is known, however, about the specific function of SUMO-2/3 and mechanistic differences that could support cellular distinctions between two SUMO subfamilies.
To gain a better understanding of the role of SUMO-2/3 and the differential regulation of SUMO-2/3 versus SUMO-1 conjugation in vertebrate cells, we have begun to characterize SUMO-2/3 at the protein level in vivo. We describe herein the development of an antibody specific to SUMO-2 and -3. Using this antibody and a complementary SUMO-1-specific antibody, we found that RanGAP1 was preferentially modified by SUMO-1, but very poorly by SUMO-2/3, despite a higher concentration of SUMO-2/3 than SUMO-1 in intracellular pools. We also demonstrated that the SUMO-2/3 conjugation pathway could be up-regulated by several protein-damaging stimuli. This is the first evidence for the involvement of a ubiquitin-like protein modification in cellular stress responses. Based on these results, we propose that there is distinct regulation of SUMO-2/3 modification as compared with SUMO-1 modification and that the SUMO-2/3 pathway may constitute an element of the cellular response to environmental stress.
EXPERIMENTAL PROCEDURES
DNA Constructs and Antibodies-Human SUMO-1 cDNA and GST-SUMO-1 constructs have been described previously (20). SUMO-2 and * The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 -3 C-terminal deletion mutants, SUMO-2-(1-92) and SUMO-3-(1-93) were generated by polymerase chain reaction from human cDNA libraries and cloned into the pGEX vector (Amersham Pharmacia Biotech) for expression of GST fusion protein in a bacterial system or into the pEGFP-C1 vector (CLONTECH, Palo Alto, CA) for transient expression of green fluorescent protein (GFP) fusion product in a mammalian cell culture system. The GST-ubiquitin construct was described previously (20). Purified rabbit ubiquitin was purchased from Sigma. Anti-SUMO-1 and anti-RanGAP1 mouse monoclonal antibodies (21C7 and 19C7, respectively) were purchased from Zymed Laboratories Inc. (South San Francisco, CA). Anti-RanBP2 polyclonal antibody raised in guinea pig was described previously (14). Horseradish peroxidase-conjugated secondary antibodies for immunoblot analysis were from Amersham Pharmacia Biotech. Rhodamine-and fluorescein-conjugated secondary antibodies for indirect immunofluorescence were obtained from Rockland Inc. (Gilbertsville, PA).
Antibody Preparation-The expression and purification of GST-SUMO-2 fusion protein were carried out as described previously (21). The purified protein was incubated with thrombin to cleave SUMO-2 from the GST moiety, followed by 4 -20% gradient polyacrylamide gel electrophoresis (Novex, San Diego, CA) to separate the SUMO-2 and GST polypeptides. The 18-kDa band containing SUMO-2 was visualized by Coomassie Brilliant Blue, excised, and used to elicit antibodies in two rabbits (Research Genetics, Huntsville, AL). The antiserum from one of these rabbits was used for the studies described in this work.
Cell Extracts-COS-7 cells were cultured in Dulbecco's modified Eagle's medium containing 10% fetal bovine serum and antibiotics at 37°C in a 5% CO 2 incubator. Cells were harvested by gentle trypsinization and centrifugation. After washing the cells and counting in phosphate-buffered saline (PBS), the cells were mixed in SDS sample buffer containing 50 mM Tris-HCl, pH 6.8, 100 mM dithiothreitol, 2% SDS, 0.1% bromphenol blue, and 10% glycerol. Fractionated interphase egg extracts were prepared as described previously (21,22). The high speed supernatant that contained membrane-free soluble proteins is referred to throughout as the Xenopus egg extract.
Stress and Drug Treatments-Cells were harvested by gentle trypsinization and centrifugation. After counting, the cells (ϳ10 7 ) were mixed in 100 l of culture medium and subjected to one of the following stimuli: heat shock (10 min at 43°C, unless stated otherwise), osmotic stress (normal medium plus 0.7 M NaCl for 15 min at 37°C), oxidative stress (100 mM H 2 O 2 for 20 min at 37°C), or exposure to 7% ethanol (20 min at 37°C).
Immunoblot and Immunoprecipitation Analyses-For immunoblot analysis, proteins were transferred to nitrocellulose membranes either in NuPAGE transfer buffer (Novex) at 25 V for 60 min for high molecular mass proteins (Ͼ30 kDa) or in 20 mM phosphate buffer at 50 V for 50 min for low molecular mass proteins (Ͻ30 kDa). Two different conditions were used because of the difficulty in transferring high and low molecular mass proteins with equal efficiency; when high molecular mass proteins were transferred well, the low molecular mass proteins were maintained in the membrane with less efficiency and vice versa. This unequal transfer efficiency can give a false impression of the relative intensity of immunoreactive bands. For example, free SUMO-1 and -2/3 at ϳ18 kDa were not detectable upon blotting with NuPAGE buffer (data not shown), suggesting that these 18-kDa proteins are poorly immobilized on the membrane under NuPAGE transfer conditions. On the other hand, the signals from high molecular mass SUMOconjugated proteins in the phosphate buffer system were not as strong as the signals in the NuPAGE system (data not shown). We tested several different transfer conditions and found that phosphate buffer is reasonably efficient in transferring a wide range of proteins. The immunoblot data described in this study were obtained using phosphate buffer transfer conditions unless otherwise stated. Anti-SUMO-1 and anti-SUMO-2/3 antibodies were diluted 1:500 and 1:3000, respectively. Other antibodies were diluted 1:1000 unless otherwise stated. Detection of the signals in the immunoblot was carried out using the Super-Signal chemiluminescent substrate system (Pierce). To enrich for the SUMO-1-conjugated form of RanGAP1 from Xenopus egg extracts, immunoprecipitation was performed using anti-RanBP2 antibody following the detailed protocol described previously (14,15).
Quantitation of Immunoblot Analysis-In the semiquantitative immunoblot analysis, GST-SUMO-1 and GST-SUMO-3 were purified according to the manufacturer's instruction (Amersham Pharmacia Biotech). The protein concentrations of these GST fusion proteins were determined with the Bio-Rad protein assay kit. For immunoblot analysis, anti-SUMO-1 and anti-SUMO-2/3 antibodies were diluted 1:500 and 1:1000, respectively, during primary antibody incubation. 125 I-Labeled protein G (Amersham Pharmacia Biotech) was also diluted 1:500 (0.5 Ci/ml) and used for the detection of the anti-SUMO-1 and anti-SUMO-2/3 antibodies. The signals derived from 125 I-labeled protein G were detected by autoradiography and quantified by InstantImager electronic autoradiography (Packard Instrument Co.).
Indirect Immunofluorescence Analysis-COS-7 cells were grown on coverslips and fixed with 3% paraformaldehyde in PBS for 10 min at room temperature and with methanol for 2 min at Ϫ20°C. For the detection of NPC/nuclear envelope-associated proteins, cells were permeabilized in 0.05% digitonin (Sigma) in PBS on ice for 5 min and washed twice in ice-cold PBS before fixation with paraformaldehyde and methanol. The coverslips were incubated with the primary antibody (1:100 dilution) followed by the rhodamine-or fluorescein-conjugated secondary antibody (1:200 dilution). During the secondary antibody incubation, 1 g/ml 4,6-diamidino-2-phenylindole (Sigma) was incubated for the detection of DNA. The cells were mounted in Vectashield (Vector Laboratories, Inc., Burlingame, CA) and analyzed with an Olympus System Model BX60 microscope using the MetaMorph imaging system (Universal Imaging Corp., West Chester, PA). Micrographs were arranged with Adobe Photoshop Version 3.0 (Adobe Systems Inc., Mountain View, CA).
Mammalian Cell Transfection-For GFP fusion protein expression, COS-7 cells cultured on coverslips were transfected with SuperFect reagent (QIAGEN Inc., Valencia, CA) according to the manufacturer's protocol. Approximately 48 h after transfection, the cells were fixed with 3% paraformaldehyde in PBS for 5 min at room temperature and with methanol for 2 min at Ϫ20°C. Cells were then incubated with PBS containing 1 g/ml 4,6-diamidino-2-phenylindole for 5 min at room temperature. Fluorescent images were analyzed as described above.
Presence of SUMO-2/3 Subfamily Members in Several Vertebrate and Insect
Species-It has been reported that there are two human cDNAs encoding for proteins that have ϳ50% identity to human SUMO-1 (16). Because these predicted proteins, designated SUMO-2/SMT3A and SUMO-3/SMT3B, are 95% identical to each other, we consider that they belong to the same subfamily and designate this subfamily as SUMO-2/3. The cDNAs encoding proteins homologous to human SUMO-2/3 were also isolated from mouse cDNA libraries (17), indicating the presence of two distinct SUMO subfamilies, SUMO-1 and -2/3, in both human and mouse. Thus, we first asked whether the two SUMO subfamilies are present in other vertebrates and in invertebrate species. Using the basic local alignment search tool algorithm BLAST, we found cDNAs encoding proteins with high homology to human SUMO-1 in the South African clawed toad (Xenopus laevis) (20) and the zebrafish (Danio reio; GenBank TM accession number AI545352). We also detected cDNAs encoding proteins with high homology to human SUMO-2 in these species (Table I). Our data base search emphasized that the two SUMO subfamilies are widely distrib- Interestingly, cDNAs coding for a protein that shows significantly higher homology to the SUMO-2/3 family than to SUMO-1 were detected in the insect species Drosophila melanogaster and Bombyx mori, respectively (Table I). This finding suggests that SUMO-2/3 polypeptides are present in at least some invertebrate species, although their function is less understood than their mammalian homologs. Development of Antibody to SUMO-2/3-To aid in our study of the function and regulation of SUMO-2/3 at the protein level, we raised rabbit polyclonal antibodies to SUMO-2. Antibody was raised against bacterially produced recombinant polypeptide corresponding to amino acids 1-92 of human SUMO-2. To establish the specificity of this antibody, we probed immunoblots of GST-SUMO-1, GST-SUMO-2, and GST-SUMO-3. As shown in Fig. 1D, the immune serum from one of the immunized rabbits recognized GST-SUMO-2 and GST-SUMO-3, but not GST-SUMO-1. No significant signal could be detected when the identical blot was probed with the preimmune serum from the same rabbit (Fig. 1C). When the immune serum was preincubated with Sepharose beads bearing either GST-SUMO-2 or GST-SUMO-3, the depleted serum did not recognize SUMO-2/3 in the immunoblot, whereas preincubation of the serum with GST-SUMO-1 did not alter the pattern of the signals (Fig. 1, E-G). These results indicate that this particular immune serum contains antibodies that specifically recognize epitopes in SUMO-2 and -3 that are not present in SUMO-1; therefore, we refer to this immune serum as anti-SUMO-2/3 antibody. Given that the amino acid sequences of SUMO-2 and -3 are almost identical (95% identity), the fact that this serum did not distinguish between the SUMO-2/3 family members under the conditions we employed throughout this investigation was expected. We also probed the immunoblot containing ubiquitin and GST-ubiquitin to exclude the possibility that anti-SUMO-2/3 antibody cross-reacts with ubiquitin. As shown in Fig. 1 (H and I), this antibody did not recognize either ubiquitin or GSTubiquitin under our experimental conditions.
For the detection of SUMO-1, we used monoclonal antibody 21C7, which was generated from BALB/c mice immunized against recombinant human SUMO-1 (1). Monoclonal antibody 21C7 detected only GST-SUMO-1 in immunoblots containing all three GST-SUMO proteins (Fig. 1B), indicating that the epitope recognized by the monoclonal antibody is absent in SUMO-2/3. Therefore, this monoclonal antibody is referred to as anti-SUMO-1 antibody in this study.
Abundance of SUMO-2/3 Versus SUMO-1-Using the SUMO-2/3-specific and SUMO-1-specific antibodies, we first compared the relative amount of SUMO-2/3 versus SUMO-1 in COS-7 cells by semiquantitative immunoblot assay using GST-SUMO-1 and GST-SUMO-3 as standards. As shown in Fig. 2, we determined conditions under which the assay exhibited fair linearity (see "Experimental Procedures" in detail). Under these conditions, we compared the signal intensity of cell lysates in SUMO-1 versus SUMO-2/3 blots.
Judging by apparent molecular mass, an 18-kDa band appeared to represent a non-conjugated pool of either SUMO-1 or SUMO-2/3, whereas bands migrating higher than 18 kDa most likely contain proteins conjugated with either SUMO-1 or SUMO-2/3. The SUMO-2/3 blot displayed a strong 18-kDa band, indicating that a large fraction of SUMO-2/3 is present as a "free" or non-conjugated form in vivo. On the other hand, a 90-kDa protein was detected as the dominant signal in the SUMO-1 blot, whereas the 18-kDa band was barely detectable. This observation suggests that most SUMO-1 exists as protein conjugate rather than in a free form in vivo and that a major target of SUMO-1 conjugation is a 90-kDa protein. Interestingly, the amount of non-conjugated SUMO-2/3 was ϳ50 times greater than that of non-conjugated SUMO-1 (Fig. 2C), indicating that the SUMO-2/3 modification system has a potentially larger capacity to modify cellular proteins than the SUMO-1 system. When the signal spanning from 18 kDa to the top of either lane 5 or 11 in A was measured and integrated, respectively, the total signal intensity of lane 11 in the SUMO-2/3 blot was ϳ4 times greater than that of lane 5 in the SUMO-1 blot (Fig. 2C). These results suggest that the total cellular pool of SUMO-2/3 is greater than that of SUMO-1.
Activation of SUMO-2/3 Conjugation by Acute Heat Fluctuation-To determine whether the large pool of free SUMO-2/3 is functional in terms of protein conjugation, we tested multiple stimuli, including anticancer drugs, enzyme inhibitors, and environmental stresses such as temperature fluctuation, for associated changes in SUMO-2/3 conjugation. As shown in Fig. 3, we found that acute temperature elevation was associated with a diminished pool of free SUMO-2/3 and an accumulation of high molecular mass SUMO-2/3 signals. Strikingly, the response to acute heat elevation was detected in as little as 5 min, suggesting the existence of a rapidly signaling cellular pathway that activates SUMO-2/3 conjugation in response to acute heat fluctuation. In addition, we found that the ratio between free SUMO-2/3 and high molecular mass conjugates returned to its basal state when cell cultures were incubated at 37°C for 60 min after acute heat stress (43°C for 10 min) (Fig. 3, B and D).
These results indicate that a substantial pool of free SUMO-2/3 is readily available for conjugation and imply that SUMO-2/3 is a reversible protein modifier in the context of heat shock. In contrast to SUMO-2/3, the pattern of SUMO-1 conjugation was not dramatically changed following heat shock, suggesting that activation of the SUMO-2/3 conjugation pathway is a more prominent feature of the cellular stress response than is that of the SUMO-1 pathway. In Fig. 3A, a 50-kDa band was observed in all lanes, and the intensity of the band appeared to be constant in the presence or absence of stimuli. We concluded that this 50-kDa band was nonspecific since the anti-SUMO-2/3 serum preincubated with either GST-SUMO-2 or GST-SUMO-3 also recognized the 50-kDa protein band (data not shown).
Given that heat stress induces denaturing of cellular proteins, we wondered whether other protein-damaging conditions would up-regulate protein modification by the SUMO-2/3 pathway. To begin investigating the response of the SUMO-2/3 pathway to various protein-damaging stresses, we monitored the activation of SUMO-2/3 conjugation during oxidative stress, ethanol addition, and osmotic stress (Fig. 4A). Immunoblot analysis of COS-7 cell extracts from cultures subjected to these stresses showed a marked accumulation of high molecular mass SUMO-2/3 conjugates and a diminished pool of free SUMO-2/3 (Fig. 4, A and B); osmotic stress indicated less dramatic changes than the other stresses. We also found that the human leukemia cell line K562 responded to these stresses 5, 6, 11, and 12), purified GST-SUMO-1 (lanes 1-4), and GST-SUMO-3 (lanes 7-10) were separated by 4 -20% SDS-PAGE and transferred to nitrocellulose membranes in phosphate buffer. Lanes 1 and 7 contain 0.44 ng, lanes 2 and 8 contain 1.33 ng, lanes 3 and 9 contain 4.0 ng, and lanes 4 and 10 contain 12 ng of either purified GST-SUMO-1 or GST-SUMO-3, respectively. Immunoblot analysis was performed using anti-SUMO-1 antibody (lanes 1-5) or anti-SUMO-2/3 antibody (lanes 7-11) as the primary antibody. The antibodies were detected by 125 I-labeled protein G as described under "Experimental Procedures." The arrowhead indicates the 18-kDa band presumed to represent the non-conjugated form of SUMO-1 and SUMO-2/3. Lanes 6 and 12 are the short-exposed images of lanes 5 and 11, respectively. 10-kDa protein size markers are indicated on the left. B, the linearity of immunoblot assay. 125 I-Labeled signals in lanes 1-4 for GST-SUMO-1 and lanes 7-10 for GST-SUMO-3 in A were quantified by InstantImager electronic autoradiography. The conditions we employed exhibited fair linearity between the amount of GST proteins and the signal intensities. C, the relative amount of SUMO-2/3 versus SUMO-1. Signals in lanes 5 and 11 in A were measured and integrated, respectively. These values were considered to represent the total amount of SUMO-1 and -2/3, respectively, and were compared with each other. 18-kDa signals in lanes 5 and 11 were separated and also measured. These values were considered as amounts of free, non-conjugated forms of SUMO-1 and -2/3 for comparison with the total signal intensities. a.u., absorbance units.
FIG. 3. Acute temperature elevation up-regulates SUMO-2/3 conjugation.
A, time-dependent activation of SUMO-2/3 conjugation during heat shock. Exponentially growing COS-7 cell cultures were incubated at 43°C for 0, 5, and 10 min and lysed directly in SDS sample buffer. Proteins from 0.5 ϫ 10 5 cells were subjected to SDS-PAGE followed by immunoblot analysis. Immunoblot analysis using anti-SUMO-2 antibody detected the accumulation of high molecular mass SUMO-2/3-immunoreactive proteins in cell extracts from heat-shocked cultures. The arrowhead indicates bands at ϳ18 kDa that may represent the free, non-conjugated form of SUMO-2/3. The asterisk indicates a 50-kDa band nonspecifically detected in all lanes. 10-kDa protein size markers are shown on the left. B, SUMO-2/3 conjugation in recovery from heat shock. COS-7 cell cultures were incubated at 43°C for 10 min, incubated at 37°C for 0 or 60 min, and lysed directly in SDS sample buffer. Lysates from parallel cultures under normal culture conditions were prepared as a control. Proteins from 0.5 ϫ 10 5 cells were subjected to SDS-PAGE followed by immunoblot analysis. Immunoblot analysis using anti-SUMO-2 antibody detected high molecular mass SUMO-2/3 conjugates at the conclusion of heat shock. These conjugates diminished within 60 min following restoration of 37°C culture conditions. The arrowhead indicates the position of the nonconjugated form of SUMO-2/3 at 18 kDa. C, time course of activation of SUMO-1 conjugation during heat shock. Cell extracts were prepared as described for A. Accumulation of high molecular mass SUMO-1 conjugates during heat shock was not as apparent as the accumulation of high molecular mass SUMO-2/3 conjugates (see A). D, the relative amount of free SUMO-2/3 during heat stress and after recovery. The signals at 18 kDa in the first through third lanes (arrowhead) in B were measured and compared with each other. with a similar redistribution of SUMO-2/3 immunoreactivity (data not shown), suggesting that prompt conjugation of free SUMO-2/3 to target protein(s) may be a stress response common to many cell systems.
Preferential Modification of RanGAP1 by SUMO-1-As described above (Fig. 2), a distinct 90-kDa band was evident in immunoblot analysis of nonstimulated COS-7 cell cultures probed with anti-SUMO-1 antibody. Immunoreactivity at this apparent molecular mass was barely detectable in semiquantitative immunoblots probed with anti-SUMO-2/3 antibody. It has been well established that RanGAP1 (which migrates at 90 kDa in SDS-PAGE) is the most abundant SUMO-1 substrate (1,4,15), but we were unable to detect a parallel modification of 90-kDa RanGAP1 by SUMO-2/3 (Figs. 2 and 3). In overexposed immunoblots, a band reactive to anti-SUMO-2/3 antibody at a molecular mass approximately equivalent to that of SUMO-1-conjugated RanGAP1 (90 kDa) can be distinguished (Fig. 5A, arrowhead). We attempted to enrich for this 90-kDa SUMO-2/3 conjugate by immunoprecipitation using anti-Ran-GAP1 monoclonal antibody 19C7 (1), but the cross-reactive signal indicating a SUMO-2/3-modified 90-kDa form of Ran-GAP1 remained difficult to detect even in this enriched immunoprecipitate (data not shown). By comparing band intensities at 90 kDa in SUMO-1-versus SUMO-2/3-stained immunoblots (Fig. 5A), we estimated that SUMO-2/3-conjugated RanGAP1 constitutes not more than 5% of the amount of SUMO-1-modified RanGAP1. In addition to COS-7 extracts, we tested hu-man HeLa, 293, SK-N-M, and NB4; mouse NIH-3T3 and liver; and Xenopus egg extracts to compare the relative amount of SUMO-2/3-versus SUMO-1-conjugated RanGAP1. None of these cell extracts contained a higher percentage of SUMO-2/ 3-conjugated RanGAP1 than did the COS-7 cells (data not shown), indicating that RanGAP1 is modified preferentially by SUMO-1, but very poorly, if at all, by SUMO-2/3 in many cell types.
Absence of SUMO-2/3 in the NPC/Nuclear Envelope-It has been proposed that SUMO-1 conjugation facilitates the localization of RanGAP1 to the NPC via interaction with RanBP2/ Nup358, a component of cytoplasmic filaments in the NPC (5,10). To test whether the limited cellular pool of SUMO-2/3modified RanGAP1 is involved in the interaction between Ran-GAP1 and RanBP2, we performed three experiments. First, we 5. RanGAP1, a principal SUMO-1 conjugation substrate, is poorly modified by SUMO-2/3. A, detection of SUMO-2/3-conjugated proteins. Exponentially growing COS-7 cell cultures were lysed directly in SDS sample buffer. Proteins from 10 5 cells were separated by 4 -20% SDS-PAGE and transferred to nitrocellulose membranes either in NuPAGE buffer for the detection of high molecular mass proteins Ͼ30 kDa or in phosphate buffer for proteins Ͻ30 kDa. Immunoblot analysis was carried out using anti-SUMO-1 antibody (lanes 1 and 3) or anti-SUMO-2/3 antibody (lanes 2 and 4) as described under "Experimental Procedures." The filters were exposed to x-ray film for short periods (ϳ30 s; lanes 1 and 2) or for long periods (ϳ10 min; lanes 3 and 4). The arrowhead indicates the 90-kDa signal. The position of the 50-kDa band detected in lanes 2 and 3 is indicated by an asterisk, and this band appears to be nonspecific. 10-kDa protein size markers are shown on the left. B, SUMO-2/3 does not appear at the nuclear pore/ nuclear envelope. COS-7 cells were permeabilized with 0.005% digitonin in PBS before fixation. Cells were double-stained for RanGAP1 using anti-RanGAP1 mouse monoclonal antibody 19C7 (upper left) and for SUMO-2/3 using anti-SUMO-2/3 antibody (upper right) or doublestained for SUMO-1 using anti-SUMO-1 antibody (lower left) and for SUMO-2/3 using anti-SUMO-2/3 antibody (lower right). Nuclear rim staining was visualized with anti-RanGAP1 and anti-SUMO-1 antibodies, but not with anti-SUMO-2/3 antibody.
Functional Heterogeneity of SUMO-1 versus SUMO-2/3
carried out indirect immunofluorescence of COS-7 cells permeabilized with digitonin prior to fixation and staining (1). Digitonin preferentially permeabilizes the plasma membranes of eukaryotic cells versus their nuclear envelopes. Thus, treatment of cells with digitonin before fixation removes large amounts of soluble proteins from the cells, allowing more sensitive detection of the NPC/nuclear envelope-associated proteins. When COS-7 cells that had been pretreated with digitonin were probed with anti-SUMO-2/3 antibody, there was no staining of the nuclear rim, whereas anti-RanGAP1 and anti-SUMO-1 antibodies clearly localized to the nuclear rim (Fig. 5B). Second, we expressed GFP-SUMO-1 or GFP-SUMO-2 fusion protein in COS-7 cells and observed a marked concentration of green fluorescence at the nuclear rim in GFP-SUMO-1transfected cells, whereas there was no significant concentration of the signal at the nuclear rim in GFP-SUMO-2-transfected cells (data not shown). Third, using anti-RanBP2 antibodies, we co-immunoprecipitated RanGAP1 that associated with RanBP2 from Xenopus laevis egg extracts (14,15) and analyzed the precipitated fraction by immunoblotting using anti-SUMO-1 or anti-SUMO-2/3 antibody. We were unable to detect a significant signal when this immunoblot was probed with anti-SUMO-2/3 antibody, despite the enrichment for RanBP2-associated RanGAP1 (data not shown). This result indicates that SUMO-1 is a dominant modifier of RanGAP1 and further implies that, if there is a minority complex containing SUMO-2/3-modified RanGAP1 and RanBP2, it must be unstable in Xenopus egg extracts. This result is not simply attributable to a lack of SUMO-2/3 in Xenopus egg extracts since we found a X. laevis cDNA coding for a SUMO-2/3 protein homolog (Table I). Also, we detected an 18-kDa protein that cross-reacted to anti-SUMO-2/3 antibody in Xenopus egg extracts (data not shown), suggesting the presence of a SUMO-2/3 homolog in Xenopus eggs. In sum, these results support the idea that RanGAP1 associated with the NPC/nuclear envelope is preferentially modified by SUMO-1 rather than by SUMO-2/3.
It should be noted that we observed an intense nuclear signal of SUMO-2/3 as well as some punctate concentrations in the nucleus (Fig. 5B); however, the number and size of these foci varied from cell to cell (data not shown). Intriguingly, some of the foci were stained by both anti-SUMO-1 and anti-SUMO-2/3 antibodies, indicating the colocalization of SUMO-1 and SUMO-2/3 in the digitonin-insoluble, nuclear punctate structures in interphase cells. DISCUSSION On the basis of sequence homology, the known small ubiquitin-related modifiers (SUMO) can be divided into two groups in vertebrates: one including human SUMO-1 and its interspecies homologs and the other comprising human SUMO-2, SUMO-3, and their counterparts in other species. Data base searches for proteins homologous to SUMO-2/3 revealed that SUMO-2/3 is highly conserved not only in vertebrate species, but also in insect species, suggesting a biologically significant role for the SUMO-2/3 conjugation pathway.
Using a newly developed anti-SUMO-2/3 antibody, we demonstrated that SUMO-2/3-and SUMO-2/3-conjugated proteins are present in vertebrate cells. One obvious difference between SUMO-1 and -2/3 was the presence of a large pool of free or non-conjugated SUMO-2/3 in COS-7 cells. This fraction of SUMO-2/3 appears to be readily available for conjugation reactions since its conjugation can be induced by cellular stresses such as acute heat elevation, oxidative stress, and ethanol addition, which are known to cause the accumulation of damaged proteins in the cells and to induce several stress-responsive kinase cascades (23,24). Recently, we found that treat-ment by the peptide aldehyde protease inhibitor MG-132, which has been reported to induce protein damage (25), also effectively up-regulates the conjugation of SUMO-2/3 to a high molecular mass protein fraction (data not shown). We are in the process of investigating proteins specifically modified by SUMO-2/3 in a stress-responsive manner and the fate of such SUMO-2/3-modified proteins in stressed cells.
It has been reported that the ubiquitin pathway responds to a wide variety of protein-damaging and environmental stresses (26). Intriguingly, heat shock (27,28) and oxidative (29) stresses have been known to induce the nearly quantitative conversion of free ubiquitin to the conjugated pool, reminiscent of the stress-induced SUMO-2/3 conjugation described in this study. Given that ubiquitin conjugation to damaged cellular proteins induces rapid degradation of the conjugates by 26 S proteasomes, the similar mode of stress response in the SUMO-2/3 pathway might represent a novel mechanism by which damaged proteins are identified and processed. For instance, SUMO-2/3 conjugation might stabilize the target protein by protecting against ubiquitin conjugation and the subsequent rapid proteasomal degradation. Recent observations suggest that IB␣, an inhibitor for the NF-B transcription factor, can be conjugated with either ubiquitin or SUMO-1 on Lys 21 (9). IB␣ conjugated with SUMO-1 remains stable, but ubiquitin conjugates are subjected to rapid proteasomal degradation. Since modifications of IB␣ with SUMO and ubiquitin are mutually exclusive, it is likely that these modifications functionally antagonize each other. Alternatively, SUMO-2/3 conjugation might enhance the denaturation process during stress and augment the ubiquitin-dependent degradation of such SUMO-2/3-conjugated, denatured/damaged protein by 26 S proteasomes. Conjugation by either SUMO-1 or SUMO-2/3, however, has not yet been reported to increase protein instability.
Whatever its role, our results suggest that the SUMO-2/3 conjugation pathway represents a new class of acute and reversible stress response. The rapid kinetics of SUMO-2/3 conjugation and deconjugation reveal highly dynamic features of the regulation of the SUMO-2/3 conjugation pathway in the context of environmental stress. A complete understanding of the SUMO-2/3 conjugation pathway in relation to environmental stress will require further information not only on the SUMO-2/3 conjugation substrate(s), but also on the regulation of the SUMO-2/3 conjugation/deconjugation enzymes during environmental stress.
Strikingly, the SUMO-1 and -2/3 modification pathways can be clearly distinguished by their differential modification of RanGAP1, which is a major SUMO-1 substrate, but is very poorly modified by SUMO-2/3. According to previously published reports, there is no indication of any ubiquitin-related polypeptide modification of RanGAP1 purified from mammalian cells and X. laevis egg extracts except SUMO-1 (1,5,14). These investigations of endogenous RanGAP1 support the hypothesis of selective modification of RanGAP1 by SUMO-1 in vivo. Others studies have shown, however, that RanGAP1 is efficiently modified by SUMO-2 and -3 as well as by SUMO-1 when cDNA carrying SUMO-2 or -3 is transiently overexpressed in mammalian cell culture (18,19). Since we identified the preferential modification of RanGAP1 by SUMO-1 not only in COS-7 cells, but also in other cells such as human HeLa, 293, SK-N-M, and NB4 and mouse NIH-3T3 and liver, a more reasonable explanation might be that overproduction of either SUMO-2 or -3 by transient transfection perturbs the specificity of the normal modification system.
The mechanism that governs the specificity of modification between SUMO-1 and -2/3 is currently unexplained, although three possibilities appear feasible. First, there may be two parallel enzymatic cascades present in cells that independently facilitate the modification of proteins with either the SUMO-1 or -2/3 family, respectively. For example, there is evidence for the existence of two slightly different isoforms of human Ubc9p (30) and three isoforms of mouse Ubc9p (31). Ubc9p is an enzyme whose activity has been demonstrated to be required for SUMO-1 conjugation to RanGAP1 (20,32). It will be interesting to compare these isoforms in terms of their specificity for SUMO-1 or -2/3. Second, similar to the ubiquitin conjugation system, one might predict the existence of a ubiquitin-protein isopeptide ligase-like activity that docks Ubc9p with RanGAP1 and determines the specificity of SUMO-1 conjugation to Ran-GAP1. One likely candidate for this putative activity is RanBP2/Nup358, as it has been shown to interact with both Ubc9p and SUMO-1-modified RanGAP1 (14,15). Third, SUMO-1 and -2/3 might both be conjugated to RanGAP1, followed by selective deconjugation of SUMO-2/3 from RanGAP1. This scenario would require SUMO-2/3-specific isopeptidase activity in the context of SUMO-1 modification of RanGAP1. The sulfhydryl proteases from yeast and bovine brain that exhibit SUMO-1-releasing activity from SUMO-1-conjugated RanGAP1 (31, 33) and the previously described deconjugation activity associated with the NPC fraction (1) represent likely candidates for the SUMO-2/3 isopeptidase, but preferential SUMO-2/3 deconjugation activity has not yet been identified.
Prolonged exposure of SUMO-2/3 immunoblots revealed multiple discrete minor bands in the range of 100 kDa (Fig. 5). Most of these immunoreactive proteins were absent in the immunoblot probed with anti-SUMO-1 antibody, suggesting that a number of cellular proteins may become selectively modified by SUMO-2/3 as opposed to SUMO-1. However, one might point out that some of the high molecular mass bands (Ͼ120 kDa) were similar in size to the high molecular mass bands that appeared in the immunoblot probed with anti-SUMO-1 antibody. Although the identities of these proteins remain to be established, some might represent common substrates for SUMO-1 and -2/3 (i.e. mixed conjugation products including both SUMO-1 and -2/3). This observation argues for the possibility of cross-talk or convergence of the SUMO-1 and -2/3 modification pathways, at least with regard to some common substrates.
Nuclear foci detected by anti-SUMO-1 antibody have been colocalized with the nuclear matrix-associated, large multiprotein complex known as nuclear domain 10 (ND10), nuclear bodies, or promyelocytic leukemia protein oncogenic domains (11, 34 -36). The functions of ND10 are not well understood, but size, number, and composition appear to be regulated throughout the cell cycle and to respond to environmental stress, interferon, and infection of several DNA viruses. We found that the nuclear foci detected by anti-SUMO-2/3 antibody appeared to represent staining patterns exhibited in common with SUMO-1 (Fig. 5B). Currently, we observed that As 2 O 3 , which has been reported to affect the assembly/maintenance of ND10 (11), induced the accumulation of both SUMO-1 and -2/3 in the nuclear punctate structures (data not shown). These results suggest that SUMO-1 and -2/3 are components of ND10 and imply involvement of the SUMO-2/3 pathway in the assembly or function of ND10.
The data presented thus far indicate that although there is functional heterogeneity between the SUMO-1 and -2/3 subfamily members, it is currently unknown whether further functional heterogeneity might be present in the SUMO-2 versus SUMO-3 pathways. It will be important in the future to generate a monoclonal antibody that distinguishes between SUMO-2 and -3 subfamily members, but this is likely to be complicated by the high homology between SUMO-2 and -3.
In summary, we have generated specific antibodies to SUMO-2/3 and have found that a number of proteins exist in SUMO-2/3-conjugated forms. The combination of this antibody and anti-SUMO-1 antibody revealed several differences between the SUMO-1 and -2/3 modification pathways. First, SUMO-2/3 is present in greater abundance than SUMO-1, and there is a large pool of non-conjugated SUMO-2/3 in COS-7 cells. Remarkably, this pool of free SUMO-2/3 was promptly activated and incorporated into high molecular mass proteins by protein-damaging stresses such as heat shock. Second, Ran-GAP1 is preferentially modified by SUMO-1, but very poorly by SUMO-2/3 in vivo, and SUMO-1-modified RanGAP1 appears to localize more abundantly at the NPC/nuclear envelope than the little SUMO-2/3-modified RanGAP1 that may be present, if any. Collectively, these results imply that regulatory and functional heterogeneity is present among SUMO-1, -2, and -3 modification systems and suggest a role for SUMO-2/3 in the response to environmental stress. | 2018-04-03T00:11:24.508Z | 2000-03-03T00:00:00.000 | {
"year": 2000,
"sha1": "650b97acd53ba8768bc69be1df9b3a8a1ded871f",
"oa_license": "CCBY",
"oa_url": "http://www.jbc.org/content/275/9/6252.full.pdf",
"oa_status": "HYBRID",
"pdf_src": "Adhoc",
"pdf_hash": "d5761b4a16d4d3021b51a422363ace50d2181e78",
"s2fieldsofstudy": [
"Biology"
],
"extfieldsofstudy": [
"Medicine",
"Biology"
]
} |
21295947 | pes2o/s2orc | v3-fos-license | Zipper-like interaction between proteins in adjacent daughter cells mediates protein localization.
Protein localization is crucial for cellular morphogenesis and intracellular signal transduction cascades. Here we describe an interaction between two membrane proteins expressed in different cells of the Bacillus subtilis sporangium, the mother cell protein SpoIIIAH and the forespore protein SpoIIQ. We used affinity chromatography, coimmunoprecipitation, and the yeast two-hybrid system to demonstrate that the extracellular domains of these proteins interact, tethering SpoIIIAH to the sporulation septum, and directing its assembly with SpoIIQ into helical arcs and foci around the forespore. We also demonstrate that this interaction can direct proteins made in the same cell to active division sites, as when SpoIIQ is made in the mother cell, it localizes to nascent septa in a SpoIIIAH-dependent manner. Both SpoIIIAH and SpoIIQ are necessary for activation of the second forespore-specific transcription factor (sigma(G)) after engulfment, and we propose that the SpoIIIAH-SpoIIQ complex contributes to a morphological checkpoint coupling sigma(G) activation to engulfment. In keeping with this hypothesis, SpoIIIAH localization depends on the first step of engulfment, septal thinning. The SpoIIQ-SpoIIIAH complex reaches from the mother cell cytoplasm to the forespore cytoplasm and is ideally positioned to govern the activity of engulfment-dependent transcription factors.
Dynamic protein localization is crucial for bacterial development, signal transduction cascades, and the cell cycle. In recent years, microbial cell biologists have discovered tubulin-like filaments involved in cell shape and chromosome segregation (Carballido-Lopez and Errington 2003a,b;Shih et al. 2003;Gerdes et al. 2004), oscillating proteins that govern division site selection (Hu and Lutkenhaus 1999;Raskin and de Boer 1999a,b), chromosome segregation, and cellular differentiation (Glaser et al. 1997;Lin et al. 1997;Autret and Errington 2003), as well as localized proteins required for pathogenesis, cell division, development, and signal transduction cascades (for review, see Lybarger and Maddock 2001;Shapiro et al. 2002;Errington et al. 2003;Ryan and Shapiro 2003). Although bacterial cells lack membrane-enclosed compartments, a growing body of evidence suggests that the activity of signal transduction proteins can be efficiently compartmentalized by being confined to a specific subcellular address. For example, in Caulobacter crescen-tus, cell division results in the production of morphologically distinct daughter cells: the motile swarmer cell and the sessile stalked cell, which is the only daughter capable of immediately initiating a new cell cycle (Mc-Adams and Shapiro 2003;Quardokus and Brun 2003;Jacobs-Wagner 2004;Skerker and Laub 2004). These divergent cell cycles are controlled by two oscillating regulatory proteins that accumulate in opposite daughter cells: one represses chromosome replication and the cell cycle in the swarmer cell while the other promotes the cell cycle in the stalked cell (Holtzendorff et al. 2004). Therefore, it is clearly important to understand the spatial and temporal distribution of bacterial proteins as well as their biochemical properties.
Protein localization is also important in B. subtilis sporulation, during which the synthesis of an asymmetrically positioned septum produces a smaller forespore, which will become the spore, and a larger mother cell, which will lyse after contributing to spore development (for review, see Errington 2003). A variety of proteins involved in morphogenesis and intracellular signal transduction localize to the septum between these cells (Driks et al. 1994;Arigoni et al. 1995;Resnekov et al. 1996;Fawcett et al. 1998;Perez et al. 2000;Abanes-De Mello et al. 2002; van Ooij and Losick 2003; Rubio and Pogliano 2004) and in many cases disrupting localization has severe consequences for development (King et al. 1999).
In theory, localized membrane proteins could either be directly inserted into their target membrane domain, or be inserted elsewhere and diffuse to their correct location. It appears that both pathways operate during sporulation. In the forespore, even membrane proteins that are ultimately randomly distributed throughout the forespore membrane initially localize to the septum, suggesting, although not conclusively demonstrating, that membrane protein insertion is restricted to the forespore septum (Rubio and Pogliano 2004). In contrast, in the mother cell, it has been proposed that membrane protein insertion is nonlocalized , since mother cell membrane proteins engineered to be expressed before division are initially randomly distributed, but then relocalize from this nonlocalized pool to the septum van Ooij and Losick 2003). Both direct and random insertion require the capture of membrane proteins at the correct location to prevent their rapid diffusion, which could be achieved if they interact with another molecule that is stationary and localized.
Bacillus subtilis sporulation generates a unique bacterial cell that has two separate membrane-enclosed compartments, a consequence of engulfment (for review, see Errington 2003). During engulfment, the membrane of the mother cell migrates around the forespore, until the leading edge meets and fuses to release the forespore into the mother cell cytoplasm (Fig. 1). After engulfment, the forespore is bounded by two membranes: its cytoplasmic membrane and a second membrane derived from the mother cell, the outer forespore membrane. The generation of a cell with two separate membrane compartments could have important consequences for the localization of membrane proteins targeted to the outer forespore membrane and is a morphological checkpoint essential for the activation of two late transcription factors: G in the forespore and K in the mother cell (for review, see Kroos and Yu 2000;Errington 2003).
SpoIIQ is an attractive candidate for a protein that senses the completion of engulfment. This forespore-ex-pressed membrane protein is conserved in all endosporeforming bacteria (Stragier 2002), required for synthesis and activation of G , and under some conditions, for engulfment (Londoño-Vallejo et al. 1997;Sun et al. 2000). Previous work has shown that SpoIIQ localizes to the septum, tracks the engulfing mother cell membrane, assembles helical arcs and foci surrounding the forespore, and is degraded after engulfment (Rubio and Pogliano 2004). Interestingly, retention of SpoIIQ at the septum requires mother cell gene expression, suggesting that its extracellular domain interacts with that of a mother cell tether (Rubio and Pogliano 2004). This interaction between SpoIIQ and a mother cell tether provides a potential route for signaling between the mother cell and forespore that is necessary for G activation (Partridge and Errington 1993).
A second conserved group of proteins required for G activity is the proteins encoded by the spoIIIA operon (Kellner et al. 1996). This operon encodes eight proteins, one of which, SpoIIIAA, shows homology to the AAA family of ATPases, whereas the rest are predicted to be membrane proteins. To investigate the role of these proteins in G activation and determine if they interact with SpoIIQ, we initiated studies of their subcellular distribution, obtaining functional fusions between the protein encoded by the last gene in the operon, SpoIIIAH and the Flag epitope. We here demonstrate that SpoIIIAH is localized to the septum by a direct interaction between its extracellular domain and that of SpoIIQ and that the two proteins colocalize in arcs and foci surrounding the forespore. Furthermore, SpoIIIAH localization depends on engulfment, suggesting that this dynamic complex plays a central role sensing engulfment.
Localization of SpoIIIAH
The final gene in the spoIIIA operon is spoIIIAH, which encodes a protein predicted by HMMTOP (Tusnady and Simon 2001) and TM pred (Hofmann and Stoffel 1993) to have one membrane-spanning segment and a large extracellular domain. As the last gene in the spoIIIA operon, it was relatively simple to construct a Flag epitope fusion to the 3Ј-end of spoIIIAH and integrate a plasmid into the B. subtilis chromosome by a single recombination event. The resulting strain expressed SpoIIIAH-Flag (but not SpoIIIAH) from its native promoter without disrupting other genes in the spoIIIA operon. This strain produced wild-type levels of spores, indicating that SpoIIIAH-Flag was functional; fractionation data demonstrated that it was membrane-associated (data not shown). We localized SpoIIIAH-Flag using immunofluorescence microscopy and used the membrane stain FM 4-64 to correlate localization with engulfment. In sporangia that have initiated engulfment and have slightly curved septa, SpoIIIAH-Flag localized to the septum, often with discrete foci at the septum and faint mother cell fluorescence ( Fig. 2A, arrow). During engulfment, SpoIIIAH-Flag tracked the engulfing mother cell mem- Stages of sporulation and cell-specific gene expression. (A) Following synthesis of the sporulation septum, F becomes active in the smaller cell, the forespore, followed by activation of E in the larger mother cell. (B) During the first step of engulfment [(i) septal thinning] septal peptidoglycan (gray) is removed, starting at the septal midpoint. (C) Subsequently, the mother cell membrane migrates around the forespore (steps iiiii), meets (D) then fuses (E) at the forespore pole to release the forespore into the mother cell cytoplasm (step iv), at which time G and K become active in the forespore and mother cell, respectively.
brane as it moved around the forespore ( Fig. 2A, arrowhead). In engulfed sporangia, SpoIIIAH-Flag localized around the forespore in discrete foci (Fig. 2B, arrow). Thus, SpoIIIAH displays a dynamic localization pattern, first localizing to the septum, and then assembling foci as the engulfing membrane surrounds the forespore (Fig. 2H).
To determine if SpoIIIAH synthesized during growth could subsequently localize to the sporulation septum, we constructed a strain in which the xylose promoter controlled expression of SpoIIIAH-Flag (P xyl -spoIIIAH-Flag). This strain was grown in the presence of xylose, then washed and resuspended in sporulation medium without xylose to prevent further synthesis of SpoIIIAH-Flag. In cells without a sporulation septum, SpoIIIAH-Flag was present throughout the membranes (Fig. 2G, arrow), but once the sporulation septum was synthesized, the majority of SpoIIIAH-Flag was localized to the septum (Fig. 2G, arrowhead). SpoIIIAH-Flag remained localized after engulfment, forming disorganized foci in ∼50% of sporangia (Fig. 2G, double arrowhead). Thus, SpoIIIAH can reach the sporulation septum by diffusion from the cytoplasmic membrane.
SpoIIIAH localization depends on SpoIIQ, but not SpoIIIAA-AF or SpoIIIJ
SpoIIIAH is required to activate the second forespore transcription factor G after engulfment, as are SpoIIIJ, SpoIIQ, and the remaining SpoIIIA proteins. We were interested in determining whether localization of SpoIIIAH-Flag would be affected by the absence of any of these proteins, or by the absence of G itself. Localization of SpoIIIAH-Flag was not affected in strains with mutations in the gene encoding G (spoIIIG) (data not shown) or in spoIIIJ ( Supplementary Fig. 1); therefore, G -dependent proteins are not required for the stages of SpoIIIAH localization described here. In contrast, SpoIIIAH-Flag appeared randomly distributed throughout the mother cell membrane in the absence of SpoIIQ (Fig. 2C,D, arrows); with a slight enrichment at the engulfing membrane (Fig. 2D, arrowhead), likely due to the presence of a double layer of mother cell membrane formed as the leading edge migrates around the forespore (Fig. 2I). SpoIIIAH localized identically in the spoIIQ null and in the absence of forespore-specific gene expression ( Supplementary Fig. 1), suggesting that SpoIIQ is the primary forespore-specific localization determinant of SpoIIIAH.
To determine if SpoIIIAH localized in the absence of the first six SpoIIIA proteins (SpoIIIAA-AF), we introduced spoIIIAH-Flag into strains containing an EZϻTN(kan) insertion in the first gene in the operon, spoIIIAA (Materials and Methods), and with in-frame deletions in each spoIIIA gene. Immunoblot analysis indicated that SpoIIIAH-Flag reached similar levels in the spoIIIAAϻEZϻTN(kan) strain as in wild type (data not shown), supporting the existence of a second spoIIIAH promoter. Immunofluorescence microscopy showed that many early sporangia had normal SpoIIIAH localization (Fig. 2E, arrowhead), while others showed little septal localization (Fig. 2E, arrow). Later, most spoIIIAAϻ EZϻTN(kan) sporangia, as well as strains with in-frame deletions in spoIIIAA, spoIIIAB, spoIIIAC-AD, spoIIIAE, spoIIIAF, and spoIIIAG showed SpoIIIAH-Flag foci apparently identical to wild type (Fig. 2F, arrow). Thus, the spoIIIAAϻEZϻTN(kan) insertion delays the initial localization of SpoIIIAH to the septum, but ultimately SpoIIIAH assembles normally.
Evidence for a direct interaction between SpoIIQ and SpoIIIAH
SpoIIQ and SpoIIIAH each track the engulfing mother cell membrane, assemble helical arcs and foci, and are predicted to contain one transmembrane domain and a large extracellular domain. Although these proteins are in separate cells, it is possible that their extracellular domains directly interact within the septal space to mediate SpoIIIAH localization. To test this possibility, we used a yeast two-hybrid system (Finley and Brent 1994), fusing the hydrophilic C terminus of each protein (amino acids 25-218 for SpoIIIAH, amino acids 41-283 for SpoIIQ) to either LexA (a DNA-binding protein) or to the transcriptional activation domain of AB. Interaction between AB and LexA fusion proteins is indicated by increased -galactosidase activity in a yeast strain carrying lacZ preceded by eight LexA-binding sites. When SpoIIQ was fused to LexA and SpoIIIAH was fused to AB, the yeast strain produce ∼25-fold more -galactosidase activity than negative controls (Table 1). In the reciprocal experiment (SpoIIIAH-LexA and SpoIIQ-AB), a small but reproducible increase in -galactosidase activity was observed.
We used affinity chromatography to further investigate the interaction between the extracellular domains of SpoIIQ and SpoIIIAH. We first constructed a plasmid encoding a GST-SpoIIQ fusion protein, and bound GST-SpoIIQ or GST (as a negative control) to GST trap columns. Extracts from sporulating B. subtilis cells expressing either SpoIIIAH-Flag or FtsH-Flag (as a negative control) or a mixture of the two were loaded onto these columns and washed, and the eluates were subjected to immunoblot analysis to determine if SpoIIIAH-Flag was retained on the column. These experiments demonstrated that SpoIIIAH-Flag but not FtsH-Flag interacted with GST-SpoIIQ but not with GST itself (Fig. 3A). Importantly, the eluates included only a few bands ( Fig. 3A), one the same size as GST-SpoIIQ. Together with the yeast two-hybrid data described above, these experiments support a direct interaction between the extracellular domains of SpoIIIAH and SpoIIQ.
SpoIIQ and SpoIIIAH are present in the same membrane-bound complex in B. subtilis
We used coimmmunoprecipitation to demonstrate that SpoIIQ and SpoIIIAH interacted in living cells. Strains were constructed that expressed either GFP-SpoIIQ or MalF-GFP with SpoIIIAH-Flag, and membrane fractions were prepared and solubilized with 0.5% NP-40 (see Materials and Methods) and incubated with anti-Flag affinity gel. The eluates were subject to SDS-PAGE followed by Coomassie staining and Western blot analysis, probing the blots with GFP-specific antibodies. The results demonstrated that GFP-SpoIIQ but not forespore-expressed MalF-GFP copurified with SpoIIIAH-Flag (Fig. 3B). A comparison of the total amount of GFP-SpoIIQ in -3,5,10-14) or FtsH-Flag (lanes 4,6,7) or a mixture of the two (lanes 8,9) and loaded onto columns with GST-SpoIIQ (lanes 2,3, [6][7][8][9]13,14), or GST only (lanes 11,12), washed, and eluted. Total protein was detected by Coomassie staining (lanes 1-3), and the presence of Flag-containing fusion proteins by immunoblots (lanes 4-14). The arrowhead indicates the position of GST-SpoIIQ, the asterisk unknown SpoIIQ-binding proteins. Lane 1 contains extract from 16 µL of original culture, lanes 4 and 5 from 166 µL of cells, while lanes 3, 7, and 9 contain the binding proteins from between 1.8 and 3.6 mL of original culture volume. The decreased amount of protein loaded and the presence of Triton in the lane 5 sample could explain the decreased level of SpoIIIAH-Flag compared to lane 9.
Zipper-mediated protein localization the membrane fraction prior to purification demonstrated that the larger of the two GFP-SpoIIQ bands was almost quantitatively recovered from the affinity gel, indicating that this protein, but not the shorter product, was completely bound to SpoIIIAH. The presence of SpoIIIAH-Flag in the eluate (Fig. 3B, asterisk) was confirmed by immunoblot with Flag-specific antibodies (data not shown). Thus, SpoIIQ and SpoIIIAH are present in the same membrane-bound complex in B. subtilis cells. Importantly, the shared topology of these two pro-teins (N terminus in, C terminus out) and the fact that they are synthesized in different cells means that this complex reaches from the mother cell cytoplasm across the septal space (where their C termini interact) to the forespore cytoplasm.
SpoIIIAH is not the primary tether for SpoIIQ
Retention of SpoIIQ at the septum requires an unidentified mother cell protein, since in the absence of mother cell gene expression, SpoIIQ moves around the forespore ahead of the engulfing mother cell membrane (Rubio and Pogliano 2004). To test if SpoIIIAH was this tether, we introduced GFP-SpoIIQ into a ⌬spoIIIAG-AH strain. In wild type, GFP-SpoIIQ tracks the engulfing mother cell membrane, often in multiple foci similar to those seen for SpoIIIAH-Flag (Fig. 4A, arrow), while later in sporulation it is degraded to release cytoplasmic GFP ( Fig. 4F; Rubio and Pogliano 2004). Early in sporulation, 31% of ⌬spoIIIAG-AH sporangia showed normal SpoIIQ localization (Fig. 4B, arrow, double arrowhead), although 69% showed a small amount of GFP-SpoIIQ ahead of the engulfing membranes (Fig. 4B [arrowhead], G; Supplementary Table 1). This defect in early SpoIIQ localization is much less severe than occurs in mutants lacking all mother cell gene expression (spoIIGB), when SpoIIQ is uniformly distributed throughout the forespore membrane, moving ahead of the engulfing mother cell membrane (Figs. 4E [arrow], 2H). Thus, SpoIIIAH is not the primary tether for SpoIIQ. The effect of spoIIIA mutations on GFP-SpoIIQ localization could be increased by either inactivating spoIIIJ or using strains that also expressed wild-type SpoIIQ. While the spoIIIJ mutation had no discernible effect on GFP-SpoIIQ localization (Fig. 4C), spoIIIA spoIIIJ double-mutant sporangia showed increased GFP-SpoIIQ fluorescence ahead of the engulfing membranes compared to spoIIIA single mutants (Fig. 4D, arrowhead). We conclude that the absence of SpoIIIAH only slightly reduces the affinity of SpoIIQ for the sporulation septum, and that another mother cell protein is the primary SpoIIQ tether.
SpoIIIAH mediates localization of mother-cell-expressed SpoIIQ
If two extracellular protein domains interact only in opposing membranes rather than in the same membrane, their interaction could mediate localization to active division sites in symmetrically dividing cells. To determine if the interaction between SpoIIQ and SpoIIIAH could mediate septal localization when each protein was made in the same cell, GFP-SpoIIQ was expressed only in the mother cell, where it failed to complement the null mutation. In strains also expressing SpoIIIAH, GFP-SpoIIQ localized as a ring at the partial septa formed when division initiates at the second potential site of polar septation (Fig. 4I,K). However, in strains lacking SpoIIIAH, GFP-SpoIIQ was delocalized, with a slight enrichment at the second division site of some sporangia and strong cytoplasmic fluorescence (Fig. 4J,M). Thus, SpoIIIAH can mediate localization of mother-cell-expressed SpoIIQ to active sites of cell division and protect it from degradation.
Colocalization of SpoIIIAH and SpoIIQ
If the extracellular domains of SpoIIQ and SpoIIIAH interact, they should colocalize at least until after engulfment, when SpoIIQ is cleaved. To test this, we used immunofluorescence microscopy to colocalize SpoIIQ-myc and SpoIIIAH-Flag. During engulfment, SpoIIQ-myc substantially colocalized with SpoIIIAH-Flag, and both formed foci along the engulfing membrane (Fig. 5A, arrow). After engulfment, many SpoIIQ-myc foci colocalized with SpoIIIAH-Flag foci (Fig. 5B, arrow), although some foci did not colocalize (Fig. 5B, arrowhead), perhaps due to loss of cell structure during preparation of the cells for immunofluorescence (which requires fixation and lysozyme treatment to permeabilize the cells). Some sporangia showed greatly reduced SpoIIQ-myc immunostaining and clear SpoIIIAH-Flag foci (Fig. 5B, double arrowhead), likely because SpoIIQ-myc (or the myc epitope) is unstable during sporulation (Rubio and Pogliano 2004). Thus, the extracellular domains of SpoIIIAH and SpoIIQ colocalize to a significant extent, although SpoIIIAH appears to persist during sporulation, whereas the extracellular domain of SpoIIQ-myc is less stable.
SpoIIIAH localization is inhibited by septal peptidoglycan
SpoIIIAH and SpoIIQ are ideally suited to couple G activity to engulfment, as they interact across the septal space and move around the forespore during engulfment. If so, then engulfment mutants might alter SpoIIIAH localization by preventing its interaction with SpoIIQ. Three mother cell proteins (SpoIID, SpoIIM, and SpoIIP) are required for septal thinning, when peptidoglycan is degraded from the septal midpoint to the edges (Fig. 1, step i), and for membrane migration (Abanes-De Mello et al. 2002). In the absence of SpoIID, SpoIIM, or SpoIIP, septal thinning is incomplete, and the growing forespore pushes through the septum, and bulges into the mother cell cytoplasm (Fig. 6K). Septal thinning is also defective Figure 5. Colocalization of SpoIIIAH-Flag and SpoIIQ-myc. Immunofluorescence microscopy was used to colocalize epitope fusions to the C termini of SpoIIQ (myc, green) and SpoIIIAH (Flag; false-colored red; strain KP868), and the membranes stained with FM 4-64 (false-colored blue), at t 2.5 (A) and t 3.5 (B). In late sporangia with ␣/ SASP/DNA rings (data not shown), SpoIIQ immunostaining was greatly reduced, while SpoIIIAH remained in foci (double arrowhead). (C) Observed colocalization pattern of the C termini of SpoIIQ (blue circles) and SpoIIIAH (green circles). Bar, 2 µm. in spoIIB spoVG strains (Margolis et al. 1993;Perez et al. 2000). Electron micrographs show that in septal thinning mutants, the septum but not the bulge contains peptidoglycan (Illing and Errington 1991;Margolis et al. 1993;Smith et al. 1993;Frandsen and Stragier 1995;Perez et al. 2000).
Most spoIIB spoVG double-mutant sporangia with no bulge showed little or no localization of SpoIIIAH-Flag (Fig. 6C, arrow; scored in Supplementary Table 2). Once the bulge had formed (Fig. 6C,D [double arrowheads], K), SpoIIIAH-Flag localized to the bulge (Fig. 6C,D; Supplementary Table 2). The SpoIIIAH assemblage in the bulge looked similar to that seen in wild type, as it was spherical and had multiple foci (Fig. 6D, double arrowhead); however, the sphere was restricted to the bulge, rather than enclosing the forespore. Thus, SpoIIIAH localization spatially correlated with the absence of peptidoglycan, as in the spoIIB spoVG mutant, it localized specifically to bulges, which have little peptidoglycan.
The spoIID-and spoIIP-null mutants also had clear localization defects early in sporulation, with increased immunostaining in the mother cell cytoplasmic membrane (Fig. 6E,G, arrows; Supplementary Table 2). However, later in sporulation, this nonlocalized material decreased, and SpoIIIAH localized mostly to the bulge ( Fig. 6F-H, double arrowheads; Supplementary Table 2). The spoIID spoIIP double mutant eliminates bulge formation, perhaps by decreasing residual septal thinning . It also showed a more persistent SpoIIIAH localization defect, with immunostaining remaining in the mother cell cytoplasmic membrane even late in sporulation (Fig. 6I,J, arrow). Together with the preferential localization of SpoIIIAH to the bulge, these results suggest that a peptidoglycan-free region of the septum facilitates the assembly of the SpoIIQ-SpoIIIAH complex.
Discussion
Most bacterial cells contain one membrane system, the cytoplasmic membrane, with localized membrane pro- teins being restricted to a particular region of the membrane, such as the cell pole or division site. Within such a cell, diffusion of these proteins away from their appropriate cellular location must be prevented by either a local reduction in membrane fluidity or interaction with another macromolecule that tethers the protein to the appropriate location. The cell wall provides an attractive candidate for tethering cell surface proteins, because it is stationary and encodes positional information. Indeed, the cell-wall-binding domain of Listeria monocytogenes murein hydrolase Ply118 mediates its localization to the septum and cell poles (Loessner et al. 2002). Here we describe a second simple mechanism to restrict membrane proteins to their appropriate subcellular address: an interaction between extracellular domains of proteins in adjacent daughter cells, in this case the mother cell protein SpoIIIAH and the forespore protein SpoIIQ (Fig. 7A-D). This interaction localizes SpoIIIAH to the sporulation septum, and without SpoIIQ, SpoIIIAH is uniformly distributed throughout the mother cell membrane. Curiously, although mother cell gene expression is essential for SpoIIQ localization (Rubio and Pogliano 2004), the absence of SpoIIIAH only slightly affects SpoIIQ tethering, indicating that another mother cell protein must be the primary tether for SpoIIQ. This interaction between proteins in adjacent cells allows each protein to remain localized to the septum during engulfment. We also propose that such interactions could me-diate localization to division sites in growing cells (Fig. 7E), since when SpoIIQ and SpoIIIAH are made in the same cell, their interaction localizes SpoIIQ to invaginating septa (Fig. 4K). We propose that the binding sites of these proteins are positioned such that interaction can occur only at site of membrane invaginations (Fig. 4L). The initial membrane insertion of localized proteins could either occur at the site where the proteins will ultimately reside (direct insertion) or throughout the membrane (random insertion) with proteins later diffusing to the appropriate location. In the forespore, random membrane proteins, such as Escherichia coli MalF, are initially localized to the septum, later diffusing throughout the forespore membrane, consistent with the direct insertion of forespore proteins into the septum (Rubio and Pogliano 2004). In the mother cell, we and others have demonstrated that sporulation-specific membrane proteins can reach the septum from a presynthesized and nonlocalized pool, van Ooij and Losick 2003), as would be required if membrane protein insertion occurred randomly. However, because membrane protein localization in bacteria requires either a tether, a diffusion barrier, or degradation of nonlocalized proteins, and because nonlocalized membrane proteins freely diffuse, even a protein normally directly inserted should be capable of localization by diffusion from a nonlocalized pool. Distinguishing between directed and random insertion will ultimately require localizing the site After the final step of engulfment, membrane fusion, SpoIIQ is degraded to yield a cytoplasmic N-terminal product (blue shading), while SpoIIIAH remains in foci and G is activated. (E) Interaction between extracellular domains could also localize proteins in symmetrically dividing cells, if the proteins only interact in opposing membranes (such as those formed during division) but not when adjacent to one another in the cytoplasmic membrane. (F) Enlarged view of the septum prior to septal thinning (as in the cell shown in A), showing the peptidoglycan (PG) between the forespore membrane (FM) and the mother cell membrane (MM). Interaction between SpoIIQ ("IIQ", blue) and SpoIIIAH ("IIIAH", green) is inhibited by septal peptidoglycan. (G) After septal thinning the proteins interact, mediating SpoIIIAH localization (as in B,C). (H) After membrane fusion (as in D), SpoIIQ is degraded by a protease (red pacman) (Rubio and Pogliano 2004) and G is activated.
at which individual proteins engage the membrane insertion apparatus.
SpoIIIAH and SpoIIQ are both required to activate the late forespore transcription factor, G , which is synthesized by the early forespore transcription factor, F but activated only after engulfment (for review, see Rudner and Losick 2001;Errington 2003). Once active, G directs its own synthesis, resulting in a positive feedback loop that allows high levels of G activity. Multiple levels of regulation are therefore required to keep G activity in check during vegetative growth and sporulation. Expression of spoIIIG (which encodes G ) is likely subject to post-transcriptional control, since there are differences in the ability of transcriptional and translational spoIIIG-lacZ fusions to be expressed by F in vivo and in vitro (Sun et al. 1991). Expression of spoIIIG also re-quires both mother cell gene expression and SpoIIQ (Partridge and Errington 1993;Sun et al. 2000), suggesting that signaling between the forespore and mother cell is required for G synthesis. In contrast, spoIIIA mutants, and mutants that fail to complete engulfment, show almost normal levels of spoIIIG expression (Kellner et al. 1996;Sun et al. 2000), indicating that the SpoIIIA proteins are more specifically required for G activation.
SpoIIIAH and SpoIIQ coassemble multiple foci and arcs that ultimately enclose the forespore in an icosahedron-like structure (Rubio and Pogliano 2004). Our data suggests that this complex is ideally suited to couple activation of late transcription factors to engulfment. First, assembly of the SpoIIIAH-SpoIIQ complex is inhibited by septal peptidoglycan (Fig. 7F-H), which is removed during the first step of engulfment (Abanes-De Mello et al. 2002). Second, given the interaction between the extracellular domains of SpoIIQ and SpoIIIAH, at least until SpoIIQ is degraded (Rubio and Pogliano 2004), the complex reaches from the mother cell cytoplasm across the septum to the forespore cytoplasm, and is thus well positioned to transmit signals from one cell to the other. It will be interesting to determine more precisely the structure of the SpoIIIAH-SpoIIQ complex, and if other proteins involved in cell-cell communication localize to this structure.
spoIIIAG-H and spoIIIJ-jag deletions
PCR amplification used primers O1 and O2 (726-bp fragment upstream of spoIIIAG) and O3 and O4 (705-bp fragment downstream of spoIIIAH); the fragments were digested and cloned to 5Ј and 3Ј of the pXJ52 kan gene. The plasmid was transformed into PY79, integrated by double recombination, replacing spoIIIAG and spoIIIAH with kan, yielding KP896. PCR amplification using primers O5 and O6 (524-bp fragment upstream of spoIIIJ) and O7 and O8 (498-bp fragment downstream of jag); the fragments were cloned to 5Ј and 3Ј of the pXJ3 chloramphenicol cassette (cat). The plasmid was transformed into PY79, integrated by double recombination, replacing spoIIIJ and jag coding regions with cat, yielding KP892.
Construction of spoIIIAH-Flag, ftsH-Flag, and P xyl -spoIIIAH-Flag SpoIIIAH and FtsH were Flag epitope tagged by amplifying the 3Ј-end of each gene (spoIIIAH-O9 and O10; ftsH-O21 and O22) to insert the Flag epitope before the stop codon of each gene. PCR products were cloned into pCR2.1 (Invitrogen) and the cat cassette from pMS38 (Zilhao et al. 2004) excised with BamHI and BglII and cloned into BamHI-digested pCR2.1. Transformation of the resulting plasmid into MB24 integrated the plasmids via a single recombination event at spoIIIAH or ftsH. amyEϻP xyl -spoIIIAH-Flag was constructed by PCR amplification of spoIIIAH using primers O11 and O12 and the 3Ј-end of spoIIIAH cloned adjacent to the BamHI site on pCR2.1. The gene was excised using BamHI, and inserted into pX (Kim et al. 1996), placing the 5Ј-end of spoIIIAH adjacent to P xyl . The plasmid was transformed into MB24 amyEϻerm and integrated at the amyE gene by double recombination, replacing amyEϻerm with amyEϻP xyl -spoIIIAH-Flag. SpoIIIAH-Flag synthesis was induced by adding 1% xylose to the growth medium.
Construction of mother-cell-expressed GFP-SpoIIQ
The spoIIQ coding region was amplified (using the primers described in Rubio and Pogliano 2004) and cloned into EagI-digested pMDS14 (Sharp and Pogliano 2002), and the resulting plasmid was transformed into the spoIIQ-null mutant strain KP575.
Functionality of GFP-SpoIIQ
Although GFP-SpoIIQ supports efficient engulfment and spore formation, and localizes identically to native SpoIIQ using immunofluorescence, expressing GFP-SpoIIQ together with wildtype SpoIIQ eliminated the ability of GFP-SpoIIQ to form foci ( Supplementary Fig. 2). In a strain also deleted for spoIIIAH, 84% of sporangia showed faint GFP-SpoIIQ fluorescence ahead of the engulfing membranes ( Supplementary Fig. 2; Supplementary Table 1). We suggest that GFP-SpoIIQ is slightly less functional than the wild-type protein, which out-competes GFP-SpoIIQ for assembly into foci and interaction with the SpoIIQ tether.
Affinity chromatography
A plasmid encoding GST-SpoIIQ was constructed using primers O19 and O20 to amplify the region of spoIIQ encoding the extracellular domain, and cloning this fragment into the EcoRI and XhoI sites of the GST-encoding pGEX-4T-3 (Amersham). DH5␣ carrying this plasmid was grown to OD 600 0.5-0.8 and induced with 1 mM IPTG. One-hundred milliliters of culture was concentrated 25-fold in PBS, passed through a French press, and centrifuged at 4000g for 10 min. The cleared lysate was loaded onto GSTrap columns (Amersham) and washed with PBS. B. subtilis strains expressing either SpoIIIAH-Flag of FtsH-Flag were sporulated by resuspension, and 100 mL of the cultures was concentrated 25-fold in PBS plus 1% Triton X-100 and 0.1 mg/mL lysozyme and passed through a French press twice. The lysates were centrifuged at 4000g for 10 min and loaded, in three column volumes, onto GST trap columns prebound with either GST or GST-SpoIIQ and incubated for 90 min. The columns were washed with PBS plus 1% Triton X-100 and eluted with 50 mM Tris and 10 mM glutathione (pH 8), and 0.5-mL fractions were collected. For immunoblot analysis in Figure 3, 6 µL of eluate was loaded on the gel (corresponding to 1.8-3.6 mL of culture). Extract corresponding to 166 µL of culture was loaded for immunoblot analysis of whole cell proteins, 16.6 µL for Coomassie blue staining.
Coimmunoprecipitation
The appropriate B. subtilis strains were induced to sporulate by resuspension and fractionated; 25 mL of culture was harvested at t 2.5 , and fractionated essentially as described , except the lysate was ultracentrifuged at 100,000g for 1 h, and the membrane pellet was resuspended in 1 mL of Buffer B (50 mM Tris at pH 8.0, 150 mM KCl, and 10% glycerol). A final concentration of 0.5% NP-40 was added, and the samples were incubated on ice for 1 h with gentle mixing every 10 min. The samples were centrifuged at 4°C at 16,100g for 15 min, and 0.5 mL of supernatant was added to 20 µL of anti-Flag M2 affinity gel (Sigma) pre-equilibrated in Buffer B. Samples were incubated overnight at 4°C with gentle rolling, and washed three times with 0.5 mL of Buffer B plus 0.5% NP-40 at 4°C. The bound proteins were eluted with 250 µL of 1× SDSloading buffer (without reducing agent) at room temperature. ME was added to 0.355 M, and 25 µL of eluate was loaded for SDS-PAGE and Western blot analysis. Mouse anti-GFP monoclonal antibody (Roche) was used to detect GFP-SpoIIQ on Western blots. | 2018-04-03T04:33:33.547Z | 2004-12-01T00:00:00.000 | {
"year": 2004,
"sha1": "6b44576bd2dfaf950e2c06aba9c3abaf46613ac1",
"oa_license": null,
"oa_url": "http://genesdev.cshlp.org/content/18/23/2916.full.pdf",
"oa_status": "GOLD",
"pdf_src": "Adhoc",
"pdf_hash": "b11660c0b2bd80829ab3b1e2bb1ae642168fefc5",
"s2fieldsofstudy": [
"Biology"
],
"extfieldsofstudy": [
"Medicine",
"Biology"
]
} |
259605707 | pes2o/s2orc | v3-fos-license | Current distribution of medical colleges in India and its potential predictors: A public domain data audit
ABSTRACT Background: A teaching hospital or medical college may help provide better health care delivery to the people of the vicinity. Hence, building new medical colleges and upgrading existing hospitals to teaching hospitals are being implemented in India. Objective: This study aimed to observe the current distribution of medical colleges in Indian states and find correlation with area, population, and net state domestic product (NSDP). Methods: We collected data from public domain websites provided by government agencies. The State-wise number of government and private medical colleges and their annual intake were obtained from the National Medical Commission website. The state-wise number of medical colleges, geographical area, and population were calculated as the percentage of total Indian colleges, area, and population, respectively. Spearman’s correlation was calculated to find any correlation of colleges and annual intake versus parameters such as area, population, and NSDP. Results: India has a total of 612 [321 (52.45%) government-run and 291 (47.55%) private] medical colleges. Tamil Nadu (70), Uttar Pradesh (67), Karnataka (63), Maharashtra (62), and Telangana (34) are the top five states with 296 (48.37%) medical colleges. States and union territories such as Karnataka, Kerala, Maharashtra, Puducherry, Tamil Nadu, and Telangana have higher medical colleges, and states such as Assam, Bihar, Odisha, Madhya Pradesh, Rajasthan, and Uttar Pradesh have lower medical colleges when compared with their population percentages. There was significant positive correlation of number of medical colleges with area (rs = 0.769, P < 0.0001), population (rs = 0.91, P < 0.0001), and NSDP (rs = 0.91, P < 0.0001). Conclusion: The current distribution of medical colleges in India is clustered over some states. Although geographical area and population are major predictors of medical colleges in Indian states, a more population-based balanced distribution of medical colleges would help distribute quality health care to the majority of the population.
Introduction
The hospitals and other health facilities along with their trained manpower make a health system to provide health-related services to the population of a particular area.In India, the health facilities are operated by both the government and private organizations.As all the medical colleges are attached to a tertiary care hospital, medical colleges are also run by
Current distribution of medical colleges in India and its potential predictors: A public domain data audit
both the government and private organizations.For the proper functioning of a health care system, sufficient numbers of physicians, nurses, pharmacists, social workers, and community workers are required.A medical college can teach and train physicians, nurses, and paramedical staff as their core curricular functions along with training social and community level health workers. [1]In addition, medical institutions help in conducting research for a better future.
To date (July 30, 2022), there are a total of 612 medical colleges in India that have the capability of educating 91,927 medical graduates in a year.Hence, India is heading toward producing a huge number of medical graduates that would surely increase the doctor-population ratio further.The World Health Organization suggested that the doctor-population ratio has already been achieved by India in 2018. [2]However, two aspects are being criticized -the unequal growth of institutions and the inadequate quality of medical education.
The unequal distribution of medical colleges may be because of several factors such as feasibility, fund, land acquisition, political factors, interest from private players, and so on. [3]or producing a huge number of doctors, many a time, both the government and private organizations may provide sub-standard infrastructure and lower numbers of manpower to the medical colleges. [4]study by Sabde et al. [5] found that a higher medical college density and a higher number of medical student intakes per year may help decrease the maternal mortality rate, which is one of the most important health care delivery indicators.Hence, it is obvious that although the colleges may have lower than adequate manpower and infrastructure and limited research facilities, upgrading or building a hospital with a teaching facility would help build a positive impact on public health.
In this context, we conducted this study to find the percentages of medical colleges according to Indian states and the relation with percentage of population, percentage of area, net state domestic product (NSDP), literacy rate, and maternal mortality rate (MMR).The finding of the study would add updates to the current knowledge of distribution of medical colleges in India according to the factors of interest.
Materials and Methods
This was a cross-sectional observational study involving the collection and analysis of data available from public domain websites.No sensitive information was collected or disseminated in this research article.Hence, ethical clearance is not necessary for this study.
The study was conducted in July 2022, and the data were collected within a span of 5 days -July 25 to July 30, 2022.We collected data from available government agencies that provide the data on public domain websites.The numbers of total colleges, colleges managed by government or government-sponsored societies or trusts, private firms, private educational trusts, and private society were obtained from the National Medical Commission (NMC) website (www.nmc.org.in). [6]The area, population, rural and urban population, and literacy rate were obtained from the Ministry of Statistics and Program Implementation (www.mospi.gov.in). [7]The per capita NSDP was obtained from the Reserve Bank of India (www.rbi.org.in). [8]The MMR was obtained from the Press Information Bureau (www.pib.gov.in). [9]atistical analysis Data were presented with numbers, percentages, median, and inter-quartile ranges.The normality of the data was tested by Shapiro-Wilk test.As the data were not following a normal distribution, non-parametric tests were used in inferential statistics. [10]For expressing the percentage of a variable in a state, the number was divided by the national total and expressed as a percentage.Spearman's correlation coefficient was calculated for finding a correlation between two continuous variables.We used Microsoft Excel 2010 and GraphPad Prism 6.01 for statistical calculations.We used a free spreadsheet template (obtained from www.indzara.com)for generating the Indian state-wise heat map presented in this article.For all statistical tests, a P value <0.05 was considered statistically significant.
Results
India has a total of 612 medical colleges including All India Institute of Medical Sciences.Among the colleges, 321 (52.45%) are run by the government or government-sponsored trust or society and 291 (47.55%) colleges are managed by private agencies including educational trusts and societies.Indian states have a median of 10 (Q1-Q3 = 1.5-29) medical colleges with 8 (Q1-Q3 = 1-13.5)government and 4 (Q1-Q3 = 0.5-12) private colleges.Indian state-wise distribution of government-run and private medical colleges is shown in Figure 1.
Among the 28 Indian states, all states excluding Nagaland have at least one teaching hospital.Among the eight union territories, Lakshadweep does not have a medical college, and data for Ladakh are to be updated as previously Ladakh was with Jammu and Kashmir.The state-wise percentage of the population and the percentage of medical colleges are shown in Figure 3.The top five states that have higher population percentages (in comparison to the percentage of medical colleges) are Uttar Pradesh, Bihar, West Bengal, Madhya Pradesh, and Rajasthan.States such as Tamil Nadu, Karnataka, Telangana, Kerala, and Puducherry have a higher percentage of medical colleges than population percentages.There was a positive correlation of 0.91 (95% CI: 0.82-0.97)between the percentage of the population and medical colleges, P < 0.0001.
The state-wise percentage of the area and the percentage of medical colleges are shown in Figure 4.The top five states with a higher percentage (in comparison with the percentage of area) of medical colleges are Tamil Nadu, Karnataka, Kerala, Uttar Pradesh, and Telangana.States such as Rajasthan, Madhya Pradesh, Odisha, Arunachal Pradesh, and Chhattisgarh are the top five states which are having higher areas but a lower number of medical colleges.There was a positive correlation of 0.769 (95% CI: 0.57-0.88)between the percentage of the area and medical colleges, P < 0.0001.
The correlation of the number of medical colleges and the number of annual student intake versus area, total population, urban population percentage, rural population percentage, population density, NSDP, literacy, and MMR are shown in Table 1.The overall number of medical colleges was positively correlated with the area, population, and NSDP.There was a negative correlation of number of medical colleges with the literacy rate of the state.The annual intake was positively correlated with the area, population, population density, and NSDP.
Discussion
India had a steep rise in the number of medical colleges in the past decade with a steeper peak in recent 2-3 years.In 2019, there were a total of 494 medical colleges.In 2022 (July), the number increased to 612 medical colleges including AIIMS.Hence, in the past 3 years, India had built new medical colleges with approximately 24% growth. [5]However, the distribution is not uniformly planned or executed in all the states and union territories.There may be several potential underlying factors for this. [11]Along with building new medical colleges, the Indian government has stressed upgrading tertiary care hospitals to teaching hospitals.Hence, in many a place, it is not possible to upgrade the hospitals to medical colleges within a short time. [12]The number of private medical colleges is also increasing day by day because of the higher demand for this professional course and the perceived scope of jobs among the aspirants.However, quality education is frequently not provided by private medical colleges.The major reason for building a private medical college is to "sell" the medical education.Hence, profit is an important aspect of private institutions, and the owner would try to cut the cost for a higher profit. [13]The previous Medical Council of India and the current NMC are trying to implement several quality checks and are coming up with strict regulations from their end to increase the quality of education in India. [14]ilding new college or upgrading tertiary care hospitals to teaching hospitals should always be planned according to geographical area and population.Although we found that the number of medical institutions and the annual intake of all the medical colleges in the states were positively correlated with the Each year, approximately 91,927 medical graduates would possess an undergraduate medical education degree and get their license after a rotatory internship.Despite this high number of trained doctors, medical colleges suffer from faculty and physician deficiency.It may be because of the lower absorption of medical graduates in the existing health care settings. [16]On one hand, the healthcare setting is having a shortage of doctors, and on the other hand, a fair number of doctors remain jobless after becoming graduates.The craze for a super specialty has crippled the system of family medicine and the primary care system. [17]However, it is high time to re-think about the number of doctors passing each year.After some years from now, severe job scarcity would be inevitable as the output would be higher than the absorption.In the coming days, the stakeholder may area and total population, the perfect linear correlation is yet to achieve.However, it is found that the percentage of urban population and rural population is not determining the number of colleges.Now, medical colleges are being built in smaller cities and villages.However, still, there is a need for more teaching hospitals for a wider coverage to provide better health care to the rural population.States such as Rajasthan, Madhya Pradesh, Odisha, Arunachal Pradesh, and Chhattisgarh still need more medical colleges in remote or difficult regions.In addition, highly populous states such as Uttar Pradesh, Bihar, West Bengal, Madhya Pradesh, and Rajasthan further require medical colleges as the current number of medical colleges is not adequate to cater the patients with optimum service with limited infrastructure and manpower. [15]hink about building colleges in rural areas with a lower number of students where limited faculty members can train them with quality education.This would solve the problems of availability of medical colleges in rural settings, can provide quality medical education, and can train a limited number of students from those areas.
This study has some limitations.The data were collected at a particular point in time.Hence, in a near future, the result may not be extrapolated.The data about the population, rural and urban percentage, NSDP, literacy, and MMR are of different times.However, we had no other option but to get updated data of a particular time.Hence, the study result would be interpreted with caution.
Conclusion
The distribution of medical colleges in India is not following a uniform distribution according to geographic area and population and is clustered over some states.Some states are having higher percentages of medical colleges, and some are having lower percentages when compared to the percentage of the population of the state.Hence, establishing medical colleges with population-based balanced distribution may be considered by the stakeholders.Special stress should be on the number of rural colleges, limited intake, providing quality medical education, and train undergraduates to become family medicine ready professionals.
Financial support and sponsorship
Nil.
Figure 1 :Figure 3 :Figure 2 :
Figure 1: State-wise number of government-run and private medical colleges in India (data as on July 30, 2022)
Table 1 :Figure 4 :
Figure 4: State-wise comparative percentage of medical colleges and area (data as on July 30, 2022) To date (July 30, 2022), the data for Ladakh are not available on the NMC website.The top five states, Karnataka (20), and West Bengal (20).These five states have a total of 142 (44.24%) government medical colleges.In comparison, the top five states with private medical colleges are Karnataka (43), Maharashtra (33), Tamil Nadu (32), Uttar Pradesh (32), and Telangana (23).They have 56.01% of the national share of private medical colleges. | 2023-07-11T16:28:51.465Z | 2023-06-01T00:00:00.000 | {
"year": 2023,
"sha1": "66a77bf0690bdfb886119d7854d81d974591c749",
"oa_license": "CCBYNCSA",
"oa_url": "https://doi.org/10.4103/jfmpc.jfmpc_1558_22",
"oa_status": "GOLD",
"pdf_src": "PubMedCentral",
"pdf_hash": "b66148c0ff9a9b8aebd951b016b85dd8f961bf05",
"s2fieldsofstudy": [
"Medicine"
],
"extfieldsofstudy": []
} |
5628182 | pes2o/s2orc | v3-fos-license | The effect of Tributyltin on thyroid follicular cells of adult male albino rats and the possible protective role of green tea: a toxicological, histological and biochemical study
Introduction Tributyltin is one of the important and wide-spread persistent organic contaminants that accumulate in the food chain. It is suspected to cause endocrine-disrupting effects in mammals, due in part to its possible transfer through marine food chains and to the consumption of contaminated seafood. Aim of the work Was to study the possible toxic effect of Tributyltin on thyroid follicular cells of adult male albino rats and to evaluate the possible protective role of green tea. Material and methods Forty-five adult male albino rats were included and randomly divided into 3 equal groups: a control group (Group I); Group II: received tributyltin chloride (TBT) dissolved in corn oil orally in a dose of 5 mg/kg for 30 days. Group III: received tributyltin chloride in the same dose with concomitant oral administration of green tea extract for 30 days. At the end of the experiment, the animals were sacrificed and blood samples were subjected to hormonal assay for T3, T4 and TSH levels. Malondialdehyde and reduced glutathione were assessed. The thyroid tissue was processed for histological and ultrastructure examination. The colloid area of thyroid follicles was evaluated morphometrically and statistically analyzed. Results A significant decrease in T3 and T4 levels and serum reduced glutathione in the group II when compared with the other groups. Furthermore, a significant increase in serum Malondialdehyde and TSH levels was recorded in group II treated group by comparison to the other two groups. Histopathological and ultrastructural changes of thyroid gland follicles were detected in tributyltin treated rats; the follicular cells appeared swollen and vacuolated. Epithelial stratification was noticed in some foci with excessive vacuolation of the colloid. Dilated rough endoplasmic reticulum filled with flocculent material and increased number of lysosomes were also detected together with variation in shape and size of the nuclei. A marked improvement in the histological features of thyroid follicles was noticed in group III. Conclusion Tributyltin induces oxidative stress in rats as well as anti-thyroid effect. The green tea extract is useful in combating tissue injury that is a result of tributyltin toxicity.
Introduction
Exposure to endocrine disrupting chemicals grabs more attention nowadays and represents international concern of many national and international health organizations, as well as being a political issue in various countries (Acerini & Hughes, 2006).
According to the World Health Organization (WHO), an endocrine disruptor is defined as "…an exogenous substance or mixture that modulates function(s) of the endocrine system and consequently causes adverse health effects in an intact organism, or its progeny, or (sub)populations" (Damstra et al., 2002).
Organotin compounds (OT), as tributyltin (TBT) and triphenyltin (TPT), are prevalent contaminants that have been widely used as biocides, agriculture fungicides and wood preservatives. Furthermore, they have been utilized as disinfecting agents in circulating industrial cooling waters, as well as, antifouling paints for marine vessels (Antizar-Ladislao, 2008).
Even though the usage of Tributyltins has been banned since 2008, they continue to persist in the environment, leading to severe contamination of the ecosystems as well as its accumulation in biological tissues (Horiguchi, 2012).
TBT at relatively high concentrations as hundreds of nanomoles has been found in human blood (Antizar-Ladislao, 2008) and the lipophilicity of organotin compounds favors their toxicity at membrane level, as well as disrupting diverse biological processes, involved in the endocrine, (Sharan et al., 2013) the immune, (Brown & Whalen, 2015) and nervous systems (Dong et al., 2006).
Moreover, TBT are reported to be a perilous factor for cardiovascular disease impairing the coronary vascular reactivity to estradiol, (dos Santos et al., 2012) as well as, altering aorta morphology and functionality (Rodrigues et al., 2014).
Accumulating data suggests that TBT can act as an endocrine disruptor. TBT has been known to produce imposex (a superimposition of male features in females) in marine gastropods through inhibition of aromatase enzyme that converts androgen to estrogen (Sousa et al., 2009;Lima et al., 2011). In mammals, nanomolar concentrations of tributyltin disturbed steroidogenesis (Yamazaki et al., 2005). Organotin compounds as endocrine disruptors are indicated as a possible cause of congenital hypothyroidism (Adeeko et al., 2003).
Recently, Tributyltin toxicity is considered as a precipitating factor for transgenerational obesity by disturbing the levels of key hormones linked to energy homeostasis (Nicole, 2013;Chamorro-García et al., 2013).
TBT has also been reported to be an environmental risk factor for Parkinson's disease, by inhibiting dopamine biosynthesis and enhancing L-DOPA-induced cytotoxicity (Kim et al., 2007).
Thyroid hormones are of crucial importance for the normal function of nearly every organ, as they are involved in normal brain development, control of metabolism, and many other substantial aspects of normal adult physiology. Therefore, adverse effects may impact development, metabolism, or adult physiology if there are changes in the function of the thyroid gland or intervention with the ability of thyroid hormone to exert its action (Brent, 2012). However, the mechanism(s) by which TBT induces toxicity have not been fully established.
Some researchers reported that the organotin can induce oxidative damage to mice cells both in vivo and in vitro . Oxidative stress, which results from the overproduction of reactive oxygen species (ROS), causes cellular oxidative injury such as lipid peroxidation, protein oxidation, and DNA damage (Ishihara et al., 2012).
Green tea, a popular well-known beverage worldwide, has captured considerable attention for its scientifically beneficial effects on human health. Most of its significant effects are attributed to its polyphenolic flavonoids, known as catechins (epicatechin, epigallocatechin, epicatechin-3gallate, as well as the major flavonoid (−)-epigallocatechin-3-gallate). The most pervasive recognized properties of green tea are their antioxidant activities, arising from their ability to scavenge reactive oxygen species (Rietveld & Wiseman, 2003;Clement, 2009).
Taken the above-mentioned considerations, the present study is conducted specifically, to investigate the probable toxic effect of Tributyltin chloride on thyroid follicular cells of adult male albino rats, and the possible impact of reactive oxygen species (ROS) on the normal function of the thyroid gland, revealing its relation to TBT toxicity. Meanwhile, to assess whether the green tea extract in low dose, simultaneously given with TBT, exerts some protective effects on the thyroid tissues.
Chemicals
Tributyltin chloride and corn oil were purchased from Sigma-Aldrich Chemical Company, St. Louis, MO, USA. Purity of TBT was 96%.
Green tea extract (GTE) was supplied in the form of tablets (200 mg) obtained from the Technomed Groups Company, Egypt; then dissolved in distilled water.
Animals and experimental design
Forty-five specifically pathogen free adult male albino rats (weighting 200-230 g) were included in the present work. All procedures followed the guidelines for the care and handling of animals and the study protocol was approved by the ethical committee of Alexandria Faculty of Medicine.
The animals were housed under the same laboratory conditions of light and temperature with free access to standard laboratory food and water. They were acclimatized to the new circumstances for one week prior to the start of the experiment. The duration of the experiment was 30 days.
The rats were randomly assigned to three numerically equal experimental groups (15 animals each) as follows: Group I (the control group): which was further, divided into 3 equal subgroups, (5 rats each): Group Ia: Each rat in this group received 3 ml of distilled water orally via orogastric-tube. Group Ib: Each rat in this group received 0.4 ml of corn oil orally via orogastric-tube. Group Ic: Rats of this group received green tea extract of 150 mg/kg. body weight dissolved in 3 ml of distilled water orally via orogastric-tube (Hamdy et al., 2012).
Group II (Tributyltin-treated group): each rat in this group received Tributyltin chloride dissolved in corn oil in a dose of 5 mg/kg for 30 days (Mitra et al., 2014).
Group III (TBT + GTE group): received Tributyltin chloride dissolved in corn oil in a dose of 5 mg/kg with concomitant administration of green tea extract (150 mg/kg body weight) for 30 days (Hamdy et al., 2012).
Hormonal analysis
Determination of serum T3, T4 and TSH At the end of the experimental period (30 days), blood samples were collected from rats' carotid arteries. Centrifugation was done at 400 x g (times gravity) for 5 min and serum was kept at −20°C for further analysis of triiodothyronine (T3), thyroxine (T4) and thyroid stimulating hormone (TSH) levels by ELISA (Kuriyama et al., 2007).
Antioxidant enzymes and lipid peroxidation
For determination of markers of oxidative stress, blood samples were collected, without using any anticoagulant and then the blood was allowed to clot for 30 min at 25°C. The blood was centrifuged at 700 x g for 10 min. A pipette was used to separate the top yellow serum layer without disturbing the white Buffy layer.
Estimation of reduced glutathione (GSH)
Reduced glutathione (GSH) content was assayed by the method reported by Miwa et al., (Miwa et al., 1989) GSH determination is based on the development of a yellow color when 5,5′ dithiobis (2-nitro benzoic acid) (DTNB) is added to compounds containing sulfhydryl groups. The values are expressed as mg/dL.
Determination of Maliondialdehyde (MDA)
The serum was added to 8.1% sodium dodecyl sulphate (SDS), 20% acetic acid solution adjusted to PH 3.5, and 0.8% thiobarbituricacid (TBA). Distilled water was added to the mixture to make a solution of 4 ml, and then subjected to heating in a water bath at 95°C for 1 h. After cooling, 1 ml distilled water and 5 ml of n-butanolpyridine (15:1; v/v) mixture were added. The absorbance of the organic layer was measured at 532 nm. A standard curve was constructed using different concentrations of 1, 1, 3, 3-Tetra methoxypropane in ethanol. The concentration of MDA in sample was determined in nmol/ml (Ohkawa et al., 1979).
Histological study
All the rats were sacrificed by decapitation under light ether anesthesia. The thyroid glands were immediately removed from animals at the time of sacrifice, and the tissues were processed for light and electron microscopic examination.
Light microscopic study One lobe of the thyroid gland was fixed in 10% of neutral buffered formalin, dehydrated and embedded in paraffin. Then the 5-μm-thickness paraffin sections were cut and stained with hematoxylin and eosin (H&E) stains (Bancroft & Gamble, 2008). Electron microscopic study Specimens for electron microscopic examination were immediately fixed in 2.5% glutaraldehyde buffered with 0.1 M phosphate buffer at pH 7.4 and then post-fixed in 1% osmium tetroxide in the same buffer. They were dehydrated and then embedded in epoxy resin. Ultrathin sections were cut, double stained with uranyl acetate and lead citrate and examined with a JEOL 100 CX electron microscope (JEOL, Tokyo, Japan) at Electron Microscopic Unit, Faculty of Science, Alexandria University (Hayat, 2000). Histomorphometric study: The colloid area of 10 randomly selected thyroid follicles were measured from the H&E-stained sections of the thyroid gland of each animal per group at magnification of 100 using the Image Analyzer (Olympus BX41TF, Tokyo, Japan) at the Cell Biology Department, Medical Research Institute, Alexandria University. Such data were subjected to bio statistical analysis.
Statistical analysis
Analysis of data was done using IBM SPSS software package version 20 and the following were calculated: Range (minimum and maximum), mean and standard deviations. For normally distributed data, comparison between the three studied groups was analyzed using F-test (ANOVA) and Post Hoc test (LSD). p-value less than 0.05 was considered statistically significant.
Biochemical results
In the present work, no statistical difference was found among the three subgroups of the control group. A significant decrease in the levels of T3 and T4 has been recorded after 30 days of TBT administration when compared with both the control and the TBT+ GTE groups (Tables 1 and 2). On the contrary, the serum level of TSH was significantly increased in the TBT treated group (Table 3). In addition, the level of serum GSH was decreased in the TBT treated group (Tables 4). Table 5 demonstrates a significant increase in serum MDA level in the TBT treated group when compared with the other two groups.
Histological results
Histomorphometric study: The mean colloid area of the thyroid follicles was significantly lowered in TBT treated group relative to the control group. On the other hand, the mean colloid area of the third group did not show any significant change when compared with the control group (Table 6).
Light microscopic results:
Group I (control group): Light microscopic examination of sections of thyroid gland showed; multiple follicles filled with acidophilic homogenous colloid. The lining follicular cells were flattened to cuboidal in shape with oval to rounded pale nuclei Fig. 1.
Group II (Tributyltin treated group):
Examination of sections of thyroid gland of TBT-treated rat revealed markedly vacuolated colloid. Most of the follicular cells appeared swollen and vacuolated. Other cells showed loss of their nuclei. Congested blood capillaries were also observed. Figure Ultrastructural examination of thyroid follicular cells of the control group (Group I) showed a flattened to cuboidal cell with a microvillous border and an oval to rounded euchromatic nucleus. The cytoplasm revealed mitochondria, rough endoplasmic reticulum and lysosomes. An intact tight junction was also encountered Fig. 5a & b.
Group II (Tributyltin-treated group)
Electron microscopic examination of thyroid follicular cells of TBT-treated rats (Group II) revealed cuboidal to high columnar cells Fig. 6. Short, blunt and disrupted microvilli were depicted in some cells Fig. 7. The nuclei of some cells were normal in shape and euchromatic while other cells showed changes varied from small irregular and dark nuclei Figs. 7 and 9 to dilatation of perinuclear cisterna Fig. 8a. The cytoplasm showed mild to markedly dilated rough endoplasmic reticulum Figs. 6, 7, 8 and 9. The lumina of some of them were filled with flocculent material Fig. 8a & b. Mitochondria with disrupted cristae were also encountered Figs. 6 and 8b. Numerous vesicles and lysosomes were also noticed Figs. 7 and 9. Some of the follicular cells were arranged in layers Fig. 9. Significance between groups p 1 < 0.001 * , p 2 < 0.001 * , p 3 < 0.001 * F: F test (ANOVA), Significance between groups was done using Post Hoc Test (LSD) p 1 : p value for comparing between Control and TBT treated groups p 2 : p value for comparing between Control and TBT + GTE groups p 3 : p value for comparing between TBT treated and TBT + GTE groups *: Statistically significant at p ≤ 0.05
Group III (TBT + GTE group)
Ultrastructural examination of thyroid follicular cells of this group that received TBT + GTE (Group III) showed marked improvement of most of the examined follicular cells. They were cuboidal in shape and exhibited a welldeveloped microvillous border. Their nuclei were euchromatic and rounded. The cytoplasm showed normal profiles of rough endoplasmic reticulum Fig. 10a. Although mildly dilated rough endoplasmic reticulum and multiple lysosomes were still encountered in some cells Fig. 10b.
Discussion
Over the last decades, a dramatic increase in hormonal disorders was reported, and it has been assumed that growing exposure to endocrine disrupting chemicals (EDCs) contributes to the burden of endocrine disorders among populations. However, a deluge of research in the field of endocrine disruption has focused on estrogenicity (Sikka & Wang, 2008;Roy et al., 2009). At present, there are scanty reports on the impact of TBT on thyroid tissues. Thereby, this spurred us to conduct an oral toxicity study to provide further inclusive information pertaining to the effect of Tributyltin on the thyroid follicular cells.
Thyroid hormone homeostasis appears to be the target of plentiful environmental chemicals either natural or manufactured. In mammals, thyroid glandular activity is commonly determined by thyroid hormone secretion rate (Zoeller, 2007). Taking into consideration that the structure of any organ closely reflects the state of its function; histological examination of the thyroid gland provides a sensitive early indicator of the glandular activity than serum T3 and T4 levels.
In this context, the primary aim of the present study was to assess the alteration in thyroid homeostasis that might occur following exposure to Tributyltin. Furthermore, this study aimed to identify potential associations between TBT and reactive oxygen species (ROS) on the normal function of the thyroid gland and the probable protective role of green tea extract when given simultaneously with TBT.
The dosing regimen and the duration selected in the current study was in accordance with the work of (Mitra et al., 2014) who studied the sub chronic toxicity of TBT in rats. He reported that one month exposure to TBT in low doses resulted in loss of cell viability in liver, kidney as well as the lungs.
The male rats were selected in the current study, as increasing evidence elucidates that estrogen influence the risk of thyroid diseases. It also plays a crucial role as a promoting factor in thyroid tumorigenesis (Derwahl & Nicula, 2014).
In the present study, the rats treated with Tributyltin demonstrated significant decrease in the serum levels of T3 and T4 along with the evident increase in TSH levels as compared with the control group. Similar disruption of the levels of these hormones were reported by previous researchers (Adeeko et al., 2003;Wang et al., 2008;Sharan et al., 2014) who attributed its occurrence to the antithyroid effect of TBT. Significance between groups p 1 < 0.001 * , p 2 < 0.001 * , p 3 < 0.001 * F: F test (ANOVA), Significance between groups was done using Post Hoc Test (LSD) p 1 : p value for comparing between Control and TBT treated groups p 2 : p value for comparing between Control and TBT + GTE groups p 3 : p value for comparing between TBT treated and TBT + GTE groups *: Statistically significant at p ≤ 0.05 This was further asserted by histological examination of follicular cells, which unveiled evident structural changes, reflecting augmented activity in thyroid follicles of this group, in response to hypersecretion of TSH to compensate the decreasing levels of T3 and T4. These changes were manifested by swollen vacuolated follicular cells, epithelial stratification, along with excessive vacuolation of the colloid. Congested blood vessels were also encountered. These changes were further bolstered by morphometric and statistical analyses that revealed a significant decrease in the mean colloid area of thyroid follicles as compared with their respective controls.
In accordance with our results, (Pereira et al., 2013) reported disorganization of follicular cell groups, with hypertrophy, hyperplasia of thyrocytes and glandular congestion compared with control thyroid gland. On the other hand, he found no changes in plasma levels of T 3 and T 4 after 15 days of treatment in his study. This could be clarified by the shorter duration of his research when compared with the present one.
It is acknowledged that prolonged stimulation of the pituitary by decreasing levels of thyroid hormones results in release of elevated levels of TSH by the thyrotrophs which may lead to thyroid gland neoplasia manifested as shrinkage of colloid area, hypertrophy as well as hyperplasia of follicular cells (Hood et al., 1999;Boelaert, 2009). Moreover, the present study demonstrated small sized follicles, empty and fused follicles accompanied by disturbance of the normal architecture of the gland. Similar findings were reported by while studying the effect of TBT on thyroid gland of Xenopus laevis. They stated that TBT can induce intense damage to the thyroid tissues. This damage manifested by reduction in follicular region, colloid depletion and malformed follicles.
Ultra-structurally, the follicular cells revealed mild to moderately dilated rough endoplasmic reticulum. Mitochondria with disrupted cristae were also encountered together with ample lysosomes and vesicles. Some follicular cells showed cuboidal to high columnar cells. Meanwhile, some of the nuclei appeared small and shrunken with peripheral clumping of heterochromatin, while other nuclei showed dilated perinuclear cisternae.
Our results are consistent with those of (Sharan et al., 2014) who studied the effect of TBT on thyroid gland, they declared that TBT possessed antithyroid effect via intervention with thyroid hormone regulation. Such disturbance occurred through decreasing the transcription of thyroid hormone receptors (TR) by disrupting the physiological concentrations of thyroid hormones, thereby increasing the ligand-dependent cooperativity of TR with the co-repressors and shedding of the co-activator. Additionally, TBT caused down-regulation of the thyroid peroxidase and thyroglobulin genes, which correlated with the decrease in T3 and T4, while boosting the thyroid stimulating hormone (TSH) levels. Furthermore, Tributyltin can cause up-regulation of thyroid-stimulating hormone receptor in the thyroid glands.
However, it is quite apparently that thyroid gland activity is positively regulated by thyroid stimulating Significance between groups p 1 < 0.001 * , p 2 < 0.001 * , p 3 = 0.023 * F: F test (ANOVA), Significance between groups was done using Post Hoc Test (LSD) p 1 : p value for comparing between Control and TBT treated groups p 2 : p value for comparing between Control and TBT + GTE groups p 3 : p value for comparing between TBT treated and TBT + GTEgroups *: Statistically significant at p ≤ 0.05 Significance between groups p 1 < 0.001 * , p 2 < 0.001 * , p 3 < 0.001 * F: F test (ANOVA), Significance between groups was done using Post Hoc Test (LSD) p 1 : p value for comparing between Control and TBT treated groups p 2 : p value for comparing between Control and TBT + GTEgroups p 3 : p value for comparing between TBT treated and TBT + GTEgroups * Statistically significant at p ≤ 0.05 hormone (TSH) synthesized and secreted from pituitary thyrotrophs, whose activity is in turn controlled by the hypothalamic TSH-releasing hormone (TRH). TSH acts on specific receptors on the membrane of follicular cells and invigorates the activity of the sodium-iodine symporter and of intracellular enzymes involved in thyroid hormone synthesis. Therefore, when the level of serum thyroid hormone dwindles, the feedback inhibition of TSH is attenuated and more TSH is secreted; this promotes thyroid cell hyperplasia and hypertrophy and stirs the function of the thyroid into the active state to sustain the body thyroid hormone needed (Chiamolera & Wondisford, 2009).
In the context, (Scanlan et al., 2004) have speculated that increased lysosomal activity in the follicular cells, is simply a reflection of augmented cellular secretory activity initiated by high levels of circulating TSH. It could also be triggered by enhanced phagocytosis secondary to the degenerative changes noticed in some cells.
Myriads of reports (Patrick, 2009;Jugan et al., 2010) suggest that thyroid disruptors can target the thyroid endocrine cascade at various levels encompassing several molecular components of the hypothalamus-pituitarythyroid-periphery (HPTP) axis as well as the functioning of the peripheral tissues including; iodine uptake, thyroid hormone production, interconversion of thyroid hormones, cellular uptake and cell receptor activation.
Thyroid follicles represent the functional subunit of thyroid tissue. Each follicle is lined by a single epithelial cell layer and is filled with a thyroglobulin (TG) containing colloidal mass formed in the rER and serving as the matrix for thyroid hormone (TH) synthesis.
In addition, TG also plays an imperative role in modulating the expression of genes involved in the synthesis of other thyroid proteins (sodium-iodide symporter [NIS] and thyroid peroxidase [TPO]) and transcription factors involved in normal thyroid physiology (Sellitti et al., 2001).
Glycosylation of TG begins in the rough endoplasmic reticulum (rER) and is completed in the Golgi complex. Within thyroid follicles, newly synthesized TG is transported along the secretory route to the apical plasma membrane of thyroid epithelial cells. After exocytosis, TG is stored within the extracellular lumen of thyroid follicles in a covalently cross-linked form (van de Graaf et al., 2001).
Because TH are iodothyronine derivatives, uptake of iodide from the blood stream represents a substantial step in their biosynthesis. During the process of generating TH, tyrosine residues on the TG molecule are coupled with iodine at the apical pole of thyrocyte. This iodination process is called organification and is controlled by the enzyme thyroid peroxidase (TPO). Thus, monoiodotyrosine (MIT) and diiodotyrosine (DIT) are formed. The coupling of MIT and DIT is also mediated by TPO as well as linking two DIT molecules to form T4. In addition to thyroglobulin and iodide, TPO requires H 2 O 2 as a third factor to carry out the above-mentioned reactions. H 2 O 2 production is presumably a rate-limiting step during TH generation (Song et al., 2007). Significance between groups p 1 = 0.012 * , p 2 = 0.8430, p 3 = .023 * .
Significance between groups was done using Post Hoc Test (LSD) p 1 : p value for comparing between Control and TBT treated groups p 2 : p value for comparing between Control and TBT + GTE groups p 3 : p value for comparing between TBT treated and TBT + GTE groups *: Statistically significant at p ≤ 0.05 Fig. 1 A Photomicrograph of a section in thyroid gland of a control rat (group I). It is showing multiple follicles filled with homogenous acidophilic colloid (Co). The follicles are lined with flattened to cuboidal follicular cells with oval (▲) to rounded (↑) pale nuclei. The interfollicular cells (Δ) and a part of a blood capillary (bc) can also be seen inbetween the follicles. H&E stain Mic.Mag. X 400 At the thyrocyte level, stimulation of the TSH receptor (TSHR) by TSH instigates several second messenger signaling cascades leading to increased iodide uptake, TH synthesis and secretion (Roger et al., 2010;Vassart & Costagliola, 2011).
It is well known that the morpho functional status of each follicle is controlled not solely by the TSH level, but rather by other factors including thyroglobulin contained within the follicle (Suzuki et al., 2011).
Thyroid hormone liberation begins with endocytosis of small amounts of colloid into vesicles that are transported inside the follicular cells. Lysosomes then fuse with these vesicles and release T4/T3. Each TG molecule stores ten times more T4 than T3 (Scanlan et al., 2004).
Although the damage of follicular cells in the thyroid seems to be a reason for impaired thyroid hormones, yet a prospective role of oxidative stress might be another reason.
The further step of the study was to evaluate the susceptibility of reactive oxygen species (ROS) to contribute in the modulation of thyroid structure.
Under normal physiological conditions, reductive power of a cell is achieved by the amount of reduced glutathione. It helps to attain oxidative damage under control by various processes and reduction in GSH leads to stress (Lushchak, 2012).
Malondialdehyde (MDA) is one of the major oxidation products of peroxidized polyunsaturated fatty acids, and thus increased MDA content is an imperative indicator of lipid peroxidation (Rahal et al., 2014).
The results of the present study depicted significant decrease in the level of serum GSH, as well as an increase in the level of serum MDA levels in the TBT treated group in relation to the other groups.
In concomitant with the previous results, ROS generation may be a radical factor underlying TBT toxicity. Multitude of studies (Demir et al., 2011;Mitra et al., 2013;Zhang et al., 2014;Bernat et al., 2014) have suggested that the generation of ROS, including the species derived from H 2 O 2 such as OH·, is one of the mechanisms involved in TBT toxicity. ROS cause damage to mitochondrial and other cytoplasmic organelle membrane structures through peroxidation of phospholipids, proteins and nucleotides. Consequently, membrane stability and integrity being disrupted resulting in osmolality changes and hydropic cell degeneration (Guo et al., 2013). Meanwhile, lipid peroxidation activates endonuclease enzymes, with subsequent breakdown of nuclear DNA and nuclear degeneration (Zhang et al., 2016).
On the other hand, (Mitra et al., 2014) reported that; reactive oxygen species but not lipid peroxidation content was observed to be significantly elevated both in the tissues and serum after a month of low dose of TBT exposure.
Similarly, (Gupta et al., 2011) on his research on thymus; clarified that oxidative stress and apoptosis are two inseparable phenomena in TBT toxicity. As a recap, it is obvious that oxidative stress as the final manifestation of a multi-step pathway, refers to cellular status imbalance between the ROS level and the cellular antioxidant defense system due to the depletion of antioxidants, or the excessive accumulation of ROS, or both, which leads to cellular damage. It has been demonstrated that exposure to TBT could yield ROS which cause various organ lesions. Approximately 0.1% of all oxygen entering the mitochondrial electron transport chain is released as ROS, which can disrupt intracellular redox status and result in homeostasis disorder (Zhang et al., 2008;Zhou et al., 2010).
Based upon the work done by (Li et al., 2015) a significant elevation of the oxidative stress indices was observed following TBT exposure, which suggested that oxidative stress was induced by TBT. Upon amalgamating previous results with the findings of this study, it is deduced that oxidative damage is one of the critical toxic mechanism of TBT.
Furthermore, (Sugawara et al., 2002) stated that reactive oxygen species can also inactivate thyroid peroxidase enzyme via two mechanisms, reversible formation of compound III due to excessive H 2 O 2 or O 2 − , and irreversible free radical-mediated thyroid peroxidase (TPO) inactivation through attacking the active site of thyroid peroxidase enzyme, causing inactivation of the catalytic site of this enzyme resulting in disruption of the level of thyroid hormones.
In the current work, co administration of green tea extract (GTE) with TBT for 30 days resulted in considerable thyroid preservation. Few cells still showed mildly dilated rER and numerous lysosomes. These histological effects correlated as well with the morphometric and biochemical results that showed significant amelioration of these attributes.
These data were on a par with who reported that green tea polyphenols (GTPP) were effective in reducing TBT-induced oxidative damage both in vivo and in vitro. They found that (ROS) production and malondialdehyde content of the liver in mice exposed to TBT were dwindled in the GTPP-treated group compared to the untreated group. Moreover, they demonstrated that the number of cells with damaged DNA in untreated mice was figured out to be significantly higher compared to GTPP-treated mice. Furthermore, damage to the nuclei and mitochondria observed in TBT-treated mice were alleviated in mice treated with both TBT and GTPP. They attributed this protective role to the powerful ability of GTPP to scavenge ROS and hinder DNA breaks.
Several studies (Narotzki et al., 2012;Giménez et al., 2013) reported that green GTE constitutes an essential source of antioxidants. Besides polyphenols, GTE contains additional antioxidants such as carotenoids, tocopherols (vitamin E derivatives) and vitamin C. Tea contains further minerals that function as co-factors in antioxidant enzymes: zinc, selenium and manganese. Polyphenols have supplementary mechanisms in which they reduce oxidation level besides direct role as antioxidants.
As antioxidants, green tea polyphenols either chelate redox-active metal ions, such as iron and copper preventing the formation of metal-catalyzed free radicals or scavenge reactive oxygen/nitrogen species or modulate antioxidant enzymes (Nakagawa & Yokozawa, 2002). On the contrary, prior researches (Chandra et al., 2011;Abulfadle et al., 2015) have declared that excessive and high doses of green tea have the potential to alter the thyroid gland physiology and architecture, that is, enlargement of thyroid gland as well as hypertrophy and/or hyperplasia of the thyroid follicles and inhibition of the activity of thyroid peroxidase and 50-deiodinase I with elevated thyroidal sodium-potassium-ATPase activity along with significant decrease in serum T3 and T4, and a parallel increase in serum thyroid stimulating hormone (TSH).
Conclusion
Thus, the current study provides novel information that accentuate the hazardous risk of TBT on the thyroid. Co-administration of green tea extract (natural antioxidant) improves Tributyltin induced morphological changes in thyroid architecture as well as restoration of glutathione activity.
The available toxicity data might provide a useful platform for further studies to clarify the human risk and to boost the global awareness about endocrine-disrupting chemicals.
These issues highlight the critical need to develop swift and robust tools to identify TBT compounds, as they are deeply interwoven with our daily lives from various sources. Thus, a holistic and comprehensive understanding of the mechanisms underlying thyroid disruption, may lead to changes in public policy and awareness, and make it possible to limit adverse outcomes for future generations. | 2017-10-17T03:04:40.633Z | 2017-07-18T00:00:00.000 | {
"year": 2017,
"sha1": "d5f4d850babd36ed5016fdbefaed2dc76264e4e7",
"oa_license": "CCBY",
"oa_url": "https://ejfs.springeropen.com/track/pdf/10.1186/s41935-017-0012-z",
"oa_status": "GOLD",
"pdf_src": "PubMedCentral",
"pdf_hash": "d5f4d850babd36ed5016fdbefaed2dc76264e4e7",
"s2fieldsofstudy": [
"Biology",
"Medicine"
],
"extfieldsofstudy": [
"Biology",
"Medicine"
]
} |
234023605 | pes2o/s2orc | v3-fos-license | Analysis of schedules and load indicators for the choice of the generation composition in the wind-diesel complex
The energy consumption of villages in the Arctic is characterized mainly by household consumers. The load schedule of such consumers has a characteristic shape and is determined by the heating season in the winter and a sharp change in external climatic conditions in the spring and autumn. The load graphs of a village in the Arctic are analyzed in an article, maximum average consumption is 369 kW in January-February. We study such indicators of load graphs as average load, rms load, variance of the load graph and standard deviation of the load from the average. As a result, it was found that the standard deviation of the load has large values precisely in the winter months and the spring-autumn period, when energy consumption changes significantly, and, therefore, this indicator must be taken into account when choosing the composition of the generation in the wind-diesel complex.
Introduction
Developing resource-rich countries face vital challenges related to economic diversification, resource replenishment, air quality, health problems, and rapidly growing domestic demand for cheap energy and water resources, which are critical factors for energy and the environment. Therefore, in order not to be vulnerable, it is necessary to take appropriate measures in the direction of developing policies and practices to promote sustainable development by harmonizing economic growth, protecting the environment and social integration.
Power supply of facilities in the Russian Arctic is carried out mainly in isolation from the Unified Energy System of Russia. The source of electricity is diesel generator sets, characterized by a large motor resource (diesel generators are oriented to long continuous operation) and low operating costs.
The maximum diesel fuel consumption in 2018 was noted in the Republic of Sakha (Yakutia) -69.3 thousand tons among all regions of the Far North and the Arctic of Russia. An indicator such as "unit actual costs for the production of 1 kWh of electric energy at a generating facility, rubles/kWh" characterizes the cost of production of 1 kWh of electricity at the generating facility. The specified indicator takes into account fuel costs, expenses on the wage fund, maintenance and repair of fixed assets and other expenses. The maximum value of this indicator in 2018 reached 42.66 rubles/kWh in the Republic of Sakha (Yakutia). This means that the average actual cost of generating 1 kWh of electricity is higher than the single-rate tariff for consumers by 37 rubles [1]. Also, the share of fuel costs varies from 30-40% in the structure of expenses for electricity generation in the Republic of Sakha (Yakutia), which is mainly related to the remoteness of these territories from large fuel production and storage centers, complex logistics and the presence of seasonality of fuel supplies, which leads to higher fuel prices than the Russian average. Therefore, more and more often in the scientific community and in the production companies the issue of using wind power plants (wind turbines) as the main source of power supply for these territories is being discussed, which will reduce the volume of deliveries and diesel fuel costs, reduce the cost of electricity generated and, moreover, reduce the impact of emissions greenhouse gases to the environment.
The demand for the introduction of renewable energy in the regions of Russia with decentralized electricity supply was formed against the backdrop of the transition to digital energy and intelligent electric networks, one of the prerequisites for which were various global and local reasons [2]. They are: Economic -an annual increase in energy tariffs and population expenses for their payment; low energy efficiency of the Russian economy; industry dependence on foreign technology and equipment [3].
Environmental -electricity is the source of 42% of anthropogenic greenhouse gas emissions (decarbonization trend); an increase in the risk of accidents by 2030 to 80% due to the deterioration of the technical condition of the equipment because of insufficient capital investments.
Technological -power shortage; the need to develop innovative technologies, devices and materials; large-scale development of distributed energy and electric transport (the trend towards decentralization); distribution of technological capabilities for the formation of the concept of an active consumer; infrastructure obsolescence (depreciation of the fuel and energy complex reaches 66%) [4,5].
Legal -the need to combine the efforts of state authorities and the industry business community, research and educational organizations [6].
Electricity consumers
Renewable energy facilities operate in only a few regions of the Russian Arctic (Murmansk Region, Nenets Autonomous Region, Republic of Sakha (Yakutia)), and foreign companies are manufacturers of components for these facilities. But it is worth noting that research in the field of application of wind turbines in the Arctic, and especially in coastal areas, is extremely popular [7,8].
The climatic conditions in these territories are very variable, the wind speed is almost always sufficient for the operation of wind turbines (at least 3-5 m / s depending on the region), it is also necessary to consider the possibility of accumulation of excess electricity.
The main consumers of electricity in the Arctic are either large industrial complexes operating in extreme climatic conditions, such as the Yamal-LNG natural gas production, liquefaction and supply plant, and hydrocarbon production on the Arctic shelf on an offshore ice-resistant stationary Prirazlomnaya platform and others [9]. Their power supply is carried out from their own sources of electricity. However, there are examples when wind turbines are additional sources in industrial facilities in auxiliary power supply systems. For example, in the Khabarovsk Territory, a wind power installation with a rated power of 100 kW (at an output voltage of 380/400 V with a frequency of 50 Hz) is integrated into the existing power supply circuit of the Unchi base of the Svetloye company to save diesel fuel and engine life of diesel generator sets.
The remaining consumers are small settlements with poorly and moderately developed production. Such facilities have characteristic load schedules, for example, in winter consumption increases because heating systems are used, in summer electricity consumption is less, but this time of year lasts in the Arctic only from late June to early August. In the spring and autumn periods, energy consumption changes significantly due to abrupt climatic changes. There is also an increase in power consumption on holidays and weekends. These patterns are basic for these consumer groups, but should be investigated more accurately at a specific object when predicting energy consumption [10,11].
Further work will be aimed at developing a method for short-term forecasting of the electric energy consumption of an industrial enterprise or facility with a household load depending on climatic conditions and weather factors, which includes a wind turbine in the power supply system, which will be possible and justified with the gradual introduction of digital technologies, such as intelligent sensors and intelligent metering, followed by the subsequent collection and processing of Big Data, demand response systems and the formation of a MicroGrid and a Smart Grid systems.
Materials and methods
The purpose of this study is to analyze the graphs and load indicators, which will subsequently affect the choice of the composition of the generating equipment in the wind-diesel complex for power supply of villages with public utility load.
Theoretical methods
In the work, theoretical methods were used, consisting in a scientific analysis of trends in the volume of electricity consumption depending on changing factors, methods of computational mathematics and mathematical statistics, statistical samples, factors and data from the weather service.
Load graph analysis
Modern approaches to modeling and forecasting power consumption are based on the application of methods for analyzing electric load graphs based on multidimensional mathematical models that allow you to find implicit relationships and patterns that exist in the process under study. The most common and studied mathematical model of power consumption over time is a model of a random process or a random graph of electrical load.
As an object of study, we consider a village in the Republic of Sakha (Yakutia), mainly with a household load of consumers. Analysis of the load curves showed that the average value of the electric load varies from 209 kW (in July) to 336 kW (in January), and its maximum and minimum value falls to January and February (369 kW) and July (201 kW), respectively.
The implementation of random graphs of electrical load obtained from the testimony of existing metering and control systems for power consumption is presented by discrete models of a real process of step type. Typical graphs for February and July are shown in Figures 1 and 2: Continuing to analyze the load schedules, we distinguished the characteristic base (white), peak (black) and semi-peak (gray) zones, which allows us to characterize the rate of change in consumption, for example, the duration of the peak zones is from 2 to 4 hours per day. The shape of the graphs in winter has a more noticeable peak zone due to the inclusion of heating devices, the power compared with the summer period increases 1.5 times.
Results and Discussion
The most important probabilities of the characteristics of a random process of electric load are the distribution function, distribution density and power variance for each moment of time.
The average load is calculated for a step graph according to formula 1: where T is the considered time period, t i is the duration of the section with power Р i . The rms load is calculated by formula 2: Variance is an important feature when choosing the composition of generating equipment, it shows how much the average values of the sample deviate on average (the average square of the deviations of the power consumption from their average value in the studied time range).
The variance of the load curve is calculated by formula 3: The standard deviation of the load from the average value is calculated by the formula 4: The calculated indicators by formulas (1-4) based on averaged data for each month of the year are presented in table 1. Based on the calculations, it is worth noting that the standard deviation of the load, showing the real average value of the deviations from the average value in the sample, is larger in the winter months and spring-autumn period, when energy consumption changes significantly due to sharp changes in external climatic conditions. For example, the largest standard deviations were found in January (σ = 35.5), February (σ = 31.0) and August (σ = 38.3). The autumn period begins in the Arctic in August and there is a sharp change of climatic conditions in the direction of lowering temperature and increasing gusts of wind. This indicator must be taken into account when choosing the composition of generation in a wind-diesel complex.
The methodology for modeling load schedules [12] using formulas (1)-(4) is widely used in applied research due to the relative simplicity and the impossibility of obtaining a sufficient number of realizations in a limited time.
The second reason for the widespread use of modeling methods based on individual implementations is a simpler model when summing up random plots, i.e. modeling a group graph P(t) according to the known characteristics of its constituent processes p(t).
Conclusion
As a result of the study, it was found that the prerequisites for the use of renewable energy sources in the Arctic were formed under the influence of the need to consolidate efforts to promote sustainable development by achieving economic growth, environmental protection and social integration.
Wind power plants have great potential for use in the Arctic together with the diesel generator sets. The article analyzed the load graphs of the village mainly with household consumption, the maximum average consumption of which is 369 kW in January and February. We studied such indicators of load curves as average load, rms load, variance of the load curve and standard deviation of the load from the average. As a result, it was found that the standard deviation of the load has large values precisely in the winter months (January, February) and the spring-autumn period (especially in August), when energy consumption changes significantly, and therefore, this indicator must be taken into account when choosing the composition of generation in wind-diesel complex. | 2021-05-10T00:03:54.905Z | 2021-02-01T00:00:00.000 | {
"year": 2021,
"sha1": "db4e219860342a0953cd8e20c1243f98eece8b23",
"oa_license": null,
"oa_url": "https://doi.org/10.1088/1742-6596/1753/1/012010",
"oa_status": "GOLD",
"pdf_src": "IOP",
"pdf_hash": "092816956870240230bd2cdf27ec5da3dd7b79ad",
"s2fieldsofstudy": [
"Engineering"
],
"extfieldsofstudy": [
"Physics",
"Environmental Science"
]
} |
55213474 | pes2o/s2orc | v3-fos-license | Assessment of ligamentum flavum thickness correlation with demographic variables and disc degeneration in Erbil governorate population sample
Background and objectives: This study aimed to investigate the correlation between the ligamentum flavum thickness and age, sex, body mass index and type of disc degeneration. Method: 126 consecutive patients were examined prospectively. The sample included all the patients who had an MRI examination after they presented to Rizgary teaching hospital complaining of lower back and/or leg pain. The LF thickness was measured by using lumbo-sacral spine MRI. Results: The mean thickness of LF was more at L4-L5 (right: 0.27 mm, left 0.25 mm), L5-S1 (right: 0.25 mm, left 0.25 mm) levels, and was thicker on the right side. LF was thicker in females than males with a statistically significant measurement at L2-L3, L3-L4 and L4-L5 levels. The mean thickness of LF was more at older age group than the younger age groups, and the results were statistically different at left side of L2-L3 and right side of L4-L5 level. Thickness of LF was more among those with higher BMI for all levels except for L5-S1. In most cases, LF was thicker in patients with advanced disc degeneration. Conclusion: Thickening of the LF is associated to some extent with sex, age, BMI and disc degeneration. Thickening of the LF is potentially due to its buckling into the spinal canal secondary to disc degeneration more than to LF hypertrophy. Further research is needed to confirm these associations.
Introduction
The ligamentum flavum (LF) is also called the yellow ligament, because the normal LF is yellow due to its greater content of elastic fiber than collagen fiber. 1 The ligamentum flavum covers most of the posterolateral part of the lumbar spinal canal extending from the second cervical vertebra to the sacral one. 2,3The LF connects two adjacent laminae and lines an important part of the osseous and soft tissue sections of the posterior epidural region. 4,5Hypertrophy of the LF is considered an important causative factor in the development of lumber spinal stenosis, compression of the dural sac and roots, and significantly contributes to lower back pain and sciatica 2,6 even in the absence of a bulging annulus fibrosus, herniated nucleus pulposus, or osseous spurs. 7,8he LF is an important anatomical structure, which might cause lower back or leg pain.Therefore, the thickness of the LF should be measured and evaluated carefully in the case of spinal stenosis 7 .This study aims to investigate the correlation between the ligamentum flavum thickness and age, sex, body mass index (BMI) and type of disc degeneration.126 consecutive patients were examined prospectively.The sample included all the patients who had an MRI examination after they were referred by a specialist Orthopedician to Rizgary teaching hospital complaining of lower back and/or leg pain Method from September 2011 to January 2012.2011 to January 2012.Patients with previous lumbar spinal surgery, evidence of lumbar or sacral tumors at MRI, diffuse bone metastasis, serious congenital anomalies, severe scoliosis, diskitis, osteomyelitis, serious lumbar vertebral fracture, spondylolysis, or spondylolisthesis were excluded from the study.The MR images were obtained with a 1.5-T unit (MAGNETOM Avanto, Tim System, Bavaria, Germany).The imaging studies include sagittal T1-weighted images (TR/TE, 590/11and slice thickness , 4 mm), sagittal T2-weighted images (TR/TE, 3500/92; and slice thickness, 4 mm), axial T2-weighted images (TR/TE, 4000/100; and slice thickness, 4 mm), and axial T1-weighted images (TR/TE, 530/17; and slice thickness, 4 mm) for all MRI examinations.The lumbar MR images were reviewed by two experienced radiologist (MD) on a digital workstation.The measured thicknesses of the LF, intervertebral disc degeneration were recorded for all subjects.The thickness of LFs were measured at L2-L3, L3-L4, L4-L5, and L5-S1 level in 126 patients on axial T1-weighed MR images at the facet joint level.All the LF thicknesses were measured by using an electronic ruler with a resolution of 0.1 mm.Thickness at the middle portion of LF was measured.The measurements were repeated twice at different times, and mean values were recorded.In cases of asymmetrical LF thickness, the thicker value was used.The intervertebral discs from L2-L3 to L5-S1 were assessed on the T2-weighted midsagittal images.The degree of degeneration was classified into five grades by using the classification of Pfirrmann et al 9 , which uses both signal intensity and disc height categories.The LF thickness of the grades I to III and grades IV to V degeneration groups were compared statistically using student t-test with P-value of ≤0.05 indicating a statistically significance difference.
One hundred twenty six patients participated in the study.Their mean ag e± S D wa s 43 .8±15 .1 yea r s.Demographic characteristics of the participants are shown in Table 1.Table 1: Demographic characteristics of the participants.
Table 2 shows type of disc degeneration at different levels.The degeneration was more at more advanced stages at L4-L5 and L5-S1 levels.The mean thickness of LF was more in L4-L5 (right: 0.27 mm, left 0.25 mm) and L5-S1 (right: 0.25 mm, left 0.25 mm) levels and was also generally more on the right side than the left side as shown in Table 3. Table 3: Mean thickness of LF at different levels and in both sides.
Thickness of LF was more in females than males.The results were statistically significant at left side of L2-L3 and L3-L4 levels and at both right and left sides of L4-L5 level as shown in Table 4.The mean thickness of LF was more at older age than the younger age groups.This difference was statistically different at left side of L2-L3 level and right side of L4-L5 level (Table 5).Table 5: Association of LF thickness with the age groups.
Thickness of LF was more among those with higher BMI for all levels except for level L5-S1.However this difference was only statistically significant for the left side of L2-L3 (Table 6).Table 6: Association of LF thickness and BMI of participants.
Thickness of LF was more among cases with advanced stages of degeneration.However, this difference was only statistically significant at left side of level L2-L3 as shown in Table 7.The LF covers a considerable part of the posterior and lateral walls of the spinal canal. 10Thickening of the LF can reduce the diameter of the spinal canal, compressing the dural sac and nerve roots, and contributes to low back pain and sciatica even in the absence of a bulging annulus fibrosus, herniated nucleus pulposus, or osseous spurs. 7Some differences were found among the LF thicknesses at each level, this finding is in agreement with previous studies. 7,11,12The thickest LF measurement was at L4-L5 and L5-S1.LF was thicker in females than males, this finding disagree with previous studies. 7,13owever, this could be attributed to the nature of the heavy work done by the women in Iraq.In our study we found that LF thickness increases proportionately with age, this observation is supported by previous studies which suggest that LF thickness is an age-dependent phenomenon. 10,12,13An association between disc degeneration and obesity had been reported; a high BMI associates significantly with disc degeneration. 14It was found that the LF thickness at most levels were greater in the subjects with BMI of 25 kg/m2.High BMI positively associated with LF thickness at the L2-L3 disc level.This observation agrees with another study. 11he pathogenesis of thickening of the LF is not well understood.Some studies have asserted that age-related fibrosis or decrease in the elastin-to-collagen ratio of the LF, along with hypertrophy of the LF, is associated with lumbar spinal stenosis, while others argued that the LF bulges inside the spinal canal and compresses nerve tissues. 1,10The "thickening" is the buckling of the LF into the spinal canal after disc collapse. 11To evaluate this theory, the relationship between the thickness of the LF and intervertebral disc degeneration, disc height at L2-L3, L3-L4, L4-L5, and L5 -S1 were examined; it was found that the LF was generally thicker in patients with grades IV to V degeneration than in those
Discussion
with grades I to III.This result was only statistically significant at L2-L3 level; this finding could be attributed to the small sample size of the study, as previous studies had shown significant difference at all levels with only one study contradicting this correlation. 10One can only verify this relationship through histopathological examination.Hence, further studies are required to confirm this association.In conclusion, thickening of the LF is associated to some extent with sex, age, BMI and disc degeneration.The thickening of the LF is potentially due to its buckling into the spinal canal secondary to disc degeneration, more than to LF hypertrophy alone.Further research is needed to confirm these associations.
Table 2 :
Type of degeneration at different levels
Table 4 :
Association of thickness of LF with gender of the patients.
Table 7 .
Association of LF thickness and stage of disc degeneration. | 2018-12-05T08:51:45.429Z | 2012-07-01T00:00:00.000 | {
"year": 2012,
"sha1": "cd3bb88772b8decf1458b641fe685fed1111b40c",
"oa_license": "CCBYNCSA",
"oa_url": "https://zjms.hmu.edu.krd/index.php/zjms/article/download/407/365",
"oa_status": "GOLD",
"pdf_src": "ScienceParseMerged",
"pdf_hash": "cd3bb88772b8decf1458b641fe685fed1111b40c",
"s2fieldsofstudy": [
"Medicine"
],
"extfieldsofstudy": [
"Medicine"
]
} |
15872228 | pes2o/s2orc | v3-fos-license | Unique diversity of acanthothoracid placoderms (basal jawed vertebrates) in the Early Devonian of the Prague Basin, Czech Republic: A new look at Radotina and Holopetalichthys
The taxonomy of Early Devonian placoderm material from the Lochkovian and Pragian of the Prague basin, previously attributed to the genera Radotina and Holopetalichthys, is revised. The Pragian species Radotina tesselata Gross 1958 shares detailed similarities with the holotype of the Lochkovian Radotina kosorensis Gross 1950, which is also the holotype of the genus; the assignation of both species to Radotina is supported. However, the Lochkovian material previously attributed to Radotina kosorensis also contains two unrecognised taxa, distinguishable from Radotina at the generic level: these are here named Tlamaspis and Sudaspis. The disputed genus Holopetalichthys, synonymised with Radotina by some previous authors, is shown to be valid. Furthermore, whereas Radotina, Tlamaspis and Sudaspis can all be assigned to the group Acanthothoracii, on the basis of several features including possession of a projecting prenasal region of the endocranium, Holopetalichthys lacks such a region and is probably not an acanthothoracid. Skull roof patterns and other aspects of morphology vary greatly between these taxa. Radotina has a substantially tesselated skull roof, whereas the skull roofs of Tlamaspis and Holopetalichthys appear to lack tesserae altogether. Tlamaspis has an extremely elongated facial region and appears to lack a premedian plate. Sudaspis has a long prenasal region, but unlike Tlamaspis the postnasal face is not elongated. Past descriptions of the braincase of 'Radotina' and the skull roofs of 'Radotina' and 'Holopetalichthys' incorporate data from more than one taxon, giving rise to spurious characterisations including an apparently extreme degree of skull roof variability. These descriptions should all be disregarded.
Introduction and Kosoraspis). The last extensive study of the Lower Devonian material from the Bohemian collections by Westoll [37] was focused on morphological terminology and phylogenetic relationships rather than taxonomy of the studied material. No other attempt to revise the Prague Basin placoderm material had been made prior to the recent studies by one of the current authors [31,38,39].
This paper examines all specimens formerly assigned to the genus Radotina either in published papers or in the catalogue of the NM. The published data have often been mentioned in studies on phylogenetic relationships [10] or systematics [25,40]. However, our current research on this material is uncovering a great deal of new information, both from the previously described specimens and from the extensive undescribed material in the NM collections, that not only augments but frequently contradicts the published accounts. This paper represents the first step in a complete revision of the Bohemian acanthothoracid material. In a later paper we will also be examining the enigmatic genus Kosoraspis [35,36], but as its taxonomic distinctness from Radotina is not in dispute it will not be considered here.
Material and methods
The majority of specimens considered in this paper belong to the Národní Muzeum (NM) in Prague (the collections of the Czech Geological Survey and Charles University contain no acanthothoracid material or specimens relevant to this study). Additional specimens from the collections of the Natural History Museum, London (NHM) and Humboldt Museum für Naturkunde, Berlin (HMN) are also discussed. The specimen numbers are listed in the Systematic palaeontology section below. No permits were required for the described study, which complied with all relevant regulations.
The collections of the NM house some 300 placoderm remains; among them 25 fragments are labelled as the genus Radotina, three as Holopetalichthys and another 30 as Macropetalichtys. 47 samples (each with a unique catalogue number; some of them counterparts or separately numbered broken pieces), catalogued either as belonging to one of these three genera or as "Placodermi indet.", were investigated for the purposes of this revision. All the studied specimens are of Devonian age and come from two localities in the Prague Basin.
All the specimens originally labelled Radotina kosorensis (most of them herein reclassified as the new genera Tlamaspis and Sudaspis) were found in the Radotín Limestone, a member of the basal Devonian (Lochkovian) Lochkov Formation (Fig 1B1) at the quarries in Černá rokle ( Fig 1A1). The bony elements are very indistinct in the dark grey fine-grained matrix of the limestone when dry. Therefore all the specimens were coated with ammonium chloride before being photographed and illustrated. Some of them were mechanically prepared in the 1950s. Preparation with acetic acid (5-10%) was also tested by one of us (V.V.) on a single specimen of an isolated dermal plate, but it was unsuccessful as the bone tended to dissolve along with the matrix.
A smaller number of specimens described herein are from the Koněprusy Limestone, a member of the Lower Devonian (Pragian) Praha Formation (Fig 1B2), which is exposed in several localities near the village of Koněprusy ( Fig 1A2). As the limestone is pure white and the bone elements have a reddish surface (caused by ferric oxide coating), the material could be photographed successfully without ammonium chloride coating. The Koněprusy Limestone is the only shallow water photic zone reef facies in the Prague Basin containing placoderms. The preserved specimens are distinctly smaller in size than the Lochkovian placoderms.
All the specimens were studied with an Olympus SZX9 optic microscope and the drawing in Fig 2A was made with a drawing attachment Olympus SZH-DA. The surface sculpture and fine details of suitable specimens were observed under the scanning electron microscope Hitachi S-3700N and Keyence VHX-2000 digital microscope.
Nomenclatural acts
The electronic edition of this article conforms to the requirements of the amended International Code of Zoological Nomenclature, and hence the new names contained herein are available under that Code from the electronic edition of this article. This published work and the nomenclatural acts it contains have been registered in ZooBank, the online registration system for the ICZN. The ZooBank LSIDs (Life Science Identifiers) can be resolved and the associated information viewed through any standard web browser by appending the LSID to the prefix "http://zoobank.org/". The LSID for this publication is: urn:lsid:zoobank.org:pub:ADCE-BED8-B1A1-42E6-B20A-C2DF1FEDB072. The electronic edition of this work was published in a journal with an ISSN, and has been archived and is available from the following digital repositories: PubMed Central, LOCKSS.
Geological and palaeontological settings
The Černá rokle (= Black Gorge) quarries are situated in the south-eastern part of the Prague Basin in the Radotín Valley ( Fig 1A1). Several quarries were operating there from the 19 th century until the 1960s. Perner [42] noted a major increase in fossil fish discoveries at the beginning of the 20 th century when closer contacts were established between the quarry workers and the local fossil collectors. The rocks were being quarried and processed by hand to make paving setts, and the workers were instructed and well trained in detecting precious finds. Those were then sold to the collectors, recruited from the local intellectual elite. The largest collections were amassed by A. Schubert (postmaster), F. J. Pecka (local teacher), W. Kolář (clerk) and R. Růžička (engineer); the collection of the late professor I. Chlupáč also contained a few finds acquired in his youth. The majority of these collections were donated to the NM, over the course of several decades. Crucially, important material was donated after Walter Gross had finished his studies of the NM placoderm collections in 1959, and is described here for the first time. After the quarrying ceased only collecting in debris was possible at the locality.
Systematic palaeontology
Class Placodermi M'Coy, 1848 [46] Order Acanthothoraci Stensiö, 1944 [22] Family Palaeacanthaspidae Stensiö, 1944 Genus Radotina Gross, 1950 [34] (Figs 2 and 3) Diagnosis. Jawed vertebrate possessing a broad, flat endocranium with an anterior prenasal expansion of the trabecular region. Skull roof composed of individual small plates separated by fields of tesserae. The rostronasal capsule in dorsal position, posterior to a trapezoidal premedian area. Dorsal margin of orbit formed by three dermal plates. The rostral plate wide but anteroposteriorly short, forming a transverse bar above the dorsal margins of the nostrils. The small pineal plate, bounded by tesserae posteriorly, has a smoothly curved posterior margin, prominent lateral corners, and a transverse anterior margin. No pineal foramen. The central plates small, widely separated and of oval shape. The ornament on the dermal plates and tesserae composed of small star-shaped tubercles, each with five to six ridges that are large relative to the central body of the tubercle.
Type species. Radotina kosorensis Gross, 1950 Radotina kosorensis Gross, 1950 Remarks. Much of the material formerly assigned to Radotina kosorensis is here split off into the new genera Tlamaspis and Sudaspis (see below). These new taxa differ from Radotina in major aspects of cranial morphology, justifying separation at the generic level. NHM P.12829 and NM Lc 461 are, along with the holotype, the only specimens that can securely be referred to Radotina kosorensis. The scales attributed to Radotina kosorensis by Gross [36] and subsequent authors are not associated with any of these specimens. For the present they should formally be regarded as indeterminate, but their characteristic tubercle morphology suggests that they may belong to Kosoraspis (pers. obs. VV).
Description. The holotype (Fig 2A) and the so-called London specimen [35] (NHM P.12829; Fig 2B) are both incomplete skull roofs, exposed in dorsal view, that extend from the premedian plate posteriorly to the anterior part of the paranuchal plate. The holotype consists of an actual dermal skeleton whereas the London specimen is a natural mould. They do not represent the same individual.
The skull roof is composed of several dermal plates separated by fields of tesserae. In contrast to the dermal plates, the tesserae are composed of a single superficial shallow layer and are very similar to body scales [35,36]. The trapezoidal premedian plate (Fig 2A and 2C) is twice as wide as long. The ossification centre lies at its anterior border. A band of tesserae borders the posterior margin of the premedian plate and reaches towards the nostrils. The nostrils face dorsally, and the orbits are positioned posterolaterally to the nostrils. In the holotype the dorsal margins of the orbits are formed by three smaller well developed plates interpreted as the postnasal, preorbital and postorbital plates. The postorbital does not reach the posterior margin of the postorbital process and does not carry the infraorbital sensory line groove. In NHM P.12829 only two plates form the orbital margin, but a possible third plate lies between these two plates and the supraorbital lateral line groove ( Fig 2B). However, it is difficult to understand the pattern as the specimen is an imperfect natural mould.
The supraorbital sensory lines emerge close to the ossification centres of the postnasal plates and continue along the mesial margins of the postnasal and preorbital plates. The supraorbital sensory lines and the nostrils outline the rostropineal area. The rostral plate is wide and anteroposteriorly short, forming a transverse bar above the dorsal margins of the nostrils. It is better preserved on the London specimen, where it touches the anterior margin of the pineal plate ( Fig 2B). The small pentangular pineal plate has a smoothly curved posterior margin, prominent lateral corners, and a transverse anterior margin. There are twin recesses for the pineal and parapineal organs, positioned side by side, but no pineal foramen. The posterior part of the rostropineal area is covered by tesserae.
The central plates are oval and show slight elevations anteriorly, posteromesially and posterolaterally that appear to correspond to the underlying auditory capsules with semi-circular canals [37]. They are separated by a narrow band of tesserae and occupy the position between the central and the posterior sensory lines. Posterior to the central plates only tesserae are present. However, Gross [36] noted some faint radiating striae resembling the middle layer of a bony plate in the posterolateral corner of the holotype near the main sensory line canal (Fig 2A). We interpret this as the anterior end of the paranuchal plate. (Note that we refer to the plate that forms the posterolateral corner of the skull roof as a "paranuchal" or "posterior paranuchal" in different taxa, depending on whether a bone identified as an "anterior paranuchal" is present. This is somewhat unsatisfactory but follows standard usage in the placoderm literature [25].) The anterior part of the skull roof, from the premedian plate as far back as the centrals, has proportions very similar to Romundina stellina [21]; the only noteworthy difference is that Radotina kosorensis has smaller orbits. However, the posterior part of the skull roof appears to be considerably longer in R. kosorensis than in Romundina stellina.
The ornament on the dermal plates and tesserae is composed of small star-shaped tubercles, each with five to six ridges that are large relative to the central body of the tubercle. On the central plates the tubercles are restricted to the marginal areas, where they are arranged concentrically. The tesserae are tiny plates, each carrying an individual tubercle or a small group.
The sensory lines have the form of deep, wide-open grooves. They do not cross the ossification centres of the bony plates. Their pattern is most clearly seen on the holotype (Fig 2A). The supraorbital sensory lines are deep, gradually widening in the anterior part. The anterior part of the main sensory line and the central sensory lines are separated from the postorbital plate by a band of tesserae. The central sensory line is gently S-shaped and runs towards the connection of the supraorbital grooves. The posterior sensory line curves anteriorly, ending just posterior to the central plate. Where a sensory line groove passes through a tesserate area, it passes between rather than through individual tesserae, and the pattern of the tesselation is somewhat disturbed. In a few areas, notably on the main sensory line posterior to its junction with the posterior sensory line, a separate lining for the sensory canal itself is preserved within the groove. This lining is composed of very thin bone with fine transverse striations and presumably lay within the membranous wall of the sensory line canal.
The median part of the endocranial base is preserved on the specimen NM Lc 461 (and its counterpart NM Lc 462; Fig 2C). This specimen does not show the dermal skull roof, but is assigned to Radotina kosorensis because of the similar proportions of the premedian region. As is frequently the case in specimens from Černá rokle, the split between slab and counterslab does not follow a single plane; in the middle and posterior parts, the split mainly follows the internal face of the perichondral bone floor of the braincase (though it jumps to the floor of the sacculus on both sides), but anteriorly it rises through the telencephalic region of the braincase before entering the basal layer of the dermal bone of the premedian region. The endocranium has a roughly rhombic shape with markedly projecting postorbital processes. The most striking feature of the endocranium is the imprint of the notochordal canal that is surrounded by a vascular web and several openings. The notochord reaches far forward, ending anterior to the postorbital processes. Other prominent features are traces of canals on the lateral margins of the endocranial base, possibly branches of the jugular vein, and anteriorly a curved transverse groove for the pseudobranchial artery. The telencephalic recesses and olfactory nerve canals can be seen in section. [38], p. 52 Diagnosis. The only known specimen is much smaller than R. kosorensis. The central sensory line canals point anteriorly and do not parallel the margins of the central plates. The supraorbital canals form a more acute angle than in R. kosorensis. Tesserae in the rostronasal area bigger than tesserae on the rest of the skull roof.
Radotina tesselata
Holotype and only specimen. Anterior part of the cranium, missing the prenasal region. Remarks. Gross [35] describes one specimen of this species only-the holotype. It is a threedimensional cranium with badly preserved ornamentation of the skull roof plates. The prenasal region and the area posterior to the centrals are missing. Both orbits and nostrils were prepared by a technician of W. Gross. Later a part of the endocranial base was prepared away by an unknown technician to expose the posterior part of the perichondrally lined brain cavity and ventral parts of the inner ear cavities. Although this specimen is much smaller than the holotype and other specimens of Radotina kosorensis, it has a partly ossified braincase (missing only the premedian region) and is thus presumably close to adult size.
Description. The pattern of the skull roof plates is similar to R. kosorensis (Fig 3A), although the premedian plate is unknown. Postnasal, preorbital and postorbital plates border the dorsal margin of the orbits. The supraorbital sensory line emerges at the posteromesial margin of the small postnasal plate and continues posteriorly along the mesial margin of the large preorbital plate. The postorbital plate is small and does not carry any sensory line canal. The pineal plate is small with prominent anterolateral corners. It covers both recesses for the pineal and parapineal organs and does not display a pineal foramen. The rest of the rostropineal area is covered by tesserae substantially larger than the ones covering the rest of the skull roof. The outline of the rostral plate is unclear, but it is evident that tesserae are present also anterior to the pineal plate. The central plates are oval with no sensory lines crossing. They are separated by tesserae.
The supraorbital sensory lines form a more acute angle (approx. 60˚) than in R. kosorensis (70˚on the holotype). The infraorbital sensory line groove runs along the posteroventral margin of the postorbital plate. A similar groove on the ventral margin of the postnasal plate may represent its anterior continuation. The central sensory line parallels the orbit and the posteromesial margin of the postorbital plate, pointing anteromesially towards the spot where the supraorbital sensory line touches the margin of the preorbital plate. This contrasts with the condition in R. kosorensis (see above), where the central sensory line is transverse and gently Scurved. All sensory lines are developed as deep and wide canals. In a few areas the grooves are covered by a thin bony lamina representing the wall of the canal itself, as described for R. kosorensis.
Two dermal plates are clearly visible on the left lateral side of the holotype (Fig 3B). Gross [35] described them tentatively as "mandibulare". A slightly curved sensory line is detectable on both plates. We interpret the larger plate as the left suborbital, which is supported by its position relative to the orbit. The second plate could be a broken and displaced right suborbital plate.
The nasal cavities, which face anteriorly, are placed medially to the anterior ends of the supraorbital sensory lines. They have an oval shape and were separated by a thin, incompletely closed septum internasale ( [35]; unpreserved at present). The posterior wall of each cavity is formed by a lamina cribrosa pierced by numerous perichondrally lined canals for branches of the olfactory nerve. Imprints of blood vessels supplying the olfactory organ are preserved on the capsulae walls.
The orbits, which are relatively larger than in Radotina kosorensis, occupy an anterior lateral position, posterior to the nostrils. Their inner space is wider than the opening and reaches beyond it towards the area of the central sensory lines. Medially the orbit reaches the level of the supraorbital sensory lines. The internal surface of the orbits shows apertures for nerves and blood vessels, as well as a small triangular eye stalk ( Fig 3B). A large opening for the optic nerve lies anterior to the eye stalk. The aperture for the jugular vein is situated in the posterolateral area of the orbit.
The otic part of the cranial cavity in the posterior ventral part of the holotype (posterior to the orbits and the dislocated suborbital plate) has been prepared by an unknown technician. The ventral and partly lateral sides of the cavities lined by a thin perichondral lamina are exposed. Anteriorly the remains of a narrow endocranial base-the medullar part of the endocranial cavity-are visible. The most prominent structures are the sacculi with the semicircular canals.
Genus Tlamaspis gen. nov. urn:lsid:zoobank.org:act: Diagnosis. Jawed vertebrate possessing a broad, flat endocranium with an anterior prenasal expansion of the trabecular region. Prenasal area very long, occupying at least one quarter of the whole length of the endocranium. Premedian area of the endocranium contains an elaborate canal network. Glenoid region of endocranium broad. Grooves of lateral aortae on ventral wall of endocranium deeply impressed, lyre-shaped, continuing forward to hypophysial fossa. Endocranial surface rugose between grooves of lateral aortae. Grooves of secondary jugular veins deeply incised, narrow, s-curved, main mesial branch attached posterior to crossing of efferent pseudobranchial artery. Dermal skull roof contains one pair of large oval central plates, meeting in the midline anteriorly but posteriorly separated by the nuchal plate. Premedian plate absent. Posterior margin of dermal skull roof forms a projecting midline point created by the nuchal. Dermal ornament consists of small, rounded tubercles with eight to nine moderately prominent ridges.
Derivation of name. After the Czech/Slovak word for large mouth-"tlama", and the Greek "aspis" (shield).
Type and only known species. Tlamaspis inopinatus sp. nov.
Remarks.
Most of the specimens we redescribe as a new genus Tlamaspis were originally included in Radotina kosorensis by Gross [36]. In fact they differ from this taxon in numerous respects including the absence of a premedian plate, very large central plates that contact each other and the nuchal plate without intervening tesserae, tubercles of a distinctive morphology, and an elongate prenasal region. Previous authors [35,36,37,10] have explained the absence of tesserae in the Tlamaspis specimens, in contrast to their presence in Radotina kosorensis, either as individual variation or as due to taphonomic processes. However, taphonomic loss can be ruled out, because the marginal areas of the central and nuchal plates of Tlamaspis (which, under the taphonomic hypothesis, would in the holotype of Radotina kosorensis lie underneath fields of tesserae) are covered with firmly attached tubercles and have clearly not been exposed by post mortem stripping-off of tesserae. Individual variation is difficult to formally disprove without a statistically significant morphometric sample, but we note that all the Tlamaspis specimens are consistently similar, and different from Radotina kosorensis, with respect to the characters mentioned above. Fig 4). Each central plate has a distinct, raised area in the middle, with a strongly radial texture and few or no tubercles. The nuchal plate is large, tapering anteriorly, and has a posterior oval projection that covers the underlying occipital process of the endocranium. The central and nuchal plates all have well-defined margins, but they are not quite as tightly joined as the bones of a typical sutured skull roof (see for example Holopetalichthys, below). Tesserae are absent between these plates. No premedian plate is present in any specimen, even though the premedian region is preserved; we infer that it is genuinely absent. The rostronasal capsule is not preserved, but its attachment area on the braincase is small. A small rhombic postpineal plate situated between the supraorbital sensory lines is known only in one specimen (NM Lc 29; Fig 4B). The supraorbital sensory lines are deep, and usually leave impressions on the dorsal side of the endocranium (Fig 4A-4C). They form a narrow V shape and meet behind the small postpineal plate. The central plates carry anteromedially directed posterior sensory lines running in the direction of the ossification centres, as well as middle and anterior pit lines. The remaining sensory lines and the openings for the endolymphatic ducts are unknown. The dermal ornament consists of small, rounded tubercles with eight to nine moderately prominent ridges.
On specimen NM Lc 7 ( Fig 5D) the inner surface of a relatively large submarginal plate is exposed adjacent to the postorbital process. Similar large oval submarginal plates are present in specimens NM Lc 11-13, NM Lc 165 and NM Lc 166 (Fig 6A and 6B). Anteriorly and posteriorly to the submarginal plate of NM Lc 7, and laterally to the endocranium, lie two other damaged plates that are difficult to identify. The posterior one could be the dorsal margin of the anterior lateral plate.
Specimens NM Lc 11-13, NM Lc 165 and NM Lc 166 (Fig 6A and 6B) display a cluster of tubular bony endoskeletal elements in various states of preservation in the area between the endocranium and the suborbital plate. We interpret them as elements of the visceral skeleton, most probably the gill arches.
The thoracic shield of Tlamaspis is incompletely known. The most frequently preserved shoulder girdle element is the anterior lateral (AL) plate with the perichondrally ossified scapulocoracoid firmly attached to its mesial side, which is seen more or less clearly in NM Lc 11, NM LC 148 and NM Lc 166 (Fig 6). Gross [36] was unsure about the taxonomic identity of the few isolated anterior lateral plates from Černá rokle that he was able to study. Their determination to the genus Tlamaspis is now confirmed by specimen NM Lc 148 (Fig 6C), which was unknown to him. The anterior lateral plate is narrow, tall and crescent shaped with the postbranchial lamina situated on the concave margin. Anteroventrally it appears to taper to a point. In overall appearance it somewhat resembles an early osteichthyan cleithrum, such as that of an onychodont [52] or porolepiform [53]. The tubercles on the postbranchial lamina are fine and dense. The ossification centre lies in the anterior area of the plate. The shape of the scapulocoracoid follows the shape of the AL plate. The scapulocoracoid is composed of an anterior coracoid process and a dorsal scapular blade (Fig 6A and 6C). Numerous imprints and openings for blood vessels and nerves are clearly visible on the surface. It is possible to recognize dorsal and ventral groups ( [47], figure 193A).
In NM Lc 166 the AL, which is preserved in approximate life position relative to the braincase (Fig 6B), is overlapped dorsally by the median dorsal plate (MD). This positional relationship is likely to be slightly disturbed, as it leaves no room for the dorsolateral thoracic plates that would be expected to carry the main lateral line canal of the trunk, separating the AL from the MD. The median dorsal plate has anterolateral projections that give the bone a butterflylike outline (Fig 6B). The dorsal spine is broken off. The remaining plates of the thoracic shield are as yet unknown or unrecognisable.
The most abundant remains of T. inopinatus are the dorsoventrally compressed, perichondrally ossified endocrania. Most commonly the rock slab containing the specimen has split horizontally along the ventral face of the endocranium, with the crack sometimes running above and sometimes below the thin ventral layer of perichondral bone, so that the bone is divided in patches between slab and counterslab. Curiously, in all specimens examined to date the crack rises dorsally in the prenasal region, so that at least part of this region ends up attached to the counterslab (Fig 5B and 5D). The dorsal surface of the endocranium is largely covered by the central and nuchal plates of the skull roof (see above), except anteriorly where the rostronasalprenasal region is exposed. (Figs 4B, 4C and 5A). In ventral view, the outline of the endocranium is clearly visible, but the actual lateral walls are difficult to reconstruct because of the strong dorsoventral compression of the specimens.
The endocranium is relatively slender. The facial region, from the postorbital processes to the anterior margin of the endocranium, is remarkably long. It comprises more than 50% of the length of the endocranium, whereas the corresponding proportion in Romundina stellina is approximately 42% (measurements taken from endocranium in [21]). The pre-and postnasal parts of the facial region appear in proportional terms to be equally elongated relative to the condition in Radotina kosorensis or Romundina stellina. In other words, the facial region of Tlamaspis appears isometrically "stretched" compared to these taxa. In general morphological terms the prenasal area is reminiscent of that in Romundina [7,21], with broad suborbital shelves separated in the midline by a raised, anteriorly flaring region that carries the attachment for the rostronasal capsule posteriorly and the premedian area anteriorly (Figs 4B, 4C and 5A). Unlike in Romundina and Radotina, there is no premedian plate in Tlamaspis, but a distinct premedian area can be recognised in the region where a premedian plate would be expected (Fig 5A). The dorsal surface of the endocranium lacks external perichondral ossification in this area, exposing a complex internal network of perichondrally lined canals.
The endocranium is widest at the level of the short anterior postorbital process (Fig 5B and 5C). The posterior postorbital process is robust but appears to be relatively short (Figs 5A and 6D). Posterior to this process, the occipital region is proportionately much shorter than in Romundina [21] and the condyles are strikingly wide. Mesial to the posterior postorbital process, the ventral surface of the endocranium carries a wide but not very deep longitudinal groove, bounded mesially by a ridge (Fig 5B, gr.eff.a). A similar groove in Romundina has been interpreted as housing a common efferent branchial artery [21].
The hypophyseal opening is slightly posterior to the rostronasal area. Anterior to the hypophysial foramen and mesial to the pseudobranchial arteries two smaller openings are situated. These can be matched in Romundina ( [20], figures 2B,3D), where they communicate with the dorsal surface of the rostronasal area through two perichondrally lined canals that pass vertically through the cartilage of the endocranium. Their function remains uncertain.
Deeply impressed imprints of paired blood vessels are visible on the ventral surfaces of all the endocrania. The grooves for the efferent pseudobranchial arteries (Fig 5B, pse.a) originate anterior to the hypophysial fossa and follow a curved transverse trajectory similar to that in Romundina [7,21]. Posteriorly there are lyre-shaped imprints of the lateral aortae. The endocranial surface between the grooves of the lateral aortae is rugose. The grooves of the secondary jugular veins (Fig 5, j.v.s) are deeply incised, narrow, and s-curved, and their main mesial branch is attached posterior to the crossing with the efferent pseudobranchial artery. The course of the notochord is visible posteromedially.
Remarks. Gross [36] described some anterior lateral plates of smaller individuals as R. kosorensis. The plates are very similar in shape to the AL of T. inopinatus, but always lack the dermal ornament and other diagnostic features and are always found in isolation. Therefore, these finds (nos. NM Lc 3, 187 and counterpart 472, 465 and counterpart 466; [36], pl. 5, f. 2, textfig. 4B) must be left in open nomenclature.
Genus Sudaspis gen. nov. urn:lsid:zoobank.org:act:4949C1DA-BFD6-4FAE-B85F-EFFED9517D8B (Fig 7) Diagnosis. Jawed vertebrate possessing a broad, flat endocranium with an anterior prenasal expansion of the trabecular region. Prenasal region long. Suborbital shelf narrows abruptly at junction with prenasal region, creating distinct"shoulder" in endocranial margin. Endocranium tapering strongly posteriorly to a narrow glenoid region with prominent condyles. Grooves of lateral aortae on ventral face of endocranium narrow, shallow, together forming hourglass shape, branching anteriorly, ending anteriorly in two clearly visible foramina at level of anterior postorbital process and efferent hyoid artery. Endocranial surface between aorta grooves not rugose. Grooves of secondary jugular veins very obvious, wide, nearly straight, giving off several minor branches laterally, main mesial branch attached anterior to crossing of efferent pseudobranchial artery. Posterior part of skull roof relatively short. Large paranuchal plates form projecting posterior corners of skull roof. Dermal ornament composed of small, close-spaced, round tubercles without ridges.
Derivation of name. After the name of the street "V Sudech" (in barrels) in Prague-Radotín, which runs parallel to the quarries, its name reflecting the characteristic shape of the upper part of the profile resembling three barrels as well as the shape of the endocranium of S. chlupaci; and the Greek "aspis" (shield).
Type and only known species. Sudaspis chlupaci sp. nov. Sudaspis chlupaci sp. nov.urn:lsid:zoobank.org:act:386A13CD-82D5-412F-9B7E-D06015C508DF (Fig 7) 1959 Radotina kosorensis Gross; Gross [36]: pl. 2, fig. 6-7; pl. 3, fig. 5; pl. 4, fig. 1-2 Description. The holotype (Fig 7A) has the posterior part of the dermal skull roof preserved, but this is not easy to interpret. Prominent paranuchal plates, similar to those of Romundina [28], form the posterolateral corners of the roof. The nuchal appears to be narrow and small. It is certainly separated from the paranuchals, but it is not clear whether the intervening space is occupied by a single medial paranuchal (posterior central), as in Romundina, or by a mosaic of smaller bones. Anterior to the paranuchals, the region occupied in Romundina by marginals, anterior paranuchals and centrals [28] appears to consist of a greater number of smaller bones. A full interpretation of the skull roof must await the discovery of additional material, but it is already clear that the skull roof of Sudaspis is diagnostically different from those of both Tlamaspis and Radotina. The dermal ornament is composed of small, close-spaced, round tubercles without ridges.
The facial region of the endocranium is differently proportioned to Romundina, Radotina and Tlamaspis. As in Tlamaspis, the length of the facial region is just over 50% of the total endocranial length, contrasting with approximately 42% in Romundina, but in Sudaspis the extra length is due entirely to the elongated prenasal region; the proportions of the postnasal region are similar to Romundina [21] and Radotina. The suborbital shelf narrows abruptly at its anterior end, creating a distinct"shoulder" in the endocranial margin. Posteriorly, the endocranium tapers strongly to a narrow glenoid region with prominent condyles. The shallow, narrow, anteriorly branching grooves for the lateral aortae form an hourglass shape. The area between these grooves is smooth, not rugose as in Tlamaspis, but further anteriorly there is a low and narrow midline ridge flanked by numerous small transverse "scratches", that extends forwards towards the hypophysial region. The secondary jugular vein grooves are wide and nearly straight, branching laterally and mesially, the main mesial branch attaching anterior to the crossing with the canal of the efferent pseudobranchial artery. A transverse vascular groove running medially from the postorbital processes and crossing the jugular vein canal is interpreted as transmitting the efferent hyoidean artery. The posterior postorbital process is visible but difficult to interpret in the available material. Posterior to this process, the occipital region is both longer and more strongly tapering than in Tlamaspis. It shows a curious double margin, with an external crista on each side that posteriorly joins with the condylar region of the occiput. This clearly reflects an anatomical feature, but it is not possible to say whether it retains its natural shape or has been modified by dorsoventral compression.
Order and Family indet. Genus Holopetalichthys von Koenen, 1895 (Fig 8) Diagnosis. Jawed vertebrate with perichondrally ossified endocranium. Endocranium with long preorbital region incorporating fused nasal capsule. Dermal skull roof entirely macromeric, tesserae absent. Supraorbital and preorbital margin of skull roof formed by single elongate supraorbital ossification. Paired rostral plates. Posterior end of postpineal plate contacts nuchal plate, separating the central plates in the midline. Central plate elongated, reaches posterior margin of skull roof. The tubercles of the dermal ornament round, composed mainly of bone. Median dorsal plate complex composed of a spine-shaped posterior median dorsal, a broad butterfly-shaped anterior median dorsal, and a posterolaterally positioned plate.
Type and only known species. Holopetalichthys primus (Barrande, 1872) Remarks. The specimens described herein as the genus Holopetalichthys had originally been split into three separate species. Barrande [32] erected the species Coccosteus primus based on specimen NM Lc 40 (Fig 8A). Its generic identification was challenged by Bayer [54]. Specimen NM Lc 136 (Fig 8B) was described by von Koenen [33] as a monospecific new genus and named Holopetalichthys novaki. Gross [35], without knowledge of the published specimens, described NM Lc 33 ( Fig 8D) as a new species of the genus Radotina with the species name tuberculata. Westoll [37] was the first to note the common characters of NM Lc 40 and 33 and united them in a single species using the name Radotina prima. He never mentioned NM Lc 136 or the generic name Holopetalichthys. Ørvig [40] pointed out this fact of priority, but suggested to dismiss the older name as a nomen nudum. Although Denison [25] repeated this suggestion, none of them applied to officially accept the younger name Radotina. However, this would not be possible, as the name Holopetalichtys had been widely used in contemporary literature (e.g. [48]; [41] and references therein). Goujet [10] doubted the determination of R. prima to the genus Radotina, based on the absence of tesserae and the premedian plate, which has been added to the reconstruction by Denison ([25] figure 22C) without supporting evidence. According to Goujet [10] the differences in histology and the shape of the dermal sculpture are strong arguments to exclude the species R. prima from the order Acanthothoraci. However, he did not suggest any taxonomic or systematic solution.
Our examination of the material historically assigned to Coccosteus primus, Holopetalichthys novaki and Radotina tuberculata shows that it represents a single species, which is so different from Radotina kosorensis and R. tesellata that assignation to Radotina is out of the question. It is also very different from Coccosteus. We establish the combination Holopetalichthys primus for this species. It has already been used by Chlupáč [55,56], with the nomination of a lectotype (NM Lc 40, Barrande's holotype of Coccosteus primus), but without a formal diagnosis; we present a diagnosis to complete the taxonomic process. The assignation of Holopetalichthys to the Acanthothoraci must be regarded as doubtful, given that there is no evidence for a projecting prenasal area or a premedian plate.
Zhu and Wang [57] described a new species of the order Macropetalichthyidae named Holopetalichthys longhuaensis. Their diagnosis is not in accordance with the former description [33], which is not even cited in the paper, and the genus Holopetalichthys is considered a genus novum. Therefore Holopetalichthys longhuaensis must be considered a preoccupied taxon and thus the determination by Zhu and Wang is invalid.
Type horizon. Lower Devonian, Pragian; Prague Formation, upper Koněprusy Limestone. Type locality. Koněprusy near Beroun, south-east of Prague, probably the area of the present day Houba Quarry.
Remarks. Gross [35] described only one specimen, NM Lc 33 ([35], pl. 1, f. 3, textfig. 6A), as Radotina tuberculata. It was known as an anterior part of the skull roof; its posterior part was thought to be lost after deposition, as glue marks were still visible on the fracture surface. We have discovered that NM Lc 33 is a part broken off specimen NM Lc 138, which contains the thoracic region and a synarcual ossification (described but not figured by Westoll [37]). The two pieces had already become separated by the 1950s when Gross started to study the material.
Specimen HMN f 633 in the Humboldt Museum für Naturkunde, Berlin, from the Lower Devonian Taunus Quartzite near Rüdesheim, Rheinish Slate Massif, Germany, is identifiable as the anterior right side of the skull roof of Holopetalichthys. It was decribed as Radotina sp. by Gross [35]. Although its attribution to Holopetalichthys is unproblematic, its incompleteness, together with the fact that it is much larger than the Koněprusy specimens and derives from another locality, means that we cannot confidently identify it as the species Holopetalichthys primus. We provisionally designate it Holopetalichthys sp.
Description. The skull roof is composed of macromeric dermal plates, tesserae are absent. The plates are clearly recognisable by their ossification centres visible in the vascular middle layer (Fig 8A, 8C and 8D). The nostrils are positioned anterior to paired rostral plates and face anterodorsally. The skull roof probably terminated at this point, as we have no record of a premedian plate. The rostronasal capsule was clearly not loosely attached as in Romundina or Tlamaspis, where it tends to become detached from the rest of the endocranium.
The dorsal rim of the orbit carries a narrow, sharply delineated encircling groove, lying close to the orbital margin anteriorly and posteriorly but diverging away mesially from it in the middle (Fig 8B). The preorbital and supraorbital area is formed by a single elongate supraorbital ossification bounded by the supraorbital sensory line mesially, by a suture with the central plate posteriorly, and by the central sensory line groove posterolaterally (Fig 8D). The central sensory line runs towards the ossification centre of the central plate. The paired rostral plates are bounded by the supraorbital sensory lines laterally, and by a suture with the round pineal plate posteromesially. In the pineal area medially to the supraorbital sensory lines lies the pineal plate. The pineal organ was originally positioned in the ossification centre of this plate, but there is no pineal opening.
Posterior to the central sensory line the ossification centres of another two plates are recognisable-the marginal and anterior paranuchal plates [37]. The marginal plate extends posteriorly separating the anterior paranuchal plate from the skull roof margin (Fig 8A and 8D). The anterior and posterior paranuchal plates are separated by the posterior pit lines, but the posterior paranuchal is not well preserved.
The central plates are elongated, oval, wider anteriorly, and do not meet mesially. The obliquely oriented posterior pit lines extend towards the ossification centres of the plates in NM Lc33 and NM Lc 40; in NM Lc 136 they appear to cross the ossification centres and continue towards the posterior meeting point of the suborbital canals. The centrals are separated in the midline by the nuchal plate and the elongated postpineal plate. The supraorbital sensory lines meet close to the ossification centre of the postpineal plate. The nuchal plate is relatively short and widens posteriorly, terminating in a blunt midline point. The sensory lines occupy deep grooves, recessed into to the dorsal surface of the endocranium. The matrix fill of the canal itself is preserved in some places, notably in the supraorbital canals of NM Lc 136 ( Fig 8B); it is surrounded by an extremely thin mineralized boundary layer that appears to represent the membranous walls of the canal. Sensory lines may follow sutures (as in the case of the supraorbital canal following the suture between the rostral and supraorbital plates), but their ends always point towards the ossification centres of bones. The endolymphatic duct opening is clearly visible on the specimen NM Lc 136 (Fig 8B).
Only one plate from the cheek area is known, the submarginal plate preserved as an imprint on the specimen NM Lc 40 (slightly displaced posteriorly; Fig 8A). It has a typical shape for this bone, widening anteriorly with an oval projection dorsally. A fragment of similar submarginal plate occurs also on the specimen NM Lc 145.
The median dorsal plate complex, which at first sight might be interpreted as a single bone, is in fact composed of a spine-shaped posterior median dorsal, a broad butterfly-shaped anterior median dorsal and an un-named posterolaterally positioned plate. The posterior dorsal has a triangular cross section, with a dorsal crest ornamented by oblong ridges (Fig 8C). Specimen NM Lc 138 (ČF 5 of Westoll [37]; Fig 8D) displays a complete thoracic complex preserved in 3D along with isolated trunk shield plates. Overall, the thoracic armour is very short anteroposteriorly, taking the form of a narrow shoulder girdle, somewhat as in acanthothoracids and ptyctodonts. The damaged anterior lateral plate resembles the shape of the AL plates found in Tlamaspis inopinatus or Palaeacanthaspis vasta [22]. Anteroventrally it appears to taper to a point. The anteroventral ramus of the AL is bounded ventrally by a spinal plate, which carries a short posterolaterally directed pectoral spine, and anterior to the spinal plate an interolateral plate with a forked posterior end that clasps the spinal plate. Remarkably, the area ventral to the spinal and interolateral plates is occupied not by a single anterior ventrolateral plate, but by two plates. We tentatively interpret them as the anterior ventral and anterior ventrolateral plates (AV, AVL; Fig 8D), resembling the condition reconstructed in Romundina cf. stellina [28]. The AL plate is ornamented with badly preserved tubercles in the mesial area and on the postbranchial lamina. Dorsally to the AL plate lie two smaller isolated plates, probably the anterior and posterior dosolateral plates. The median dorsal plate complex has the dorsal spine broken off and its inner bone layer is exposed along with the spine cavity (compare the preservation of P. vasta [22]). The median dorsal plate is connected to a robust bony element in the area of the former dorsal spine, the synarcual [37], which appears to be slightly displaced. The synarcual has a joint surface on the posterior end. Medially, there is a band of small openings -the accreted vertebrae. In the vicinity of the MD plate lies a larger, oval plate, probably a displaced suborbital plate.
The surface sculpture of specimen NM Lc 136 ( Fig 8B) is mostly abraded, broken off or destroyed by polishing. Specimen NM Lc 33, also strongly polished, has the tubercles most clearly visible on the rostral plates. They are poorly preserved, but they evidently were rather bulky, not star shaped or striated. Similar tubercles are very well preserved in the imprint of the submarginal plate of the holotype (Fig 8A) and have been examined on a latex peel. They seem to be built mostly of a vascular bone layer, the semidentine is restricted only to the top. This type of tubercle occurs also on the probable spinal plate of the specimen NM Lc 134, which was found in the same piece of rock as the imprint of a median dorsal plate complex of Holopetalichthys. The counterpart of the MD complex is found on specimen NM Lc 135. Median dorsal plate complexes of the same shape can be found on specimens NM Lc 139 ( Fig 8C) and NM Lc 141; both have a distinctive dermal ornament. Specimen NM Lc 136 ( Fig 8B) has a fragment of another plate attached to the posterior border of the skull roof. Its position suggests that it may be part of a median dorsal complex, but it is too incomplete to identify.
Phylogenetic and evolutionary implications
We do not present a phylogenetic analysis in this paper because it would be premature at this stage. Detailed anatomical investigations of all the Lochkovian and Pragian placoderms of the Prague Basin are under way, on the basis of synchrotron microtomography, and will produce large amounts of new phylogenetic data within the next few years. However, our taxonomic findings already have a major impact on the phylogenetic significance of the Prague Basin placoderms, because they show that past interpretations have been based on multi-taxon chimaeras. This is especially important because they have been widely recognized as very primitive placoderms, potentially informative about questions such as the origin of a macromeric skeleton. For example, Moy-Thomas and Miles [48] consider at some length the significance of dermal bone variability in "Holopetalichthys kosorensis", which is described (following [37]) as having a skull roof that shows individual variation between fully macromeric and largely tesselated conditions. But as we have shown above, this "Holopetalichthys kosorensis" is a multitaxon composite that includes aspects of Radotina kosorensis, Tlamaspis inopinatus and Holopetalichthys primus, three very different placoderms, each with its own characteristic (and apparently stable) skull roof pattern. The extreme individual variability posited by Westoll [37] and Moy-Thomas and Miles [48] simply does not exist. Given this unusual situation, with its potential for seriously corrupting phylogenetic and evolutionary data sets relating to early gnathostomes, we urge that all previous descriptions and interpretations of the Prague Basin placoderms be excluded from the current discussion of these questions.
Our taxonomic revision reveals that the Early Devonian of the Prague Basin contains a remarkable richness of acanthothoracids, especially in the Lochkovian where the placoderm fauna can now be seen to consist of at least four acanthothoracids (Radotina kosorensis, Tlamaspis inopinatus, Sudaspis chlupaci and Kosoraspis peckai) with no other placoderm groups known to be represented in the assemblage. This is interesting from an ecological perspective (see below) but also with regard to the evolution of dermal bone macromery.
The last few years have seen a remarkable about-turn as regards the evolution of macromery, from an almost universally held belief that the macromeric skeletons of placoderms and osteichthyans are independently evolved (e.g. [14,49]) to widespread acceptance that they are homologous (e.g. [6,13]). This extends the homologue set of our own macromeric skeleton down into the upper part of the gnathostome stem group, with the implication that the absence of homologous bones in chondrichthyans is secondary [13]. The lower part of the gnathostome stem group comprises jawless bony vertebrates known as ostracoderms. Among these, thelodonts and anaspids are entirely covered with scales, and are thus micromeric in the same sense as chondrichthyans. Galeaspids and osteostracans have dermal head shields that are normally solid one-piece structures in the adult, but are composed of tesserae that fuse late in ontogeny [58,59]. This can also be considered as a type of micromeric skeleton, because the tesserae do not appear to have conserved individual identities, but in osteostracans at least the anterior rim of the head shield is composed of larger, morphologically distinctive bones [59]. Heterostracans have macromeric dermal headshields, but these show no pattern similarities with either placoderms or osteichthyans [49]. However, bands of tesserae are sometimes present between the macromeric bones (notably in the psammosteid Drepanaspis [49]), in a manner very reminiscent of Radotina.
The lack of obvious homologies between the macromeric patterns of heterostracans and placoderms suggests that the macromeric bones that characterize placoderms and osteichthyans evolved de novo within the gnathostome stem group, at approximately the same time as the origin of jaws. However, the head-shield tesserae of osteostracans, galeaspids and some heterostracans are strongly reminiscent of those in acanthothoracid placoderms such as Radotina, raising the intriguing possibility that their presence in acanthothoracids could be a primitive character retained from agnathan ancestors. The fact that many recent phylogenetic analyses recover acanthothoracids as among the most basal jawed vertebrates [7,8,13,19] makes this hypothesis all the more interesting.
Given our limited understanding of the skull roof patterns in some Prague Basin placoderms, especially Sudaspis, and the current lack of microanatomical data for the tesselated regions, it is too early to venture on a detailed interpretation of these patterns and their significance. However, one striking observation that is worth highlighting is the relationship between the premedian plates of Radotina kosorensis and Romundina stellina. Romundina has a unitary premedian plate without obvious sutures, but synchrotron microtomography reveals that its internal vasculature divides into two discrete domains ( [20], figures 1C and 2B). The thick anterior part of the plate contains several superimposed layers of internal vasculature, all with a radial pattern originating at a midline point near the anterior margin. By contrast, the much thinner posterior half of the plate has a non-radial vasculature with a largely anteroposterior orientation, principally occupying the contact surface between the dermal bone and the underlying endoskeleton. In Radotina kosorensis (Fig 2A), the premedian dermal bone cover consists of two discrete components: a premedian plate anteriorly and a field of tesserae posteriorly. The premedian plate shows a clear radial structure, originating from the same place in the anterior midline as the radial vascular pattern of Romundina. This suggests that the seemingly unitary premedian plate of Romundina incorporates the same combination of an anterior plate and a posterior field of tesserae as the premedian dermal skeleton of Radotina, but that the sutures have become obscured. Further data on dermal bone microanatomy, which should emerge from the synchrotron microtomography investigation of the Prague Basin acanthothoracids, will provide a more robust basis for future hypotheses about the evolution of macromery.
Taphonomy
The large number of morphologically diverse and well preserved placoderm specimens from the localities Černá rokle (Lochkovian) and Koněprusy (Pragian) provides a good database for studies of taphonomy and subsequent diagenetic effects. The strongly dorsoventrally flattened skulls with partly exposed endocranial structures represent the best preserved specimens from the Černá rokle quarries. In life, the endocrania were built of unmineralized cartilage, covered by a thin layer of perichondral bone; after death the cartilage decayed and was replaced with sedimentary infill. The endocrania are flattened to a thickness of only a few millimetres [36], contrasting with the three-dimensional preservation of similar endocrania of Romundina in the Lochkovian of Prince of Wales Island [7,28,40] and Radotina tesselata in the Pragian Koněprusy limestone (see below). The endocrania display openings and imprints of nerves and blood vessels. The slabs containing the specimens were usually split in the horizontal plane, so the inner cast of the specimen represents the base of the endocranium, i.e. the inner ventral surface. Although the compression is strong, some prominent features (e.g. the prenasal area of T. inopinatus) have retained their threedimensionality. No endocranium is laterally compressed, even in semi-articulated specimens where the adjacent shoulder girdle is preserved in lateral view (Fig 6). This probably reflects the fact that acanthothoracid endocrania, like those of most placoderms, were rather broad and flat even in life (e.g. [21]) and thus tended to settle in a horizontal position in the sediment.
In most cases, the dorsal endocranial surface is covered by at least the central and nuchal plates of the skull roof. The cheek plates (e.g. the submarginal) are preserved separately. They were not attached to the endocranium as firmly as the skull roof plates and are found associated to the endocranium or the thoracic plates with their ventral surfaces exposed. Also the anterior lateral plates are preserved isolated with the strongly compressed scapulocoracoid always firmly attached. Other thoracic plates are found isolated as well; the median dorsal plate has the spine broken off as a rule.
A number of specimens of Tlamaspis inopinatus show a specific form of preservation where the specimen, despite being flattened, is complete with the head closely associated to the shoulder girdle and the visceral skeleton pushed from beneath the head laterally (Fig 6A and 6B). The process can be reconstructed as a dorsoventral or semi-lateral compression of a complete anterior body of the fish, e.g. head and thoracic shield. The ventral thoracic plates are unknown; the complete posterior body has not yet been discovered. Some specimens show scattered disarticulated body scales associated to separate dermal plates or their fragments.
The postmortem transport of placoderms found in the Černá rokle quarries was apparently only over a short distance. The model of the prevailing taphonomic processes can be reconstructed from the patterns of fragment distribution. The carcasses were being transported to the anoxic basin from a supposed platform by turbidite currents. During the process of decay, before the specimens were covered by sediment, another short distance transport of smaller and lighter parts (e.g. fragments of dermal plates or scales) may have occurred. The strong compression was a part of the subsequent diagenetic process. However, the accompanying fauna, including other fish (e.g. fin spines of Machaeracanthus [44]), does not display such strong compression [41]. In rare cases the specimens display a slight horizontal displacement of the dermal plates (e.g. NM Lc 29; Fig 4B). The overlap increases but the plate position is constant.
The Koněprusy material is preserved three-dimensionally. The most common specimens are skull roofs along with median dorsal plate complexes. The dermal plates are split through their middle vascular bone layer; the surface sculpture was preserved in the missing counterparts, but can be reconstructed from a few areas of natural mould present on the specimens. The recently associated specimens NM Lc 33 and NM Lc 138 (Fig 8D) display a fully preserved head situated inside the thoracic region with adjacent separate plates and vertebral column, all deposited within the infill of an orthocone cephalopod shell.
Palaeoecology
The Černá rokle quarries yielded the most abundant placoderm material, greatly exceeding the number of specimens from the remaining localities of the Prague Basin [31]. It is uncertain whether this abundance is a taphonomic effect, the result of intensive collecting of all fragments during the period of active quarrying, or if the placoderms were genuinely more abundant in the Lochkovian of the Prague Basin. Perner [42] and Gross [36] supposed the existence of a "fish bearing" layer or accumulation exposed at the time of active quarrying, nowadays inaccessible, but probably belonging to a relatively short overlap of the Paranowakia bohemica and P. intermedia tentaculite zones (for more details see [31]). Only scarce fragments of placoderm fossils can be found nowadays. Contra King et al. [12], who argue that phylogenetically basal placoderms were benthic, we suggest that the Lochkovian acanthothoracids were most probably nektonic active swimmers, considering the shape of their crania and trunk armours, and the lateral position of the orbits [25,28]. The environment in the Lochkovian of the Černá rokle is supposed to be relatively deep water [43].
In the bioherm environment of the Koněprusy quarries in the Pragian the small placoderms may represent prey for larger predators (e.g. the acanthodian genus Machaeracanthus found in the same strata [44]). Often the placoderm remains are preserved in the infill of large orthoconic cephalophod shells (e.g. Fig 8B and 8D). The prominence of acanthothoracids in these assemblages is remarkable, especially in the Lochkovian Radotín Limestone where at least four morphologically divergent acanthothoracids (Radotina, Tlamaspis, Sudaspis and Kosoraspis) occur side by side. We are not aware of another example of such an acanthothoracid-dominated placoderm fauna. By contrast, Emsian and later placoderm assemblages from the Prague Basin contain only arthrodires [31].
Conclusions
This paper has significantly changed the view of the systematic structure of the Prague Basin Lower Devonian (Lochkovian and Pragian) placoderm fauna, finally resolving the long prevailing confusion about the variable character states in individual taxa. Four genera (Radotina, Tlamaspis, Sudaspis and Holopetalichthys) have been established and provided with unambiguous diagnoses based on well documented unique characters. The species name Radotina kosorensis Gross, 1950 is now limited to a few specimens that share unique characters with the holotype. Radotina tesselata Gross, 1958 remains a single specimen species. Radotina has a skull roof with large tesselated areas separating the macromeric bones. Tlamaspis is characterised by a very long facial region and a dermal skull roof lacking tesserae. Sudaspis has a long prenasal region with a distinctive, stepped lateral profile. The skull roof is essentially macromeric, including large and prominent paranuchals. We consider all the previously published attempts to reconstruct or interpret the "radotinid" material, apart from references to holotypes, as invalid because they inadvertently combine information from more than one taxon.
We have also resolved the taxonomic confusion following the generic determination of the so-called "Radotina" prima (previously known as Coccosteus primus, Holopetalichthys novaki and Radotina tuberculata) and its problematic position within the acanthothoracids. We establish the name combination Holopetalichthys primus (Barrande, 1872) for a number of specimens displaying the same unique characters. The genus Holopetalichthys is excluded from the acanthothoracids on the basis of the probable absence of the prenasal area as well as other distinct characters listed above. However, it displays many unusual features in its dermal skeleton. We interpret Holopetalichthys as a basal placoderm of uncertain affinities. | 2018-04-03T02:13:10.572Z | 2017-04-05T00:00:00.000 | {
"year": 2017,
"sha1": "f7b3204115237b7319bad4545885de6a655073e1",
"oa_license": "CCBY",
"oa_url": "https://journals.plos.org/plosone/article/file?id=10.1371/journal.pone.0174794&type=printable",
"oa_status": "GOLD",
"pdf_src": "PubMedCentral",
"pdf_hash": "f7b3204115237b7319bad4545885de6a655073e1",
"s2fieldsofstudy": [
"Environmental Science"
],
"extfieldsofstudy": [
"Biology",
"Medicine"
]
} |
235419344 | pes2o/s2orc | v3-fos-license | Characteristics and retention of emergency department patients who left without being seen (LWBS)
A retrospective observational study was conducted for patients 18 years or older presenting to a Midwestern emergency department (ED) in the United States during February 2019–January 2020 to characterize associated subsequent care utilization in patients who left the ED without being seen. Patients were classified as left without being seen (LWBS) based on documented ED disposition. The healthcare system’s records were reviewed for any associated utilizations within 3 weeks following the initial ED encounter. During the study period, 45,456 patients presented to the ED, with 2269 (5.0%) classified as LWBS. The median documented time until patients left the ED was 112 min. Of these patients, 1257 (55.4%) had a subsequent encounter within the health system within 3 weeks and 920 (73.2%) of these visits were determined to be related to the LWBS chief complaint. These visits included 67.5% of patients returning to ED or hospital, 27.5% to primary care or an urgent care clinic, and 5.0% to a specialty or other provider appointment. Of patients returning to ED, 78.1% did so within 72 h. Patients without a subsequent health system associated encounter tended to be younger, female, non-White, and present with possible lower-acuity chief complaints. At least one-half of LWBS patients sought care related to the concerns by a health system provider within 3 weeks of the initial encounter within the same system. The high prevalence of ED returns within a narrow turnaround window highlights a missed opportunity to provide services to these patients during their initial encounter. Supplementary Information The online version contains supplementary material available at 10.1007/s11739-021-02775-z.
Background
The Emergency Medical Treatment and Labor Act (EMTALA) was introduced in the United States in 1986 and states that patients are entitled to a medical screening/ examination by a qualified provider when presenting to an emergency department (ED). This examination includes diagnosis, stabilization, and treatment of any emergent medical concerns and precludes referral to an urgent care or primary care office before ED evaluation [1]. Given the wide range of concerns that patients may present with, personnel working in the ED attempt to identify and triage patients based on their acuity. Unfortunately, when coupled with high patient volumes and/or acuity, this process can create substantial wait times for many patients-especially those with lower acuity of illness [2,3].
While waiting for care in the ED, patients may opt to leave before being evaluated by a qualified provider. These patients are commonly referred to as 'left without being seen' (LWBS). In the literature, Johnson et al. interviewed 72 LWBS patients and found 77% of these patients left due to wait time, 12% due to concern self-resolving, and 11% for reasons such as staff demeanor, boredom, or other [4]. While this research highlights some of the reasons a patient may choose to leave the ED, the impact of LWBS 1 3 rates on subsequent healthcare utilizations and patient retention has not been well studied.
Importance
The rate of patients who LWBS from an ED is a metric that represents the accessibility of emergency care [5,6]. Given the wide range of reasons why patients may leave without being seen, it is unclear if leaving necessarily impacts the patient's health or their subsequent access of medical care in a particular healthcare institution. For example, if patients presenting to an ED choose to leave without being seen, they may seek care elsewhere within the network or potentially go without care. A particularly problematic scenario is the patient who leaves without being seen with an undiagnosed and untreated emergent medical condition may experience adverse health outcomes due to the delay [7,8]. However, if patients present to an ED with a lower-acuity complaint and leave to seek care at another facility or because their concern has self-resolved, their leaving should have no direct impact on their health outcomes. Similarly, patients who present with lower-acuity complaints may come to the ED if it is the only available option given the day/time but would otherwise have sought treatment from a primary care provider or in an urgent care setting. For patients in these scenarios, they may be less likely to seek subsequent care as it simply may not be necessary. Because of this, a greater understanding of LWBS patients would be helpful in determining how common these various scenarios are for patients and for institutions which track LWBS due to their concern for accessibility-to-emergency care and the financial implications of patients' subsequent patronage.
To this last point, LWBS patients in the United Stateswhich emphasizes a private institution-based business-model for healthcare delivery-also represent a unique population of patients who may seek care at an alternative healthcare institution in a competitive healthcare marketplace; because of this, it is certainly possible for LWBS patients to directly impact the financial situation for EDs and health systems. For these reasons, a greater understanding of the characteristics of LWBS patients, the frequency of their retention within the same organization, and the care they subsequently receive is beneficial to EDs, especially EDs in the United States.
Goal of the study
To characterize associated subsequent care utilization within a healthcare system for patients who left the ED without being seen.
Study design and setting
A retrospective observational study was conducted involving the review of electronic medical record (EMR) data for patients 18 years or older presenting to a Midwestern ED in the United States between February 2019 and January 2020. The facility is a level I trauma center in an urban community-setting, consisting of 41 acute ED beds and an annual adult ED volume of 45,000 visits. The ED is part of a multi-state health system, with all facilities using EPIC (Epic Systems Corporation, Verona, WI) as the centralized EMR. The local community also has five other public EDs, three of which are not in the health system and had inaccessible EMRs.
Study protocol
Charts were reviewed for patients with an ED disposition of LWBS and reviewed for any ED, hospital, or clinic visits within the same health system during the 3 weeks following the initial ED encounter. The health system includes a network of clinics and two additional smaller EDs in the immediate area; while patient records include visits to these other facilities, subsequent visits to other non-health system local clinics/EDs could not be captured. The first documented subsequent visit in the health system within the 3-week period was recorded and reviewed by study personnel to determine whether the care was associated with the reported chief complaint at the initial ED LWBS encounter. These visits were then categorized into three groups: returns to the ED; family medicine/urgent care visits; or specialty/other provider appointments. Also documented was whether the patient was admitted to one of the three local network hospitals within the reviewed 3-week period, as well as if those with a subsequent ED visit did so within 72 h.
Recorded patient data included demographic information such as age, gender, and race, as well as individual wait times and the presenting ED chief complaint. Chief complaints were originally recorded as a free text field and were reviewed and collapsed into nine categories: abdominal pain/gastrointestinal (GI); chest pain/dyspnea; gynecological/genitourinary (Gyn/GU); HEENT (head, eyes, ears, nose, and throat); immune response/infection; injury/musculoskeletal pain; neurologic/headache; psychiatric; and other. Categories were established by the research team and were comparable to those used in prior research [10,11]. When a nursing triage assessment was conducted, emergency severity index (ESI) scores (i.e., 1 3 1-5; lower scores representing greater severity) and pain scores (i.e., 10 point analog scale, higher scores representing greater pain) were collected.
Outcome
Primary outcome was an associated local healthcare system visit within 3 weeks of the LWBS encounter.
Data analysis
Patient and encounter data are presented using descriptive statistics, with continuous variables presented as measures of central tendency and dispersion, while categorical data are presented as counts with percentages. Comparisons between LWBS patients with and without an associated encounter were examined using generalized linear regression modeling with a binomial distribution and logit link. Models controlled for all other reviewed covariates. Estimates are reported as adjusted odds ratios (aOR) with 99% confidence intervals (CI). Lastly, an exploratory analysis was conducted to examine the possible relationship between daily numbers of LWBS patients versus ED daily census and counts of daily ED inpatient boarding using multiple linear regression. Visualizations were generated via individual heatmaps of LWBS counts, ED census, and ED inpatient boarding by day of the week versus week of the year. This study received Institutional Review Board approval.
Results
There were 45,456 ED encounters at the study hospital during the 1-year reviewed period, with 2269 (5.0%) of these patients having an ED disposition of LWBS. Descriptive information for these patients is provided in Table 1. Of note, 302 (13.3%) of these LWBS patients left the ED prior to receiving an initial nurse triage assessment and did not have ESI scores, pain scores and vital sign data. Such patients registered with clerical staff at the ED entrance, but due to multiple simultaneous ED check-ins had to wait to see the triage nurse and left prior to this initial clinical assessment being recorded. These likely represent the busiest times in the ED, as the standard practice is to take the checked-in patient immediately to the triage room for nursing assessment. The LWBS encounters included 2098 unique patients, with 112 having 2 encounters and 26 having more than 2 encounters during the sample period.
Of patients seen in the ED, median wait time was 9 min [interquartile range (IQR): 0, 50]. In patients who left the ED without being seen, the median documented time until the patient left was 112 min (IQR: 58, 177). Compared to patients who did not leave the ED, LWBS patients were more likely to be younger, female, Black, and arrive at the ED on weekdays between 8:00 AM and 5:00 PM; however, Hispanic patients were less likely to leave without being seen. While undocumented/unknown race was also significant, this could be due to LWBS patients leaving before all demographic information could be collected. Finally, the chief complaint category of abdominal/GI pain had the greatest association with LWBS status compared to the other categories.
Main results
Of the 2269 LWBS patients, 1257 (55.4%) were found to have a subsequent visit within the same health system during the next 3 weeks, whereas 1012 (44.6%) did not; among those individuals with subsequent visits, 920 (73.2%) were identified as having the subsequent visit associated with their initial LWBS encounter based on comparable chief complaints (Table 2). When examining these first subsequent visits in greater detail, 67.5% of patients returned to the ED, 27.5% visited a family medicine or urgent care clinic, and 5.0% went to a specialty or other provider appointment in the health system. Furthermore, 52.7% of the 920 patients were considered to have returned to the ED within 72 h with a similar chief complaint, while 13.9% were admitted to the hospital for an associated concern at some point during the subsequent 3 weeks.
These 920 patients with an associated visit in the health system during the subsequent 3 weeks were compared with the 1349 LWBS patients who did not have a documented associated visit (Table 3). Older LWBS patients were more likely to have an associated subsequent visit (aOR: 1.08; 99% CI 1.01, 1.15), while patients who were Black (aOR: 0.68; 99% CI 0.50, 0.93), came to the ED during the weekend (aOR: 0.63; 99% CI 0.45, 0.89) or presented with a chief complaint of HEENT (aOR: 0.41; 99% CI 0.19, 0.87) were less likely to have an associated visit during the following 3 weeks.
Among all LWBS patients, 485 (21.4%) had an associated return visit to the ED within 72 h of the initial ED encounter (Table 4). When comparing these patients with all other LWBS patients, differences were comparable to those regarding associated subsequent visits. Specifically, patients who were relatively older (aOR: 1.09; 95% CI 1.01, 1.18) were more likely to return to the ED for an associated concern within 72 h; however, patients who initially came to the ED over the weekend (aOR: 0.64; 95% CI 0.41, 0.97) and initially presented with a chief complaint of HEENT (aOR: 0.30; 95% CI 0.10, 0.86) were less likely to have an associated return to the ED.
In total, 128 LWBS patients were admitted into a hospital within the same health system during the 3 weeks following the initial ED encounter. The median length of stay for these The exploratory examination on the relationship between the number of daily LWBS patients and ED daily census and/ or ED daily inpatient boarding revealed positive correlations. Overall, initial results suggest that every additional patient in census would increase the number of LWBS patients by 0.21 (95% CI 0.18, 0.25); similarly, every additional patient in boarding would increase the number of LWBS patients by 0.27 (95% CI 0.21, 0.34). Estimates controlled for both SD standard deviation, IQR interquartile range, CC chief complaint a Variables collected during nurse's triage were not available for the 301 patients who left prior to assessment. Presented adjusted odds ratios were based on fitting two models: one excluding variables with 'a' superscripts; and one including those variables. Estimates with 'a' superscript included those variables in the model b Adjusted odds ratio based on 10-unit increase in heart rate, 5-unit increase in respiratory rate and O 2 saturation c Precision bounds excluded the null value variables. These findings suggest that roughly five additional patients in census would lead to an additional patient leaving without being seen, as would roughly four additional boarding patients. These three variables are presented using heat maps in Supplemental Figures 1, 2, 3.
Discussion
This analysis by patients who LWBS from a single Midwestern ED over a year found a substantial number of patients continue to seek healthcare within the same organization, and specifically go on to have their LWBS concern addressed through other venues in the same organization.
While there remain concerns with LWBS effects on patients' care quality and outcomes, among other reasons to continue to reduce LWBS rates, these data can help inform administrative strategies to optimize health systems and improve efficiency. This finding builds on the limited data that exist from previous studies on subsequent healthcare utilization of LWBS patients [2]. The effect of specific efforts to mitigate LWBS rates as well as future studies comparing these retention rates to that of patients who did not LWBS are still necessary.
Patients who returned to the ED within 72 h were of particular interest in this study. These patients represent the subset of the LWBS population that should be especially concerning, as the multiple ED visits for the same unevaluated Table 3 Characteristics of emergency department (ED) left without being seen patients stratified by documented associated visit within health system within 3 weeks following ED encounter status, n = 2269 SD standard deviation, IQR interquartile range, CC chief complaint a Variables collected during nurse's triage were not available for the 301 patients who left prior to assessment. Presented adjusted odds ratios were based on fitting two models: one excluding variables with 'a' superscripts; and one including those variables. Estimates with 'a' superscript included those variables in the model b Adjusted odds ratio based on 10-unit increase in heart rate, 5-unit increase in respiratory rate and O 2 saturation, and 1-unit increase in age, pain and severity c Precision bounds excluded the null value concern could result in preventable medical complications and high costs; however, they also represent a degree of confidence in the same institution by the patients to return and not seek emergency care elsewhere. The predictive factors of ED returns found in this study will be important considerations moving forward for EDs wishing to minimize the impact LWBS can have on their health system. Patients who were admitted were much more likely to have their post-LWBS visit be to the ED rather than to a clinic, perhaps indicating the severity/progression of the index ED concern. Patients who LWBS prior to nursing triage subsequently had a lower rate of returning to the ED and being admitted to the hospital. While this may seem to suggest that these patients may be accurate in believing themselves to be well enough to leave prior to triage, we cannot conclude this given our inability to account for subsequent care received at other facilities within the area.
Given that a majority of patients who leave the ED are likely to seek subsequent care within the same system, this could impact providers in a variety of ways. Specifically, recent changes in health policies in the United States have increased support for Accountable Care Organizations (ACOs) and in turn incentivized healthcare systems to not only mitigate costly ED visits but also retain patients within their networks [9]. Patients leaving the ED without being seen may have historically represented a potential loss of revenue, but the opposite may be true in an ACO structure. Thus, substitution to non-ED care after LWBS may Table 4 Characteristics of emergency department left without being seen patients stratified return to the ED within 72 h for an associated concern, n = 2269 SD standard deviation, IQR interquartile range, CC chief complaint a Variables collected during nurse's triage were not available for the 301 patients who left prior to assessment. Presented adjusted odds ratios were based on fitting two models: one excluding variables with 'a' superscripts; and one including those variables. Estimates with 'a' superscript included those variables in the model b Adjusted odds ratio based on 10-unit increase in heart rate, 5-unit increase in respiratory rate and O 2 saturation, and 1-unit increase in age, pain and severity c Precision bounds excluded the null value reduce total medical costs for a health system participating in an ACO. While this scenario is speculative, it becomes important to evaluate patient behavior after LWBS visits in order to understand the likelihood patients will still receive care and whether they will do so within the same healthcare system. Furthermore, this study may be a valuable baseline from which health care organizations' future ventures and responses to LWBS can be gauged in light of changing healthcare economics. Further research is needed to compare the behaviors of the LWBS population to those who are fully evaluated in the ED. Differences in retention rates between these two populations could identify potential incremental effects of LWBS on a health system's market share, but the findings presented here remain valuable for identifying the post-LWBS course within a health system.
Limitations
This study focused on the health system's perspective on patient retention and subsequent provided healthcare after a patient left the ED without being seen, meaning that data could not be captured for patients who received care elsewhere. It did not intend to capture outcomes for all the LWBS patients, but rather focused on the characteristics and predictors surrounding those who continued to return to the same organization. Similarly, the presented study took place within a single region and therefore limits the generalizability of findings, as the demographics, infrastructure, and resources of other communities may impact a patient's post-LWBS care utilization differently. For example, personnel at the study ED do not routinely attempt to contact LWBS patients for follow-up.
Patients who left prior to an initial nurse triage had a limited interaction with the study ED and often provided no data beyond a chief complaint. A nominal amount of patients contributed more than one LWBS encounter to the dataset. The potential dependencies between these encounters were not directly controlled for in models, but indirectly combatted using a smaller alpha value in calculated estimates.
This study looked at data pertaining to ED visits between February 1, 2019 and January 31, 2020, a period just months before the COVID-19 pandemic struck the United States. It is important to consider that EDs and health systems have gone through substantial changes during these unprecedented times and could potentially shift many dynamics such as LWBS rates and subsequent healthcare utilization.
Lastly, the impact of daily ED census and boarding on LWBS counts were examined as daily totals. This means the hour-to-hour variability throughout the course of the day was not examined and could contribute significantly to the relationship between ED inpatient boarding, which tends to peak in the afternoon-to-evening, and LWBS. Hourly data and possible correlations could be a valuable area of further study.
Conclusions
At least one-half of LWBS patients did not go untreated as they sought care related to the concerns by a provider within 3 weeks of the initial encounter. The high prevalence of ED returns within a narrow turnaround window highlights a missed opportunity to provide services to these patients during their initial encounter. | 2021-06-14T13:43:55.799Z | 2021-06-13T00:00:00.000 | {
"year": 2021,
"sha1": "8aae2aaa1b2202a43b7241fbfd6b5b7fe13de242",
"oa_license": null,
"oa_url": "https://link.springer.com/content/pdf/10.1007/s11739-021-02775-z.pdf",
"oa_status": "BRONZE",
"pdf_src": "PubMedCentral",
"pdf_hash": "8aae2aaa1b2202a43b7241fbfd6b5b7fe13de242",
"s2fieldsofstudy": [
"Medicine",
"Political Science"
],
"extfieldsofstudy": [
"Medicine"
]
} |
269036929 | pes2o/s2orc | v3-fos-license | Changes in the list of over-the-counter drugs containing biologically active substances of plant origin which are intended to be applied among pediatric patients in Bulgaria
Over-The-Counter (OTC) drugs constitute a large category found in pharmacies. They are intended to be applied among adult patients and infants as well. They are also used for prophylaxis. The number of OTC drugs containing biologically active substances (BAS) of plant origin which are intended to be applied among children is insignificant. The changes in the list of OTC medicines which are applied among children mainly affect products influencing cold and flu symptoms. These medicines often contain decongestants which are considered to be the main reason for the emergence of a number of unwanted drug reactions. In addition, the list of OTC drugs has been supplemented with new medicines, most of which contain the same BAS of plant origin, but the product has been produced by a different manufacturer.
Introduction
Over-the-counter (OTC) drugs are widely accessible in pharmacies, drugstores and also on various Internet websites.In some pharmacies, these medicines are placed in the free-access area.The specified conditions and other similar conditions serve as a prerequisite for their increased use.
The category of a given medicine, be it a medicine prescribed by a doctor or an over-the-counter medicine, is determined by its safety and effectiveness profile (Khan et al. 2013).The main features that OTC drugs shall have been the following: proven safety; convenient use; the number of the dose units shall be conformed with the duration of the intake; the information contained in the leaflet shall be accurate and complete.
The U.S. Food and Drug Administration, the OTC/ Nonprescription drugs department, the pediatric and maternity health unit and the pediatric therapy department have set the following criteria for the OTC products: they can be used by consumers for self-treatment in an appropriate manner; no medical specialist is required for their safe and effective use; they have a low abuse rate (American Academy of Pediatrics 2020).
In Bulgaria, the Medicinal Products in Human Medicine Act is the normative document regulating the regime of the over-the counter medicines, which has been stipulated in Chapter Seven entitled "Classification of medicinal products" (Ministry of Health Republic of Bulgaria 2023).
In addition, these medicines are easily accessible and the patients/parents themselves become active participants when choosing a medicine and the treatment (Cooper 2013).
Over-the-counter drugs are usually used in cases of non-serious up to medium conditions and also before the provision of medical aid (Siopen et al. 2013).
Over-the-counter drugs are used not only for prophylaxis, but also in the treatment of a wide range of conditions such as a headache, cold, musculoskeletal pains, allergies, nicotine addiction, stomach acid and others (Sansgiry et al. 2017;Lebanova et al. 2020;Schifano et al. 2021).
A number of examples have been found in literature which show that an increasing number of parents give OTC drugs to their children in case of various diseases (Halim et al. 2010), as the reasons and the factors for this have not been completely established (Andritsou et al. 2017).
A conducted study has shown that the most commonly used medicines prescribed by a doctor to be used among children aged 0-18 years are analgesics, antibiotics, dermatological, respiratory and antihistamines.
Some of the over-the-counter drugs are analgesics and also those medicines used to relieve cold symptoms (Kesti et al. 2023).
Aim
Our study analyzes the aspect of the changes that have occurred for a period of three years in the list of the OTC drugs intended to be applied among children.
Documentary method
A documentary analysis of the list of over-the-counter medicines in the Republic of Bulgaria dated 23 rd August 2023.The list has been published on the official website of the Bulgarian Drug Agency.We have examined the OTC drugs with reference to the following indicators: active substance content; age differentiation of the pediatric patients; distribution of the registered OTC drugs in terms of indications for use /nosology.Comparison of the obtained data with those from the list of OTC drugs dated 25 th January 2020.
Statistical method
Statistical data processing was performed with IBM SPSS v.23.The variables with non-normal distribution were compared with the Kruskal-Wallis test and the Mann-Whitney test.The relationship between categorical variables in cross tables was analyzed using χ 2 test.
For the significance level of the null hypothesis, we assumed at P<0.05 at 95% confidence interval.
MS Excel 2016 was used for graphical representation of the results.
Results
The list of over-the-counter drugs is accessible on the official website of the Bulgarian Drug Agency.It is periodically updated.Based on official data, 1077 over-the-counter drugs are included in the list as on 23 rd August 2023.As a result of the conducted analysis of 1077 OTC drugs, we have established that 868 (80.6%) can be applied among pediatric patients, of which 234 drugs (27%) contain biologically active substance/s of plant origin (individually, in combination with another one, in combination with a synthetic substance or a derivative + a synthetic substance), 93 (10.7%) are homeopathic medicines, 525 (60.5%) are synthetic substance (Fig. 1).
We classified the remaining 16 drugs (1.8%) as others for which the summary of product characteristics (SmPC) contains the following information: "To be carefully applied among children!"; "Children aged 6-12 years shall take half a dose, i.e. one tablet".We have also included in the group of the others those OTC drugs with reference to which the following has been stated: "The use of Drotaverin among children has not been assessed in clinical surveys", which has been written about NO-SPA 40 mg tabl, and with reference to DRO-SPAZ 40 mg.tabl, the summary of product characteristic (SmPC) states the following: "The use of Drotaverin has not been assessed in clinical surveys but if it is necessary to use Drotaverin: children aged 12 can be given …".
The distribution of non-prescription drugs containing in their composition only BAS from plant raw materials, a combination of BAS from plant raw materials, a derivative or another version of the composition in which BAS of plant origin is present has been shown in Fig. 2. Compared to the list of OTC drugs as of 25 th January 2020, the number of registered over-the-counter plantbased drugs administered to children was 198 out of a total of 999 (Petkova and Hadzhieva 2021), or their number has increased by 36 products.
According to the active substance content, the number of OTC drugs containing only a plant component, which are intended for use among pediatric patients in the 2023 list was 62 (26.5%) and in the 2020 list it was 57 (29%), with which we have proven that there is no significant difference regarding these over-the-counter drugs.We have also established that among the studied medicines, there has been an increase in the number of those that mostly contain the same plant active substance (e.g.dry extract of the leaves of Hedera helix L., folium), but the product is from a different manufacturer, and also those intended to affect cold and flu symptoms.
We have examined the number of OTC drugs containing BAS of plant origin, a derivative or and in a combined composition that are used for cold symptoms among children based on their age (Fig. 4).
We have established that there was no significant difference in the number of OTC medicines containing a plant component that could be administered in the infancy-early childhood period (0-3 years), the number being as follows: 25 medicines for 2023 and 21 for the year 2020.
In addition, beyond the scope of our study, we have established that in the list of medicinal products without a doctor's prescription in Bulgaria as of 23.08.2023, updated on 20.12.23, there are medicinal products that lack the summary of product characteristics (SmPC) in the register of the official page of the Bulgarian Drug Agency.Also, one medicinal product was entered twice, and in three cases, the order of entry was not respected according to the chronology of the Latin alphabet.
Discussion
Our study has shown that for the year 2020, and also as of August 2023, in the list of over-the-counter drugs used among children, the largest is the share of those intended to affect the symptoms of the common cold and flu.We could define as a positive change the addition of medicines for flu and cold to the OTC list, since children are most often ill with colds and coughs.
Substances of different structure and origin are present in the composition of non-prescription drugs.Herbal medicines have always been the focus of the pharmaceutical industry for a number of reasons.Traditional medicines are generally known to be harmless, more effective with minimal side effects, and easily metabolized and absorbed by the body.Synthetic drugs, on the one hand, provide quick relief, but have many adverse effects and are also more expensive, making them financially unaffordable (Nasim et al. 2022).
Examining the composition of OTC drugs used for the symptomatic treatment of children with flu and colds, we have established that a number of them contain a decongestant and antihistamine (Sharfstein and Serwint 2007).
It should be noted that a number of studies have reported adverse effects following the use of the decongestant and antihistamine combination in children (Isbister et al. 2012;Şahin and Gülen 2015;De Sutter et al. 2022).Antihistamines (such as pheniramine maleate), decongestants (pseudoephedrine, phenylephrine hydrochloride, dimethidine) contained in OTC drugs lead to an increase in the frequency of adverse reactions (Lokker et al. 2009), therefore, the application among children under 6 years of age is limited and should be done only under medical supervision.The use of over-the-counter cough and cold drugs among pediatric patients is widespread in the United States, although patterns of use are unknown, leading to adverse reactions and death of children (Wingert et al. 2007;Vernacchio et al. 2008).Also, the U.S. Food and Drug Administration (FDA) and the Healthcare Consumers' Association recommend that these drugs not be given to children under 4 years of age due to the potential risk (Briars 2009;Shefrin and Goldman 2009).
At the end of 2023, the Medicines Safety Committee (PRAC) of the European Medicines Agency (EMA) made new recommendations for drugs containing pseudoephedrine (European Medicines Agency 2023).Due to the risk of toxic manifestations resulting from the use of OTC drugs containing the combination of decongestant + antihistamine, paracetamol is given to children since birth to affect the symptoms of pain, fever and cold (Meremikwu and Oyo-Ita 2002;Kanabar 2017).
Conclusion
OTC drugs given to children to treat cold and flu symptoms are widely available on the pharmaceutical market in Bulgaria and their number has grown over the past three years.Most of these OTC products contain substances such as pseudoephedrine and, according to the short description of these medicines, they should not be given to children under 6 years of age.
The challenge for the pharmaceutical manufacturers is to find mechanisms to implement OTC drugs that con- tain BAS of plant origin with an anti-inflammatory and antiviral activity so that they can be administered from an early age.
Through our research, we would like to draw the attention of the responsible institutions to correcting certain omissions in the Register of the short descriptions of medicinal products authorized for use, as well as in the list of OTC products which are available on the official page of the Bulgarian Drug Agency.
Figure 1 .
Figure 1.Distribution of the OTC drugs based on the active substance, which are intended to be applied among pediatric patients.
Figure 2 .
Figure 2. OTC drugs containing BAS of plant origin, a derivative or and in a combined composition applied among children aged 0+ -18 years.
Figure 3 .
Figure 3. ОТС drugs containing BAS of plant origin, a derivative or and in a combined composition applied among children with various indications.
Figure 4 .
Figure 4. ОТС drugs with BAS of plant origin applied among children with a cold and flu. | 2024-04-11T15:12:37.489Z | 2024-04-09T00:00:00.000 | {
"year": 2024,
"sha1": "7b7498ba9aa88b4f493758475873cc1fbe188777",
"oa_license": "CCBY",
"oa_url": "https://pharmacia.pensoft.net/article/121793/download/pdf/",
"oa_status": "GOLD",
"pdf_src": "ScienceParsePlus",
"pdf_hash": "45e5c8fb4a4cc8cd9770d11e820dfc9c9f65020e",
"s2fieldsofstudy": [
"Medicine"
],
"extfieldsofstudy": []
} |
249967966 | pes2o/s2orc | v3-fos-license | Critical theory in use: Organizing the Frankfurt School
The Frankfurt School was an interdisciplinary grouping of left-wing thinkers whose contributions to the social sciences and humanities made them one of the most influential groups of scholars from the last century. Their work has inspired decades of critical organizational research. Yet, across this body of thought, few organization theorists have considered the Frankfurt School as an organization. This article argues that we cannot apply Frankfurt School theories to organizations unless we understand how the School managed its own activities. Reading the School’s texts and examining its working practices through historical documents, we show that Frankfurt School thinkers did not ignore everyday organizational tasks, nor did they grudgingly accept them as a practical necessity. Rather, they embraced them as components of a dialectical theory of organizing and society – which we term critical theory-in-use. Defining what it means to be a critical scholar today is, we conclude, not just a matter of reading Frankfurt School theory but also understanding how this research institute endured for so long and had such a significant influence.
Introduction
What does it mean to be critical?In organization theory and the wider social sciences, this question has been asked repeatedly since the 1960s.In the more recent literature on organizations, it has been discussed as a question of identity (Reedy, 2008), political affiliation (Rowlinson and Hassard, 2011), tactics (Spicer et al., 2009), institutional location (Bristow et al., 2017), and the rise and fall of labour markets (Parker, 2015).But could it also be what Burrell (2013) calls a style of organizing?Can we define 'critical' not only in terms of who you think you are, where you work, or what you think, but also in terms of how your thinking is organized?That is, might critical theory also involve the practices that are necessary to create a context for the generation, development and dissemination of critical thought?
It is no coincidence that the Frankfurt School, a key inspiration for critical organization theory, has an organization in its name.Despite being treated in the organizational literature as a collection of 'closely related' writers (Alvesson and Willmott, 1996: 67) with little concern for 'the mundane world of management and organization' (Alvesson and Willmott 1992b: 437), it conceived its own project in organizational terms.It described itself as 'a collective entity and not merely a more or less artificial and haphazard gathering of scientists working in related fields' (Institute, 1944: 10) and aimed, in the words of its second director, to be 'a new type of scientific work organization' (Grünberg, in Jeffries, 2016: 73).
In this sense, the School's theory of organizing is revealed not only in its written texts but is also embodied in the theories-in-use that shaped what it did.In order to demonstrate this, we return to historical and biographical literature as well as primary historical sources that explore the organization of the School, including declassified FBI records (see Tables 1 and 2).Far from being simply lofty theorists, these reveal that the School's membership was trained in business and deeply embedded in administrative affairs.In fact, many of the founding salaried members wrote very little theory but spent their time ensuring the smooth running of the organization.It could be said that the School's practices were focused on organizing and that the production of specific ideas or texts was an outcome of this focus.
To make sense of the apparent contradictions of radical critical theorists spending their time discussing investment opportunities and rates of pay for secretarial staff, we develop a theory of organization from Adorno's (1951Adorno's ( , 1973) ) writing on dialectics.We contend that the School worked with a dialectical theory of organizations based on three premises.First, organizations can be dialectical mechanisms for social progress.Second, organizations can be dominating and emancipatory at the same time.Rather than trying to purify organizations of dominating elements, the route to emancipation involves pushing constraining features of organizing such as hierarchy, contracts and finance 'to the point where they turn back on themselves' (Adorno, 1951: 86).Third, organizations can be designed with these processes in mind, and those engaged in a critical project should be self-aware of this.These three principles call for a shift from what critical theory is about to how it is organized.
Underpinning this critical theory of organizing is a theory of the relationship between critical ideas, organizations and social change that has implications for contemporary debates.It is now common to propose that a new world can be found in the present (Spicer et al., 2009) or in the imaginaries of counter-hegemonic and prefigurative organizations (Parker et al., 2014).The lesson we draw from the Frankfurt School is, rather, that we cannot imagine new forms of organizing within existing conditions, nor individually overcome the current moment.We can collectively create the conditions in which new thinking and new forms of organizing may emerge -without being certain what these will look like at the outset.
To support our argument, we begin by outlining the ways in which the School has been understood, both positively and negatively, as a body of theory by critical organization theorists.This leads to a section in which we read Adorno to frame how the School's organizational theory manifested in its practices.Then, using various historical sources covering the first 25 years of the School's history, we document a dialectics of leadership and collective labour, administration and research, and independence and alliance.We conclude with reflections on what contemporary critical theories of organization can learn from the practical lesson of the Frankfurt School.
The Frankfurt School and critical organization theory
Spreading through the humanities and social sciences from the 1960s onwards, Frankfurt School thinking was put to use in organization theory from the 1980s.In 1985, for example, setting out the 'points of departure for a critical organization theory', Alvesson suggested that 'critical' means 'Frankfurt-inspired' (1985: 117).Likewise, in the foreword to the edited collection that inaugurates Critical Management Studies (CMS), Alvesson and Willmott (1992a: 9) stated that 'the primary focus . . . is upon Critical Theory, in the sense of the Frankfurt School and its followers'.1933-1938 (1938)
Columbia University 38
Notes on the Institute's Activities (1941) British Library 3 Ten Years at Morningside Heights: A Report on the Institute's History 1934History -1944History (1944) ) British Library 36 Early CMS inherited from the School a belief in 'the feasibility and desirability of greater autonomy for individuals, who . . .are able to master their own destinies through collaboration with peers' (Adler et al., 2007: 139), a negative assessment of positivism, mass production, consumerism and technocratic governance (Alvesson and Willmott, 1996) and a desire to question the 'adherence to the ideologies of managerialism, technicism and consumption' within organization theory itself (Alvesson, 1994: 309).It provided early critical theorists of organizations with a body of thought with which to analyse management.
CMS has since developed three broad positions on the Frankfurt School.First, it is used to evidence pluralism.As Granter (2014: 550) points out, the 'first generation of the Frankfurt School' such as Adorno, Marcuse and Horkheimer 'were not the essential touchstone for all'.Rather, Foucault, Laclau and Mouffe, Butler and many other theorists have taken centre stage since.Second, the School has been challenged for overlooking political economy and class conflict (Hassard et al., 2001) and characterized as elitist, needlessly intellectual and pessimistic (King and Land, 2018).In an influential contribution, Spicer et al. (2016: 226) argue that the wave of CMS 'largely inspired by Frankfurt School critical theory' is 'increasingly moribund, offering increasingly little in the way of claims that are academically rigorous, intellectually interesting and practically relevant'.The antagonistic relationship between the School's ideas and management practice, they argue, impedes the impact of critical organization theory.Nevertheless, recent work has found new value in Frankfurt School concepts (see Granter, 2014, for a review).McCann et al. (2020: 435), for example, turn to Marcuse's reflections on university administration to make sense of their experiences in higher education.Granter (2017) argues that Adorno and Horkheimer's ideas about 'rackets', a theory they developed to make sense of their experiences with research funding bodies, illuminates the behaviour of corporate and political actors.Summarizing this position, Granter writes: Although the Frankfurt school appear in critical organization studies texts less frequently than they once did, it is the case that they still offer useful critical perspectives . . .critical theory is uniquely positioned to take account of the ways in which capitalism evolves, but this potential is currently under explored.(2014: 555) Despite these different perspectives, wherever one looks in the literature, critical organization theory has paid very little attention to the organization of the Frankfurt School.In fact, a claimed absence of practical organizational concerns in the School's work was a starting point for the CMS version of critical organization theory.In their influential Academy of Management Review piece, Alvesson and Willmott (1992: 437) argue that the Frankfurt School theorists' preference for lofty theorizing and lack of concern for 'the microdynamics of everyday life, including the mundane world of management and organization' leave their ideas too obscure for managers to understand or translate into their practices.In response, Alvesson and Willmott (1992b: 437) suggest that bringing management knowledge to critical theory and focusing on micro-practices and micro-emancipations will allow 'the ideals of CT [to] be fully realized'.
It seems to us that such interpretations of the Frankfurt School risk misrepresenting their work, even though they have profoundly influenced the development of critical organization theory.From the beginning, thinkers associated with the School researched management and organizational issues.The School began by documenting 'economic history and the development of the labor movement, assembling a unique library of 60,000 volumes and a collection of letters, pamphlets, newspapers and posters on the history of the labor movement in Europe' (Institute, 1944: 2).Later, it innovated largescale field research utilizing focus groups, depth interviews, content analyses and questionnaires to understand the political attitudes of German and American workers and contributed major works on the social psychology of authority and leadership; the organizing principles of nazism and hydraulic societies; and the material conditions of cultural production.Frankfurt School historian Thomas Wheatland (2009: 204) tells us: 'Far from being appendages to the theoretical writings . . . the empirical research projects are inextricable from the social theory that the Institute had been crafting in Europe in the early 1930s'.
Even more compelling for contemporary debates in organization theory is the Frankfurt School members' own engagements with organizational practice.The Frankfurt School is not merely a label for a collection of thinkers but names a single formal organization: The Institute for Social Research.This organization was not just a salon of aloof and detached aesthetes.It did not run itself.Members had formal, salaried posts and led, managed and administered it.This might seem obvious to organizational scholars but it makes the absence of a sustained organizational analysis of the Frankfurt School in organization studies even more surprising.One consequence of this is that, as we have seen, critical organization theory has divided critical theory and organizational practice.Such an interpretation has not only framed developments in the field but also been used to accuse critical organization theorists of hypocrisy (Willmott, 2006).Critical theory, they say, has no place in contemporary organization and management theory or education because it is not practical.This has been a damaging line of attack against the critical project and it has cost some scholars their livelihoods (Parker, 2021).Looking at the organization of the Frankfurt School, then, not only offers the possibility of a more productive reading of the historical record but also reframing critical theory's relationship to organizational practice.So, what was the organization theory of the Frankfurt School?
Dialectics and organizing
Piecing together a single account from the School's written work is very difficult because there were many members over a long period of time and they wrote many things.But, in this section, we use Adorno's (1951) collection of aphorisms, Minima Moralia: Reflections on a Damaged Life, to guide our thinking.This text was published in honour of Max Horkheimer, the long-time Director of the School, and offers ruminations on everyday life as well as trends in administered societies.Our reading of this text is inspired by Claussen's (2008) emphasis on the interplay between biography and theory in Adorno's writing.Based on the idea that Adorno's theory was constructed with a second meaning -a compositional technique Adorno learned from Alan Berg that 'conveys an additional meaning to the connoisseur' (Claussen, 2008: 142) -Claussen argues that references to real experiences and events included in Adorno's texts conveyed definite meanings to fellow travellers.In particular, Adorno's texts Minima Moralia and Negative Dialectics can be read as attempts to theorize Frankfurt School members' intellectual experience.Claussen (2008: 321) notes that Adorno even 'toyed with the idea of giving Negative Dialectics the title On The Theory of Intellectual Experience'.He tells us that Minima Moralia 'can be read as a sustained effort to interpret the experience of the human subject as a source of knowledge ' (2008: 241).
Yet, this is not an abstract subject nor an individual subject.Jäger (2004: 169) suggests that Adorno is not 'identical with the "implied author"' of the text and it is not meant to offer 'genuine observations' of his 'empirical existence'.Rather, the text refers to 'an exiled group existence' shared by members of the School (Claussen, 2008: 239).It 'begins with a reflection on the role of the intellectual, who is introduced as the son of well-to-do parents -the autobiographical element is barely concealed' (Jäger, 2004: 167-168).As it develops, we are presented with 'the bustle of a modern university such as Columbia in New York', where the Frankfurt School was housed during its exile in America, 'and the offices of the screenwriters in Hollywood', where friends and contemporaries of the School took roles in the culture industries (Claussen, 2008: 197).So, although we are not claiming that Adorno's text was written with an explicit theory of organizing in mind, we believe that it reflects the shared experiences of members of the School and offers an account of the organization of their academic labour.
Organizations as sites of dialectics
The first principle we read in Adorno's thinking frames organizations through a dialectical theory of society and knowledge.Following Hegel and Marx, members of the Frankfurt School believed that social and philosophical progress occurs when two opposites are combined in a higher totality.Socially, they conceived of progress as a movement towards greater autonomy of thought and action and, philosophically, they saw it as a process of closer identification between conceptual understandings and the true nature of the social world (Adorno, 1973).Their account of progress depends absolutely on understanding theory and practice as being entwined.In both cases, progress (affirmation) depends on opposition (negation).This is why members abhorred 'anything isolated' and refused to 'affirm individual things in their isolation and separateness' (Adorno, 1951: 16, 71).Indeed, they were highly critical of any form of 'one-dimensional' analysis, existence and action (Marcuse, 1964(Marcuse, /2013)).
Critical theory is, in this sense, not simply critique for the sake of critique.It is a way of achieving affirmation through negation.The critical theorist supports the processes of social and philosophical progress by developing what founding member of the School, Leo Lowenthal, called 'the negative phase of the dialectical process' (in Dubiel, 1981: 146).In this regard, critical theory is what Horkheimer called an act of 'conscious opposition'.It is aware of its social and philosophical purpose.Its aim: . . . is not simply to eliminate one or other abuse . . .its purpose is not . . . the better functioning of any element in the structure.On the contrary, it is suspicious of the very categories of better, useful, appropriate, productive, and valuable, as these are understood in the present order, and refuses to take them as nonscientific presuppositions about which one can do nothing.(Horkheimer, 1937: 207) For Adorno, an individual may adopt a critical attitude but, as a product of their society, they cannot see beyond the current moment and articulate what a better society should look like.Gestures towards freedom, such as promoting democratic organizations or insisting on intellectual autonomy, affirm one aspect of contemporary understandings by negating another, such as hierarchy or collectivity.Social and philosophical progress can only happen through the negation of the negation, or the refusal of seductive but partial ideas.It is not simply a matter of proposing a better practice but of questioning what 'better' might mean.
On this point, Adorno (1973: 32) argues against a 'bureaucratic way of thinking' that engages with alternatives as if they were proposals going to a committee for approval.He suggests that when we are dealing with social and philosophical progress, accepting an alternative is an acceptance of the system that made it alternative.Adorno's view of dialectics postpones such judgements.The legitimacy of a practice or of knowledge, he tells us, only 'emerge[s] from it at the end' of a synthesis not at the start.As Adorno wrote to Horkheimer, 'only if the entire system were to change could change be approved of ' (cited in Claussen, 2008: 357).This is one reason why members of the School were hesitant to advocate particular policies and is also one of the key distinctions between Adorno and Hegel's dialectical thinking.Instead of seeing a universal truth being revealed through dialectical processes, Adorno's view is that this itself needs to be negated.This is reflected in one of the most oft-quoted aphorisms in Minimia Moralia: 'The whole is false'.
Adorno's treatment of solidarity illustrates this view.He explains that although solidarity appears to represent 'the most honourable mode of conduct' in which the individual both finds themselves and rises above themselves, in the version produced in 20th-century Germany, Russia and the USA, it 'polarized into desperate loyalty'.Far from offering security, solidarity negated itself into a 'permanent fear', with the result that 'the strength that might have been used to test the enemy's weakness [was] wasted in anticipating the whims of one's own leaders, who inspire more inner trembling than the old enemy' (Adorno, 1951: 51-52).
If progress is to be found when contradictory objects combine into a new form, and it cannot be found in the isolated individual nor some declamation about a new future, what hope is there?We think that this is why the Frankfurt School had such an enduring interest in organizations and, as we will demonstrate, why they were so reflexive about their own organization.Organizations are an intermediate category that combines individuals into collectives.They are sites of dialectics between individuals and society.This may help explain Horkheimer's suggestion that the distinguishing feature of critical theory is its subject, not its object.This subject is not to be understood as: . . . the isolated individual nor of a sum-total of individuals [but] a definite individual in his real relation to other individuals and groups, in his conflict with a particular class, and, finally, in the resultant web of relationships with the social totality and with nature.(Horkheimer, 1937: 212) Frankfurt School theory emphasizes the importance of organizations as spaces for dialectical processes that allow individuals to combine into new social arrangements and produce new ways of knowing and being.It positions organizations as both objects and subjects of critical theory.
Self-contradiction
The second principle we read in Adorno builds on the School's idea of 'one-dimensionality'. Accepting that social forms such as organizations, solidarity and so on are both emancipatory and regressive, Adorno reframes the dialectic as a process based on 'a potential that waits in the object ' (1973: 14).'We are not', he continues, 'to philosophize about concrete things, we are to philosophize, rather, out of these things' (Adorno, 1973: 33).Instead of negating an object from the outside, then, Adorno's dialectic revolves around the notion of self-contradiction.Adorno writes that we should 'strive, by way of the concept, to transcend the concept ' (1973: 15).For him, 'the dialectic advances by way of extremes, driving thoughts to the point where they turn back on themselves' and this results in 'fresh concepts not yet encompassed by the general pattern' (Adorno, 1951: 86, 67-68).This is the basis of Adorno's (1973) 'negative dialectics'.Although traditional 'dialectics meant to achieve something positive by means of negation', his conception frees 'dialectics from such affirmative traits' (Adorno, 1973: xix).
This pushes against any notion that the School operated at a critical distance.Rather, it saw being 'in the matter' that you are thinking about and trying to act upon as a precondition for action (Adorno, 1951: 16).Adorno writes: He who stands aloof runs the risk of believing himself better than others and misusing his critique of society as an ideology for his private interest . . .The detached observer is as much entangled as the active participant; the only advantage of the former is insight into his entanglement . . .His own distance from business at large is a luxury which only that business confers.(Adorno, 1951: 26) Accepting that organizations can be sites of social progress, the principle of self-contradiction suggests that progress does not come about by denying elements that tend towards domination.It occurs by using them and negating them simultaneously.Attempting to create an organization free from constraint and inequality, for example, is likely to produce organizations that are even more constrained and unequal.Or, refusing to respond to social and historical context in the name of intellectual freedom is likely to make thought even more enslaved to its own history because 'the very movement of withdrawal bears features of what it negates' (Adorno, 1951: 26).The second principle we read from Adorno is self-contradiction and working through of constraints.If social progress occurs through organizations, this principle tells us it occurs through the limiting features of organizations as much as their emancipatory ones.
The organization of thought
The third principle we read from Adorno's thinking emphasizes the construction of knowledge through specific organizations.Generally, Adorno was critical of academic practices which separated the thinking subject from the object of their study.Far from providing objectivity, he believed they obscured the theorists' view of the object.Prominent in his writing here are thoughts about the organization of academic work.
Adorno criticizes the tendency to formalize knowledge into specialist departments which encourages thinkers to become ever more expert in less and less.For Adorno, 'those who throw in their lot with salaried profundity are compelled . . . to be at each moment as naïve as the colleagues on whom their careers depend ' (1951: 66).He also criticizes academic rules and conventions that standardize and decontextualize academic practices.The injunction 'to show explicitly all the steps that have led (to one's) conclusions, so enabling every reader to follow the process through and, where possible -in the academic industry -to duplicate it' is, for Adorno, tantamount to the 'sabotage of thought ' (1951: 80).Genuine knowledge arrives not through the execution of plans and conventions, but through a mixture of 'prejudices, opinions, innervations, self-corrections, presuppositions and exaggerations, in short through the dense, firmly-founded but by no means uniformly transparent medium of experience' (Adorno, 1951: 80).
Adorno's criticism of these practices is not directed at the practices themselves.But towards the idea that a separation of the subject from the object of study is both possible and desirable.Adorno rejects both in typically aphoristic terms: 'So great is the advancing organization of thought, that those who want to keep outside it are driven to resentful vanity, babbling self-advertisement and, finally, in their defeat, to imposture ' (1951: 67).True autonomy of the thinking subject is only possible, for Adorno, through its integration with the object of study.He writes: The more critically we see through the autonomy of subjectivity . . .and the clearer our awareness of its own mediated nature, the more incumbent is it upon our thinking to take on what lends it the solidity it does not have in itself.(Adorno, 1973: 39) Here, Adorno's idea has particular resonance for thinking about organizations as it emphasizes the need for the organization theorist to be aware of the emancipatory and restrictive potential of the organizations they work in.Like Horkheimer (1937), Adorno emphasizes that thinking together with others allows an individual to be 'in the matter' of social relations at the same time as viewing them as an object of critique.It provides a sort of specialization that can focus thought as well as the material support necessary for thinking to take place.As Adorno puts it: 'People who belong together ought neither to keep silent about their material interests, not to sink to their level, but to assimilate them by reflection into their relationships and so surpass them ' (1951: 45).Sharing material interests, and consequently bonding individual interests together into a higher totality such as a formal organization, is a way to support forms of thought that surpass rather than merely reproduce the thinking of any given age.
It is helpful here to turn to Karl Wittfogel, who was at the conference that established the Institute and was a salaried member for decades but is now generally considered part of the outer circle.In his 1957 study Oriental Despotism, he suggests that 'total alienation' occurs when one resigns oneself to reality as it exists and cuts oneself off from it.'Partial alienation', in contrast, is neither complete adaptation nor complete isolation.Wittfogel (1957) explains that: an intellectual may feel himself out of tune with his co-nationals, or in times of crises he may completely reject a social order that apparently has no use for him.In such situations he may know loneliness.But as long as he can join with others of like mind, his alienation from society will be only partial.(1957: 156-157) The difference between total and partial alienation is related to the ability to organize collectively.Partial alienation allows one to be both within and without the object of study, to be simultaneously detached and engaged.Academic organizations can crystallize the definite relations one has with others and, through this, combine theory and practice.We are part of organizations, and at the same time are aware of how they shape our thought.
These three principals -organization as a site of dialectics, dialectic as a process of self-contradiction, and awareness of the organization of thinking -suggest a view of the School that challenges conventional assumptions in critical organization theory.In place of critical distance (Fournier and Grey, 2000) and 'distal judgement' (King and Land, 2018: 1536), Adorno calls for theorists to be embedded in the things they are analyzing.Further, his ideas about the mediation of thinking suggests we can embrace formal organizations to support our work to the point where our thinking partly frees itself from them.Far from demanding that theorists speak from positions of personal and organizational purity, Adorno sees critical theory as a practical activity that embraces tension and contradiction as a route to emancipation.In what follows, we use these principles to make sense of the School's own organizing practices as evidenced in a range of historical sources.
Organizing the Frankfurt School
Substantive discussions of the workings of the School abound in 'a number of good historical overviews' (Scherer, 2009: 30), but as Habermas (in Marcuse et al., 1978) points out, such histories rarely centre on its management and organization.To develop a picture of the working of the School in its first 25 years, evidence has been sought from this historical literature as well as written accounts, published interviews with members and reports produced by the School.Additional primary material covering the School's time in the US comes from declassified FBI records secured through electronic Freedom of Information Requests.Wheatland (2009) notes that the Institute was a frequent target of government investigations as a result of anonymous accusations regarding Communist sympathies among the group's members.Its members were aware of this.Where primary documents are referenced, accompanying document references are included if possible (see Tables 1 and 2).
These sources have been read through an organizational lens.That is, rather than seek to write an authoritative history of the School, references to organizational practices, working arrangements, decision-making, leadership, personnel, accounting and financial arrangements were identified and compared across sources to reveal features of the School as a formal organization.To illustrate them, we begin with an account of its leadership and collective labour, followed by sections on its administration and research, and relationships with other organizations and the wider environment.For both pragmatic and conventional reasons, we have limited our analysis to the School's history up to their return to Germany in the 1950s.This limits the scope of the historical material, but also reflects a break in the School's history.
Leadership and collective labour
The Frankfurt School was originally housed at the University of Frankfurt and was sponsored by a wealthy grain merchant, Hermann Weil, who offered an endowment for equipment, a building and a yearly grant.Frankfurt University was receptive to this offer as 'the citadel of progressive thought in the German university system' (Institute, 1944: 1).It was also suffering 'a period of poverty and financial restriction' (Wiggershaus, 1994: 19).The details of the new research institute were laid out by Felix Weil, son of the School's benefactor.He 'conceived' the organization 'in the fall of 1919' having been politicized by the First World War (Institute, 1944: 2).
Weil insisted that the Institute must have a single leader who had 'dictatorial control' (Wiggershaus, 1994: 21).Here, Marcuse tells us that he found the School to be 'rather hierarchic and authoritarian' (Marcuse et al., 1978: 127).Yet, although the School was hierarchical, it aimed at interdisciplinarity and collaboration.After the death of the first Director, the second Director Carl Grünberg removed existing academic divisions of labour and staffed the School with political activists and academics from every area of the human sciences (Institute, 1938).They were directed to work collectively on common problems.
In 1927, Grunberg resigned because of ill health and was replaced by Friedrich Pollock, who in turn passed the role to Horkheimer in 1930.Lowenthal reported that for the year prior to Horkheimer's promotion, 'a large part of the activity at the institute was devoted to strategic planning, as it were.And we were successful' (in Dubiel, 1981: 141).Horkheimer stated his desire for the Institute to 'organize research projects stimulated by contemporary philosophical problems, in which philosophers, sociologists, economists, historians and psychologists [work] together in permanent collaboration' (cited in Müller-Doohm, 2005: 135).He called for empirical research using statistics, expert reports, content analysis, media studies, close document reading and large-scale field research utilizing observation and questionnaires.
To cement the relationship between the Institute and the University, the Weils funded a professorship for Horkheimer located in the Faculty of Economics and Social Science.This meant the Director not only sat at the top of the School hierarchy but was also the only member with formal connections to the wider academic system.Indeed, according to Wheatland, Horkheimer's leadership position 'resembled the type of authoritarian family that occupied the imaginations of so many Institute members ' (2009: 80).Adorno reflects on this in a letter to Horkheimer.'You have never denied having patriarchal features, but they were sublimated into an extraordinary flair for power relations, and hence for the ability to ensure that you and yours are in position to assert their rights by resisting' (cited in Claussen, 2008: 361).Wheatland (2009) cites Daniel Bell's recollections of the School's interactions in research seminars: The group assembled around a long rectangular table during the seminars . . .Horkheimer, as paternal authority figure, occupied the table's center seat and never smiled . . .After each paper, Institute members would address it in turn.Horkheimer would speak first, followed by Pollock, then Adorno, and so it would proceed.(2009: 80-81) This combination of seemingly opposing tendencies, hierarchy and collaboration, was mirrored in the physical manifestation of the School.A dedicated building, designed by Franz Röckle, a modernist architect, opened in Frankfurt in June 1924.Later described as a 'Fortress of Science' (Roesler, 2012), it included a 36-seat reading room, 16 small workrooms, four seminar rooms and a library with space for 75,000 volumes, and combined modern, open spaces and bare materials with classic styling.Rather than seeking to minimize these architectural differences, the building exaggerated them, each style being pushed to its extreme.Hans Eisler, who later wrote Hollywood film scores for Fritz Lang and co-authored a book with Adorno, commented on this building in a personal letter: 'A stunning building with a large number of rooms in it.It simply cries out for scholarship . . .The ideas that are being produced there are quite different ' (in Claussen, 2014: 70).
Rather than instruct the staff what to research, the Director only insisted that they worked together.Written texts, though attributed to individual authors, were the result of collective labour.As Lowenthal commented: 'This is how the language of critical theory began.In common theoretical work a collective opinion emerged within our group' (Dubiel, 1981: 150).Marcuse provides a more concrete illustration: The problems and the selection of articles were discussed more or less in Horkheimer's office.Anyone who happened to be there participated, Pollock, Lowenthal, they were always there, Adorno came later, I too . . .Every one of the colleagues shared the area here reserved for Horkheimer . . .We discussed it and made the decision.Horkheimer did not dictate: Now you will work on this.(Marcuse et al., 1978: 128) This sentiment is reflected in a 1944 report produced by the School: From its inception, the Institute has held that overspecialization is an ever-present danger to the social sciences.We therefore have found it necessary to work at an integration and mutual fructification of their various branches.[. ..]It has been a standard practice of the Institute, since the Frankfurt days, to meet regularly for discussion of the various problems arising out of separate branches of investigation.Every contribution by any member of the staff, has, prior to publication, had the advantage of frequent discussion and criticism by members representing different disciplines.Thus the Institute has constantly been a collective entity and not merely a more or less artificial and haphazard gathering of scientists working in related fields.(Institute, 1944: 10) So, the School might be remembered through individual theorists but its work practices were self-consciously organized as cooperative and empirical, facilitated through a dictatorial leader who did not dictate.Indeed, it is striking to compare the formal structure of the School around a Director with dictatorial control and Marcuse's insistence that Horkheimer did not dictate in practice.Studies in Prejudice provides a vivid example of this approach in action.Developed with the Berkeley Public Opinion Study Group after the School had moved to California in the late 1940s, it was a deeply empirical project that not only utilized but also innovated quantitative and qualitative social research methods.It resulted in a new construct, 'the Authoritarian Personality', measured through the F-Scale.The social psychologist Gordon Allport (1963: 433-434) explains its influence: 'Literally hundreds of researches have employed the F-scale . ... The findings have been so timely and so impressive that psychologists have devoted much zeal to analysing, checking, criticising, and modifying both the original scale and the theory involved'.According to Adorno, the School's collaborative working style was decisive to the project's success: The conjecture is hardly too far-fetched that whatever The Authoritarian Personality exhibits in originality, unconventionality, imagination, and interest in important themes is due precisely to that freedom.The element of playfulness that I would like to think is essential to every intellectual productivity was in no way lacking during the development of the F scale.We spent hours thinking up whole dimensions, variables, and syndromes as well as particular questionnaire items of which we were all the prouder the less apparent their relation to the main theme was.(Adorno, in Müller-Doohm, 2005: 296) Although there are now debates about the validity of the study and the precise nature of the Institute's contribution, there is no doubt that, organizationally, the project would not have happened without the Institute.As Sanford (1986), the study's main Berkeley contributor noted, 'Dr.Horkheimer was interested in seeing some of the quantitative methods of American social psychology brought to bear upon the theories developed in this institute.In the fall of 1943 he made funds available to our project' (p.211).Institute researchers played an essential role in the research design, data gathering and eventual publication.Sanford, again, writes: Adorno was a most stimulating intellectual companion.He had what seemed to us a profound grasp of psychoanalytic theory, complete familiarity with the ins and outs of German fascism and [. ..] was very helpful when it came to thinking up items for the F-Scale.(1986: 211) This sentiment is reflected in Adorno's own comments on the project.Although he was attributed lead authorship of the project, Adorno wrote that 'everything occurred in consummate teamwork, without any hierarchical aspects' (in Müller-Doohm, 2005: 292).Pollock confirms this in an interview recorded by the FBI.He stated that the study, although 'published by one of our members . . .was really written in our Institute' (FBI 100-106126).
Looking at the structure and working practices of the Frankfurt School, then, we can see that, far from being unconcerned with questions of organization, the School paid a great deal of attention to its own workings.Indeed, when asked whether Horkheimer's position as leader relegated other members to support roles, Marcuse commented that such an interpretation presents 'an unthinkable split, a completely undialectical split' which goes against the way the School worked (Marcuse et al., 1978: 128).In a nice example of self-contradiction, the School embraced authoritarian leadership to promote collaboration.The Director might not tell everyone what to think but could insist they work together across conventional academic divisions.
Administration and research
The Institute was an organization with a salaried membership.Scholars such as Marcuse and Adorno worked for the School for several years before being granted full membership.Erich Fromm was awarded $20,000 in severance payments when he gave up his membership (Wheatland, 2009: 70).As well as its formal members, the School also employed a staff of secretaries.Lowenthal explained that in America the School paid its secretaries $14 a week: 'This is why we had good secretaries: at that time, in Wall Street, they would have earned only twelve dollars' (in Dubiel, 1988: 144).It also formally contracted research teams 'to gather its empirical data' (Wheatland, 2009: 212).In the 10 years after 1934, for example, approximately $200,000 was distributed among 116 doctoral candidates and 14 postdoctoral researchers.Within the membership, there was a formal division of labour.During the period we are concentrating on, Horkheimer was the Director, Pollock acted as business manager, and Lowenthal organized the School's journal.
One reason the Institute could work this way was that many members had a grounding in business and administration.Horkheimer's father was a millionaire who owned several textile factories, and Adorno described Horkheimer's character as having 'the duality of a theoretical and practical talent' (cited in Claussen, 2008: 226).Pollock noted that Horkheimer and he were both 'originally meant to become businessmen and take over their fathers' factories' (in Wiggershaus, 1994: 21).They had undergone commercial training including lengthy tours of factories in England.An anonymous long-time employee of the Institute told the FBI that Herman Weil 'entrusted POLLOCK with a good deal of financial authority' and consciously chose Pollock, 'rather than his son, with the administration of the considerable funds which he gave to the Institute' (FBI 100-26504).Associates of the School also had administrative experience.FBI reports claim that Julian Gumperz, a research assistant who facilitated the School's move to America, made investments for Weil and also 'represents the investments of certain Dutch interests.He is a treasurer and director of Marlow Equipment, Inc . . .which acts as sales agent for a motor-driven exercising machine' .
Crucial to our argument here is that, in executing these administrative functions, the School did not make a strict division between administration and research activities but saw them as part of a dialectical whole, just as Adorno (1951Adorno ( , 1973) ) saw explicitness about material arrangements as the way of giving thought solidity.Certainly, the School's research informed one of their most important administrative decisions: the move to America.Their empirical studies of German workers revealed a latent authoritarianism in manual and white-collar workers that led the School to take the threat of nazism more seriously than some contemporaries.In an interview recorded by the FBI in 1944, Pollock explained that this insight led the School to reorganize their financial arrangements.He stated that 'as a precaution they took [their] funds out of Germany just in time' -this amounted to 'six or seven hundred thousand dollars' (FBI 100-106126).
An FBI report details the administrative work needed to support this move.A new research institute, the International Institute for Social Research, was incorporated 'pursuant to the statutes of Switzerland' with an 'Advisory Committee of American Scientists' from institutions including Yale, Johns Hopkins, Harvard, Duke, Cornell and Chicago (Institute of Social Research, 1944: 3).The constitution of the new Institute was prepared by a New York based law firm (FBI 100-26504).It prohibited 'dipping into principal', meaning that funds had to be secured through investments (Wheatland, 2009: 82).In the late 1930s, these suffered 'substantial decreases' -losing 'approximately four hundred thousand dollars in 1937' (Wheatland, 2009: 215).This led members to 'pray to all the saints that the New York stock market will rise again' (Horkheimer, in Müller-Doohm, When we look at the way the Frankfurt School was administered, then, we see that its members spent a great deal of their time engaged in everyday organizational activities.They sought new sources of income, managed their finances, engaged in human resource management and built networks.Indeed, Claussen (2008) suggests that Lazarsfeld and Merton modelled Columbia's highly-successful Bureau of Applied Social Research on the School administrative structure.So, it is clear that members were not all the ivorytower theorists that they have been portrayed but neither were all members engaged in management, administration and finance.A deliberate division of labour meant that some concentrated more on research and writing, and other members such as Horkheimer, Lowenthal and Pollock acted as 'the Institute's administrators' (Wheatland, 2009: 233).Just as the School's organizational design can be interpreted as a dialectic between hierarchy and collaboration, the School framed administration and research as dependent on each other.This may be one reason why texts such as Minima Moralia and Dialectic of Enlightenment carry dedications to the School's leading administrators.Without this organizational and material support, the texts would not have been written.It granted their theoretical work what Adorno (1973: 39) called 'the solidity it does not have in itself'.
Independence and alliance
From its inauguration, independence from the state and other academic institutions was a continuing theme for the School.It was officially launched with a ceremony as an institute 'at' (not 'of') the university.Claussen (1973) explains: The Weils wanted to secure the survival of the institute both against changes in political majorities and also against any interference by the Faculty of Economics and Social Science, which was far from being well disposed toward the new foundation.(p.77) For Jay, this strategic choice, 'although entailing certain disadvantages, was one of their primary reasons for the [School's] theoretical achievements ' (1973: 4).
For its first two decades, Weil's endowment allowed the Institute to be substantially independent from other institutions.But, although formally independent, the School never pursued a strategy of complete isolation.In addition to its research activities, it established its own journal in 1932 -the Zeitschrift für Sozialforschung.It was 'the mouthpiece of critical theory' (Lowenthal, in Dubiel, 1981: 149) and helped the School to synthesize its work with that of others.Habermas and Marcuse described it as 'the organizational centre' of the School (Marcuse et al., 1978: 129) as it was in editorial meetings for the journal that members engaged in dialogue.As Wiggershaus concludes: 'Exhaustively evaluated and criticized by the other members of the Institute before they appeared, many articles were almost as much collective productions as individual works ' (1994: 26).Indeed, the journal was designed to promote engagement with wider academic communities.It not only published original contributions, some from people outside the university system such as Walter Benjamin, but also included 'an unusually large review section' (Müller-Doohm, 2005: 150).
The School also connected to wider communities through international offices in Geneva, London, Paris and New York -themselves necessitating administration and management.After the School relocated to America, it formed an association with Columbia University, which provided it with a building on 117th Street, utilized its staff for teaching duties and formed research collaboration.However, Horkheimer felt that formally integrating the Institute into Columbia would mean 'destroying the Institute . . .both materially and theoretically' (in Müller-Doohm, 2005: 270).Instead, the School built a network of influence.Paul Lazarsfeld introduced the Institute's studies to luminaries including Robert Merton, David Riesman, C. Wright Mills, Everett Hughes, Alvin Gouldner and Edward Shils.The latter introduced the Institute's work to Harvard social scientists, most notably Talcott Parsons.In 1945, Horkheimer requested a meeting with Gordon Allport because he had taken an interest in their studies on the psychology of anti-Semitism.According to Wheatland: 'By May of the same year, this collaboration was formalized.At a meeting on 18 May, accompanied by Robert MacIver and Hadley Cantril, Allport became a codirector of the anti-Semitism project ' (2009: 250).
The Institute put these networks to good use.Recognizing the importance of connections in securing research funding, a recognition that led Adorno and Horkheimer to formulate their racket theory (Granter, 2017), they included testimonials and advisory boards on grant bids.Wheatland observes: The testimonials, in particular, performed the impressive function of highlighting both the importance of the project and the Institute's capabilities.Especially when one considers the reputations of those being quoted . . .Here were some of the country's prominent social scientists praising both the Horkheimer Circle and its research plans.(2009: 235) Likewise, members of the School did not withdraw from their broader social context.During the War, Kircheimer took on a post at the Office of Strategic Service (a forebearer of the CIA); Marcuse worked at the Office of War Information and the Office of Strategic Service; Neuman acted as Chief Consultant to the Board of Economic Warfare.Adorno worked on the Radio Research Project with Lazarsfeld as a part-time research assistant and conducted empirical studies, including a content analysis of NBC Music Appreciation Hour.He was 'a salaried scholar in an environment with specific performance criteria as well as values and forms of cooperation to which he had to adapt' (Müller-Doohm, 2005: 172).
There was a similar engagement in cultural matters when the School moved from Columbia to California in the mid-1940s.Members lived in the vicinity of émigré artists including Thomas Mann, Bertolt Brecht and Arnold Schoenberg.Adorno wrote extensive notes for Mann that were later used unattributed, but almost word for word, in the text of Mann's Doctor Faustus.Through these connections they mingled with other cultural producers.The Adornos, for example, held 'a large party in honour of Davison Taylor, the director of programming at CBS' (Müller-Doohm, 2005: 300).They met Charlie Chaplin, then probably the most famous star in the world and who satirized Adorno at a party, and were invited to a private showing of his 1947 film 'Monsieur Verdoux' (Claussen, 2008: 165;Müller-Doohm, 2005: 312).
In order to ensure that the School was not isolated from its cultural context it was also willing to self-censor to adapt to its surroundings.The largely Jewish background of its main members was downplayed by editing 'Jewish-sounding names on the Institute's roster' (Jay, 1973: 34) , and: . ..theZeitschrift scrupulously avoided using words like "Marxism" or "communism," substituting "dialectical materialism" or "the materialist theory of society" instead.Careful editing prevented emphasising the revolutionary implications of their thought.[. ..]These changes were doubtless due in part to the sensitive situation in which the Institute's members found themselves at Columbia.(Jay, 1973: 44) The School also modified the presentation of its work during their time in California.The findings of the Studies in Prejudice had painted American workers and society in a negative light and, as early as July 1944, Horkheimer had worried about the reaction of domestic opinion to 'a bunch of foreign-born intellectuals sticking their noses into the private affairs of American workers' (in Jay, 1973: 225).Similar fears led Adorno to insist in 1947 that his name was removed from the cover of Composing for the Filmsa book that was written with composer Hans Eisler.He did not want to be publicly associated with an 'orthodox supporter of Soviet Marxism' (Müller-Doohm, 2005: 314).Horkheimer even embargoed publication of Dialectic of Enlightenment until the 1960s, despite writing it with Adorno in Hollywood in the 1940s (Claussen, 2008).
So, throughout the period we are discussing, the School provided a mechanism through which individual members could exist both separately from wider interests but also engage with them.It was through the School that the members connected with other academic institutions, through the journal that they engaged with wider thinking, and through the branches that they engaged with other academic communities.But they always maintained their organizational and financial independence.They never assimilated fully into other institutions or communities.The organization allowed them, in short, to perform Wittfogel's partial alienation (1957).Looking at the School's relation with other organizations and its wider environment, then, we can see this third dialectic at work.The School was set up as an independent organization yet partial connections were engineered with other institutions.It seems that they understood, as Adorno wrote to Horkheimer, 'not just life's difficulties but also its entanglements ' (in Claussen, 2008: 355).
Discussion: Critical theory-in-use
Describing the organizational practices of the Frankfurt School helps us view their theories in new ways.The first point to note is the extent to which members of the Frankfurt School were tied, personally, financially and psychologically to the Institute.The Frankfurt School was not simply a pragmatic arrangement that allowed a loose collection of thinkers to pursue their individual interests.From the start, it involved a formal membership that was attentive to the organization's structure and working relations.Indeed, we could even reframe the Frankfurt School's theoretical texts as contributions, produced strategically and pragmatically, for the benefit of the organization rather than seeing the organization as a mere background in which individual theorists worked.
This illustrates, for us, that the first premise of our reading of Adorno appears as a theory-in-use in the Frankfurt School (Arygris and Schon, 1974).We call this "critical theory-in-use".It positions organizations, rather than individual academics, as the subject of critical thinking.This is based on the idea that organizations such as the Frankfurt School are sites for progress because definite social relations and shared material interests between individuals can open up space for new ideas and new values to emerge.
So, although it may be common to attribute Frankfurt School texts to specific authors, our interpretation of the historical record tells a different story.Recognizing that texts were coproduced between members, and that prominent intellectual contributors to the School postponed their scholarly activities to administer it, we suggest that protecting the autonomy and possibility of the School was a primary aim of the School's activities.This was often difficult and came at a cost to individual academics.They could, for example, have formally integrated into Columbia, with individuals taking tenured positions, but they chose independence even though this brought them into contact with the racket society of research funding.
Leading on from this, the second point to critical theory-in-use revealed in the organization of the Frankfurt School concerns the practicality of critical theory.They did not experience theory and practice as separate realms.Rather, the Frankfurt School embraced the practical challenges of organizing even when this brought them into contact with what they might consider repressive elements of society.This approach was predicated on the idea that self-consciously dialectical organizations can attain a form of autonomy necessary to produce new, progressive social relations -whatever those new relations might look like.Such means might produce unpredictable ends, not the rolling out of some sort of political strategy which has already been determined in advance.As Lowenthal put it, 'one must always say no to what is happening because it is not happening in freedom . . . the synthesis is to be made by the subjects themselves' (Dubiel, 1981: 146).
As such, when we consider that critical theorists of culture industries sat alongside Hollywood stars at a private screening, critics of state capitalism joined government agencies, abstract thinkers spent their time worrying about the stock market, and influential analysts of authoritarianism worked under a dictatorial leader, it would be easy to dismiss the Frankfurt School as hypocrites.But these were not experiences they denied, hid or rationalized away.Rather, they reveal a critical theory-in-use that valued potentially repressive elements of organizing as the routes to emancipatory practices.This extended to see theory and practice in a dialectical relationship with each other based on self-contradiction.The Frankfurt School embraced the 'living paradox' in the matter of organizing (Adorno, in Claussen, 2008: 355).
Learning from the Frankfurt School here may help contemporary theorists address charges of hypocrisy (Willmott, 2006) and intellectual arrogance (Clegg et al., 2006).Indeed, the Frankfurt School's organizational practices could suggest a set of organizing principles to guide critical theorists of organizations today.But we do not think critical theory-in-use offers a straightforward blueprint to copy.It is easy to imagine a situation in which such a form of organization could prove highly repressive, especially if its various elements did not work dialectically.Hierarchy, for example, can easily lead to blind loyalty and exploitation.Forced inter-disciplinarily could end up being a cacophony, not collaboration.Independence could become no more than aloofness.It is not enough, in other words, to valorize the Frankfurt School's practices as ends in themselves.
On this point, our view of critical theory-in-use challenges the grounds for dismissing the Frankfurt School posited within recent organization theory and recent innovations in critical organization theory.In terms of the depiction of the Frankfurt School in organization theory, we are told that Frankfurt School theorists were only interested in radical macro-emancipation (Alvesson and Willmott, 1992b), that they adopted a position of distanced negativity towards mundane organizational practices (King and Land, 2018) and, consequently, that they support 'little in the way of claims that are academically rigorous, intellectually interesting and practically relevant' (Spicer et al., 2016: 226).These views are accepted because critical organization theory has, from its earliest interpretations to more recent discussions, largely overlooked the formal organization that produced critical theory.For example, contemporary readers are often told that, to be critical, they should challenge 'established, mainstream conceptions of management' by embracing heterdox theories (Adler et al., 2007: 124-125).The problem with this approach is that it fetishizes the theory of critical theory (Cluley, 2014).It assumes that critical scholars can think differently about management and organizations because of a theory, and that their own organizational contexts and working practices are somehow just a background.
Reflecting on his experiences of leading what at the time was a critically-oriented department, Gibson Burrell illustrates the seduction of this sort of position.He concludes that 'it is always easier to write something that is different, than it is to do something that is different ' (2009: 555, emphasis in original).This, of course, is only true if the two things are not related.The view of critical theory-in-use presented here sees such a split as undialectical.Simply presenting a new theory falls into the trap of aloofness, of being outside the matter of the world.Instead, we need to be aware of the integration of theory and practice in the very organization of thinking and be willing to critique the theories revealed through our own practices.In this regard, a fundamental distinction between the Frankfurt School and contemporary critical organizational theory is not a lack of interest in organizing and management but their focus on their own organization as the outcome of their thinking.The evidence we have presented demonstrates the Frankfurt School theorists' deep and ongoing reflections on the organization of their work.
Turning to the challenges critical theory-in-use poses for recent innovations in critical organization theory, we can turn first to what is perhaps the most prominent framing of the relationship between critical theory and practice in contemporary thinking: critical performativity.Spicer et al. (2009Spicer et al. ( , 2016) ) suggest that individual scholars enact social change by engaging specific critical research tactics.They call for 'active and subversive intervention into managerial discourses and practices' through the three tactics: circumspect care, progressive pragmatism and present potentialities (Alvesson and Spicer, 2012).Employing these tactics marks a researcher as critical.If we ask the right questions, reflect on the right issues and address the right audiences, we are critical-even if what we do looks like 'mainstream' theory and supports existing managerial practices.
Our understanding of the Frankfurt School's critical theory-in-use leads us to a very different position.Critical performativity reframes critical theory as an individual practice and implies, in turn, a clear distinction between theory and practice.If we look at the tactics set out in the critical performativity literature, they are largely addressed to the individual researcher.There is little reflection on the wider organizational contexts in which academics perform their labour.This is manifest in guidance for 'would-be critical performativists' such as the need for them to 'ask themselves: "Am I addressing an issue of wider public importance?"'and examples of successful critical interventions cited in the critical performativity literature, such as 'the publication by one of us of an opinion piece in one of the major national newspapers' (Spicer et al., 2016: 234, emphasis added).
Ironically, this focus on the individual as enacting critical practices means critical performativity is relatively silent about the organization of academic work.This prompted Fleming and Banerjee (2016) to observe that the 'silence about CMS's relationship to its own institutional conditions is a significant oversight ' (p, 268).We would go further.Critical performativity might be performative, but it is not critical in Frankfurt School terms.Assuming that an individual academic can go beyond the constraints of the current moment diverges significantly from the School's view of their project as a dialectical one and their organizational practices, which aimed at forming a collective voice and protecting an autonomous organization capable of producing new ways of thinking and acting.
Another strand of the CMS literature that has addressed the relation of critical theory and practice is thinking on alternative organizations.This emphasizes that organizations rather than individuals drive social change.It is explained by Parker and Parker (2017), who tell us that 'the exploration of alternative forms of organization and management, themselves already involved in struggle against a hegemonic present, should be the proper task of a discipline that wishes to engage with the present and remain "critical"' (p.1366).
From the point of view of the Frankfurt School, this idea supposes that we can find new social relations that prefigure, or imagine, the future within the current moment, something that this version of critical theory insists is impossible.Indeed, there is much literature on alternative organizing from the 1960s onwards that shows how prefigurative practices that appear to challenge mainstream practices can end up reinforcing them.King and Land (2018), for example, show how supposedly democratic forms of organizing may end up being used to produce highly undemocratic organizations.This finding would not surprise Frankfurt School theorists, who, as we have seen, would prefer to look for autonomy and emancipation within hegemonic practices.For them, the celebration of 'the alternative' makes the same mistake as those who seek to import novel theories as the basis for their critical interventions.Whereas the former are limited by existing theory, the latter are limited by current practices.The objective of critical theory is, though, to look beyond the current moment.
So, instead of looking outside the research organization for counter-hegemonic practices, or concentrating on our individual research practices, the School's critical theoryin-use encourages us to think about how we organize research itself -as both a practical and theoretical matter.This means thinking about the conditions of possibility for the collective development and application of critical theory and engaging with organizations through and within definite social relations.
Here, critical theory-in-use differs from a final theme in contemporary organization theory.Fleming and Banerjee (2016), pushing back against what they see as the cooptation of real critique by critical performativity, argue: . . . to make a meaningful contribution, the CMS community needs to fight for spaces that enable scholars to 'uselessly' reflect, imagine inconceivable utopias, take their good time to read and reread the canons, lose themselves in lofty theorising and patiently study minute empirical details and texts in order to ask 'big' questions, even those without obvious practical answers.(2016: 273) The argument in this article suggests that such attempts to isolate theory from context are doomed to fail.As Adorno (1951: 57) explains, 'thinking in the forms of free, detached, disinterested appraisal' is 'unable to accommodate' the repressive trends that it claims to critique and this 'annuls such thinking'.Rather, the critical organization theorist must be 'in the matter' of the organizations they make and are made by, embracing its constraining elements and using the contradictions it produces to move towards something new, and not theoretically or even materially cutting themselves off from the organization of their work.This suggests that a space that seems hostile to critical concepts, such as the contemporary business school, could prove emancipatory if organized differently (see Hancock and Tyler, 2005;Parker, 2018).
The Institute achieved this by a leadership focused on collective intellectual labour that promoted multi-method empirical projects as well as dense theory.It saw researchers engage in administration and finance.It operated with an autonomy that brought it into contact with networks and constraints in the administered society it sought to understand.All this was shaped by an understanding of dialectics which insisted that the present must be both inhabited and refused in order to produce a different future.Its legacy today is massive, arguably greater than that of any other social science research institute but ironically often understood as a pantheon of lofty individuals.These are theorists we can learn from, and they have been important to those who have built and institutionalized CMS over the past 30 years, but learning from how they organized themselves is arguably the most important lesson the Frankfurt School has for us today.
Table 1 .
List of primary sources.
Table 2 .
List of secondary sources. | 2022-06-24T15:10:29.417Z | 2022-06-21T00:00:00.000 | {
"year": 2023,
"sha1": "b7a5ea48dac15da9404fc1f3eb433734a4ba93ab",
"oa_license": "CCBYNC",
"oa_url": "https://doi.org/10.1177/00187267221111219",
"oa_status": "HYBRID",
"pdf_src": "Sage",
"pdf_hash": "89f77d4b800135bf0ec9341acedde9b96675d3e0",
"s2fieldsofstudy": [
"History",
"Sociology"
],
"extfieldsofstudy": []
} |
Subsets and Splits
No community queries yet
The top public SQL queries from the community will appear here once available.